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SF 253 .A55

1978

STANDARD METHODS FDR THE EXAMINATION OF DAIRY PRODUCTS

/'y5"3ISSUED TO

SF 253 .A55

1978

STANDARD METHODS FOR THE EXAMINATION OF DAIRY PRODUCTS


/^
,

^1

standard

Methods

for the Examination of

I3mty JPtoduts
14th edition

standard

Methods

for the Examination of

naitp Products
14th edition

Elmer H. Marth, Ph.D.


Editor

Publication Office:

American Public Health Association


1015 Eighteenth Street, N.W.
Washington, DC 20036

Interdisciplinary Books & Periodicals


For the Professional and the Layman

Fourteenth Edition

Copyright

1978

AMERICAN PUBLIC HEALTH ASSOCIATION,

All rights reserved.


ly

No

part of this publication

Inc.

may be reproduced,

graphical-

or electronically, including storage and retrieval systems, without the

prior written permission of the publisher.

lOM 10/78
Number: 78-72892
Book Number: 0-87553-084-2

Library of Congress Catalog


International Standard

and bound in the United States of America


Typography: Byrd Pre-Press, Inc., Springfield VA
Set in: Times Roman, Helvetica
Text and Binding: R. R. Donnelley & Sons Company, Crawfordsville IN

Printed

Cover Design: Donya Melanson Assoc, Boston

MA

INTERSOCIETY COUNCIL ON STANDARD METHODS


FOR THE EXAMINATION OF DAIRY PRODUCTS

Elmer H. Marth, Ph.D., Chairman


Professor of Food Science and Bacteriology, Department of Food

Sci-

ence, University of Wisconsin-Madison, Madison, Wisconsin 53706

A. Richard Brazis,' Ph.D.


Corporate Microbiologist, Research and Development, Fairmont Food
Company. 4123 S. 67th Street, Omaha, Nebraska 68117
Representative: American Dairy Science Association

Warren

S. Clark. Jr., Ph.D.


Executive Director, American Dry Milk Institute and Whey Products
Institute, 130 N. Franklin Street, Chicago, Illinois 60606
Representative: The Dairy Industry

Jim L. Dizikes, Ph.D.


Chemist-in-Charge, Dairy Division Laboratory, U.S. Department of
Agriculture, 610 S. Canal St., Chicago, Illinois 60607
Representative: U.S. Department of Agriculture
William

J.

Hausler,

Jr.,

Ph.D.

Director, State Hygienic Laboratory, University of Iowa,

Iowa

City,

Iowa 52242
Representative: American Public Health Association (Editor, 13th Edition)

Robert T. Marshall, Ph.D.


Professor, Department of Food Science and Nutrition, 203 Eckles Hall,
University of Missouri-Columbia, Columbia, Missouri 65201
Representative: International Association of Milk, Food and Environmental Sanitarians

Don W. Mather, Ph.D.


Manager, Cheese and Dairy Laboratory, Research and Development,
Kraft, Inc., Glenview, Illinois 60025
Representative: The Dairy Industry

'Dr. Brazis served as the representative of the American Dairy Science Association during
1975-1976 while Dr. Richardson was on leave from Utah State University. While serving on the
Council. Dr. Brazis was with the Food and Drug Administration.

INTERSOCIETY COUNCIL

iv

George W. Reinbold.^ Ph.D.


Vice President-Research and Development, Leprino Cheese Company,
1830 W. 38th Ave., Denver, Colorado 80211
Representative: The Dairy Industry

Gary H. Richardson, Ph.D.


Professor, Department of Nutrition and Food Sciences, Utah State University. Logan, Utah 84322
Representative: American Dairy Science Association
William

W. Ullmann,^ Ph.D.

Director, Dairy Control Services Laboratory, 1400

Bamum

Ave., Strat-

ford, Connecticut 06497

Representative: Association of State and Territorial Public Health Laboratory Directors


Project Director

Howard

L. Bodily, Ph.D.

Staff Associate for

sociation, P.O.

Laboratory Programs, American Public Health As128, Midway, Utah 84049

Box

Project Officer

Ralston B. Read,

Jr.,

Ph.D.

Acting Director, Division of Microbiology, Bureau of Foods, Food and

Drug Administration, 200 C

^Dr. Reinbold

was with

the

Street,

S.W., Washington, D.C. 20204

Department of Food Technology, Iowa State University during a

portion of the time he served on the Council.


'Dr.

Uiimann was with the Connecticut State Board of Health and with Diamond-Shamrock

Health Sciences, Inc. during the time he served on the Council.

CONTRIBUTORS

Robert F. Anderson, B.S.


Executive Director, National Cheese Institute, Inc. and American Butter Institute, 110 N. Franklin St., Chicago, Illinois 60606
William L. Arledge, B.S.
Director of Quality Control and Related Services, Dairymen, Inc.,
604 Portland Bldg., 200 W. Broadway, Louisville, Kentucky 40202
Frederick

J.

Babel, Ph.D.

Professor of Food Microbiology, Department of Animal Science, Smith


Hall, Purdue University, West Lafayette, Indiana 47907
Calvin E. Beckelheimer, M.S.

Associate Director of Laboratory, Hygienic Laboratory Division, West


Virginia Department of Health, 167-llth Ave., South Charleston,

West

Virginia 25303

Harry M. Behney, Jr., B.S.


Principal Laboratory Survey Officer, Pennsylvania Department of Agriculture, 2301 N. Cameron St., Harrisburg, Pennsylvania 17042
R.A. Belknap, M.P.H.
Chief, Sampling Surveillance Unit, Division of Microbiology, Food and
Drug Administration, 1090 Tusculum Ave.. Cincinnati, Ohio 45226
Richard H. Bell, Ph.D.

Manager, Quality Control Laboratory, Difco Laboratories,


1058A, Detroit, Michigan 48232

Inc.,

Box

Harold K. Bengsch, B.S.


Chief. Environmental Hygiene, Springfield-Greene County Public
Health Center, 227 E. Chestnut Expressway, Springfield. Missouri
65802
Louis J. Bianco, B.S.
Administration,
Assurance
Quality
Director
of
500 Peshtigo Ct., Chicago, Illinois 60690

Kraft,

Inc.,

D.A. Biggs, M.S.


Professor of Food Science, Department of Food Science, University of
Guelph, Guelph, Ontario, Canada

CONTRIBUTORS

vi

A. Richard Brazis, Ph.D.


Corporate Microbiologist, Research and Development, Fairmont Food
Company, 4123 S. 67th Street, Omaha, Nebraska 68117
Jerry A. Burke, B.S.

Food and Drug AdS.W., Washington, D.C. 20204

Chief, Analytical Chemistry and Physics Branch,

ministration, 200

Street,

Robert Y. Cannon, Ph.D.


Professor of Animal Science, Department of Animal Science, Auburn

Alabama 36830

University, Auburn,

Warren

S. Clark, Jr., Ph.D.


Executive Director, American Dry Milk Institute and
Institute, 130 N. Franklin St., Chicago, Illinois 60606

Whey

Products

Larry L. Claypool, Ph.D.


Vice President, Research and Quality Control Services, Mid-America
Dairymen, 800 W. Tampa, Springfield, Missouri 65805

Roger W. Cochran, B.S.


Chief, Radiological Health Division, State Hygienic Laboratory, University of Iowa,

Iowa City, Iowa 52242

Roger Dabbah, Ph.D.


Microbiology
Associate
Director,
Research and Development,
Travenol Inc., 6301 Lincoln Ave., Morton Grove, Illinois 60053

Jim L. Dizikes, Ph.D.


Chemist-in-Charge, Dairy Division Laboratory, U.S. Department of
Agriculture, 610 S. Canal St., Chicago, Illinois 60607
Lyle E. Eckberg, B.S.

Manager, Analytical Laboratory, Land O'Lakes,


Place, N.E., Minneapolis. Minnesota 55413

Inc.,

614 McKinley

Tinsel L. Eddleman, M.S.

Drug and Dairy Laboratory Division, Indiana State


Board of Health, 1330 W. Michigan St., Indianapolis, Indiana 46112

Director, Food,

J.C. Flake, Ph.D.

Executive Vice President, Evaporated Milk Association,

15

W. Mont-

gomery Ave., Rockville, Maryland 20850


James L. Fowler, D.V.M., M.S.
Director, Live

Oak

Diagnostic Laboratory, Live Oak, Florida 32060

Daniel Y.C. Fung, Ph.D.

Department of Animal Science and Industry, Call Hall, Kansas State


University, Manhattan, Kansas 66502
Stanley E. Gilliland, Ph.D.

Associate Professor, Animal Science Department,


versity, Stillwater,

Oklahoma 74074

Oklahoma

State Uni-

CONTRIBUTORS

Roy

vii

E. Ginn, B.S.
General Manager, Dairy Quality Control Institute,
St., St. Paul, Minnesota 55113

Inc.,

2353 N. Rice

Kathryn Glynn, M.S.


Assistant Director (Retired), Laboratory Division, Connecticut State

Department of Health, P.O. Box 1689, Hartford. Connecticut 06101

Wesley L. Green, A.B.


Milk Laboratory Evaluation Officer (Retired), Indiana State Board of
Health, 1330 W. Michigan St., Indianapolis, Indiana 46206
Lester Hankin, Ph.D.
Biochemist, Connecticut Agricultural Experiment Station, 123 Huntington St.,

New

Claude Harper,

Haven, Connecticut 06504

Jr.,

B.S.

Director of Quality Control

Foods Company, 1526

Eastern and Canadian

S. State St.,

Chicago,

Illinois

Regions, Beatrice

60605

Paul A. Hartman, Ph.D.

Distinguished Professor and Chairman, Department of Bacteriology,

Iowa State University, Ames, Iowa 50011


G.A. Houghtby, Ph.D.

Food and Drug Administra45226


Ohio
1090 Tusculum Ave., Cincinnati,

Microbiologist, Division of Microbiology,


tion,

Charles N. Huhtanen, B.S.


Microbiologist, Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Philadelphia, Pennsyl-

vania 191 18

Glen E. Huskey, Ph.D.


Group Vice President-Processing and Manufacturing, Pet Incorporated
Dairy Group, Box "O", C.R.S., Johnson City, Tennessee 37601
Jane P. Jensen, M.S.
Administrative Assistant, State Hygienic Laboratory, University of
Iowa, Wallace State Office Bldg., E. 9th and Grand Sts., Des Moines,

Iowa 50309
C. K. Johns, Ph.D.

Director (Retired), Dairy Technology Research Institute, Canada Department of Agriculture, R.R. 4, Imperial Harbor, Bonita Springs.
Florida 33923

William Johnson, Ph.D.


Associate Professor, Department of Microbiology, College of Medicine,
University of Iowa, Iowa City, Iowa 52242

Dick H. Kleyn, Ph.D.


Associate Professor of Food Science, Department of Food Science,
Rutgers University, New Brunswick, New Jersey 08903

CONTRIBUTORS

viij

William S. La Grange, Ph.D.


Extension Food Technologist, Department of Food Technology, Iowa
State University,

Ames, Iowa 50011

W.C. Lawton, Ph.D.


General Manager, A & L Laboratories, Inc., 1001 Glenwood Ave.,
Minneapolis, Minnesota 55405

D.A. Lehman, B.S.


Product Specialist
Fishkill,

New

Dairy Division, Foss America,

Inc., P.O.

Box

504,

York 12524

Donald T. Liden, M.S.


Dairy Products Marketing Specialist, U.S. Department of Agriculture,
14th and Independence, Washington, D.C. 20250
Robert T. Marshall, Ph.D.
Professor, Department of

University of Missouri

Food Science and

Nutrition, 203 Eckles Hall,

Columbia, Columbia, Missouri 65201

Elmer H. Marth, Ph.D.


Professor of Food Science and Bacteriology, Department of Food Science, University of Wisconsin-Madison, Madison, Wisconsin 53706
James H. Martin, Ph.D.
Professor and Head, Department of Dairy Science, Clemson University. Clemson, South Carolina 29631
James W. Messer, Ph.D.

Food and Drug AdministraTusculum Ave., Cincinnati, Ohio 45226

Microbiologist, Division of Microbiology,


tion, 1090

Emil M. Mikolajcik, Ph.D.

Food Science and Nutrition, Ohio


Road, Columbus, Ohio 43210

Professor, Department of
versity, 2121 Fyflfe

State Uni-

R.L. Morris, Ph.D. (Deceased)


Associate Director, State Hygienic Laboratory, University of Iowa,
Iowa City, Iowa 52242

George A. Muck, Ph.D.


Vice President, Research and Development, Dean Foods Company,
1126 Kilburn Ave., Rockford, Illinois 61101

Gopala K. Murthy, Ph.D.


Assistant Chief, Microbial Biochemistry Branch, Division of Micro-

Food and Drug Administration, 1090 Tusculum Ave., CincinOhio 45226

biology,
nati,

F.

Eugene Nelson, Ph.D.


Professor Emeritus, Department of Nutrition and Food Science, University of Arizona,

Norman

Tucson, Arizona 85718

F. Olson, Ph.D.

Professor, Department of

Food Science, University of Wisconsin-Madi-

son, Madison, Wisconsin 53706

CONTRIBUTORS

ix

Z. John Ordal, Ph.D.

Professor

of

Food

Microbiology,

580 Bevier Hall, University of

Department of Food Science,


Urbana, Illinois 61801

Illinois,

Vernal S. Packard, Ph.D.

Extension Specialist-Dairy Products, Department of Food Science and


Nutrition, University of Minnesota, 1354 Eckles, St. Paul, Minnesota

55108

John

J. Redys, B.S.
Director, Laboratory Division, Connecticut State Department of
Health, 10 Clinton St., Hartford, Connecticut 06101

George W. Reinbold, Ph.D.


Vice President-Research and Development, Leprino Cheese Company,
1830 W. 38th Ave., Denver, Colorado 80211

Gary H. Richardson, Ph.D.


Professor, Department of Nutrition and Food Sciences, Utah State University, Logan, Utah 84322
Michael H. Roman, A. B.S.
Supervising Dairy Products Specialist, New York State Department of
Agriculture and Markets, Lowville, New York 13367

Gene W. Ronald, M.S.


Manager, State Hygienic Laboratory, Des Moines Branch, 405 E. 7th
and Court Sts., Des Moines, Iowa 50309
William Roth, Ph.D.
Director, Washington State

Blakeley

St., Seattle,

Food and Drug Laboratory, 2900 N.E.

Washington 98105

Arnold C. Salinger, B.S.


Assistant Chief, Division of Microbiology, Laboratories Administra-

Maryland State Department of Health and Mental Hygiene,


201 W. Preston St., Baltimore, Maryland 21203
tion,

Walter D. Schultze, Ph.D.


Microbiologist, Agricultural Research Center-East, Bldg. 173, Agricultural Research Service, U.S. Department of Agriculture, Beltsville,

Maryland 20705
Robert L. Sellars, Ph.D.
Vice President and Director, Culture Research Center, Chr. Hansen's
Laboratory, 9015 W. Maple St., Milwaukee, Wisconsin 53214

John W. Sherbon, Ph.D.


Professor, Department of Food Science, 211 Stocking Hall, Cornell
University, Ithaca,

New York

14853

George Sherman, M.S.


Supervisory Microbiologist, Dairy Laboratory, U.S. Department of Agriculture, 610 S. Canal St., Chicago, Illinois 60607

CONTRIBUTORS

E.L. Sing. M.S.


Executive Director, Moseley Laboratories. Inc., 3862 East Washington
Street, Indianapolis, Indiana 46201
Snyder, Ph.D.
Professor and Chairman, Department of Microbiology, 2095 Basic Sciences Bldg.. West Virginia University, Morgantown, West Virginia

Irvin S.

26506

Donald

I.

Thompson, M.S.
Water Laboratory Evaluation Program, Wisconsin
Laboratory of Hygiene, 465 Henry Mall, Madison, Wisconsin

Chief, Milk and


State

53706
William

W. Ullmann, Ph.D.

Director. Dairy Control Services Laboratory. 1400

Bamum

Ave., Strat-

ford. Connecticut 06497

Carl Vanderzant, Ph.D.

Professor of Food Microbiology. Animal Science Department, Texas

A& M

University. College Station. Texas 77843

Ebenezer R. Vedamuthu. Ph.D.


Chief Research Microbiologist.

Microlife

Technics,

1833-57th

St.,

P.O. Box 3917, Sarasota, Florida 33578

Donald Vesley, Ph.D.


Associate Professor, School of Public Health, 1158
versity of Minnesota, Minneapolis,

Richard

W. Webber,

Mayo

Bldg., Uni-

Minnesota 55455

B.S.

Assistant Chief, Dairy Section, Standards Branch,

PDQD, FSQS,

U.S.

Department of Agriculture, Washington, D.C. 20250


David B. Weddle, Ph.D.
Director of Product
Development, Mid-America
800 W. Tampa, Springfield, Missouri 65805

Farms,

Inc.,

Kenneth W. Whaley, M.S.


Laboratory Evaluation Officer, Division of Laboratory Services, Tennessee Department of Public Health, 425 Cordell Hull Building, Nashville.

Tennessee 37219

Charles H. White. Ph.D.


Director.

Quality Assurance.

Ave.. Rockford.

Illinois

Dean Foods Company, 1126 Kilbum

61101

Robert L. Winslow. Ph.D.


Manager, Corporate Quality Assurance Department, Safeway Stores,
Inc., Oakland, California 94660
Earl O. Wright, M.S.

Executive Secretary and Managing Editor, International Association of


Milk, Food and Environmental Sanitarians, Inc., P.O. Box 701,
413 Kellogg, Ames, Iowa 50010

CONTRIBUTORS
Albert F.

xi

Zimmerman, B.S.

Laboratory Director and Vice President, Quality Control Laboratory,


Industrial Highway, Southhampton, Pennsylvania 18966

Edmund

A. Zottola, Ph.D.

Professor, Department of

Food Science and

Minnesota, 1334 Eckles Ave.,

St.

Nutrition, University of

Paul, Minnesota 55108

PREFACE

The

14th edition of Standard

Methods for

the Examination of Dairy Prod-

Counbetween editions of the book, b.)


making changes in methods between editions, c.) improved coordination between chapters so that instructions in one do not disagree with those in another, d.) an adequate scientific background for making decisions on methods to be included, and e.) doing some laboratory work to develop a needed
method or to verify the adequacy of an existing method. Activities of the
current Intersociety Council were made possible by funds provided by the
Food and Drug Administration (FDA) via contract to the American Public

ucts
cil.

is

the second to be prepared with the guidance of an Intersociety

This approach allows

for: a.) continuity

Health Association (APHA).


After the contract was developed, the editor was selected by APHA and
was asked to organize the Intersociety Council which he would chair. Representatives of appropriate professional societies, industry, the academic

com-

munity, and governmental agencies were appointed and the newly formed
Council had its first meeting late in 1972. Several members of the current

Council had served on the earlier Council that prepared the 13th edition and
thus provided continuity between the two editions. Some of the major activities

of the Council will be described

in the

following paragraphs. Ultimately

their activities resulted in the 14th edition of

Standard Methods for the Ex-

amination of Dairy Products. As was true of earlier editions, this edition of


Standard Methods presents the best currently available methods to test milk
and milk products.
Activities of the Intersociety Council

The

first

edition of

major activity of the Council was to obtain reactions to the 13th


Standard Methods which, in part, would serve as guidance for

changes that are evident in the current edition. Presentations of Council


members followed by often lengthy discussions at several meetings of professional organizations were a major source of suggestions which were considered by the Council at its deliberations. Additional input came from personal contact by Council members with different persons who used the 13th
edition and from interested persons who attended some meetings of the

PREFACE

xiv

Council.

The Council

also

made

a conscientious

eflFort

to

keep potential

users of the 14th edition informed as to major changes that were being considered and offered an opportunity for response by interested persons before

decisions were finalized.

due course responsibility for chapters was assigned to members of the


in turn, with the Council's approval, developed the committees needed to revise the several chapters of Standard Methods. Members of the Council and the chairperson of each committee met before chapters were developed to consider needed changes and to establish a working
policy for the 14th edition. That policy was established for the 12th edition,
was operative for the 13th edition, and guided preparation of the 14th edition
of Standard Methods. It is as follows:
In

Council who.

No new method or modification of an old method should be introduced unless it has undergone careful comparative testing in several laboratories with the data being made available
to the Council and to other interested parties, preferably by publication in a recognized
scientific journal. Notice of intention to include or modify should appear in print, with
enough time allowed for any interested party to submit evidence for or against and to make
recommendations.

Although this was the general philosophy for the I4th edition of Standard
Methods, pragmatic considerations sometimes dictated that decisions about
some methods be made on other bases.
Manuscripts of chapters were submitted to the Council, reviewed, and
returned to authors for needed revisions. All manuscripts were revised once
and some several times before they were finally accepted by the Council.
Two matters relating to manuscripts of chapters were given considerable
attention by the Council. First, the Council seriously considered changing
conditions of incubation for the Standard Plate Count from 32 C-48 hr to
30 C-72 hr. After much discussion the Council concluded that the proposed
method probably would not result in improved products for the consumer
but would cost more than the present method and thus elected to retain
32 C-48 hr for the 14th edition. Second, the Council considered methods
using BaciUus stearothermophdus var. caUdolactis to determine penicillin in
milk and decided that presently all methods using this bacterium should appear in Appendix A. As more information on these methods becomes available, one or several may become "standard."
The Council established a precedent by publishing, in the Journal of Food
Protection (then still Ihe Journal of Milk and Food Technology), a change in
the 13th edition of Standard Methods. This procedure to eflfect changes in
Standard Methods should be useful in the future and should serve to extend
the interval between editions of this book.
Limited funds to support laboratory work were available through the contract between the FDA and APHA. Studies supported with these funds include: use of discs of various sizes for the sediment test, use of a plastic

PREFACE

XV

to enumerate anaerobes in cheese and to prepare samples of hard


cheese for bacteriological analysis, collaborative study on the microtiter
count method, the flooded plate method, comparison of 30 C- 72 hr with
32 C-48 hr for the Standard Plate Count, vapor pressure osmometer to mea-

pouch

sure freezing point of milk, acid injury in coliform bacteria, automated colo-

ny counters, and several others.


The Council considered but did not resolve the problem of approving or
accepting automated equipment which is subsequently modified by the manufacturer. This problem will continue to occur as more automated equipment is developed. The Council must deal with this before the 15th edition of
Standard Methods is developed.
Highlights of the 14th Edition

Changes

in the

following chapters are essentially limited to editorial modi-

improve clarity and readability: Chapter 6, "Coliform Bacteria";


Chapter 7. "Thermoduric. Thermophilic and Psychrotrophic Bacteria";
Chapter 10. "Microbiological Methods for Concentrated Milk and Dry
Milk"; Chapter 11. "Microbiological Methods for Butter, Margarine and
Related Products"; Chapter 13, "Microbiological Methods for Ice Cream
and Related Food Products"; Chapter 14, "Direct Microscopic Method for

fications to

Bacteria"; Chapter 15, "Reduction Methods"; Chapter 16. "Microbiological Tests for Equipment. Supplies, and Water"; and Chapter 20, "Radionuclides

in

Milk".

Major changes

remaining chapters will be mentioned briefly in the


1, "Quality Tests", is largely philosophical in
now contains limited guidelines for doing collaborative

in the

following sentences. Chapter


nature; however,

it

program in the laboratory. "SignifPathogens in Dairy Products", Chapter 2, continues as a review of the
subject and does not provide methods for isolating and handling the pathogens that are discussed. Each section has been updated, the section on
mycotoxins has been completely revised and sections on yersiniosis and toxoplasmosis have been added.
Since sampling of milk and its products is so important if meaningful results are to come from a laboratory. Chapter 3 ("Sampling Dairy and Restudies and for an over-all quality control
icant

been extensively revised to provide suitable guidelines


so adequate sampling can be done. Procedures for sampling milk in bulk
tanks are given in detail. Instructions for sampling of products have been

lated Products") has

to include various coffee creamers or whiteners; margarine and


related foods; and processed, dried and imitation cheeses. The metric sys-

expanded

tem has been introduced in this chapter.


Chapter 4, "Culture Media and Preparation", now contains a section on
quality control in preparation of media and on suitability of water for microbiological application. Other major changes include: a.) buffered dilution water will

now

contain

MgS04

besides KH.PO.,. b.) the interval plating proce-

PREFACE

xvi

dure has replaced the distilled water suitability test, c.) the expression
"microbiologically suitable water (MSW)"' is introduced, and d.) formulae

commercially available culture media have been eliminated.


Major changes in Chapter 5. "Standard Plate Count Method", include: a.)
merit of preliminary incubation (PI) of raw milk samples is mentioned but
the method is described in the Appendix, b.) instrumental colony counters
can be used under certain conditions, and c.) drawings of pipets have been

for

clarified.

The chemical method

to disperse fat in electronic counting of

somatic

cells

has been added to Chapter 8. "Screening and Confirmatory Methods to Detect Abnormal Milk". Hallmarks of Chapter 9. "Detection of Antibiotic

Residues

in

Milk and Milk Products", are retention of disc assay methods

using Bacillus suhtilis and addition of the cylinder plate method that uses

Sarcina lutea. All methods that use Bacillus stearothenuophilus var. calidolactis appear in the Appendix.

Chapter 12, "Microbiological Methods for Cheese and Other Cultured


Products", allows use of a plastic pouch technique to prepare samples of
cheese for subsequent analysis. Discs that expose a filtering area, in di1
Vs, 0.4, 0.2, 0. 14, or 0. 10 inch can now be used for the sediment

ameter, of

"Sediment in Milk". The Rutgers Phosphatase


of the remainder of Chapter 18, "Phosphatase
Methods", has been rewritten. Changes in Chapter 19, "Chemical Methods"
are too extensive to enumerate in this preface. The interested reader should
consult this chapter and Appendix B, "Supplemental Chemical Control
Methods".
Chapter 21, "Alternate Viable Count Methods", now includes the spiral
plate method and a statistical protocol for use by analysts.
It is evident from this brief discussion that a substantial number of changes
were introduced into the 14th edition of Standard Methods even though it
appeared just 6 years after the 13th edition was published. Authors, members of the Intersociety Council, and the editor hope that users of this book
will find the changes beneficial as laboratory workers and others continue in
their efforts to insure that consumers receive safe, palatable, and nutritious
test,

test

according to Chapter

has been added and

17,

much

milk products.

Acknowledgments

More than 80 persons contributed directly toward development of this


book. Their efforts are recognized and appreciated. A special word of appre-

members of the Intersociety Council, to the project direcH. L. Bodily, and to the project officer. Dr. R. B. Read. Jr. Their
help, support, and guidance were immeasurable.
Thanks also go to the secretarial staff of the Department of Food Science
at the University of Wisconsin, and in particular to Ms. Judy Brickner and
ciation goes to the
tor. Dr.

Ms. Nancy Stewart, for help

in

handling manuscripts and correspondence.

PREFACE

xvii

Dr. Elizabeth D. Robinton served as copy editor and prepared the index for
this

book. Her

lications for

Phyllis, for

efiforts

and those of Mr. Allen

J.

Seeber, Director of Pub-

word of thanks to my wife,


her patience and understanding while this project was in prog-

APHA,

are appreciated. Finally, a

ress.

Madison, Wisconsin
October, 1978

Elmer H. Marth, Ph.D.


Editor 14th edition and Chairman of the
Intersociety Council on Standard Methods
the Examination of Dairy Products

for

TABLE OF CONTENTS

Intersociety Council

iii

Contributors
Preface

1.

xiii

Quality Tests
Introduction

Parameters of Quality and Acceptance

Function

//

Standard Procedures 2/ Adopting New Procedures i/ Collaborative Studies on Methods i/ Uniformity of Procedures 41 Split-Sample Tests 51 Integration of Farm and Plant Inspection with Laboratory Control 6/ Relative Accuracy of Methods
for Measuring Sanitary Quality 61 Consideration of Specific Methof Standard Methods

ods
2.

71

//

Quality Control

in the

Significant Pathogens

IN

Laboratory 81 References 9

Dairy Products

11

Introduction /// Bacterial Infections and Intoxications 131 Mycotoxins 221 Viral, Rickettsial and Other Diseases 231 Protozoan Infections 261 References 27

3.

Sampling Dairy AND Related Products


Fluid Milk and

Cream Samples

33

331 Other Dairy Products 421

Sam-

pling for Specific Laboratory Procedures 481 References 53

4.

Culture Media AND Preparation

55

Introduction 551 Basic Steps in Medium Preparation 561 Adjustment of Reaction (pH) 581 Sterilization and Storage 581 Quality
Control 60/Suitability of Water for Microbiological Applications 611

Preparation of Phosphate-buffered Dilution Water and Testing for


Toxicity 62/Cleaning Glassware and Testing for Detergent Residues 641 Formulas of Culture Media and Directions to Prepare Media for Use 651 Physical Standards for Standard Methods Agar 681
Productivity Tests for Standard Methods Agar

ences 74

Medium

691 Refer-

XX

5.

Table of Contents

Standard Plate Count Method

77

Introduction 771 Equipment and Supplies 781 Materials

8,21 Steri-

Examination of Samples 831 Preparing Samples 841 Diluting Samples 851 Plating 871 Sterility Controls of Medium, Dilutions and Equipment 871 Incubation 881 Counting Colonies on
Plates and Recording Results 55/ Computing and Recording Counts
921 Reporting and Interpreting Counts 931 Personal Errors 931 Reflization 831

erences 93

6.

CoLiFORM Bacteria

95

Introduction 951 Definitions 961 General Interpretations 961 Sam-

Media 971 General Procedure 971


Tube Methods 98i Coliform Test with a

pling 971 Equipment, Supplies and

Relative Value of Plate and

Solid Medium 991 Coliform Test with a Liquid Medium 991 Confirmed Test from a Solid Medium 991 Completed Test from a Liquid

Medium

lOOl Reporting Results lOOl Table of

MPN

Coliforms

1021 References 104

7.

Thermoduric, Thermophilic, and Psychrotrophic Bacteria

107

Thermoduric Bacteria 1071 Thermophilic Bacteria 1091 Psychrotrophic Bacteria IIOI References 112

8.

Screening and Confirmatory Methods for the Detection


OF Abnormal Milk
115
Introduction 1151 Sampling 1151 Screening Tests 1151 Confirmatory

Tests 725/ References 139

9.

Detection of Antibiotic Residues


Products

in

Milk and Dairy


141

Introduction 1411 Bacillus suhtUis Disc Assay 1421 Modified Sarcina lutea Cylinder Plate
fat

Dry Milk 1451

Method

NonMethods 1491

for Detection of Penicillin in

Specific Antimicrobial Cylinder

References 149

10.

Microbiological Methods for Concentrated and Dry


Milk Products
151
Evaporated Milk 1511 Concentrated and Sweetened Condensed
Milks 1521 Dry Diary Products 1531 References 156

Table of Contents

11.

xxi

Microbiological Methods for Butter, Margarine and

Related Products

157

Introduction 1571 Microbiological Methods 1571 Sampling Procedure 1581 Bacterial Counts 1581 Yeast and Mold Counts 1591 References 159

12.

Microbiological Methods for Cheese and Other Cultured Products


161
Introduction 161 1 Collection and Preparation of Samples 1621 Microbiological Analyses 1631 References 164

13.

Microbiological Methods for Ice Cream and Related


Food Products
165
Introduction 1651 Equipment and Supplies 1661 Sampling Proce-

dures 766/ Preparation of Samples for Plating 1661 Standard Plate

Count 1671 Test for Coliforms 1681 Psychrotrophic Count 1681


Yeast and Mold Count 1681 References 168
14.

Direct Microscopic Method for Bacteria


Introduction 1691 Applications to

Raw

169

Milk to be Pasteurized 1691

Applications to Pasteurized Milk 1691 Applications to Dry Milk 1701


Sources of Error in Method 1701 Bacterial Estimates 1701 Interpreting and Reporting Bacterial Counts or Grades 1711 Procedure
for Collecting

Samples 1721 Equipment and Supplies 1721 Materials

1781 Illumination Adjustment 1781 Adjustment and Calibration of

Microscope 1801 Calculation of Microscopic Factor 1811 Derivation


of Working Factor 1811 Using Transfer Instruments and Preparing
Films 1821 Handling of Slides during Staining 1831 Preparation and

Use of

Stain 1831

Examining Films 1841 Standards 1851 References

185

15.

Reduction Methods

187

Methylene Blue Reduction Method 1871 Resazurin Reduction

Method
16.

1911 References 194

Microbiological Tests for Equipment, Containers, Wa197


ter and Air
Introduction 797/ Sample Collection 797/ Tests for Sanitation of
Equipment 797/ Standard Tests for Water Supplies 2031 Tests for
Microbiological Quality of Air 2041 References 204

Table of Contents

xxii

17.

Sediment

IN

Milk

207

Introduction 2071 General Methods 2081 Equipment and Supplies


2081 Preparation and

Use of Standard Sediment Discs 2101 Proce-

dure 2101 References 211

18.

Phosphatase Methods

213

Introduction 2131 General Precautions 2141 Controls Applicable to


All Phosphatase Procedures 2151 Scharer

Rapid Phosphatase Test

Method 2201 CorPhosphatase Test 222/ Rutgers Phosphatase Test 225/ Phosphatase Reactivation in Dairy Products Heated by High-heat Shorttime and Ultra-pasteurization Methods and Ultra High-temperature
Methods 2261 References 228

2171 Modified Spectrophotometric Phosphatase


nell

19.

Chemical Methods
Introduction 2311

231

Added Water

in

Milk 2311 Available Chlorine:

Thiosulfate Titration 2i5/ Fat 2i6/ Moisture and Solids 252/ Organin Milk (Examination for Multiple
Residues)26y/ Protein269/ Fat, Protein, Lactose: Infrared Spectrophotometry 2771 References 281

ochlorine Pesticide Residues

20.

Radionuclides

IN

Milk

283

Introduction 25i/ Sampling 254/ Milk Preservation 254/ Determination of ^^Sr and ^^'Sr in Milk by Ion Exchange 2841 Determination of
89Sr and "^^Sr in Milk by

'"Cs

21.

in

Milk by

TCA295/ Determination

Gamma Ray

of '^'I, '^"Ba and


Spectrometry 3041 References 309

Alternate Viable Count Methods

311

Introduction i/// Collection and Storage of Samples Before Testing

3111 Statistical Protocol for an Analysti/// Oval Tube Methodi/2/


Plate

324

Loop Method i 75/

Spiral Plate

Count Method i/ 7/ References

Table of Contents

xxiii

Appendixes
A.

Supplemental Microbiological Control Methods

327

Aureomycin-Rose Bengal Agar Method to Detect Molds and


Yeasts 327/ Citrate-Azide Agar Method for Enterococci in Butter
328/ Cylinder Count Method to Determine Plate Count of Pasteurized Milk Products 329/ Detection of Sulfa Drugs and Antibiotics
in Milk 330/DetecUon of Penicillin in Milk by a Disc Assay TechniqueInternational Standard Fil-IDF 57: 1970 ii// Modified IDF
Disc Test for Penicillin in Milk ii5/ Delvotest F338/ BR Foss Test
341 / Disintegration Method to Determine the Microbial Content of
Paper Container Materials i44/ Moseley Keeping-Quality Test 347/

Raw

Milk 347/ Screening Method


(Nutrient Broth Modification) for Retail Milk Containers 349/
Screening Tests for Microbiological Quality of Air 349/ Swabbing
Methods: Calcium Alginate, Dacron (Polyester) and Rayon Swabs
351/ Testing of Sterilized or Microbiologically Stable Milk ProdPreliminary Incubation (PI) for

Automated Plate Loop Count Procedure 353/ Detection


and Enumeration of Lactic Culture Bacteriophage 353/

ucts 352/

B.

Supplemental Chemical Control Methods


Acidity: Titratable 355/ Acidity: Potentiometric,

355

pH 358/

Alkalinity

Dry Buttermilk, Nonfat and Whole Dried Milki6// Chlorides J6i/ Extraneous Matter i67/ Fat: Modified Methods i69/ Fat:
Gerberi75/ Fat, Moisture and Salt in Butter and Margarine (Modified Kohman Method) 376/ Hydrolytic Rancidity 379/ Lactose in
CheesQ 381/ Moisture and Solids i<^5/ Somatic Cell Couni 388/ Ash
of Ash

in

399/

Index

403

CHAPTER

QUALITY TESTS
E. H.

Marth

Introduction Parameters of Quality and Acceptance


Numerous characteristics of a food determine its acceptability to consumers and to regulatory officials. In the United States and many other areas
1 .1

where food

is

relatively abundant, not only safety

involved, but also

many

aesthetic considerations.

and nutritional value are

Food must be

attractively

presented, have desirable odor and flavor characteristics, and possess other
qualities that

have

little

to

do with whether the product

is

free

from patho-

genic microorganisms or other potentially harmful agents. In actual practice,


the public generally gives little thought to the possibility that consumption of
any food may cause illness by infection or poisoning. Much of this attitude
undoubtedly is attributable to the outstanding job which industrial and regulatory groups have done in providing the public with wholesome food processed under sanitary conditions.
Those qualities of dairy products that are discernible by the consumer on
careful examination will generally not be considered here, no matter how
important they may be in determining acceptability of the foods. The same is
true of most tests for specific nutrient constituents. The major concern of
Standard Methods for the Examination of Dairy Products is those tests on
dairy products which can be done by laboratory procedures. Subjective tests
for such attributes as body, texture, and flavor generally are not adaptable to
the type of standardization required for Standard Methods. Some procedures described in this book may be applied more or less routinely for laboratory evaluation of products. Other methods are for special situations

where information of a type beyond


trol

practices

is

that ordinarily obtained in routine con-

needed.
to provide a product in
appearance
have been preand

The ultimate aim of control procedures should be

which the original nutritive qualities, flavor,


served and no harmful organisms or substances are present to
consumer adversely.

affect the

Function of Standard Methods


Because milk and milk products commonly move through several hands
from producer to consumer and may move beyond the regulatory juris1.2

ISC Liaison: E.H. Marth

QUALITY TESTS

diction within

which they are produced or processed, uniformity

in

proce-

dures for examination becomes essential for orderly marketing. Uniformity


of standards for acceptance
It

is

also desirable.

should be emphasized that Standard Methods

is

compendium of

rec-

ognized methodology and is not an attempt to establish for either government or industry legal standards for acceptability of products. For example,

maximum permissible Standard Plate Count on pasteurized milk, the


maximum permissible amount of pesticide residue, and the minimum

the

amount of milkfat in whipping cream are legal decisions outside the scope of
Standard Methods. A change in methodology involving incubation temperature or time, composition of a culture medium, or preparation of dilution
blanks may change significantly the count obtained on a given sample. This
in turn may determine whether the product will be acceptable within the
current limits established by government or industry. In some instances a
change in procedure may have the effect of appreciably increasing the stringency of a standard because more of the microorganisms are enumerated or
a smaller amount of a chemical contaminant is detected than was possible by
earlier methods. The background for many of these tests and discussions of
standards to be applied can be found in standard texts on dairy microbiology '- '^and in ordinances and codes such as those suggested for milk
by the U.S. Public Health Service.
'**

''^^

1.3

Standard Procedures

Standard Methods provides two types of procedures. One type is the stanthe basis for official control actions. The second type of
procedure may be used for quality control of milk and other dairy products
because of its greater speed, greater simplicity, lower cost, or similar advantages over the standard procedure or other alternative methods. A method
also may lack adequate testing and use to permit its substitution for a presently used standard procedure, even though preliminary trials may indicate
that the new procedure has certain advantages over the current standard
method and eventually may replace it.
Although one method to enumerate those organisms which produce
countable colonies under a specific combination of conditions may be designated as a standard procedure, this does not mean that other methods for
this type of determination do not require standardization if they are to be
used by several laboratories for control purposes. Some tests which may
have value for industry and regulatory personnel but are not considered as
standard procedures are given in the two appendixes in this book. Presence
of methods in an appendix should not be construed as an indication that such
procedures are of limited value, but rather generally they have not been
evaluated to the extent needed before they can be considered as a standard
method. A procedure completely adequate for routine testing of many sam-

dard procedure

ples for satisfaction of

some requirement might very possibly not be

the

Collaborative Studies on

1.5

Methods

procedure that one would want to use if a court case develops or even for
routine acceptance of the product by a governmental regulatory agency.
Thus the procedure would be appropriate for inclusion in this edition, but in
hence its placement
a manner which will prevent confusion as to its status
in an appendix. While not all people concerned with examination of dairy
products may be satisfied by this approach, such distinction is believed to be

essential.

wide acceptance for testing a variety of products, the Stanat 32 C, with incubation for 48 hr 3 hr, has been retained
as the standard method to enumerate viable organisms in dairy products.
One of the major problems of the past, as well as the present, has been
determination of equivalence between methods, or between two or more
alternative procedures for essentially the same test. Unfortunately, equivalence under one set of conditions does not necessarily mean equivalence
under all other conditions encountered. One example is the considerably
greater effect of suboptimal conditions on enumeration of physically
stressed microorganisms than on enumeration of the same microorganisms
which have not been subjected to stress. ^- ^- '^' -*- -' -^ For this reason, alternative methods generally have not been presented.

Because of

its

dard Plate Count

1.4

Adopting

New Procedures

Although procedures to designate a method as ''Standard'" are far removed from due legal process. Standard Methods, in effect, often has quasilegal status. Regulatory ordinances and codes frequently contain stipulations that procedures in Standard Methods shall be used to determine
whether a product satisfies certain aspects of the regulations. Some jurisdictions have sought protection from capricious changes in Standard Methods by specifying a particular edition of that publication as a reference. Under these conditions, when a new edition appears, it will not replace the old
until new procedures have been examined thoroughly and the law has been
changed to adopt that particular edition.
Decisions relative to change in Standard Methods, whether of inclusion or
omission, have been made only after interested parties have had an opportunity to study the

impact of the proposal and make appropriate recommenda-

tions.

The Intersociety Council on Standard Methods for the Examination of


Dairy Products generally has required publication of the method and of a
collaborative study on the method before accepting a new method or modification of old ones and has sought to establish criteria for retaining previously
accepted methods. Insofar as seemed possible within the realms of available
facts and funds, this has been done.
1

.5

Collaborative Studies on Methods

published method should be collaboratively studied before acceptance


'"
have given details for planning
as a standard method. Youden and Steiner

QUALITY TESTS

a study and analyzing the results. The format for a study is not rigid but the
following are essential ingredients of a collaborative study:
A. A minimum of five collaborators should participate.
B.

A minimum

of three samples

in

duplicate should be analyzed.

suggested that the number of collaborators multiplied by the number of


determinations per collaborator equal 60.) Samples should cover the concen(It is

tration range

should be

expected of the method. Duplicate portions of the sample

unknown

to the collaborator.

C. Estimates of experimental error should be computed from the duplicate observations.

D. Estimates of variation are computed among analysts. Youden and


^' point out that this is the determining factor in evaluating a method.
Computations for components of variance are detailed by Youden and
Steiner.'" A summary of the components of variance can be expressed in the
Steiner

standard deviation for reproducibility below.

ax =

Vo-'l

a'is

0-5

where,
cr't

= component

cr^

= component of variance between

als

= component of variance

of variance

among

analysts (laboratories)
replicates

for the analyst (laboratory)

x sample

inter-

action

computed

compared to preal.^ was not a


collaborative study, they did estimate components 0-5 = 0.005 and
(t'I = 0.007 for the Standard Plate Count (SPC). These values were computed from the logio SPC. An alternate testing method '^ was recently colValues of

ctx

in

a collaborative study can be

viously reported results. Although the report by Donnelly et

laboratively studied for milk

These values were similar

^^

and foods. "^

to those of the 1960 study.

used to examine the reaction of cr| estimated

in

An F

a previous study

test

can be

(i.e.,

0.012)

and one computed for a new method. Chapter 21 gives a procedure for an
analyst to test his performance on an alternate procedure after it has been
accepted and on the SPC.
1.6 Uniformity of

Procedures

Emphasis must be placed on uniformity of procedures employed in various laboratories." If products are to be moved from one factory to another,
or from one regulatory jurisdiction to another, uniformity of testing procedures is essential so that a reasonable degree of agreement can be achieved
among different laboratories. Many people do not realize the extent to which
minor variation in technique may influence results. The Laboratory Development Program of the Food and Drug Administration, in cooperation with

Split-Sample Tests

1.7

the several states, has been an important factor in standardization of labora-

American Public Health Association appointed


methods for bacteriological examination of
milk.-'' Studies by this and subsequent committees have resulted in successive editions of Standard Methods for the Examination of Dairy Products

tory procedures. In 1905, the

a committee to standardize

and,

in turn,

much

standardization of laboratory methods. Despite standard-

emphasize the importance of adhering


closely to recommended procedures - and of improving current methods.
Observations show that where milk laboratories are licensed and supervised by some central authority, operations are more uniform than in the
absence of such control." Approvals for licensing are usually based on qualifications and experience of personnel, on adequate facilities, and on operational compliance with standard procedures. Currently, following request of
approval, all qualifying industrial, independent commercial, and governmental laboratories in many states are licensed to examine milk. Official
agencies may accept results of analyses from such licensed laboratories or
from laboratories that have been approved in accordance with requirements
of the National Conference on Interstate Milk Shipments. ^- -** Acceptance of
ization, there

is

determinations
gate

much

a constant need to

made

in

such laboratories enables control agencies to dele-

routine quality control

work

to dealers

who employ

ties.

these

facili-

The need for manufacturers to standardize culture media not only as to


formulas used, but also between lots prepared from the same formula has
become increasingly apparent. This applies not only to the medium for the
Standard Plate Count but also to media employed for selective enumeration
of specific types of microorganisms important

in

various dairy products.

1.7 Split-Sample Tests

Mindful of inherent limitations of methods, administrators have sought to


determine reproducibility of tests on identical samples in diff'erent laborato''
i'- ^^
In 1950, the first National Conference on Interstate Milk Shipries.
'''

ments approved a provision that States may accept results of local laboratories which comply with Standard Methods and which check closely with
results on split samples a minimum of twice a year.

Use of the

split-sample technique

is

not distinctively a routine procedure,

but it does offer the administrator an opportunity to determine how satisfactorily work is being done in laboratories under his supervision. For example,
determinations on split samples have revealed many causes for irregularities
in different laboratories.

When

such causes are found,

it

becomes

relatively

easy to change practices so that these irregularities do not recur.


The program of the National Conference on Interstate Milk Shipments

recommends that an acceptable split-sample program "shall consist of a


minimum of 10 to 12 samples to be analyzed each 6 months by all laboratory
methods for which the laboratory

is

approved, representative of

all

types of

QUALITY TESTS

milk and milk products

certified'^ for interstate

shipment, including samples

yielding a normal range of results as well as representative high and low

samples, with replicate samples." Coordination of the split-sample program


a function of the

is

Food and Drug Administration under agreements with

the National Conference on Interstate Milk Shipments.

1.8 Integration of

Farm and Plant Inspection with Laboratory

Control

The importance of farm inspections is recognized because the quality of


is determined initially where it is produced.*^ However, laboratory test

milk

improving milk
and correcting sanitation failures. In short, farm and plant inspections should go hand in hand with laboratory tests.
results serve as a valuable adjunct to guide sanitarians in

quality

1 .9

Relative Accuracy of
Quality

Methods

for

Measuring Sanitary

Determinations on individual samples by any one method should be

inter-

preted solely on the merits of that method. Regardless of the method by


which the limit for acceptability is officially established, enforcement officers as well as dealers or producers,
to several conclusions.

They can

tell

when shown

of safety in terms of sanitary codes, (b)


(c)

marginally violates codes, or (d)

results of tests, can

whether a product
is

falls just

(a)

come

has good margins

within "acceptable" limits,

grossly contaminated.

The

cardinal

samples into one of these groups.


Dairy foods are often held refrigerated for extended periods before reaching the consumer and this has increased the importance of bacteria which
cause undesirable changes in the foods during storage at refrigeration temperatures. Some of these microorganisms produce colonies poorly, if at all,
when incubated at 32 C, and may reduce indicator dyes very slowly. Under
some conditions, these organisms may stain less satisfactorily for the direct
microscopic count than do many of the organisms which predominate at
higher holding temperatures. While some psychrotrophic microorganisms
cause rancidity and bitterness after growth to large numbers, most pathogenic bacteria grow slowly or not at all at these lower temperatures.
Since laboratory procedures to evaluate dairy products cover materials of
widely different physical and chemical composition, as well as products in
which certain constituents of milk are present in amounts representing concentration in some instances and dilution in others, details of procedures for
sample preparation applicable to one product may be quite unsuitable for
another. Various constituents may be added in the formulation of certain
products, also adding to the complexity of examination procedures. Not only the final product, but also the individual materials used to make the final
product, must therefore be examined. For some products these examination
procedures are mostly chemical. In past editions of Standard Methods,
objective

is

to classify

Consideration of Specific Methods

1.10

many

of these chemical procedures were reproduced from publications of

Chemists (AOAC), but in the


are given in one of the appendixes. In several instances chemical procedures which have not been considered for official action by AOAC are incorporated in this edition oi Standard
Methods because of their particular applicability to a situation or product.
There is constant change in recommendations and regulations for pesticides, antibiotics and similar compounds which may be encountered in dairy
products. Improvements in analytical procedures lead to greater sensitivity
and considerable reduction in the amounts of residue detectable. In this state
of flux, a procedure accepted today may be outdated tomorrow. Because of
this, precedent has been established to publish (in the Journal of Food Protection) changes in Standard Methods after one and before another edition is
printed.-" Furthermore, a method proposed for addition to the fourteenth
edition of Standard Methods was published so reaction from persons in the
field could be obtained before a final decision was made on inclusion of the
the

Association

of Otficial

fourteenth edition,

Analytical

some chemical procedures

method.-'

The methodology of microbiological

tests differs

somewhat among prod-

ucts because of variations in processing, holding conditions, and other factors. In

some circumstances presence of

yeasts or molds; "total" bacterial

population; or thermoduric, thermophilic or psychrotrophic bacteria in a

product

may be

of greater significance than

in others. In this edition, there-

some specialized presentations are made for the


with numerous cross-references to basic procedures.

fore,

Decisions as to which

test

different products,

procedure to use for control of milk and milk

products, frequency of testing, and values considered satisfactory for compliance must be

made by

the local group, whether industrial or govern-

The Grade A Pasteurized Milk Ordinance '" recommends maximum


permissible numbers of bacteria, maximum permissible amounts of certain
undesirable chemicals, and minimum amounts for some desirable constitumental.

ents, as

determined

in

most instances by procedures outlined

in

Standard

Methods
1.10 Consideration of Specific

Methods

Routine determinations for the presence in milk and milk products of pathogenic bacteria, rickettsiae, protozoa, and especially viruses are impractical
with available techniques. Therefore, procedures to detect conditions under
which these agents of disease might be present in the product are used widely. Since pasteurization is almost universally employed to kill pathogens,

phosphatase tests for determining effectiveness of pasteurization are frequently employed. Adequacy of protection against post-pasteurization contamination commonly is evaluated by testing for coliform bacteria, which
are destroyed by proper pasteurization. Presence of non-sporeforming psychrotrophic bacteria also

may be

interpreted as the result of post-pasteuriza-

QUALITY TESTS

most of these organisms are unable to survive


psychrotrophic bacteria (including some
However,
this heat treatment.
in milk and many milk products held
grow
organisms)
strains of coliform
tion contamination because

refrigerated.

may

Numbers

of these organisms found in dairy products so held

not be indicative of the

amount of post-pasteurization contamination

found immediately after pasteurization.


An assumption that care in excluding microorganisms might, in general,
result in exclusion of potentially harmful types appears to be justified. Based
on the validity of this assumption, determination of the "total" microbial
population on raw milk by the Standard Plate Count, microscopic count, or
dye-reduction test probably has some public health significance.
Current levels of radionuclides in milk and milk products are considered
well below those which should cause concern.'^ However, the possibility
exists that there could be increases in contamination levels.

For

this reason,

monitor radionuclides

in

dairy products must be investigated

continuously so they are available

in

optimum form,

methods

to

current methods are presented

in

if

needed. Therefore,

Standard Methods.

Concern over small amounts of pesticide residues in milk and milk prodbecomes more sensitive. Efforts are being
made to minimize pesticide contamination through better education and regulation of those who use them in the environment of dairy animals. A com-

ucts continues as methodology

mittee of the National Academy of Sciences has stressed the futility of notolerance requirements. Analytical procedures are improving and modern

instruments can detect ever smaller amounts of widely used pesticides. Because of the interest in procedures of this type, a standard method to detect
certain pesticide residues is given in Chapter 19.
Other chapters give methods to properly sample dairy products, estimate
"total" microbial population as determined by the Standard Plate Count,
detect specific types of organisms, determine several chemical constituents,
and detect materials foreign to the several products. Another group of experts might have

material but

made

other selections and chosen other ways to present the


in the fourteenth edition of Standard Methods

methods given

number of scientists chosen as


members of the Intersociety Council because

represent the consensus of a considerable


contributors to chapters or as

of their special background and interests.

1.11

Quality Control

Decisions

made by

in

the Laboratory

a regulatory agency or a

company

are often based on

Furthermore, funds are expended for personnel to staff the laboratory and for supplies and equipment to operate the
laboratory. Hence, management needs and expects the most accurate results obtainable with currently acceptable methods. This requires an ongo-

results obtained in the laboratory.

ing

program of quality control in the laboratory.


quality control program for the laboratory includes

suitability of the

References

1.12

work environment, including


quality of results

safety precautions, because this impacts on the


and because the Occupational Safety and Health Act of

1970 deals with this matter.

Other aspects of a quality control program include (a) adequacy of steriprocedures, microbiological media, and reagents (discussed in
Chapter 4), (b) purity and viability of microbial cultures to be used for tests
or that are isolated for study, (c) control of contamination within the laboratory, (d) proper handling of samples, (e) doing tests properly according to
prescribed procedures, (f) proper reading of test results, and (g) proper recording and reporting of results.
The split-sample program is an attempt to assure that high-quality results
are obtained from a laboratory. However, supervisors and laboratory personnel should institute and use daily a quality control program that will insure the adequacy and reliability of data obtained regularly with various test
lization

procedures.

References

1.12
1.

2.

Anonymous.

1958. National

conference.

Milk Food Technol. 21:164-171.

Black,

J.

Conference on Interstate Milk Shipments. Background of the

L. A. 1943. Surveys of milk laboratories in

war areas

in the

United States. Reprint

2522. Pub. Health Rep. 58:1605. 1641, 1681.


3.
4.

1956. Split sample evaluations of milk laboratories. Pub. Health Lab. 14:94.

Black, L.A.
Black, L.A.

1958. Status of interstate milk shipment laboratory certification.

J. .Milk

Food

Technol. 21:187-191.
5.

Blac

K,

L.A. 1960. Recent progress

in certification

of milk laboratories.

J.

Milk Food Tech-

nol. 23:13-18.
6.

1976. Practical implications of injured microorganisms in food.

Busta, F.F.

J.

Milk Food

Technol. 39:138-145.
7.

Busta, F.F. and Jezeski, J.J. 1963. Effect of sodium chloride concentration in agar medion growth of heat-shocked Staphylococcus aureus. Appl. Microbiol. 11:404-407.
Dahlberg. A.C. Adams, H.S.. and M.E. Held. 1953. Sanitary milk control and its rela,

um
8.

tion to the sanitary, nutritive

and other qualities of milk. National Academy of Science-

National Research Council Pub. 250.


9.

Donnelly, C.B.. Harris, E.K., Black, L.A., and K.H. Lewis.


of standard plate counts of milk samples

split

1960. Statistical analysis

with state laboratories.

J.

Milk Food Technol.

23:315-319.
10.

Donnelly, C.B.,

Bi ack. L.A., and K.H. Lewis. 1958. Containers, refrigerants and

sulation for split milk samples.


11.

Donnelly, C.B., Peeler.

J.

J.T.,

in-

Milk Food Technol. 21:131-137.

and L.A. Black. 1966. Evaluation of state central milk


J. Milk Food Technol. 29:19-

laboratories by statistical analyses of standard plate counts.


24.
12.
13.

14.

15.

Book Co., New York.


Taylor, D.W., and LB. Schialman. 1963. The United States Public
Health Service method of rating milk supplies and its use in the interstate milk shipper
program. J. Milk Food Technol. 26:277-281.
Foster, E.M., Nelson, F.E., Speck, M.L.. Doetsch, R.N., and J.C. Olson, Jr. 1957.
Dairy Microbiology. Prentice-Hall, Inc., Englewood CliflFs, N.J.
Fries, G.F., Burmann, F.J., Cole, C.L., Sims, J. A., and G.E. Stoddard. 1%6. Radioiodine in milk of cows under various feeding and management systems. J. Dairy Sci. 49:24Ft

IKER, P.R. 1949. Practical Dairy Bacteriology. McGraw-Hill

Faulkner,

27.

J.D.,

QUALITY TESTS

10

16.

Gilchrist. J.E.. Donnelly. C.B.. Peeler, J.T.. and J.E. Campbell. 1977. Collaboracomparing the spiral plate and aerobic plate count methods. J. Assoc. Offic.

tive study

Chem. 60:807-812.

.'\nal.

17.

Gilchrist. J.E.. Campbell. J.E.. Donnelly. C.B.. Peeler. J.T.. and J.M. Delaney.

18.

Hammer, B.W.. and

1973. Spiral plate

Inc.,
19.

New

method
F.J.

for bacterial determination. Appl. Microbiol. 25:244-252.

Babel. 1957. Dairy Bacteriology. 2nd

John Wiley

&

Sons,

1957. The eftect of the plating medium on the surPxeudonionas fluorescens Food Res. 22:164-169.
Marth. E.H., Reinbold. G.W.. Marshall. R.T.. Mather. D.W.. Clark, Jr.. W.S..
DiziKES. J.L.. Halsler. Jr., W.J., Richardson, G.H., and W.W. Ullmann. 1974. An
addition to the thirteenth edition of Standard Methods for the Examination of Dairy Prodnets: Single-service plastic vials for samples of raw milk and cream. J. Milk Food Technol.

Heather. CD., and

C.

Vanderzant.

vival of heat-treated cells of

20.

ed.

York.

37:159.
21.

22.

Messer, J.W., Cla^pool, L.L., Holghtby, G.A., Mikolajcik, E.M., Sing, E.L., and
E.H. Marth. 1977. Request for comments on method to detect penicillin in raw milk as
proposed for inclusion in Standard Methods for the Examination of Dairy Products. J.

Food Prot. 40:270-271.


Mickle. F.L. 1938. The need

for official supervision of laboratories.

J.

Milk Techno!. 1:3-

8.

23.

Nelson. F.E.

1943. Factors

parison of four agar media.


24.

Peeler.

J.T..

which influence the growth of heat-treated bacteria.


J.

1.

A com-

Bacteriol. 45:395-403.

Gilchrist. J.E., Donnelly, C.B., and J.E. Campbell. 1977. A collaboramethod for examining milk samples. J. Food Prot. 40:462-464.

tive stud\ of the spiral


25.

Prescott. S.C. 1905. The need for uniform methods


246. Public Health Papers and Reports of the

26.

27.

the sanitary examination of milk. p.

31 (Part

2).

RUPPERT. E.L.. Taylor. D.W.. and 1. Schlafman. 1965. The interstate milk shipper certification program-a cooperative accomplishment. J. Milk Food Technol. 28:379-382.
Stiles. M.E.. and L.D. Witter. 1965. Thermal inactivation. heat injury, and recovery of
Staphylococcus aureus.

28.

in

APHA

J.

Dairy Sci. 48:677-681.

Thomas. W.R., Reinbold, G.W.. and F.E. Nelson.

1963. Effect of temperature and time

of plate incubation on the enumeration of pasteurization-resistant bacteria

29.

Food Technol. 26:357-363.


U.S. Department of Health, Education
tories.

30.

U.S.

USPHS

Youden,

milk.

J.

Milk

1965. Evaluation of milk labora-

Pub. No. 999-FP-3, Washington. D.C.

Department of Health, Education & Welfare.

ordinance
31.

& Welfare.

in

Recommendations of the Public Health

Grade A pasteurized milk


USPHS, Washington, D.C.

1965.

Service.

W.J., and E.H. Steiner. 1975. Statistical manual of the

Official Analytical

Chemists. Washington. D.C.

AOAC,

Association of

CHAPTER
SIGNIFICANT PATHOGENS
I.S.

2.1

IN

DAIRY PRODUCTS

Snyder, W. Johnson, and E.A. Zottola

Introduction

Pasteurized fluid milk products

in

the United States are unquestionably

is supported by the fact that


foodborne disease caused by pasteurized fluid milk has not been reported for
several decades. The incidence of milkborne outbreaks is at such a low level
that the Center for Disease Control has not maintained a separate category
of milkborne disease sihce 1968 but puts such outbreaks into the general
category of foodborne outbreaks. This record is remarkable when one considers that over 100 billion pounds of milk are consumed each year in the

the safest foods to consume. This statement

United States.
This excellent record can be attributed, in part, to the effect that Standard
Methods for the Examination of Dairy Products has had on standardizing
laboratory techniques throughout the United States and the influence that
this standardization has had on improving the quality of milk and milk prod'-''
and the
ucts. Development of the Grade A Pasteurized Milk Ordinance
'-"
also had an influence on the high
Interstate Milk Shippers Agreement
quality of pasteurized milk products available to the American consumer.

However,

the success of the

program should not lead

to

complacency

in

the

activity of regulatory agencies in this area. Regulatory activity should re-

main constant to assure the continued safety of milk and milk products. With
the air travel and individual mobility that prevail today, there are no longer
any geographic borders around infectious diseases and potential dangers
posed by any infection are considerable.
In spite of the excellent public health record demonstrated by pasteurization of milk and milk products, there is some interest by food faddists to
return to the "good old days" and do away with pasteurization of milk. The
statement that pasteurized milk is not as nutritious as raw milk because pasteurization destroys nutrients in milk is incorrect. The pasteurization process has little, if any, eflFect on the nutritive value of milk and the safety
achieved by pasteurization far exceeds any effect on nutrients."- If this concept, i.e., drinking raw milk because it is presumably more nutritious, becomes more popular, the probability that an increase in milkborne epidemISC Liaison: W.J. Hausler,

Jr.

11

SIGNIFICANT PATHOGENS

12

ics will return is great.

McCoy "

reported that between the periods of 1941/

1948 and 1962/1972 outbreaks of Salmonella associated with milk increased

from 8 to 20%. Most of the milk outbreaks occurred on farms or among


consumers purchasing raw milk. Those milk-related outbreaks which have
occurred have been caused largely by manufactured dairy products, ice
cream, cheese, non-fat dry milk and whey powder where recontamination
had occurred after pasteurization.
Although the automatic bulk milk-vending machine has not been implicated in food-associated illness, it most certainly represents a tremendous
potential for spread of disease if organisms are present in plastic feeder
used widely in cafeterias, dormitories and other
tubes. These machines
foodservice areas must be properly and routinely sanitized, as mishandling
could contaminate the tubing, and thus each glass of milk drawn off.

Salmonella

diihlin infections associated with certified

raw milk have been

reported and the organism was isolated from both milk and cattle.

'''^

This

is

problem which has reoccurred since 1958 and indicates the difficulty in controlling these infections in animals and the consequent contamination of
milk.

The report oi Brucella

London

'"-

and in cheese imported


from Mexico, Spain and Italy
along with the epidemic in Trinidad '"* caused by ice cream contaminated by Salmonella typhi should serve
to emphasize the importance of pasteurization and sanitation in preparation
of milk and milk products.
Staphylococci are still the organisms most commonly involved in dairy
product-associated food poisonings. Since the last edition (13th) of Standard
Methods for the Examination of Dairy Products, outbreaks of foodborne
disease caused by enteropathogenic Escherichia coli in cheese have been
'-'' '^^' '''"
reported.^'*'"*''
Increased interest in disease caused by E. coli will
probably result in identification of other outbreaks of infection by enteropathogenic . coli. Animals are not the only source of organisms implicated
in milkborne disease. Man can be a source of contamination and subsequent
infection at any point in the processing until the product is actually consumed. The most critical stages relating to man-milk-man transmission of
in

raw milk
'''''

^'^^

in

"'-

'^-

disease are those following pasteurization.

This chapter will serve only to provide information on agents that


find their

way

into milk or milk products

of their importance.

No

techniques

will

may

and to provide a current assessment


be presented in view of the paucity

of standard methods for isolation and identification of pathogenic micro-

organisms. Studies on the standardization of methods for specific pathogens


dairy products must be initiated and pursued to include rapid micromethods such as fluorescent-antibody techniques, Immunoelectrophoresis,
gas chromatography and other automated or semi-automated methodology
for enumeration and identification of pathogens. Techniques such as microin

calorimetry

should be investigated further as a tool for monitoring bacterial

2.2

13

Bacterial Infections and Intoxications

load.

The report of endotoxin

tive tests

in dried

milk

^^

suggests that rapid and sensi-

such as the Limulus amebocyte lysate

^^

might be evaluated for

detecting gram-negative rods.

Studies showing presence of Pseiidomonas


cereiis

^^'

portance

in

,'^'^

E.

coli

'"^

and Bacillus

milk need to be evaluated particularly with respect to their im-

in the

The report of
the Salmonella

compromised host fed milk or milk products.


variability in lactose fermentation
^"

again emphasizes

by both E.

coli

^^' ^^^

and

part of the difficulty in standardizing

methodology and at the same time the need for good methods. Although
standard methods are not yet available, several reference books can be recommended for current methodology in clinical immunology and micro'^' ^^'
^*"'' ^^
There are also several publications that can be
biology.^'
used to review the history, incidence, diagnosis and treatment for as well
foodborne infections and intoxications.
as the types of food implicated in
Books by Dack '^^ and Riemann ^"" are excellent examples of this type of
review reference on problems of food poisoning.
Laboratory studies on control of microbial growth and toxin production in
milk and milk products are too numerous to be cited here. Continued research in this area will certainly lead to even better control of diseases transmitted by these products. Studies of viruses in milk and milk products, how-"'^'' '""
Techniques are available to isolate, detect, and
ever, are limited.'^"
determine survival of viruses and further studies along this line should be
'^'^'

'^*''

encouraged.-*'^''^'^-'^'''

'^

2.2 Bacterial Infections


A. Anthrax:

and Intoxications

Caution Specialized safety procedures are mandatory.


Transmission of Bacillus anthracis from milk of an infected animal to man
is not likely. In most instances the disease is transmitted by the oral route via
meat from infected animals." ^' Even though milk from animals with acute
Infection may contain 5. anthracis, the milk is usually so altered in appearance and its volume so reduced that it would not be used. However, in barns

where anthrax has occurred,"'

'"'

contamination of milk during handling

is

possibility.

Isolation of 5. anthracis

is

best accomplished by injecting milk or

cream

and sediment of centrifuged milk samples into guinea pigs. The animals are
highly susceptible to 5. anthracis and will die within 72 hours. Demonstration of large gram-positive bacilli in smears from the blood and spleen of the
dead animal can be followed by cultivation of the organism, from the tissues,
on nutrient or blood agar. Identification of the cultures asB. anthracis may
^^^'^

be made by appropriate laboratory procedures.


B. Bacillus cereus infections:
Vlad '^^ in 1972 reported an outbreak of food poisoning due to ingestion of
milk contaminated with 5. cereus. B. cereus is found in soil and air. Disease

SIGNIFICANT PATHOGENS

14

is

due to production of enterotoxin and

is

not the result of tissue invasion. In

became

the outbreak just cited 819f of the children involved


Isolation

and identification of B. cereus

is

ill.

easily accomplished.''^'*'*'

C. Botulism:

an intoxication that rarely occurs from ingestion of milk and


a single incident caused by ingestion of a commercial
1
cheese spread was investigated. Two simultaneous outbreaks of botulism in
France and Switzerland, associated with ingestion of Brie cheese, were reported in 1974.''"' Botulinum B toxin was identified in blood of 12 (37.5%)
patients from the outbreak in France. Another outbreak of botulism associBotulism

is

milk products. In 195

persons was recently


Type A botulinum toxin was detected and type A Clostridium
botulinum was isolated from the cheese.
ated with a commercial cheese spread and involving

1 1

reported.''*'-

Botulism results from ingestion of a neurotoxin formed during growth of


the anaerobic sporeforming microorganism C. botulinum The microorganism does not usually cause an infection in animals or humans but is
.

normally found

in soil.

Milk or other dairy products

may be contaminated

during the course of production or processing and the microorganism then

can grow in the food and produce toxin.


Laboratory identification is usually directed toward detection of the toxin
rather than isolation of the microorganism, although the latter may be accomplished. Detailed procedures to isolate and identify C. botulinum are
^'' '*^'
available and are well documented.'^"
***'

D. Brucellosis:
Caution
Infection of

Specialized safety procedures are mandatory.

man

with any of the three species of Brucella {abortus, suis or

melitensis) occurs by ingestion of unpasteurized dairy products, by contact

with infected animals or by inhalation of aerosols containing firwce// orga-

human infections in the United States are traced to cattle infected with B. abortus or to swine infected with B. suis. Cattle in contact with
infected swine may become infected with B. suis and disseminate the organism in milk. In other parts of the world, goats and sheep, which harbor 5.
nisms. Most

melitensis, are the major source of

human

infection.

Other animals may also

^^
be a reservoir for Brucella organisms.

Unpasteurized milk or butter, unfermented cheese, and other products


made from untreated milk constitute a serious health hazard in areas where
Brucella infection is widespread on dairy farms. This has recently been emphasized by the report of brucellosis contracted by drinking unpasteurized
milk. The source of the outbreak was confirmed by isolation of the organisms from milk and infected cows.'"

Outbreaks of brucellosis resulting from ingestion of unpasteurized goat


cheese imported from Mexico were reported recently. ''' "" '*''5. melitensis
was isolated. Another report associated brucellosis with cheese imported

from Spain.

'''

Bacterial Infections and Intoxications

2.2

15

from cream and sediment of centrifuged milk samfrom cheese or other milk products is possible by direct culture on
selective media or by inoculation of guinea pigs.''''" The ring test for antibodies in milk is widely used to screen for presence of Brucella infection in
dairy herds. To determine which animals are infected, the blood agglutination test is most commonly used, or milk samples from individual animals
may be examined by the whey agglutination test. Details of the serological
and bacteriological methods used in diagnosis and control of brucellosis can
be found in a monograph by Alton and Jones,'* in Diagnostic Procedures for
Isolation of brucellae

ples or

in the Manual of Clinical Miand Parasitic Infections


crobiology,^'^" and in the Manual of Clinical Immunology .^^"
E. Clostridium peifringens infections and intoxications:
,''''

Bacterial, Mycotic,

Clostridium perfringens

is

one of the most ubiquitous of the pathogenic


It has been isolated from air, dust, soil,

anaerobic sporeforming bacteria.

water, vegetables, fresh meat, intestinal tracts, seafood, etc. Since

it

is

pres-

forms are resistant to


drying, it is not surprising that the organism is so widespread. As a pathogen, it manifests itself in many ways, causing gas gangrene in humans, human food poisoning, lamb dysentery, sheep enterotoxemia, and various other diseases, often intestinal.-' Foodborne disease caused by C. perfringens is
ent in soil in large numbers, and because the spores

due

it

to elaboration of an enterotoxin.'-'

is found in the alimentary tract of nearly all warmblooded animals, fecal contamination of milk is one source of the organism;
mastitis is another, as is the presence of excessive dust and dirt during
milking. Naguib ^^ in a study on occurrence of Clostridia in milk reported
that 809f of milk samples obtained from collecting centers near Cairo, Egypt
contained Clostridia. Of the 150 isolates, 108 were C. perfringens, 30 were

Since C. perfringens

Clostridium butyricum and 12 were Clostridium sporogenes.

from cheese in Norway '^^ and from homemade


cheese in this country has been documented.'-^ Nelson **" described an outbreak of diarrhea accompanied by fever, in children who ingested milk later
found to be heavily contaminated with C. perfringens. Changing the diet to a
noncontaminated acid milk quickly restored the children to normal. Nelson
stressed the ability of clostridial spores to survive pasteurization which
Isolation of C. perfringens

would kill most other acid-forming bacteria. Thus pasteurization actually


improves conditions for growth of sporeforming bacteria by eliminating
competing bacteria. These few reports indicate the rarity with which C. perfringens food poisoning is associated with dairy products, although this organism is frequently associated with food poisoning caused by meat and

meat products.
Isolation of C. perfringens

is

not especially difficult, and typical colonies

of C. perfringens on agar plates should be subcultured for further identification. The various biochemical, serological, and toxicological tests used for
"-'**'''
^ found elsewhere in the literature.'''this purpose may
'

SIGNIFICANT PATHOGENS

16

F. Diphtheria:

The usual pasteurization of milk


diphtheriae

This organism

is

is

adequate to destroy Corynebacterium

the least heat-resistant of those pathogenic

agents found in milk. The diphtheria bacillus is primarily a parasite of man


and therefore milkborne outbreaks can occur through post-pasteurization
contamination." The corynebacteria grow in tissue and produce an exotoxin
which inhibits protein synthesis." Primary isolation and identification may

be obtained by following procedures given

in

various reference sources.

'* ^*^

G. Enteropathogenic Escherichia coli infections:


The coliform group of bacteria is defined and discussed in Chapter 6. Information in that chapter is for identification of coliform organisms as a
bacterial recontamination following pasteurization.

means of detecting

At

times, the coliform count in raw milk is used to ascertain the quality of sanitation maintained in milk production. Certain serological groups of ". coli
are associated with severe diarrhea in infants and

young

children.'''-

"*'^

Both animals and man are carriers of enteropathogenic E. coli; the organisms have been recovered from the milk of healthy animals as well as from
those with mastitis." Enteropathogenic E. coli is found in milk and
*^^' '^^' ^^^
Jones not only reported E. coli in retail milk
cheese. ^^' ^^' ^^' ^^samples but also showed that some of the isolated strains were of serotypes
associated with enteropathogenicity and that some were resistant to one or
^'*'

more

antibiotics."'^

were 96 outbreaks of foodborne disease involving 227 pereaten imported French Camembert or Brie cheese. Enteropathogenic E. coli serotype 0124 was isolated from stools of patients and
from cheese. This was the first outbreak of invasive E. coli disease in adults
In 1971 there

sons

who had

in the

United

States.'^-

'''^-

'-" '^"

An

outbreak of disease

in

children associat-

ed with milk has also been reported.'*'


Since approximately 10*" cells are required to establish disease in man,
control in production should suffice to keep foodborne infection caused by

minimum. Park et al. have shown that production of Camembert


cheese containing few if any E. coli cells can be achieved. *" Fantasia et al.''^
reported that in assaying 2,000 wheels of cheese, over 10% contained E. coli
belonging to six serogroups associated with diarrhea. Also, the count of
E. coli increased from < 10' E. coli/g to lOVg at room temperature.

E. coli at a

Isolation of E. coli

may

follow procedures outlined

procedures given
sitic

Infections

/-^

in

Chapter

6. Identifi-

may be made by

following
Diagnostic Procedures for Bacterial, Mycotic and Paraand in the Manual of Clinical Microbiology J'*' Fluorescent-

cation of the specific enteropathogenic strains


in

antibody techniques have proved quite applicable for direct "presumptive"


identification of enteropathogenic . coli in fecal material.''

However,

appli-

cation of this procedure to examination of milk and milk products has not

been reported. The invasive E. coli involved in the outbreak described by


al. were non-lactose fermenters and reacted with Shigella anti-

Tulloch et

2.2

Bacterial Infections

and Intoxications

17

serum. '-^ This emphasizes the importance of competent microbiologists

in

accurately defining food-associated diseases.


Gastroenteritis caused by E. coli may be related either to production of an
enterotoxin or to invasion of the intestinal tract. Illness due to the enterotoxin (food poisoning)

is

a cholera-like disease whereas invasion of the colon

mucosa results in a Shigella-hke disease."*'' "


The demonstration that strains ofE. coli cause disease through
'"^^

properties
of invasiveness or toxin production has cast doubt on the significance of
serotyping as an indicator of enteropathogenicity. Not all strains of entero-

pathogenic E. coli belong to the serogroups associated with enteropatho-

A number

genicity.

of methods for assay of both the heat stable and heat

labile enterotoxins as well as for invasiveness

have been described. ^"^

H. Leptospirosis:
Caution Specialized Safety procedures are mandatory.

Many

serotypes of Leptospira cause disease

in

dairy cattle in

all

the world. In the United States Leptospira interrogans serovar,

parts of

pomona

has been reported as the major cause of leptospirosis in dairy cattle. Although this organism may be present in the milk for infected animals,
there is no clear-cut evidence that Leptospira has been transmitted to
man via infected milk.^' The organism is labile in milk and does not survive because milk contains a heat-stable factor that inactivates and lyses
Leptospira. This factor will withstand pasteurization. The methodology for
""^
isolation and identification of leptospires is presented elsewhere.'^/.

Listeriosis:

monocytogenes causes bovine mastitis and bovine abortion; the


'"^- ""
microorganism is excreted in milk from the infected animal. ^~-''''Evidence for the role of milk in transmission of L. monocytogenes from
infected dairy cattle to man has been presented by a number of workers. The
microorganism has been isolated from raw milk where listeriosis occurred in
dairy cattle and in man.'*^^ The microorganism grows well in both raw and
pasteurized milk and has been isolated from cheese.'"'' L. monocytogenes
in unpasteurized cheese milk at counts of 240/ml was recently reported.""*
After 7 and 14 days of brining, counts in cheese increased to 7.8 x lO"^
Listeria

'""'

and
1.3

L.

X IC/g. After 28 days the fully ripened cheese


10^

still

contained

organisms/g.

monocytogenes

specimens;

is

being isolated with greater frequency from clinical

this in all probability relates

more

to techniques

and awareness

than to increased incidence. Perhaps the same results would be forthcoming

on milk and milk products if proper awareness and techniques


were exercised.
J. Mycobacteria:
Improved control measures, including mandatory tuberculin testing in the
United States, have almost eradicated tuberculosis in cattle and pasteurization of milk has reduced the importance of milk as a vehicle for transmission
for studies

SIGNIFICANT PATHOGENS

18

human tubercle bacilli in milk is


human discharge from a tuberculous

of bovine tuberculosis to man.^' Presence of


usually the result of contamination with

milker or milk handler. Aural

human

tion of unpasteurized milk, has

Bovine tuberculosis localizes

tuberculosis, possibly related to inges-

been reported
in the

cases so large numbers of tubercle

udder

in
in

South Africa.'""
only a small percentage of

found

bacilli are rarely

in

market milk.

Kells and Lear^** have stated that the present pasteurization standards pro-

vide a safety margin of approximately 28.5 min at 61.6


71.7

(161

F),

(143 F) and 14 sec at

according to thermal death-time studies on three bovine

strains.

Atypical acid-fast mycobacteria have been isolated from raw milk.

Of

351 samples of raw milk tested, 175 were positive for atypical acid-fast bacil-

The source of contamination

is probably milking machines, collecting conand storage and transport tanks. The highest frequency of isolation
was in the winter months and this preceded the highest incidence of cervical
lymphadenitis in children by 2 months.'" Since atypical acid-fast bacilli can
survive 140 F for 30 min, the margin of safety in the pasteurization process
(holding method) is markedly diminished.
Cheese products may be contaminated with mycobacteria by the use of
contaminated milk powder.''^ Mycohacterium tuberculosis does not multiply
in cheese but may survive up to 220 days in Cheddar cheese. The aging
period used for commercial production of some cheeses may not be sufficient to insure destruction of all mycobacteria.
Cultivation of tubercle bacilli from dairy products is often difficult because
of the presence of other bacteria which overgrow colonies of tubercle bacilli.
These non-acid-fast organisms may be destroyed by one of several methods
Media containing
without harming many of the tubercle bacilli present.
isolation
of
M. tuberculosis
are
useful
for
primary
egg yolk, serum or both
agar in a CO2
Cultivation
Middlebrook's
7H10
other
mycobacteria.
on
and
atmosphere has markedly assisted the microbiologist working with clinical
specimens. Other enriched media, such as Lowenstein-Jensen or Peizer,
can also be used but they usually require a longer incubation time. Directions to prepare and use these media can be found in Diagnostic Procedures
for Bacterial, Mycotic and Parasitic Infections^''' and in the Manual of Clinical Microbiology .""
li.

tainers

''

'*''

Growth of acid-fast organisms from milk is only presumptive evidence of


presence of the tubercle bacilli, as it might well be one of the atypical mycobacterial strains

whose

role in the disease processes of

man

is

not clearly

defined.

Although involved cultural and biochemical

tests

may be

required to de-

termine the species of mycobacterial isolates, primary presumptive evidence


might be obtained by judicious use of fluorochrome stains (fluorescent dyes

such as Auramine O) on direct smears of the milk centrifugate. Applicability


of these methods to milk quality control studies has not been reported, but

it

2.2

Bacterial Infections

and Intoxications

should be determined. Methods utiUzed


detailed in several texts.

19

in

studies on clinical material are

''*''

K. Pasteurellosis:
Although various species of the genus Pastenrella have been isolated on
occasion from bovine infections, those most often found are Pasteurella
multocida and Pasteurella haemolytica. These organisms are quite similar,
differing only in certain biochemical and serological properties. Both species
have been associated with hemorrhagic septicemia (shipping fever) in cattle.
Shipping fever usually takes the pneumonic form and is frequently fatal.
P. haemolytica and P. multocida do not appear to be the primary agents causing shipping fever, but are involved as secondary invaders

and undoubtedly

contribute substantially to the fatal pneumonia.

There are a few reports in the literature o^ Pasteurella mastitis. Some of


these involve only sporadic cases, but others concern herd outbreaks.

^'

''

''--'

Although some species o^ Pasteurella are highly pathogenic for humans,


P. multocida and P. haemolytica are normally considered as opportunistic

pathogens that can produce severe upper respiratory infections, especially


in the debilitated

or

compromised patient."

Milkborne infections of man have not been reported, probably because


adequate pasteurization destroys these bacteria and there is little opportunity for recontamination by infected handlers.
Isolation of the causative agent in suspected cases oi Pasteurella mastitis
is

the only

method

to detect

bers of the genus Pasteurella

may be made

if

desired.

its
is

presence. Identification of isolates as

mem-

usually sufficient, but further differentiation

''"''

L. Salmonellosis:

The salmonellae continue

to be important organisms in foodborne and waUnited States. Most of the foodborne outbreaks
have not been attributed to milk or milk products. Most have implicated
foods containing eggs. Nevertheless, there have been several outbreaks of
salmonellosis for which milk or milk products were responsible.

terborne diseases

in the

Salmonella typhi. Salmonella reading and S. dublin were implicated two


decades ago. In the last dccdde Salmonella heidelherg and Salmonella newbrunswick were responsible for outbreaks of foodborne disease traced to
24.50, oo^
heidelherg and Salmonella
fluid milk and dry milk, respectively.
newport were isolated from unpasteurized milk from cattle with fever and
enteritis but no signs of mastitis. Reports of infection of cattle with Salmo*'*-4'''-'
The source of the infection
nella paratyphi-B appeared recently.
was a stream polluted with human effluent. Milk was not a vehicle of infection but the level of environmental contamination was considerable as evidenced by isolation o^ Salmonella for 2-3 months after removal of the last
infected cow.

Recently, S. duhlin infections associated with consumption of certified

SIGNIFICANT PATHOGENS

20

raw milk have been reported. '^^ Confirmation by culture of milk was obtained. No source of 5. dublin was identified, but some of the cattle were
shedding Salmonella typhimurium and Salmonella livingstone. The report of
5. dublin is a recurrence of a problem which previously existed and shows
the difficulty in control of these infections. Only a small percentage of cattle
actually are infected and thus detection is difficult. Three episodes of milkborne infection caused by Salmonella virchow were reported in persons
drinking unpasteurized milk.'^^ Salmonella were isolated from feces of
cattle, milk filters, and from the farmer, his family and patients. More recently, typhoid fever involving 132 persons in Trinidad, was attributed to
ingestion of contaminated ice cream.

'^'^

Cheese and cheese products have also been implicated in Salmonella outbreaks. Marth has written a detailed review of the Salmonella problem
as related to milk and milk products." The problem of Salmonella in milk
products continues and an outbreak associated with ingestion of pasteur^^^
ized Cheddar cheese containing S. Heidelberg was recently reported.
Salmonella does not ferment lactose. However, Blackburn and Ellis *" reported that approximately 16% of the Salmonella isolated from milk and milkdrying plants could ferment lactose. This emphasizes the need for proper
microbiological procedures in identification of isolates.

tamination of milk or a milk

product may be man,

The source of con-

animal, or animal products

such as frozen eggs. Thus, the potential for food-associated outbreaks of


disease is vast, and with the rise in popularity of convenience foods, especially desserts,

may

create a whole

new problem

ology for Salmonella detection employed

area.

The

specific

method-

by various industries and govern-

mental agencies should be evaluated to develop standard methods for milk

and milk products.


study of food specimens suspected of harboring
enteric disease organisms that they be maintained at the proper temperature
during transit to the laboratory. Chilled products must be kept chilled and
It

is

imperative

in the

frozen products must not be allowed to thaw.

M.

Shigellosis:

Shigellae are organisms which cause disease only in

man and some

pri-

mates. For this reason, outbreaks of shigellosis due to ingestion of milk or


milk products results from contamination by

humans and

are not the result

of infection in the animals, thereby accounting for a low incidence in foods

because of excellent hygienic practices.


Shigella sonnei was implicated in an outbreak of milkborne disease in
1954.'^^ An outbreak of disease after ingestion of contaminated cheese has
also been reported. '"" ''**

N. Intoxication by staphylococcal enterotoxin:


Staphylococcus aureus may be found in milk and milk products as a result
of contamination from the bovine udder or from human sources. In the ab-

2.2

Bacterial Infections

and Intoxications

21

organism may grow and produce potent engrow rapidly at 27-49 C (80-120 F), and
prolonged holding of dairy products at these temperatures after pasteurization should be avoided. Current knowledge of the composition of the enterotoxin and methods used to detect them in foods is too complex to present
properly in this introductory type of discussion. However, the most current
methodologies and concepts are reviewed by Montie, Kadis and AjP'^ and by
Minor and Marth. ^^' **- ^^ Although the exact number of cells needed to generate the minimal concentration of enterotoxin required to produce illness is
not known, recent studies have shown that 1/xg of enterotoxin in 20 g of
cheese produced symptoms in human volunteers. Zehren and Zehren were
able to detect approximately 12 ng of enterotoxin in 100 g of cheese.*'*'* Although proper pasteurization will kill staphylococci, the enterotoxin is heat
stable and dairy products may still contain sufficient amounts of enterotoxin
to cause a food poisoning episode.
Enterotoxin production in raw milk rarely presents a health problem
because of competition by other microorganisms. Food poisoning traced
to dry milk and whipped butter has been reported. Wieneke*^^ reported that
6-18% of 5'. aureus isolated by routine sampling from cheese and raw milk
produced enterotoxin. Most of the strains produced enterotoxin D. Growth
of staphylococci can occur either before or during cheesemaking and
cheeses have been implicated in several food poisoning outbreaks in recent
years. ^^' *^'* High populations of staphylococci in cheese are grounds for suspicion, but should not be taken as presumptive evidence for presence of
enterotoxin. This can only be determined by analyzing food for the enterotoxins. Although most enterotoxin-producing strains are coagulase-positive,
enterotoxigenic coagulase-negative strains have been reported. This problem is discussed in detail in Microbial Toxins. ^^ Minor and Marth have published excellent reviews of the outbreaks which occurred after ingestion of
*^^' ^^
milk, non-fat dry milk and cheese.
There are many techniques available to enumerate staphylococci in a given food product. These methods are detailed in the reference literature.
In outbreaks of staphylococcal food poisoning, bacteriophage typing of
sence of proper cooling,

this

terotoxins. Similarly, S. aureus will

the staphylococcal isolates

may

assist in tracing the source of the organisms.

Only pure cultures of coagulase-positive staphylococci should be submitted


phage typing and then submitted only to a laboratory experienced in
doing and interpreting the test. Methods used are detailed by Blair and Williams.'* Several investigators have supplemented the standard phage set
with others isolated from staphylococci of bovine origin and thus have ob-

for

tained a higher percentage of typable strains.


O. Streptococcal infections:

Streptococci

commonly

'^^-'^^

responsible for bovine mastitis are Streptococcus

agalactiae (Lancefield's Group

B), Streptococcus dysgalactiae

(Lance-

SIGNIFICANT PATHOGENS

22

field's

Group C) and Streptococcus uteris, and it is currently recommended


from cows with mastitis be withheld from the market.^" Strepto-

that milk

cocci have been isolated from bulk milk tanks.-'- 5. agalactiae


pasteurization since

some

strains are resistant to heating at 70

may survive
C for 10 min

and have been isolated from pasteurized milk in Sweden, Denmark, Norway, France. Germany. England and the U.S.A.''" It has been suggested""
that S. agalactiae found in pasteurized milk may be responsible for rheumatoid arthritis although this association has not yet received wide acceptance.
Fecal streptococci have been isolated from six of six commercial samples
of instant malted milk powders.'" The source was most likely contaminated
malt kernels used to prepare the product.
Occurrence in the udder of streptococci pathogenic for humans is rare.
Adequate pasteurization will kill these organisms but they may be introduced into pasteurized milk by infected human handlers.^" Streptococcus
pyogenes (Lancefield's Group A) and Streptococcus equismilis (Lancefield's
Group C) have been isolated from the bovine udder. In Rumania, Streptococcus zooepidemicus (Lancefield's Group C) was responsible for an outbreak of sore throats in which one-third of the individuals later developed
acute glomerulonephritis.' The outbreak was attributed to a temporary fault
in the pasteurization process. It has been recognized for many years that
foods serve as a vehicle for transmission of streptococcal infections such as
sore throat and scarlet fever. Such infections should not be confused with
food poisoning caused by streptococci an association which has been
reported frequently but still appears to be open to debate. ^^' ^*^' ^"' '"^

P.

Yersiniosis:

Yersinia enterocolitica has recently received increased attention as an

agent of disease in man.

It

has been isolated from a number of animals,

including cattle, and from ice cream. ^ In man,

it produces gastroenteritis,
and mesenteric lymphadenitis. The symptoms commonly resemble
those of appendicitis. An outbreak in New York in the fall of 1976 incriminated chocolate milk as the vehicle of transmission of an intestinal ill-

ileitis,

ness attributed

to

Y.

enterocolitica

although the actual

route of con-

tamination was not discovered.'"

2.3

Mycotoxins

Certain pathogenic fungi can survive pasteurization temperatures

"

but

human infections caused by such


has been shown in the possible role of

there have been no reports of milkborne


fungi.

However, considerable

interest

and food products.


Aflatoxins are metabolites produced by Aspergillus flavus and Aspergillus
parasiticus, two molds which grow on peanuts, rice, sorghum, corn, soybeans, wheat, cotton and cotton seed meal, and other substrates under appropriate conditions of temperature and humidity. Since aflatoxins have
been implicated as tumor-inducing agents in rats''' and in rainbow trout, '"**
aflatoxins in food

2.4

Viral, Rickettsial

and Other Diseases

no tolerance for aflatoxins

23

any food."^ Routine confirmation of


made by chemical
and toxicologic means. ''*^ Aflatoxin M, can be detected at the parts per trillion level in milk.^-^ Several methods have been described for chemical and
biological identification and quantitation of aflatoxin. 29,51,78. im
Experimental feeding studies have shown that cows fed aflatoxins B, or Bo
metabolize these compounds to produce aflatoxins M, and M2 (the 2-hydroxy derivative of the parent compound) which are excreted in the urine,
feces and milk."- The quantity of aflatoxin M, and M2 found in milk is proportional to the quantity of aflatoxins Bj and B.^ ingested.- Aflatoxin Mj is as
carcinogenic in rainbow trout as its parent compound aflatoxin B, ^^- '"** and
both have similar LD50 values in ducklings.^"
Aflatoxin M, appears in milk 12 to 24 hr after feeding aflatoxin-contaminated feed to cows and disappears from milk 3 to 4 days after removal of the
contaminated feed.''^'^ Cows fed 1.5 mg of aflatoxin B, daily yielded milk with
aflatoxin Mj levels of 0.2 and 0.3 mg per liter and it has been suggested that
0.l9c to O.Wc of the aflatoxin fed to cows is found in milk."- It has been
calculated that 16 fxg of aflatoxin B, per kg of feed would give aflatoxin M,
levels in milk at the limits of detection permitted by present analytical methods. ^^ Since December, 1977 the FDA will take action if milk contains > 0.5
ppb of aflatoxin Mj.
Aflatoxins are not completely destroyed by pasteurization or spray
''"
drying
so contamination of commercial milk and milk products is possible.
Extracts of freeze-dried milk from cows fed aflatoxin-contaminated feed
produced liver damage when fed to ducklings.' No aflatoxin was found in
but
19 samples from eleven collection areas in England, Wales and Ireland
was found in five of 21 milk samples collected in South
aflatoxin
Africa. ^^ The source of aflatoxins was moldy groundnuts and groundnut-hay
mixtures which had been fed to cows. In one study in the United States, no
aflatoxin was detected in milk from tank trucks, dried skim milk, canned
tested milk and
evaporated milk or bottled whole milk.''^ However, Jung
milk products and did find aflatoxin M2 in dried milk products. Lie and
Marth have experimentally produced aflatoxins in Cheddar cheese and in a
casein substrate resembling cottage cheese.*^"- "" Purchase et al.^" found aflatoxin in whey but not cottage cheese made from aflatoxin-contaminated
milk. Soybean protein is used in many protein-rich infant formulas, so the
potential exists for contamination of these products with aflatoxin.
there

is

in

aflatoxin in foods at levels of 20 parts per billion can be

'

'''

2.4 Viral, Rickettsial

Caution

and Other Diseases

Specialized safety procedures are mandatory.

A. Adenovirus infections:

Kaplan

et al.^' believe that

adenoviruses should be considered with en-

teroviruses as possible agents of milkborne disease. There

is

serological evi-

dence of human infection by bovine adenoviruses and evidence that

cattle

SIGNIFICANT PATHOGENS

24

can be infected by human adenoviruses.-" Adenoviruses


milk pasteurization."*^

will not

survive

B. Enterovirus infections:

Enteroviruses
mals.

may be

isolated

As human pathogens,

from the

intestinal tract of

man and

ani-

polioviruses, coxsackieviruses, echoviruses and

those viruses causing diarrhea in infants are the ones of greatest importance.
Several bovine enteroviruses have been isolated," but no evidence exists

man. However, enteroviruses of human origin

that they are pathogenic for

that find their

way

into milk

may be

transmitted via dairy products. Polio-

and 3 were isolated from samples of bulk tank milk and milk from
mastitic cows. Ten outbreaks of poliomyelitis which occurred during
1914-1949 were due to contamination of raw milk by a worker.'^ Cliver '^^ reported that 98% of poliovirus type 1 and 100% of influenza A and vesicular
stomatitis virus were lost during Cheddar cheese-making. The poliovirus perviruses

sisted during aging but

was inactivated a

million-fold

by prior heat treatment

of milk used in making the cheese. Potter'^ points out that current methods

of pasteurization were not designed to inactivate viruses and refers to studies

which show

that poliovirus

and other enteroviruses can

persist in cottage

cheese and sour milk products.

and

Isolation

identification of these

human

enteroviruses are accom-

plished by the use of tissue culture and serological techniques described in


the fourth edition of Diagnostic Procedures for Viral
tions

.'^'-

and

Rickettsial Infec-

Procedures to isolate and identify bovine enteroviruses can be


the report by Cliver and Bohl.'^"^ Kostemba "' has reported an

^^^-^

found in
improved method for recovery of enteroviruses from food.
C. Infectious hepatitis:

Since infectious hepatitis virus


drink

may

is

excreted

in

the patient's feces, food and

serve as vehicles for transmission of this viral agent. Epidemio-

by milk was first noted in


Seven of the eight households involved
were customers of the same dairy. Other epidemics of infectious hepatitis
associated with milk and cream have been reported. '"-^^
Practical methods to study the infectious hepatitis virus are not available.
Thus, all inferences or implications are drawn from epidemiologic studies of
logic evidence that this virus might be transmitted

a small outbreak

in

Georgia

in 1945.

*^^

infectious hepatitis outbreaks.

D. Tickhorne encephalitis:

Evidence for the role of milk from goats, sheep and cows

in

of the virus responsible for tickborne encephalitis (one of the


boviruses) to
Pogodina."''

man

The

has been discussed by Kaplan

virus

is

et

al.,'''

transmission

Group B

Blaskovic,''^

ar-

and

present in Eastern Europe and Asia and usually

is

transmitted by ticks from animal to animal to man. Goats, cows, and sheep

develop a viremia and excrete the virus in their milk. Pasteurization of milk
inactivates the virus. Milkborne disease in man has been named biphasic
meningoencephalitis or biphasic milk fever.'- The tickborne disease is

2.4

Viral. Rickettsial

25

and Other Diseases

as Russian spring-summer fever. The virus is not present in the


United States, but an antigenically related virus has been isolated in North
America.'-" A similar virus (louping ill) is present in sheep and cattle in the
United Kingdom. There are no known human infections with louping ill virus caused by milk transmission.-^"
These viruses may be isolated and studied by tissue culture and sero-

known

logic techniques as described in


sial Infections

Diagnostic Procedures for Viral and Rickett-

.^'^

D. Mycoplasma infections:

Mycoplasma has never been defined in food-infection outHowever, isolation of Mycoplasma from milk was reported in
1967.^' Since that report, isolation of Mycoplasma from milk and milk products seems to have received little attention but isolation of Mycoplasma
The

role of

breaks.

from various types of human illnesses continues to increase. Failure to cause


mastitis in cows by intramammary injection of human, simian and canine Tmycoplasma would suggest that Mycoplasma contamination would have to
occur during processing for these agents to be incriminated in disease transmitted by dairy products. ^^ The ubiquitous distribution and the importance
of these organisms in disease suggest that further efforts must be made to
determine the role, if any, of dairy products in transmission of Mycoplasma.
E. Q-Fever:
Bovine infections with the rickettsia of Q-fever, Coxiella burnetii, occur
"^'^"
This rickettsia causes an undetermined
in all parts of the world.''amount of human infection and illness. In one outbreak, men in a detention
center became infected after drinking unpasteurized milk.*^ The organisms
are excreted in milk and are found in very large quantities in placental tissues and fluids. Goats and sheep may also be infected and excrete the orga-

nisms. Asymptomatic animal infections, which are widespread among dairy


cattle, are spreading and may constitute an ever-increasing public health

problem. Wisniewski and Krumbiegel have shown that the incidence of Qfever in cattle in Wisconsin increased from 32% in 1957 to 73% in 1962.'-*"
These workers also reported that in a random sampling of 50 seroreactive

84%

those herds were shedding C. burnetii in their


milk. However, in another phase of this study of the Milwaukee milk, only
.^"^^
3.4% of the humans examined had detectable antibody to C. burnetii
herds,

study

of the

in

cows

California

in

showed

that

82%

of the dairy

cows

tested from sites

toC. burnetii
Northern and Southern
Ferris et
their
milk.^
in
burnetii
and 23% of the animals were shedding C.
'"
milk
samples
tested.
bulk
the
more
of
al.
found C. burnetii in 10.5 to 60% or
or
occupationcasual
with
associated
usually
Human cases of Q-fever are
al exposure to infected livestock and contaminated premises, from residence
'^' '^'
It should be
near infected premises, or from consumption of raw milk.*^ingestion
of raw
follow
not
did
disease
clinical
study,
noted that, in a recent
in

parts of the state contained antibody

milk naturally infected with C. burnetii.

Human

volunteers consumed milk

SIGNIFICANT PATHOGENS

26

one month and none experienced any clinical illness. Similarly, no antibodies to Q-fever could be demonstrated in these individuals. In naturally
acquired infections the correlation between antibody and clinical syndrome

for

''-

is poor.-'*^

by either animal inoculation


Animal inoculations may be
done by injection of guinea pigs with the suspected milk. After 4-6 weeks the
animals are bled and complement fixation tests are done on the

The Q-fever

rickettsiae are usually detected

or a capillary-tube agglutination test (CAT).*''^

sera

^^' ^^'

*'''

'"'^

The capillary-tube agglutination test, the method of choice for testing milk
specimens, may also be used on bovine or human sera. This simple method
uses a colored antigen, is reproducible and highly specific, and is ideally
suited for rapid screening of milk from individual cows, or of pooled milk
from entire herds in ascertaining the infection status of large groups of cows.

been demonstrated between agglutinating antibody


Two other techniques which can be
used to detect inapparent infections of Q-fever are the radioisotope precipitation test (RIP) and a relatively sensitive skin test. These procedures and
the CAT were used to reevaluate the presence of inapparent infections
among dairy workers in the Milwaukee area.'^'C. burnetii survives in raw cream " and sometimes withstands inadequate
pasteurization.*^'' Pasteurization of milk at 63 C for 30 min or at 71.6 C for
direct correlation has

of milk and presence of rickettsiae.

titers

15 sec

destroys the agent.

30 min or 74.3

''^

An

additional 2.7

for 15 sec) has

(to a

temperature of 65.7

for

been recommended for processing products

such as ice cream mixes and chocolate milk.'^


2.5

Protozoan Infections

A. Toxoplasmosis:

Toxoplasma is a parasite widely distributed in nature. It develops in a


number of vertebrate hosts but undergoes a typical coccidian life cycle only
in

the intestine of the cat.

Most human infections are benign and the disease is asymptomatic. Congenital toxoplasmosis affects largely the central nervous system. Acquired
toxoplasmosis may resemble infectious mononucleosis or it may be an acute
or chronic disease. In the acute disease there
chills,

prostration

with

may be

a skin rash, high fever,

meningoencephalitis, hepatitis,

myocarditis and

pneumonitis. In the chronic form, symptoms are vague and indefinite. Retinochoroiditis or posterior uveitis occurs. Diagnosis is by isolation of the

organism or by serological methods. Transmission of Toxoplasma from


animal to animal via infected milk has been well documented. ^^ A single case
of toxoplasmosis in a child who drank unpasteurized goat milk has been
reported. Toxoplasma was not obtained from the goat milk, but four of the
ten goats studied

were seropositive.'"'

27

2.6

References

2.6

References

1.

Allcroft,
toxin in

2.

R., and R.B.A.

human food

Carnaghan.

1963.

Groundnut

toxicity; an examination for

products from animals fed toxic groundnut meal. Vet. Rec. 75:259-263.

Roberts. 1968. Toxic groundnut meal. The relationship between


cows and excretion of aflatoxin M, in milk. Vet. Rec. 82:116-119.
Alton, G.G., and L.M. Jones. 1967. Laboratory techniques in brucellosis. WHO
Monogr. Ser. No. 55.
American Public Health Association. 1976. Compendium of Methods for the Microbiological Examination of Foods. American Public Health Association. Washington, D.C.

Allcroft,

R., and B.A.

aflatoxin B, intake by
3.

4.

5.

Barnum, D.A.
Comp. Med.

J.

6.

1954.

herd outbreak of mastitis caused by Pasteurella miiltocida. Can.

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Beck, M.D., and R.J. Huebner. 1950. Epidemiological studies of Q fever in


J. Am. Med. Assoc. 142:868-872.
Bernescu, E., Feld, C, and C. Rosca. 1969. A nephritogenic streptococcus. J. Hyg.
Bell,

J.

A.,

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7.

67:691-692.
8.

Berridge, N.J., Cousins,

CM.,

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J. Dairy Res. 41:203-215.

certain species of bacteria growing in sterilized separated milk.


9.

10.

Biberstein, E.L., Behymer, D.E., Bushnell, R., Grenshaw. G., Riemann, H.P.,
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Am. J. Vet. Res. 35:1577-1582.


Blackburn, B.O., and E.M. Ellis.

1973. Lactose-fermenting Salmonella

from dried

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11.

Blair, J.E., and R.E.O. Williams. 1961. Phage typing of staphylococci.

WHO

Bull.

24:771-784.
12.

Blaskovic, D.
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Press,
13.

New

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In Biology of Viruses of the Tick-borne Encephalitis

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Bodily, H.L., Updyke, E.L., and J.O. Mason, Eds. 1970. Diagnostic Procedures for
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New
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16.

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Braun, J.L. 1962. The epidemiology of Q fever in Iowa. Pub. Health Rep. 77:171-176.
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17.

18.

J.

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19.

20.

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21.
22.

Cliver, D.O. 1973. Cheddar cheese as a vehicle for viruses. J. Dairy Sci. 56:1329-1331.
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23.

24.

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Coles, E.H., and A. Eisenstark. 1961. Staphylococci phages. 111. Typing of Staphylococcus aureus cultures isolated from the bovine udder. Amer. J. Vet. Res. 20:838-840.
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SIGNIFICANT PATHOGENS

28
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Dack, G.M.

1952.

Food and water-borne disease outbreaks. Pub. Health Rep. 67:1089-

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1975. Brucellosis linked to

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Am. Med. Assoc.

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637.
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The pasteurization of cream, chocolate milk and ice cream mixes


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containing the organism of


35.

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J.B.

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and

its

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F.J.

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Hammond, D.M.,

and P.L. Long. 1973. The Coccidia. University Park Press, Baltimore,

Md.
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Harbourne,
paratyphi

J.F.,

Randall,

C.J.,

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J.G.

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1972. Salmonella

Vet. Rec. 91:112-114.

48.

The isolation of Mycoplasma (PPLO) from cattle milk samples. MonMed. 22:22-25.
Holdeman, L.V., and W.E.C. Moore. 1973. Anaerobe Laboratory Manual, 2nd ed.,

49.

Howard,

47.

Hauke, H.

1967.

atshefte fur Vet.

Virginia Polytechnic Institute, Blacksburg, Va.

isolated

C.J.,

Gourlay, R.N., and

J.

Brownlie.

1973.

The virulence of T-mycoplasma

from various animal species assayed by intramammary inoculation

Hyg. 71:163-170.

in cattle. J.

2.6

50.

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29

Hutchinson,

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52.

Jacobson, W.C. Harmeyer, W.C, and H.G. Wiseman. 1971. Determinations of aflatoxin B, and M, in milk. J. Dairy Sci. 54:21-24.
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53.

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54.

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55.

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56.

Jung, M.
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57.

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Raw

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58.

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63.

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65.

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SIGNIFICANT PATHOGENS

30
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76.

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80.

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Outbreak of infectious hepatitis

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97.

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99.

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Hyg. Epidemiol. Microbiol. Immu-

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100.
101.

RiEMANN. H. 1969. Food Borne Infections and Intoxications. Academic Press. N.Y.
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102.

Rogers, B.T.. Keir, P.M., and W.G. Henderson.


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104.

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Acute bmcellosis with unex-

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Method

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32

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J.

Dairy Sci. 51:635-649.

CHAPTER

SAMPLING DAIRY AND RELATED PRODUCTS


R.A. Belknap, W.L. Arledge, K.W. Whaley,
E.O. Wright, and A.F. Zimmerman

Fluid Milk

3.1

and Cream Samples

Analytical results are no better than the sample that is tested. The sample
must be representative of the mass of material being examined. It must be
collected in such a way that it is not contaminated microbiologically or
chemically. Protection against change in the sample during the period between collection and analysis is essential.

3.1

Equipment

for Collecting

Samples

Regardless of the type of sample being collected and/or the designated


laboratory procedure, the following items of equipment are basic and required:

A. Thermometer: Mercury-filled or dial type, graduation intervals not to


exceed 1 C (2 F).* Accuracy of a mercury-filled thermometer must be
checked at least biennially with a thermometer certified by the National Bureau of Standards [5.2(D)] or one of equivalent accuracy.

When

a dial type

once during each


themiometer is used, its
should be
thermometer
the
test
of
the
accuracy
record
of
6-month period. A
with
an approthermometer
dial-type
A
maintained by the sample collector.
or balstorage
from
collected
are
samples
priate range is suggested where
thermometers
glass
wherever
or
bulk
tanks,
farm
or
ance tanks, vats, cans
accuracy must be checked

at least

might be broken.
B.

Sample

transfer instruments:

Individually

wrapped

sterile

or pre-

sterilized plastic single-service sampling tubes are preferred. Straight tubes


or stainless steel metal dippers with long handles, capacity 10 ml or greater,

may be

used. Other equivalent

means may be used, but

onstrating equivalence rests with the user. For

line

the burden of

dem-

sampling, the needle of a

disposable plastic sterile hypodermic syringe may be inserted through a sanitized gasket into a pipeline, or the needle may be inserted into a sterile rub-

ber serum cap

*Use of metric
found

at the

fitted to

units:

end of

a stainless steel nipple that

chart to convert units of

this chapter.

ISC Liaison: G.W. Reinboid

33

measurement

is

welded onto a standard

for temperatures

and volumes

is

SAMPLING DAIRY AND RELATED PRODUCTS

34
line fitting.*'

Collection of an

initial

All

sample from appropriate sampling cocks,

before sampling, may be satisfactory.


sampling instruments must be protected from contamination before use.

properly sanitized and purged (2


C.

Sample containers:

1.)

liters)

Multiple-use containers holding a

minimum

of

ml but of sufficient capacity to permit thorough mixing of contents may be


used. These containers shall be sterilized by dry heat [3.12(A)], or steam
[3.12(B)], and have leakproof closures that have been demonstrated to be
nontoxic. Containers and closures must be phenol-free. 2.) Sterile, evacuated sampling equipment may be used for collecting at least 10-ml portions.
15

3.)

Presterilized polyethylene or other suitable non-toxic plastic containers

of adequate size are acceptable.

4.) Single-service,

non-sterile leak-proof

raw milk and cream may be used provided that: a.)


maximum viable bacterial counts in rinse tests of containers do not exceed

vials

^^

for samples of

made according to each different formulation


and are not bacteriostatic or bactericidal; and c.) the closure is
designed so the container can be opened and closed easily without con1/ml of capacity; b.) containers

are non-toxic

tamination of the

lip

of the vial or the inner surface of the closure.

Users of non-sterile, single-service plastic vials must determine if such


containers are manufactured and prepared for shipment and are inspected
and certified by State Milk Sanitation Rating Authorities as being in compliance with established requirements '^ and are listed in the FDA Quarterly
Publication.'*'

D. Sample case: Should be of

rigid

metal or plastic construction, com-

and with ample space for cracked ice or


other refrigerant to cool and maintain samples at 0-4.4 C until removed from
the sample case at the laboratory. When a mixture of ice and water is used
for rapid cooling, the sample case should be provided with racks, compartments or baffles to 1.) hold sample containers vertically, 2.) keep necks
of containers above the surface of the cooling medium at all times, and
3.) maintain the cooling medium no higher than the level of liquid in sample
containers. To protect sample(s) from moisture, the sample container may
be inserted into a moisture-proof metal box or can, or a properly closed
pletely insulated with a tight cover,

plastic bag.

E. Shipping case:

When

samples are transferred to the laboratory from

receiving station, plant, or other point of collection via

common

carrier, the

shipping case must be provided with a tamper-proof sealing and locking device,

and with handles. Attach warning labels "This Side

Up" on

top of

shipping case to insure proper handling during transportation to the laboratory.

F. Inner shipping case: When inner shipping cases are needed to protect
samples in small vials or containers, optionally provide boxes with tightly
fitted

covers, just large enough to hold bottles upright and to prevent con-

tainer breakage

when

the

box

is

placed

in a larger

G. Agitators: The 3-A Sanitary Standards

ment or mechanical

"

'

shipping case

'"

[3.1 1(E)].

specify types of equip-

agitators acceptable for use in bulk tank operations,

3.13

35

Sampling Procedures

weigh vats, and storage tanks. Air systems used for agitation must comply
with the appHcable 3-A Sanitary Standards " for odor-free, filtered air. To
sample from cans or weigh-tanks, use 1.) a metal disc, diam about 6 in.
(15.24 cm), on the end of a metal rod long enough to reach the bottom of the
container; or 2.) a bowl-type agitator-sampler combination consisting of a
solid handle welded to the outside of a bowl with a pouring spout.
3.12 Cleaning, Sterilizing and Sanitizing Equipment

equipment used

All multiple-use

oughly before

it

must be properly protected

pling devices

instruments

to collect

in

samples must be cleaned thor-

sanitized or sterilized. Single-service containers and sam-

is

at all

times before use. Rinse

all

tap water immediately after use and clean with a suitable

detergent solution. Avoid excessive exposure to strong alkali and, immediately after treatment, rinse thoroughly to

remove detergent

residues.

A. Hot-air sterilization: Glassware and metal equipment may be sterilized


at a temperature not lower than 170 C for not less than 1 hour [4.4(B)].
B. Autoclave (steam) sterilization: Glassware and metal equipment may
at 121 C for not less than 15 minutes [4.4(A)]. Equipment
must be dry before use.
C. Hot water sanitization: Use two stainless steel containers of approximately 40 liters capacity (or two 10-gal milk cans in good condition)
equipped with threaded fittings to provide inlets of metal (do not substitute
rubber hose) for steam and water connections. The first container is for cold
water rinse of the agitator and sampling instrument. The second container,
with a bottom inlet for steam under pressure, is for hot water (not under
82 C) treatment of instruments. Equip the second container with a thermometer that is immersed in water to determine the temperature at all times
during treatment. Minimal time for exposure in hot water should be one
minute. Minimize the risk of burns and excessive noise by installing in the
second container a steam coil with a condensate trap on the exhaust side. To
insure against possible back-siphonage in potable water lines, the cold water
line should terminate not less than one inch above the lip of the can.
D. Chemical sanitization: Use two containers as in 3. 12(C), except that in
the second container, replace hot water with a solution of an approved

be steam-sterilized

germicide,'^ bactericidally equivalent at


(100

ppm) of

all

times to

available chlorine as hypochlorite.

at least

100

mg

per

liter

Submerge sampling equip-

for at least 30 sec in the chemical sanitizer. Sampling dippers carried


on bulk tank trucks shall be stored in a tightly stoppered tube of sanitizer
solution. Maintain and determine daily the strength of sanitizing solution
with applicable test kit. Wash dipper and tube holding sanitizer solution each

ment

day after use.


3.13 Sampling Procedures
Perishability of

raw milk and pasteurized milk and milk products requires


and sub-

special handling to prevent their direct contamination with bacteria

SAMPLING DAIRY AND RELATED PRODUCTS

36

sequent growth of such contaminants during sampHng, transportation and


storage of samples before analysis. Collection procedures are a fundamental
consideration

when samples

are to be used as a basis for

payment or

to

determine compliance with legal bacteriological or chemical standards.


A. General requirements: Use the applicable items of equipment [3.11] to
collect samples. Thoroughly agitate or mix the volume of milk or randomly

products to be sampled.
Meaningful results cannot be obtained unless representative samples are
collected without contamination and tested within 36 hours. Cool samples
immediately to and maintain at 0-4.4 C; do not freeze samples. Promptly
identify each sample legibly with a number, label, or tag corresponding to
inspection records. Record time and date of sampling. Record temperature
select

of milk at

all

sampling locations.

If

milkfat or other chemical tests are to be

remove portions of samples for microbiological


analysis after suitable agitation [3.14]. The temperature of all samples reand 4.4 C.
ceived at the testing laboratory should be between
Always include an extra sample of milk or dairy product in the sample
case or shipping case for use as a temperature control. The temperature
done on the samples,

first

control sample shall be collected at the

perature control sample to the

first

sampling point. Subject the tem-

same storage conditions as other samples

during completion of the sampling schedule and subsequent transit to the


laboratory.

Do

not use the temperature control sample for microbial tests,

but otherwise treat

it

exactly like

all

other samples [3.14].

When

samples are

be examined by the Direct Microscopic Count method for bacteria within


36 hr after collection, and cooling is impracticable, preserve samples by adding 0.08% formaldehyde to the milk.''- ^^

to

B.

Raw
1.

milk:

Farm bulk tank

milk:

The farm bulk milk hauler

tant in the current structure of

milk marketing.

When

is

critically

impor-

the hauler's obligation

includes collection and delivery of samples to the laboratory for analysis, he

becomes a

vital part of quality control and regulatory programs. Deviation


from the following acceptable sample collection practices could cause a milk
producer to lose his market or payment for his product,
a. Preparations for collecting the milk sample:
(1) Wash and dry hands after connecting the milk transfer hose and
before measuring and sampling milk. Keep hands clean during the sam-

pling operation.

Observe milk do not sample frozen, partially frozen, lumpy,


curdled, or churned milk for regulatory purposes. If such milk is sampled for other purposes, record any abnormalities that are present.
(3) Dry the measuring stick with a clean, single-service sanitary paper towel. Measuring sticks stored outside the bulk tank should first be
sanitized, then wiped dry with a sanitary paper towel.
(4) Agitate milk stored in farm holding cooling tanks at least 5 min
(2)

immediately before sampling.'" Agitate contents of larger farm storage

3.13

Sampling Raw Milk

37

tanks (1,500 gal or more) (567.7

min or according

liters) for 10

to tank

specifications.*^
(5) Check temperature of milk. Use an approved-type of test
mometer [3.11(A)], the accuracy of which has been determined.

tize test

thermometer before insertion

ther-

Sani-

into milk.

Compare accuracy of bulk tank thermometer


month with the test thermometer.
(6)

at

least

once a

and information needed for each sample:


Determine and record temperature of milk sampled at all sampling

Identification

b.
(1)

locations.

Record time

(2)

in

edge of the time milk

hours and date at each sampling location. (Knowlis picked up is helpful when planning milk hauler

inspection schedules.)
(3) Legibly and indelibly identify each sample container by producer
name, number, official tag, or label.
(4) Record all data on either laboratory report forms or weight slips
and submit to the laboratory with samples.
c.

Special precautions:
Protect sampling instruments from exposure and contamination

(1)

before and during use.

When

(2)

drain,

using a dipper, remove it from the sanitizing solution,


and rinse at least twice in milk before transferring the sample.
Handle sample container or caps aseptically do not drop, lay

(3)

down, or otherwise contaminate.

Do

(4)

not carry presterilized plastic bags or plastic vials

of clothing. Clipboards, covered containers, or boxes

in

pockets

may be used

to

carry empty single-service sample containers.

Pre-cool multi-use sterile glass containers in sample case as

(5)

needed.

Do

(6)

not hold or

fill

sample container over top of tank while trans-

ferring sample.

sample container not more than 3/4


sample at the laboratory. Do not expel

Fill

(7)

ing of

full to

air

permit proper mix-

when

folding or whirl-

ing plastic bags.

Collect representative sample of each producer's total milk sup-

(8)

Sample

all tanks individually and label samples.


Provide a temperature control by collecting two samples at the
first collection point (one sample to be used as the temperature control).
Identify temperature control and show producer number, date, time of

ply.

(9)

and temperature of sample on the container. If there is no


temperature control, the laboratory is required to use the first official
collection,

sample collected [3.14].


do not delay. If tem(10) Place sample in sample case immediately
perature of sample is above 4.4 C, use ice and water to lower its temper-

ature.

SAMPLING DAIRY AND RELATED PRODUCTS

38

Rinse dipper

(11)

in

tap water immediately after use before returning

to sanitizing solution,
d.

Storage and transportation of sample:

Use

(1)

ice or other refrigerant at all times to cool

required temperature of 0-4.4 C.

Keep

ice level in

and hold samples at


sample case slightly

above the milk level. Do not freeze fluid samples before or during shipment to the laboratory. Use ice or refrigerant all year to insure uniform
storage temperature of 0-4.4 C at all times. Do not rely on air temperature in winter to keep samples cold.
(2) Protect samples from all contamination. Contamination by ice
water in the sample case may be prevented by using racks, compartments, or other means [3.11(C, D)]. Ice water should be no higher
than the level of milk in sample containers. Do not bury tops of containers in ice.

Deliver samples to the laboratory promptly. Microbiological and

(3)

somatic

cell

examinations must be started within 36 hr after collection

[3.14].
(4) Samples shipped to a laboratory by common carrier should be
packed in a shipping case with a tamper-proof lock or seal [3.11(E)].
2. Milk cans and weigh tanks: After agitation [3.11(G)], sample milk
with a sanitized dipper or sterile sampling tube [3.12]; then cool to and hold
sample [3.13(A)] at 0-4.4 C. Place sample in a sample case [3.11(D)] and
deliver promptly to the laboratory [3.13(A)]. If the weigh vat is not large
enough for the producer's total volume of milk, collect proportionate
amounts of milk m a single sample container from each filling.
3. Mobile lank trucks (farm pickup and over-the-road tankers) and dairy
plant storage tanks: Collection of representative samples from a transport
tank or storage tank is dependent on adequate agitation of milk. The time

interval required to agitate a tank of milk until

it

is

homogeneous

is

deter-

and shape of the tank, volume of product held, type, location,


force of agitation, and time allowed for creaming before starting agitation.
Because of the importance of these variables, it is necessary to determine
how much agitation time is needed to assure homogeneity of contents of a

mined by

size

given tank.

The agitation time for milk storage tanks and milk tank trucks may be
determined by taking a series of milkfat samples at specified intervals (e.g.,
3 min, 4 min, etc.) of time during mixing until at least five milkfat tests stabilize at a definite value. From a full tank, take samples from the top and
bottom, and from nearby and extreme distances from source of agitation. If
construction of a tank does not permit direct removal of subsamples, with
agitator in operation,
tation"

is

draw samples through

that degree of agitation

which

the outlet valve.

at full

Adequate

tank capacity will result

variation in milkfat content of the product in the tank of not

0.1%

as determined by an Official

AOAC

Milk Fat Test.

When

agiin

more than
the appro-

Sampling Raw Milk

3.13

priate time interval has

39

been determined for each tank

installation or truck,

record and use this time as the guide for proper agitation time for taking

samples.
Studies

show

that inadequate agitation of bulk tank milk (farm tank, tank

truck, and/or plant storage tanks) before sampling

is

accompanied by the

presence of more bacteria and milkfat in the sample than in milk lower in the
tank.^ This increase in bacteria is not caused by growth but by the rising of
globules, which serves to "sweep'' microorganisms and somatic cells
toward the surface, thus concentrating them in the cream layer.
The following methods may be used to agitate milk in tankers or storage

fat

tanks:
a.

mechanical agitator

built into the

tank and driven by an electric

motor.
b.

propeller or agitator driven by an electric motor or by an air-

driven motor and placed on the manhole with the agitator suspended

in

the milk.

An

oil-free filtered 3-A air supply


injected through a quick coupling device on the rear of the tanker or through a sanitary air hose
c.

'

inserted through the

manhole of the tanker.

An approved

sanitary tank washer/air agitator system, permanently installed in the tank, through which 3-A-quality air ^ enters
and agitates the milk. This method of agitation is not recommended und.

an approved CIP system has been installed at the receiving plant.


e. The milk transfer hose, attached to the tanker unloading pump and
inserted through the manhole, permits recirculation and mixing of the
milk. A well-agitated sample may also be obtained by pumping milk
less

from the tank truck into an empty plant storage tank. The
sample may be collected through the sampling cock located in the tank
access door. Do not collect the sample until the sampling cock has been
adequately sanitized on the outside and at least 2 liters of milk have
directly

been purged through

An approved

automatic

it

[3.11(B)].

line

drip-sampling device

may be used

to provide

a representative sample from a tanker as milk is unloaded. Samples collected


by this method for bacteriological analysis must be cooled to and maintained

0^.4 C during the collection period.


To sample over-the-road tankers, collect two samples through
truck dome immediately after filling. The first sample is to be used

at

fat,

the tank
for milk-

bacteriological, or chemical analysis by the shipper; the second sample,

cooled to and held

at

0-4.4 C, should

accompany

the over-the-road tank

truck to the final destination for delivery to the laboratory for analysis. Cool
and transport samples to the laboratory so that testing can be started within

36 hr after collection [3.13(A)].


To sample commingled milk

in

dairy plant storage tanks, collect the

sample directly from the balance tank before pasteurization or

at

some other

SAMPLING DAIRY AND RELATED PRODUCTS

40
processing step [3.13(B.5)].

When

collecting samples of

raw milk through

the sampling cock in the storage tank door, use proper sanitization proce-

dures to prevent contamination of the sample.


4. Line samples: To collect representative samples of raw milk from
silo

and/or dairy plant storage tanks, or to locate sources of contamination

raw or pasteurized milk, collect samples aseptically at suitable locations


between the storage tank and the first processing step, or at different points

for

after pasteurization. Sterile or sanitized metal tubes or dippers

able transfer instruments at certain dairy plant locations.


pling technique involves use of a disposable sterile

needle of which
stainless

steel

is

may be

suit-

special line-sam-

hypodermic syringe, the

inserted into a pipeline gasket or the rubber closure of a

The nipple can be clamped on or perany desired spot. Cool samples and deliver promptly to

nipple [3.11(B)].

manently located

at

the laboratory [3.13(A)].

C. Pasteurized packaged products, dispenser, coffee creamers, and presRandomly select representative samples of all pasteur-

surized containers:

and milk products regardless of age while they are still in possesNote processing code date of sample on sample
identification form submitted to the laboratory. To avoid sample contamination, preferably submit an unopened container of retail product to the
laboratory. If necessary, after thoroughly mixing product in container,
aseptically transfer a representative portion to a sterile sample container.
Cool, store, and transfer samples to the laboratory as rapidly as possible so
ized milk

sion of the processor.

that testing will be started within 36 hr after collection, acidic dairy products

excepted. Collect and examine acid and cultured milk and milk products
within 24 hr after processing [3.24(B)].

When

shipping samples to the laboratory by common carrier, protect confrom contamination by using tight-fitting waterproof bags. Do not
crush or damage plastic and paper containers [3.11(E)].
Always transmit a temperature control sample [3. 13(A)] to the laboratory.
1. Dispenser milk: Collect official sample from dispenser tube into
sample container [3.11(C)] without flushing milk through tube. Optionally
collect a second sample, after drawing off about V2 liter of milk, to determine
if the tube is contaminated. Cool samples to 0-4.4 C [3.13(A)]. When applicable, transfer samples to sample cases [3.11(D)], and delivery promptly to
tainers

the laboratory [3.13(A)].


2.

Coffee creamers-pasteurized, sterilized, dried, or frozen: Follow the

general procedures for milk and

(container of product)

is

cream

[3.11-3.12]. Since a single

sample

usually of insufficient quantity for analytical proce-

dures, randomly select sufficient samples for testing requirements and also
to represent a lot [3.2].

unopened containers
a.

bag

To avoid sample contamination, submit

original

to the laboratory.

Pasteurized and sterilized products: Place samples in a waterproof


sample case, cool, and maintain at 0-4.4 C until tested. Transfer

in

3.14

41

Receiving Laboratory's Responsibilities

samples to the laboratory as rapidly as possible so that testing

may be

started within 36 hr after collection.


b.

Dried creamers: Place dried product

tamination.

Do

not carry in clothing.

in a bag or box to avoid conHold and transport samples at

room temperature. Samples of dried products may be

transferred to the

laboratory without refrigeration.


c. Frozen creamers: Follow general sampling procedures for ice
cream [3.25]. Place in plastic bag or container to avoid contamination.

Keep samples frozen


3.

at all

times until analyzed.

Pressurized containers: Randomly select commercial-size pressur-

ized containers.

Shake containers 25 times through complete inversions

within 30 seconds. Aseptically dispense contents of container into sterile

sample container. Cool samples to 0-4.4 C, and deliver promptly to the laboratory [3.13(A)]. Consumer-size pressurized containers should be

randomly

selected and delivered directly to the laboratory.

D. Special samples:
1. Milk from the udder: Collect samples immediately before a regular
milking. Do not discard any streams of milk since the foremilk usually contains the greatest number of infectious or other microorganisms.*^ Immediately before obtaining samples prepare the cow by thoroughly washing the
teats, udder, and adjacent flank area. Use clean paper towels to wash and
dry the animal. After washing, thoroughly cleanse the end of each teat with a
cotton ball or cloth gauze pledget saturated with 70% alcohol to insure that
the sphincter area is free of any soil. To prevent recontamination of teat
ends, first cleanse the teats farthest away from the sampler, then proceed to
the teats nearest the collector. Following the above protocol, collect samples from individual quarters as needed using pre-cooled sample containers
where applicable. Cool and hold samples at 0-4. 4C [3.13(A)] until tested.
4 Responsibilities of Laboratory Receiving Samples
As samples arrive at the laboratory, determine temperature of each group
of samples by inserting a precooled (0-4.4 C) thermometer [3.11(A)] into
temperature controls [3.13(A)] treated exactly like other samples. If a temperature control sample is not received, determine the temperature of one of
the official samples (preferably the first sample collected) and label it as the

3.1

temperature control. The temperature of


laboratory should be between
this section
is

and 4.4

all

samples received at the testing


Requirements posed by

[3.13(A)].

occasionally are unattainable. For these occasions, the following

recommended:

When

arrival of pasteurized samsamples should be accepted by the


testing laboratory, provided the sample temperature control meets the temperature requirements of the current Grade "A" Pasteurized Milk Ordinance,^^ and the sample temperature on arrival at the laboratory does not

the elapsed time

ples at the laboratory

is

between collection and

less than 3 hr,

SAMPLING DAIRY AND RELATED PRODUCTS

42

exceed that of the sample temperature at the time of collection. Samples of


pasteurized milk above 7.2 C should be rejected.
Record temperature of sample(s), time, and date of receipt on sample
identification form. Store samples at 0-4.4 C until tested. When samples are

and date of analysis and temperature of sample control


each set of samples. If chemical tests are also to be done on samples, first
aseptically remove a portion of samples for microbiological analyses, after
suitable agitation of samples [5.6(B)].
tested, record time

for

Record and transmit with each series of samples the time, date, and temwhen samples are transferred at an interim
sample transfer point, from the time of collection until receipt at the testing
laboratory. (Interim transfer points are locations where samples are transferred to a temporary cold storage box or cabinet, and held before transfer to
the examining laboratory.)
perature of the control sample

3.2

Other Dairy Products [See

When

3.1]

several chemical and microbiological analyses are to be done, larger

samples may be required. Specific procedures should be reviewed before


sampling so adequate and proper samples may be taken for the required
analyses.

sampling bulk or large quantities


may not be representative, but may be the best available sample under certain conditions. It is
necessary to consider the condition or type of product to be sampled, e.g.,
dry powder, solid, semi-solid, or viscous, before sampling so that the number of units will be representative and/or statistically significant.
It

should also be considered that

where numerous units comprise a

Unfortunately,

it

is

may

in

a single sample

not possible to present and use one set of tables,

graphs, or instructions for


ditions

lot,

all

sampling situations. Each

set of physical

con-

require the use of diff"erent techniques. Consequently, there are

number of diversified plans for particular applications presented in the


The mode of measuring the items determines two general sets of
plans. An item may be examined by taking data on its physical, chemical, or

literature.^

it may be inspected by its


appearance (attribute sampling). More than one measurement of attribute or
variable can be observed for one item.
^ can also be arranged in several ways to insure the same
Sampling plans
level of consumer or producer protection. The number of samples can be

biological characteristics (variable sampling), or

'*-

taken

all at

once

(single sampling), or in

groups (multiple sampling), or one

item or unit at a time (sequential sampling).

Economic consideration often

one or the other of these arrangements. Some


measurements destroy samples, and others leave them unharmed. The added cost of destructive sampling is an example of an economic consideration.
An individual wishing to devise a sampling plan for inspection of items or
indicates the usefulness of

units should obtain the services of a statistician to evaluate specific situations.

3.22

Dry Milk, Whey, Dry Whip Topping

43

is a compromise between the desire


consumer protection and the cost of providing the

Current practice
level of

to achieve a high

service. Available

experimental data indicate that at least 10 units are necessary for representative sampling where a large lot or large number of units are to be sampled.^ A
lot

may be

defined as an identifiable unit of production such as one day's


in any other suitable manner.'^

production or

Sometimes an

alternative procedure to reduce laboratory workload,

if

substantiated by backgroLind data, would be to composite groups of samples

within a given lot or series of units.

Evaporated, Concentrated and Condensed Milk


A. Equipment and supplies: Follow the general procedures for milk and
cream [3.11-112 and 3.21(B)].

3.21

B. Procedure: Collect samples in original, unopened, hermetically sealed


containers. Transferring portions from hermetically sealed cans in the field

is

When

necessary to take samples from opened containers, e.g.,


bulk tanks, or industrial-size containers, provide equipment for transferring
discouraged.

representative portions to sample containers 13.11(C)]. Promptly identify

samples legibly and indelibly with their


to the inspection record.

official

number or

all

tag corresponding

Cool perishable samples to 0-4.4

C and

deliver to

the laboratory as soon as possible [3.13].


1. Retail-size-cans: Where sterility of retail-size cans is to be determined, take the original unopened package to the laboratory, using refrig-

eration

when

appropriate.

Bulk tanks and industrial-size containers: To determine the bacterial


count of evaporated or concentrated milk in bulk tanks or industrial-size
containers, thoroughly agitate the product and aseptically transfer a representative portion (not less than 30 ml) to a sterile sample container
2.

[3.11-3.12].
3.

Field sampling of

unopened containers:

When

necessary to obtain

representative samples of unopened or hermetically sealed containers out-

minimize contamination by insuring sterility of the samand by working in a room as free as possible from dust and
air currents. Shake the container until contents are homogeneous, sponge
one end of the container with a 70% alcohol swab, and expose the surface to
flame just long enough to dry. With a presterilized can-opening device and
with the surface of the container cleaned and sanitized, puncture the container, making an opening large enough to insert a sterile straw or pipet.
Promptly remove sample (not less than 30 ml) and transfer to a sterile sample
container. Close the sample container, cool to and maintain at 0-4.4 C. and
transport promptly to the laboratory [3.13].
side the laboratory,

pling apparatus

3.22 Dry Milk, Whey, Casein, Dry Whip Topping, and Related Products
A. Equipment and supplies: Provide sterile or sanitized trier(s), spoons or
spatulas for sample collection.

SAMPLING DAIRY AND RELATED PRODUCTS

44

B. Procedure: Follow the general procedures, using equipment specified

With sterile tube or trier, transfer at least three plugs or


columns from each large bulk-type container; or with sterile spoon or spatula, transfer not less than 30 g of product from retail-type containers to sterile
sample jar. Since dry milk absorbs moisture quickly, make transfers rapidly
after opening container, and seal containers immediately after completing
transfer. Sample contents of stitched bags by opening them at the top. Minimize contamination when untying bags; collect sample and reseal bags.
Where necessary, aseptically prepare a composite sample in a larger sample
container and after proper mixing, transfer representative portions to sample
container. If some portions of the package appear to be damp, caked or
soiled, sample such portions separately, noting such observations in the
sampling record and the bacteriological report.
for milk [3.11-3.12].

3.23 Butter, Margarine, and Related Products

Follow the general procedures for sampling milk and cream [3.11-3.12]
and butter [3.23(A, B)]. Cool samples at once to 0-4.4 C, record temperature, time and date of collection, and deliver as soon as possible to the
laboratory [3.13]. Although butter is mentioned in the discussion that follows, procedures are applicable to margarine and related products.
A. Equipment and supplies:
1. Butter trier, sanitized: Under commercial conditions where numerous samples must be taken daily, use of a sterile trier for each sample may be
impractical. For microbiological examinations satisfactory results may be

obtained with a single

trier if

it

is

a polished stainless steel trier that

thoroughly after each sample with clean tissue paper, dipped


(and the adhering alcohol burned

off),

in

is

70%

and tempered by plunging

it

wiped

alcohol
into the

For
between each

butter to be sampled at least twice before transfer of the official sample.

composition or chemical examination, clean and dry

trier

sample.
2.

Wrap

Triers, spatulas or spoons, sterilized:

spatulas or spoons in kraft paper or place

them

in

stainless steel triers,

metal or glass containers

before sterilization.
3.

Sample

jars or containers, sterilized:

Size 240 ml or larger, with

screw or glass tops; or pre-sterilized plastic containers.


B. Procedure:
1. Butter from chums: Transfer three 120-g portions of finished butter,
with sterile stainless steel trier, from center and end locations of batch of
butter or from successive sampling points in the stream of butter from continuous churns.
2.

Butter in boxes or bulk packages: With a stainless steel

trier,

remove

of butter from three different corners of the container by inlong,


diagonally through the butter. Take plugs at least 75

at least 120 g

serting trier

including,

when

mm

appropriate, surface portions. With a sterile spatula or

spoon, transfer butter to sterile sample container. Use a small portion of the

3.24

45

Cheese and Other Cultured Products

sample plug to seal the hole from which the plug was removed. (NOTE: A
sample from the corner of the butter box, which includes the outside surface
on three sides, is more effective for detecting surface mold contamination
than

is

subsurface sampling.)
Print butter:

3.

Because of differences

in the ratio

between surface area

and weights of V4-, V2-, and 1-lb prints, preferably remove samples from
print with trier to insure uniformity in surface area per sample. Use a No. 8
cheese trier to take at least a 75-mm plug (approximately 120 g) from end of
print and transfer it (including surface portion) with sterile spatula or spoon
to the

sample container. To sample !4-lb

print,

send entire print

(Va lb) to the

laboratory.

Cheese and Other Cultured Products


A. Equipment and supplies: Follow general procedures for milk and
cream [3.11-3.12] and cheese [3.24(A.l, 2)].
sterile: Cheese trier, cork borer, spoon,
1. Sampling instruments,
3.24

wooden tongue depressors,

knife or spatula to

remove sample. Wrap

the

covered

sampling equipment in kraft paper, parchment or foil, or place


metal or glass containers and sterilized [3.12(A, B)]. Where insufficient
laboratory-sterilized sampling equipment is available, sanitize instruments
it

in

[3.12(C, D)and3.23(A.l)].
2.

clean,

Sample container:

Sterile glass or presterilized plastic containers,

wide-mouth, dry, leakproof, with a suitable

label area for

sample

identification [3.11(C)].

B. Procedure: Collect samples in original unopened containers, or otherwise transfer representative portions from opened containers, using apparatus as outlined for milk and

cream [3.11-3.12] and cheese

[3.24(A.l)]. Sub-

cultured products to the laboratory in the original

mit cottage cheese and


container except where container size makes this practice impractical. Since
numbers of coliform organisms tend to decrease rapidly in cultured products
because of the acid environment, examine samples within 24 hr after products are manufactured. Protect samples

from potential contaminants

at all

times. Promptly cool samples to 0-4.4 C and transport or ship to the laboratory for examination within 24 hr after processing for all acid-treated or cul-

tured products.

Surface samples: Surface portions of cheese are subject to diverse


forms of contamination. Surface microorganisms may differ from those in
the interior because of the different environment. Surface layers should be
sampled only as required to establish presence or absence of surface con1.

taminants, or as necessary for other special purposes.


2. Natural, processed, dehydrated and imitation cheeses: Sample these

types of cheese with sterile or sanitized sampling instruments [3.24(A.l)].


Transfer at least five samples, each consisting of not less than 5 g, from
different parts of the individual cheese to the sample container. Discard the
outer 2-cm portions of each sample.

When

sampling soft cheese, remove the

SAMPLING DAIRY AND RELATED PRODUCTS

46
surface portions as noted.

With a

sterile

instrument, transfer

at least five

samples, each consisting of not less than 5 g from different parts of the
cheese, from freshly exposed areas to the sample container. Cheese can be

sampled by the following techniques depending upon the size and shape of
make two cuts radiating from the
center of cheese, if the cheese has a circular base, or parallel to the sides if
the cheese is rectangular. Place resultant piece of cheese into sample concheese. Using a knife with a pointed blade,

tainer.

Sampling can be done with a cheese

trier

by the following techniques de-

pending upon the shape, size and type of cheese: a.) insert trier obliquely
towards center of cheese once or several times into one of the flat surfaces at
a point <10 cm from edge, b.) insert trier perpendicularly into one face and
pass through center of cheese to reach opposite face,
tally into vertical

face of cheese

c.) insert trier

midway between two plane

horizon-

faces,

toward

center of cheese, d.) with cheese transported in barrels, boxes or other bulk
containers, or cheese which

is

formed

into large

compact blocks, sample by


Use

passing trier obliquely through contents of container from top to base.

outer 2 cm or more of plug containing rind for closing hole made in cheese.
Remainder of plug constitutes sample. Close plug holes and, if possible, seal
over with suitable sealing compound such as one made by mixing molten
paraffin, beeswax and white petrolatum (1 +
2) or white petrolatum and
1

paraffin (1

-I-

1).

Small cheeses and cheese packaged

in

small containers are normally sam-

pled by taking the entire cheese. Crumbled, grated, and dehydrated cheese

can be sampled by transferring at least five samples, each consisting of not


less than 5 g, from a single lot of cheese to a sample container.

Cream and Related Frozen Products


Observe the general sampling procedures for milk and cream 13.11-3.12].
Ice cream ingredients such as evaporated milk, condensed milk, dry milk,
whey, butter, fluid milk, and cream are sampled according to applicable pro-

3.25 Ice

cedures 13.21-3.22].

Keep samples of frozen products frozen until arrival at the laboratory and
is begun. Dry ice is the refrigerant of choice. Store samples of

the analysis

semi-frozen

products

(mix,

soft-serve)

at

0-4.4 C.

Start

examinations

within 36 hr after collection for mix and soft-serve products. Samples of dry
materials

may

be transported to the laboratory without refrigeration.

A Equipment and
.

supplies: 13.11-3.12].

Sample cases for transporting samples. [3.11(D)].


2. Sampling instruments: Sterilize in the laboratory or sanitize immediately before use [3.12(A-D)] and protect against contamination. Collect samples of semi-frozen products (mix or soft-serve), with a sanitized dry spoon
1.

or sampling tube, or a stainless steel dipper with a capacity of approximately

50 ml. For frozen ingredients, use a sanitized dry knife, spoon, butter

trier,

or spatula. Samples of eggs or fruits, frozen solid in large blocks or contain-

3.25

ers,

Ice

Cream and Related Frozen Products

may

such as a

47

consist of composite borings taken with a sanitized boring device


bit

or hollow tube driven by an appropriate electric

drill.

Where

cans or other large-volume containers or unfrozen or melted products are to


be sampled, agitate the product until contents are thoroughly mixed
[3.11(G), 3.13(B)].
3.

Sample containers: These should be clean,

sterile,

dry and leak-

proof, have a wide mouth, and suitable space for sample identification
[3.11(C)].

B. Sampling procedures:
1. Packaged samples of ice cream and frozen desserts: If possible coland submit samples in their original containers, keeping the samples
frozen at all times until they are to be analyzed. Otherwise, follow the procedure [3.21(B)] for samples not in original containers.
2. Bulk samples of ice cream and frozen desserts: Aseptically transfer
representative portions of not less than 50 g to sterile sample containers
[3.11(C)] for bacteriological examination. If the container has been opened
previously, use sterile instruments to remove and discard the surface portion
to a depth of 2 cm around the area from which the sample is to be taken.
With a second sterile instrument, aseptically transfer to the sample container
a sample of at least 50 g from the newly exposed surface.
If the object is to determine the care used by the retailer in vending operations, remove surface portions only to a depth of 1 cm of the sample, using a

lect

minimum of 50

g of material. Sample individual servings of frozen dairy


products as dipped by the retailer by taking representative portions of not
less than 50 g with a service dipper or other dispensing device, and treat such

samples as regular bulk samples. When high counts (total or coliform) have
been observed previously on samples from the retailer's cabinet, take additional samples from unopened packages to determine whether the responsibility for high counts rests with supplier or retailer.
3. Process samples: When a particular part of the processing operation
is being evaluated, take samples of unfrozen or partially frozen mixes by
passing a sterile sample bottle or dipper under properly sanitized orifices at
intervals or by withdrawing samples with properly sterilized sampling instruments. Thoroughly agitate contents of cans or vats before aseptically
removing 50-g portions to sterile containers.
4. Coloring and flavoring materials: Mix individual solutions of coloring
materials, extracts, flavors, etc., until homogeneous and pour directly and
aseptically into sample container; or transfer with a sterile sampling tube or
pipet not less than 30 g from the original container into a sterile sample
container. With a sterile spoon, aseptically transfer not less than 30 g of wellmixed coloring powders, fruits, fruit preparations, nuts or nut preparations,

cakes, pastries, or confections to a sample container. Where cold-pack or


frozen fruits are not thawed, use a high-speed electric drill with sanitized
auger, hollow coring tube, or other suitable sharp, sanitized instrument to

SAMPLING DAIRY AND RELATED PRODUCTS

48

obtain enough material for the sample. Aseptically transfer representative

portions to the sample container. Cool perishable unfrozen samples to

0^.4 C and

place under refrigeration. Maintain frozen products in frozen

condition and transport as soon as possible to the laboratory.

With sterile trier, transfer at least three


columns (total not less than 120 g) from each container, or with
sterile spoon or spatula remove not less than 120 g of powder from various
places and transfer to sterile sample container. Make transfers as rapidly as
possible after opening containers and immediately seal the sample container
after transfer is completed. If some portions of package contents are believed to be more highly contaminated than others, sample such portions
5.

Stabilizers and emulsifiers:

plugs or

separately.
6.

Sweetening agents: Transfer not

less than 30 g of

sweetener to

sterile

sample container, cool to 0-4.4 C, and transfer to the laboratory.


7. Eggs and egg products: Sampling procedures and methods for preparing samples are described in the current edition of Official Methods of
Analysis of the Association of Official Analytical Chemists.
8. Sample handling: Maintain frozen samples in frozen condition until
they are to be examined. Samples collected locally that are to be tested
promptly may be packed in cracked ice and water when no other refrigeration is available. Store perishable, semi-frozen, or unfrozen samples at
0-4.4 C until examined. Keep records of the time when samples are collected

and shipped or delivered to the laboratory. Test unfrozen samples as soon as


possible and always within 36 hr after collection. Keep frozen samples frozen until tested. Test within 72 hr after collection. Prevent the thawing and
refreezing of frozen samples. Avoid freezing frozen dessert mixes and softserve products during storage.
3.3
3.31

Sampling

for Specific Laboratory

Direct Microscopic

Procedures

Count Methods

A. Procedure for collecting samples:

To

collect

samples for direct micro-

scopic examination, follow the general procedures for milk and cream
[3.11-3.12 and 3.13(B)1.

3.32 Reduction Methods

A. Equipment and supplies: See 3.11-3.12; use transfer dipper or pipet to


deliver 10-ml portions.
1.

Tubes,

vials,

preferred length 150

or suitable nontoxic plastic bags: Tubes without

mm

similar diameter with

x 15-18

mm

lips,

inside diameter, or special tubes of

10-ml graduation. Uniform diameter and uniform

length of tubes or vials permit interchangeable use of closures and possible

when inverting a rack of tubes.


bags [3.11(C)1 and transferred later to

use of rigid cover to hold firmly over closures

Samples may be collected


tubes or vials.

in plastic


3.33

49

Containers, Equipment, Water and Air Tests

2.

Racks, sheet metal or wire: Corrosion-resistant, for holding tubes

vertically in single or multiple rows.

B. Procedure: Using sterile equipment [3.12(A, B)], transfer 10-ml por-

mixed sample directly from


the tube in which the test is to be
3.13, 3.13(A) and 3.13(B.1,2), but

tions of well-stirred or otherwise adequately

can, weigh tank, or other container to


made. Optionally take samples, as in
before subsequent transfer of 10-ml portions to tubes or vials, shake samples
by rapidly inverting each container 25 times [5.6(B)]. Use a sterile pipet for
each sample, or sanitize the dipper or pipet between samples [3.12(C, D)].

Test sample(s) within 36 hr after collection.


lar

To avoid

intervals, store tubes containing samples at 0-4.4

tested. This permits a choice of test periods

starting tests at irregu-

until

they are to be

that will fall within the usual

working hours of the laboratory or milk plant.


3.33 Microbiological Tests of Containers, Equipment, Water, and Air

A. Equipment and supplies:


1. Tubes or vials, sterile:

Same as 3.11, plus the following


Same as tubes and vials under 3.32(A.l).

B. Rinse test procedures:

Firm-walled capped items: Remove three containers from the conveyor line without touching lips or interiors before containers reach the filler
valves. Aseptically introduce 20 ml of sterile buffered rinse solution
[16.3 1(A. 5)] into each container, then insert the containers into the conveyor
lines beyond the filler valves to be capped mechanically. For containers with
a capacity greater than 4 liters, introduce 100 ml of rinse solution. Should
1.

inspection

show

mechanical cappers are unsanitary, apply laboratorytaken from the conveyor before
valves. Test samples within 36 hr of collection.

that

sterilized closures to three other containers

reaching the

filler

Firm-walled uncapped items: Paper, laminate, or plastic containers


that are formed, filled, and sealed in a single device must be obtained after
sanitization but before the product to be packaged is introduced. Swab a
2.

portion of the side surface of each of three containers with

70%

alcohol,

a sterile hypodermic syringe with 20 ml of sterile, buffered


rinse solution, and inject into the container. For a container larger than
4-liter capacity, introduce 100 ml of rinse solution [16.31(A.5)]. Aseptically
aseptically

fill

apply a piece of cellophane tape to cover the area punctured by the needle.
3. Flexible-walled items: Swab all caps on fill tubes of some collapsed
liners with a soft paper towel moistened with 70% alcohol, then remove and
introduce 20 ml of buffered rinse solution [16.31(A.5)] aseptically with a sterile pipet. For containers larger than 4-liter capacity, introduce 100 ml of

tubes are sealed, swab an area of the tube adjacent to


alcohol and introduce rinse with a sterile hypodermic

rinse solution.

Where

the liner with

70%

syringe; apply cellophane tape to the puncture area.


scissors, cut a corner off uncapped packages to obtain contube of flexible liner before removing closure (or cutting tube
with sanitized scissors) to avoid losing rinse solution. Then lower tube to

With sanitized

tents. Lift

fill

SAMPLING DAIRY AND RELATED PRODUCTS

50

permit aseptic transfer of rinse to sample container. Immediately after clos-

sample container, place

ing

in

shipping case and store

at

0-4.4

until solu-

tions are plated. Sample(s) shall be tested within 36 hr of collection.


4.

be

Storage and shipment: Since large containers with rinse solution

cumbersome and

difficult to refrigerate

and transport

transfer rinse solution to sterile tubes or vials.

Swab

may

to the laboratory,

closures and adjacent

areas of firm-walled capped and uncapped containers, as well as closures

and
in

fill

tubes of flexible liners, with 70% alcohol. Rinse containers as shown


Remove closures from capped firm-walled containers, open the

16.31.

oudet from uncapped, firm-walled containers in the customary manner and


aseptically transfer rinse solution to sample containers.
5. Modification for "cleaned in place" (CIP) equipment: The rinse solution method may be employed on individual items of equipment or on systems designed for "cleaning-in-place." This method is not practical for other
equipment because the volume of rinse solution required would be equivalent to the capacity of the entire unit and adequate agitation to flush surfaces
thoroughly might not be available.
The minimum amount of solution needed to thoroughly flush the productcontact surfaces of CIP items is much greater than can be autoclaved conveniently. Instead, determine the volume of water needed to completely fill the
CIP system ^^ and sanitize the rinse solution by superchlorination (chlorine
added to 25 mg of residual per liter and held for 10 minutes). Then neutralize
hypochlorite by adding an excess of sterile 10% sodium thiosulfate solution
(0.2 m! per 100 ml of rinse solution).
liter)
Use a suitable wide-mouth container of sufficient size (at least
made of borosilicate glass resistant to heat shock for use in the autoclave.
Keep 1 liter of the chlorine-treated water that has been neutralized for use as
1

a control sample.

Introduce neutralized chlorine-treated water into the "ready for use"

CIP

and continue for 20 times the period required to move


the same volume of water with the pump. Stop the pump and open the outlet
valve at the lowest point in the system. Fill separate sterile sample containers at the beginning, middle, and end of the unit-emptying step.
For large volumes of neutralized chlorine-treated rinses, the membrane
filter procedure may be used to analyze both rinse and control samples.
Cool rinse and control samples to 0-4.4 C and transport to the laboratory.
unit. Start circulation

6. Modification for line sampling: The sanitary condition of components of various assemblies can be determined by sampling milk progressively at specific sanitized access points 13.11(B)]. Samples may be analyzed
using the Standard Plate Count to determine where significant changes in
counts occur. Specific flora can be determined by employing appropriate

differential

media and incubation temperatures. To determine the presence


may be laboratory-pasteurized and then

of thermoduric bacteria, samples


plated.
7.

Modification of rinse test for ice cream freezers:

The

sanitary condi-

3.34

Sediment

in Fluid

51

Milk

tion of scrapers and interior surfaces of "batch" freezers may be determined


by a modified rinse method.
Prepare sterile rinse solution [16.31(A.5)] in 100-ml quantities per 10-qt
(9.46 liter) capacity of batch freezer barrels to be tested. With outlet valve of
the barrel closed, pour sterile rinse into inlet. Operate the scraper for 2 min,
collect sample through outlet valve into sterile bottle, cool to 0-4.4 C, and

transport to laboratory.

Swab

procedure: Use the swab

test method to determine the saniequipment and large containers that can not be tested by
the rinse method. Open a sterile swab container, grasp end of stick (being
careful not to touch any portion that might be inserted into the vial), and
remove aseptically. Open a vial of buffered rinse solution [16.31(A.5)], moisten swab head, and press out excess solution against the interior of the vial
with a rotational motion. Hold the swab handle to make 30-angle contact
with the surface. Rub the swab head slowly and thoroughly over approximately 20 cm^ of surface. Rub the swab three times over this surface, reversing direction between successive strokes. Return the swab head to the solution vial, rinse briefly in solution, and then press out excess. Swab four more
20-cm^ areas of surface of the item, as above; rinse swab in solution after
each swabbing and remove excess moisture. After the fifth area has been
swabbed, position head in vial and break or cut with sterile scissors or other
device, leaving swab head in vial. Replace screw cap, put vial in waterproof
container, cool to 0-4.4 C and deliver to the laboratory; begin test within

C.

test

tary condition of

36 hr of collection.

D. Procedure for water supplies: Procedures and equipment for sampling


water supplies are detailed in the current edition of Standard Methods for
the Examination of Water and Wastewater.^
E. Procedures for air supplies: Sampling for airborne microorganisms in
dairy plants involves collecting samples in air pathways by which organisms
may be introduced onto processing equipment. Samples may be taken 1.) at
openings in equipment subject to potential contamination from organisms
transported by air currents, 2.) at selected points for testing quality of room
air, e.g.,

where product

is filled

into containers,

ployees are concentrated. Because of

sampling by volumetric methods

will

air

and

3.) in

areas where em-

turbulence during operating hours,

be more effective and dependable than

use of the sedimentation technique.


Sampling time for all methods of collection usually

is

standardized

at

and 60 min, with duration determined by experience or estimation


(see Chapter 16). Samples may be taken monthly to detect specific microorganisms unless more frequent samples are necessitated by emergency con15, 30,

ditions or suspected contamination.

3.34 Sediment

in

Fluid Milk

A. Mixed-sample method: For retail containers, 5- to 10-gal cans, and


bulk storage tanks, a Vi-liter, 120-ml, 60-ml, or 30-ml sample may be used

SAMPLING DAIRY AND RELATED PRODUCTS

52

depending on the diameter of the

filtering disc

being used. Before mixing the

sample, with a small strainer transfer any floating extraneous contaminants


such as flies, hair or debris to a separate disc and properly identify. Agitate
contents of bulk storage tanks for at least 5 min or according to manufacturer's recommendation before test portions are removed. Avoid con-

tamination of samples with foreign matter.


B. Off-bottom method: For 5- to 10-gal cans, take Vi-liter sample with an
oS'-bottom tester from an unstirred can of milk. Before withdrawing sample,
remove any floating extraneous matter with a small strainer as directed in
3.34(A). Refer to Chapter 17 for testing and grading.

3.35 Phosphatase Method


A. Equipment and supplies:
1.

Trier:

Thoroughly clean, phenol-free and

sterile for

sampling solid

and semisolid products.


2. Tubes, bottles, plastic bags: Clean and phenol-free.
B. Procedure: Collect not less than 10 ml of fluid milk products or not less
than 10 g of solid or semisolid dairy products. Obtain samples of solid products from the interior with a clean trier. Avoid taking samples from exposed
surfaces of solid or semisolid products. Take all samples in phenol-free containers [3.11(C)] and refrigerate at 0-4.4 C until tested. Examine the sample
promptly. Preferably take samples for phosphatase before addition of nuts,
fruits, candy, etc. However, in testing flavored products, strain out nuts,
fruits, etc., before testing. If preservative is necessary, add 1-3% chloroform.

3.36 Other Ghemica! Methods

A. Equipment and supplies:


1. Tube, vial, or plastic bag: Airtight, nonabsorbent.
B. Procedure: Size of sample needed varies with analyses required. For
the usual analyses, collect approximately Vi liter of sample; for milkfat determination, about 120 ml. Collect one or more containers of bottled or packaged milk as processed for sale. Thoroughly agitate milk until homogeneous
by pouring back and forth from one container to another. If a cream layer
has formed, detach from sides of the container and stir until evenly distributed.

Place sample(s) in nonabsorbent, airtight container(s). Cool to and store at


C until examined. Use preservative as needed. Tablets containing

0-4.4

KaCrgO: or other suitable preservative at least 0.5 g of active ingredient per


each 240 ml of milk, but total weight of such tablet should not be
more than 1 g; or 36% solution of HCHO, 0. ml (2 drops) per 30 ml may be

tablet for

used unless the presence of preservative

chemical tests that must be

made

is

objectionable in physical or

in addition to the

determination of milkfat.

53

References

3.4

3.37 Radionuclides

in

Milk

A. Equipment and supplies [3.36(A.l)].


B. Procedure: To avoid spoilage, immediately after collection add 3 ml of
36% formaldehyde per liter of milk and hold the sample at 0-4.4 C until

A 4-liter sample is required when gamma spectrometry is


used to determine ^^4, ^^^Ba, and ^^Ts. This same sample may be used for
^^Sr and '^^'Sr analyses following gamma spectrometry.
Carefully note time and date of sampling. Ship samples for ^^4 determinaready for analysis.

tion to the laboratory with all possible dispatch.

CONVERSION CHART
The following metric measurements used in Chapter 3
the U.S. System for the reader's convenience:

are converted (approximate-

ly) into

U.S. Measure

Metric Quantities

30 g
50 g

30 ml
Vi liter

oz

pt

(fluid)

2qt

4 liters

gal

2 gal

8 liters

40

oz

Iqt

liter

2 liters

20

oz

1.5

4.0 oz

120 g

1.0

liters

5 gal

liters

10 gal

Temperature Conversion:

OC

32

C
C
C

40

4.4
7.2

82.0

3.4
1.

References
1976. Standard Methods for the Examination of
American Public Health Association. Washington, D.C.

American Public Health Association.


Water and Waste Water.

2.

45
180

F
F
F
F

14th ed.

Brazis, A.R. 1962. Interstate shipment of milk,

J.

Milk Food Technol. 25:172-175.

SAMPLING DAIRY AND RELATED PRODUCTS

54
3.

Claiborne, F.B. and K.E. Cox.

1951.

The

direct microscopic count

samples: an effective measure for uniform statewide milk control.

J.

on preserved milk

Milk Food Technol.

14:105-108.
4.

Dodge, H.F., and H.G. Romig.

1959.

Sampling Inspection Tables. 2nd ed. John Wiley and

Sons, N.Y.
5.

Duncan, A.J. 1959. Quality Control


Homewood, 111.

in Industrial Statistics.

Revised ed. Richard D. Irwin,

Inc.,
6.

Elliker, P.R., Sing, E.L., Christensen,

L.J.,

bacteria and keeping quality of pasteurized milk.


7.

8.

and W.E. Sandine. 1964. Psychrophilic


J. Milk Food Technol. 27:69-75.

International Association of Milk, Food and Environmental Sanitarians, Inc.


U.S. Public Health Service, and the Dairy Industry Committee. 1972. 3-A Accepted Practices for Supplying Air Under Pressure in Contact with Milk. Milk Products and
Product Contact Surfaces, Serial #60403. J. Milk Food Technol. 35:378-382.
International Association of Milk, Food and Environmental Sanitarians, Inc.
U.S. Public Health Service, and the Dairy Industry Committee. 1973. 3-A Sanitary Standards for Farm Milk Storage Tanks, Serial #3000. J. Milk Food Technol. 36:341348.

9.

International Association of Milk, Food and Environmental Sanitarians, Inc.


U.S. Public Health Service, and the Dairy Industry Committee. 1974. 3-A Sanitary Standards for Storage Tanks for Milk and Milk Products Serial #01-06. J. Milk Food
Technol. 37:56-61.

10.

International Association of Milk, Food and Environmental Sanitarians, Inc.


U.S. Public Health Service, and the Dairy Industry Committee. 1975. 3-A Sanitary Standards for Farm Milk Cooling and Holding Tanks. Serial #13-06. J. Milk Food
Technol. 38:646-653.

11.

Levine, B.S. 1950. Effect of formaldehyde on the direct microscopic count of raw milk.
Publ. Health Rep. 65:931-38.

12.

Marth, E.H., Reinbold, G.W., Marshall,

R.T.,

Mather. D.W., Clark, Jr., W.S.,


W.W. Ullmann. 1974. An

DiziKES, J.L., Hausler, Jr., W.J., Richardson, G.H., and


addition to the Thirteenth Edition of Standard
ucts. Single-service plastic vials for

Methods

for the

Examination of Dairy Prod-

samples of raw milk and cream.

J.

Milk Food Technol.

37:159.
13.

National Mastitis Council, Research Committee.


for the Diagnosis of Bovine Mastitis.

NMC,

1969. Microbiological Procedures

910 Seventeenth Street,

NW,

Washington,

D.C.
14.

Health, Education and Welfare. Fabrication of Single-Service Conand Closures for Milk and Milk Products. PHS/FDA, Washington, D.C.
U.S. Dept. of Health, Education and Welfare. Grade A Pasteurized Milk Ordinance-Recommendations of the Food and Drug Administration, Washington, D.C.
U.S. Dept. of Health, Education and Welfare. Sanitation Compliance & Enforcement Ratings of Interstate Milk Shippers, Quarterly Publication, PHS/FDA, Washington,
U.S. Dept. of

tainers
15.

16.

DC.

CHAPTER

CULTURE MEDIA AND PREPARATION


Jane

4.1

P.

Jensen, C.N. Huhtanen, and R.H.

Bell

Introduction

Accuracy and precision of results obtained from microbiological


dairy products are dependent to a large extent on culture media

testing of

employed

and care exerted by technologists in their use. Therefore, it is important that


media always be prepared and handled properly.
The following culture media are those mentioned in other chapters of this
book; names given them are for the most part those in the literature. In most
instances media listed here can be obtained in dehydrated form, and it is
strongly recommended that such media be employed. Directions for making

them are found on the bottle labels and in manufacturer's technical bulletins.
However, two of the media listed below* may be unavailable commercially.
Section 4.9 of this chapter provides the necessary information for preparation of these media.

A K Medium #2

''

Medium #1
Green Lactose Bile Broth, 2%
Casein Soy Peptone Agar
Casein Soy Peptone Agar with Polysorbate (Tween) 80 and Lecithin
*Citrate Azide Agar '^- '' [See 4.9]'
Eosin Methylene Blue Agar, Levine '"
Antibiotic
Brilliant

Lactose Broth
Broth
Mueller-Hinton Agar

MF-Endo

'^

Nutrient Agar
Nutrient Broth

Potato Glucose Agar (acidified)


*Rose Bengal Agar with Chlortetracycline

Standard Methods Agar:


Pancreatic digest of casein

ISC Liaison: W.J. Hausler,

Jr.

55

CULTURE MEDIA AND PREPARATION

56

Yeast extract
Glucose

Agar
Standard Methods Agar with Polysorbate (Tween) 80 and Lecithin
Violet

Red

Bile

Agar

A. Dehydrated media:
Dehydrated media should be stored

in

sealed containers in a cool, dry

place and protected from light. If the environment is hot and humid, media
may be stored in a refrigerator or freezer, as preferred. Properly stored,
most media should be stable for at least three years; however, purchases
should be planned to permit a complete turnover within a year or two.
After the first usage, when the seal has been broken, quality of the medium may depend upon the storage environment. It is recommended that a
suitable package size be purchased to minimize repeated use of material
from an unsealed container.
The unsealed container may admit air and moisture which can initiate reactions that result in reduced productivity of the medium. Some chemical or
microbiological contamination may also take place if unclean spatulas are

used to transfer the material.


B. Prepared media:

The

"life" of any prepared

medium, whether

in

tubes, bottles, or plates,

depends on the condition of storage and the type of medium. Prepared media
should not be stored unless protected against water loss. This may be
accomplished by using screw-capped tubes and bottles instead of cottonplugged containers. Prepared plates should be stored in moisture-proof
containers, such as plastic bags, to minimize moisture loss. Because of the
instability of prepared culture media, it is advisable to use prepared plates no
more than one week old and media in screw-capped tubes no more than six
months old. If dehydration has occurred, media should be discarded. All
prepared media whether in plates or tubes, should be stored between 2-8 C.
4.2 Basic

Steps

in

Medium Preparation

A. Weigh carefully the proper amount of the dehydrated base medium or


the correct proportion of constituent ingredients.
B. Place the requisite amount of microbiologically suitable (distilled, deionized or otherwise suitably treated) water into a suitable container (e.g.,
borosilicate glass or stainless steel).

C.
rod.

Add the weighed material(s) to part


Add remaining water and mix again.

of the water.

Mix with

a stirring

necessary to effect complete solution, by boiling on an asbestos-centered wire gauze over a free flame or over an electric hot plate,
agitating frequently to prevent burning of medium at the bottom of the container. A non-pressurized free-flowing steam unit may also be used. Media

D. Heat,

if

4.2

Medium

57

Preparation

containing agar should be boiled to insure solution of the agar. Prolonged

may cause

undesirable foaming; this can be reduced by holding the


water for a few seconds after initial boiling has been accomplished. Restore water, if necessary, to compensate for loss by evaporation.
An alternative method of preparing agar is to add the dry ingredients to the
requisite amount of water in the flask, mix to disperse lumps, and autoclave
boiling

flask in cold

for the required time. After autoclaving, leave the flask in the autoclave with

the door open for 5 min, then place in a water bath and carefully agitate until

any superheated steam has escaped. Then mix vigorously. Care must be
taken to avoid agitating the freshly autoclaved agar as the superheated solution

may

boil violently.

Determine pH of the medium and adjust


media is best determined before boiling.
E.

if

necessary. The

pH

of agar

F. Distribute medium into suitable containers. This can be most easily


accomplished for tubed liquid media by using automatic pipettors commercially available through laboratory supply houses. A handheld adjustable
pipettor is most suitable for small numbers of test tubes, whereas an electrically operated machine is more satisfactory for larger numbers. In each instance, before use, machines must be flushed with microbiologically suitable

medium until the water has been completely removed


from the system. After use, the apparatus must be flushed with warm water,
detergent solution, tap water, and distilled water. If any residues accumulate
the apparatus should be disassembled and scrubbed. A small amount of water should be left in the syringe portion to prevent drying out and subsequent
water, followed by

''freezing" of the plunger and barrel.

G. The amount of medium per container should be limited so that no point


volume of medium is more than 2.5 cm from the top surface of

within the

medium

or area of

medium

equilibration of temperature

interfacing with the container (to insure rapid

when placed

in a

waterbath).

min or according to recommended


procedures for each medium. Rechecking of pH before use of medium is
H.

Sterilize at 121

recommended

(149.8 F) for 15

[4.3].

Autoclaving efficiency is greatly reduced by overloading the autoclave


or by improper spacing of containers. For good results, separate containers
by at least one-half inch in all directions and do not use volumes of media in
/.

excess of

3-4%

of the volume of the autoclave.

If

larger

volumes are used,

sterilizing times should be increased [see 4.4(A)].

use, melt and hold solid media at 44-46 C until used, but not exceeding 24 hours. If a precipitate forms, discard media. Do not re-melt
J.

To

media.

NOTE

Chemicals and substrates such as carbohydrates must be of reagent grade unless otherwise specified. Follow manufacturer's instructions
for storing stock reagents. Discontinue use of chemicals showing any evidence (color change, for example) of contamination, decomposition, or
1.

hydration.

CULTURE MEDIA AND PREPARATION

58

NOTE 2. Automatic agar makers with a complete sterilization cycle are


on the market and they may be of value to some laboratories.
4.3

Adjustment of Reaction (pH)

Determine the hydrogen-ion concentration (pH) of culture media at 25 C


(77 F) electrometrically. This temperature is used in commercial production
of media and should be used in the laboratory to determine the pH of media
before use. Determinations made at 45 C (113 F) to take advantage of the
fluid state of agar are not accurate, differing significantly from those obtained
at

25 C.'"

A. Electrometric procedure (potentiometer):

Allow electrodes of the instrument

to equilibrate

(some models may reis to be made,

quire 30 min) to the temperature at which the determination

and adjust the buffer solution to the same temperature before testing the
instrument. Only buffers referenced to the National Bureau of Standards are
recommended. The pH of the buffer solution should be in the range of the
pH of the medium to be tested. The temperature of test solution and buffer
should be the same; room temperature (25 C) is generally used for convenience. For solid media, macerate a suitable aliquot thoroughly with a glass
rod before inserting the electrodes. Be sure that the temperature is maintained until the reading

is

complete.

If in

doubt, repeat the determination.

Do not dilute test solutions or buffers.


NOTE: Plating media are not highly buffered and this can cause confusion.
Meters are designed only to show a difference in pH between two solutions
at the

same temperature. Completely anomalous observations

will

be ob-

used under other conditions. Temperature compensators of pH meters do not permit correction of a temperature difference
between test solution and reference buffer. The reason for this is that the H^
concentration of the buffer changes with temperature. Temperature com-

tained

if

the meter

is

pensators should be set at the temperature at which measurement is made.


Since the emf/pH ratio is less at 25 C than at 45 C, for example, a correction
factor must be used; this

mine the correct

pH

4.4 Sterilization

is

built into the

and Storage

Before sterilization, bring the


of a

medium may

medium

to boiling temperature, stirring fre-

necessary [4. 2(D)] and autoclave. Because the


change during sterilization and because of possible
is important not to exceed the recommended temper-

quently. Restore lost water

pH

instrument and allows one to deter-

directly.

if

browning reactions, it
ature and time. Reduce pressure with reasonable promptness (but no less
than 15 min) to prevent undue changes in the nutritional properties of the
medium and remove from the autoclave when atmospheric pressure is obtained. Preferably use flasks or bottles from which melted medium may be

4.4

Sterilization

and Storage

59

poured into plates. Optionally use


for pouring melted

medium

test

tubes containing 15-20-ml amounts

into plates.

Prepare medium in quantities so that, if stored, it will be used before loss


of moisture through evaporation becomes evident. To prevent contamination and excessive evaporation of moisture from a medium in flasks

and bottles during storage, optionally fit pliable aluminum foil, rubber, plastic, parchment or heavy kraft paper, or viscose caps securely over closures
before autoclaving. Use of screw-cap or crown (cork-and-seal type) closures
on containers appreciably reduces such risks. If tubed media are used within
a short time, commercially available polypropylene or stainless steel closures may be used. Media should be stored at 2-8 C in a dry dust-free area
and should not be exposed to direct sunlight.
A. Steam sterilization:
Steam-sterilize media, water and materials (such as rubber, cork, cotton,

paper, heat-stable plastic tubes and closures) that are likely to be charred in
the dry-air sterilizer by autoclaving at 121

for not less than 15 minutes.

Autoclave media and dilution blanks within an hour of preparation. Slightly


loosen stoppers to allow passage of steam into and air from closed containers when autoclaved. Place one or more spore controls in the center of the
load, preferably within a container similar to those being processed. Make
certain that the load is loosely packed [4.2]. Before allowing steam pressure
to rise, automatically or manually expel all air from the sterilizer through an
exhaust valve of suitable size. If manual means are used, make sure free
steam is being exhausted before pressurization begins. Because temperature
obtained at a constant pressure of saturated-steam will vary according to
atmospheric pressure, rely only on a properly operating and calibrated thermometer rather than a pressure gauge to insure sterilization.
Avoid overloading autoclaves so that the rate of air exhaust or heating is
not appreciably delayed [4.2]. The autoclave should reach 121 C slowly but
within 20 min after starting the air-exhaust operation. Contrary to popular
belief, a rapid come-up time in an autoclave does not necessarily result in
more efficient autoclaving. This results from failure of steam to replace air
when steam enters the autoclave too quickly. A steam flow which is too slow
also results in decreased efficiency because air-steam mixtures are forming.
Where non-liquid materials with slow heat conductance are to be sterilized, or where the packing arrangement or volume of materials otherwise
retards penetration of heat, allow extra time for materials to reach 121

before beginning to time the sterilization period and,

if

necessary, use longer

sterilization periods to insure sterility.

After sterilization, gradually reduce pressure within the autoclave (no less

than 15 min

since liquids may be at temperatures above


atmospheric pressure. This is necessary because liquids
through boiling when lowering the pressure too rapidly. When
is

recommended)

their boiling point at

can be

lost

CULTURE MEDIA AND PREPARATION

60

dry materials, such as sampling equipment or empty sample botpressure may be released rapidly at the end of the 15-min holding peri-

Sterilizing
tles,

od at 121 C. This prevents collection of condensate and speeds up drying of


paper-wrapped equipment.
Decontamination of used plates, pipets, tubes, etc. should be done routinely in all microbiological laboratories. Dairy products, especially raw
milk, may harbor potential pathogens such as Staphylococcus, Streptococcus, etc. The same principles apply for loading the sterilizer and sterilization as for media preparation. Plastic petri dishes are most conveniently
sterilized by placing them in heat-resistant autoclaveable bags. When large
numbers of petri dishes are autoclaved, sterilization time should be at least
30 minutes. Insufficient sterilization of plastic petri dishes

some degree

they retain

is

indicated

when

of their original shape; properly autoclaved plates

become amorphous. This property by

itself,

however,

is

not an indication of

sterility.

B. Hot-air sterilization:
Sterilize

equipment with dry heat

in hot-air sterilizers

so that materials at

(338 F) for not less

the center of the load are heated to not less than 170

than

hour

(this usually requires

sure sterility, do not

exposure for about 2 hr

crowd the oven, and when the oven

preferably use a longer period or


packs or thermocouples should be used

city,

at 170 C).

is

slightly higher temperature.


in

To

in-

loaded to capa-

Spore

doubtful situations.

4.5 Quality Control


This chapter on preparation of culture media

is

in itself

a discussion of

day-to-day quality control practices; however, specific mention should be


made concerning the advisability of routine adherance to simple quality control

measures involved with preparation of culture media for use

in

standard-

ized procedures.

The following items represent minimal but adequate

quality control proce-

may be used by laboratories using standard methods to analyze


products. Many of them are mentioned elsewhere in this chapter, but

dures that
dairy

summarized here to emphasize their contribution to quality control in


media production. A portion of the items was taken from a DHEW pubare

lication.'^

A. Date the label of each bottle of dehydrated

was received and when

medium

indicating

when

it

was first opened.


B. Store dehydrated media in tightly capped bottles in a cool, dry place
protected from light, or in a refrigerator if necessary. Keep no more than a
6-month to a year's supply on hand, being sure to use older stocks first. Dehydrated media should be free flowing powders; if a change is noted in this
it

property, or in color, discard.


C. Whenever possible, commercial dehydrated media should be used.
D. Complete mixing of medium to form a homogeneous solution in water

4.6

is

Suitability of

Water

iSl

necessary before sterilization and dispensing. Stirring which causes foam-

ing should be avoided.

Performance of autoclave should be monitored by either a continuous


in combination with properly placed indicator
strips or discs, or spore strips or suspensions. The record for each "run"
should be dated, numbered, and filed.
E.

temperature recording device

Limit heating of the

F.

and

tion

sure,

it

sterilization.

medium

When

to the

minimum necessary

to insure solu-

the autoclave has reached atmospheric pres-

should be opened immediately following the recommended cycle.

Prolonged storage

in a

water bath should be avoided.

G. Check the final pH of each lot of medium, which should be cooled to


room temperature. The pH of agar is obtained using a slurry of the medium
in a

beaker.

H. Aseptic technique should be followed strictly during dispensing of


sterilized material. Hands should not touch any part of the dispensing tubing
which comes in contact with sterile material. During interruptions in a dispensing cycle, the spout of a dispenser train should be placed
glass container.

in

a sterile

dispensing cycle should not be interrupted except in an

emergency.
/.

Moving

parts of any dispensing apparatus should be oiled or greased as

indicated at least once a month.

Any

leaks should be corrected immediately.

Accuracy of dispensation should be checked with a graduated container at


the beginning of each dispensing cycle.
J. Media containing dyes should be protected from light by storage in a
dark room, in a dark glass bottle, or by wrapping with brown paper.
K. Each container of autoclaved medium should be labeled with the name
of the medium and the autoclave "run number."
L. Inclusive dates during which each lot and container of dehydrated medium was used should be recorded for possible future troubleshooting.
M. Each lot should be inspected visually before use for volume, tightness
of closures, clarity, color, consistency, and completeness of label.
4.6 Suitability of

Water for

IVIicrobiological Applications

Only water that has been treated to free it from traces of dissolved metals
and bactericidal and inhibitory compounds should be used to prepare culture
media, reagents, and dilution blanks. However, for routine use in dairy
product analysis it is the growth medium or dilution system prepared with
the water which is of concern, not the water itself. Culture media provide
considerable protection from toxic agents present in water;^^ therefore, the
primary interest in water quality for routine applications is in its use as a
dilution fluid. A procedure to determine if dilution of a sample may have a
toxic effect

However,

is

given in Section 4.7(C).

if

special circumstances arise

be examined, specific resistance

in

dication of distilled water quality,

which suggest that water per se


as a rough in-

ohms may be determined


if

the apparatus

is

available. Generally,

CULTURE MEDIA AND PREPARATION

62

between 400,000 and 500,000 ohms specific resistance is the breakpoint between acceptable and unacceptable distilled water. A procedure to determine specific resistance is given in a publication of the American Society of
Medical Technologists (ASMT) entitled Reagent Grade Water.
However,
only ionic concentration is measured by specific resistance; presence of organic substances is not detected by this procedure. If organic contamination
'^'^

is

suspected an experienced chemist should be contacted to

make

the deter-

mination.

from chlorinated water occasionally contains significant


Distillate
amounts of free chlorine, even when passed through an ion-exchange resin
column before use. If distilled or treated water gives color with an orthotolidine test,'^ it should be neutralized with sodium thiosulfate before use for
milk dilution blanks.

4.7 Preparation of Phosphate-buffered Dilution


Testing for Toxicity

Water and

A. Stock phosphate buffer and magnesium sulfate sohitions:


1. Phosphate buffer: To prepare stock solution, preferably use previously adjusted dehydrated buffer. Alternatively, dissolve 34 g of potassium dihydrogen phosphate (KH2PO4) in 500 ml of distilled water, adjust to

pH
If

7.2 with

N NaOH

solution,

and make up to

desired, place in small vials, sterilize at 121

refrigerator to prevent microbial

Magnesium

2.

MgS04

make

liter

7H2O

sulfate:

To

growth

liter

with distilled water.

for 15 min,

in the buffer

and store

in

before use.

prepare stock solution, measure 50 g of


and add distilled water to

into a 1-liter volumetric flask

of solution.

B. Buffered dilution water:

To prepare

dilution water, add 1.25 ml of stock phosphate buffer solution


ml of stock MgS04 7H2O solution to distilled water and make up to
liter. Dispense as desired and autoclave at 121 C for 15 minutes. Addition
of magnesium sulfate improves recovery of organisms with metabolic injury
which may be induced by toxic properties in the dilution water. ^' "

and

C. Interval Plating Procedure:

Some

data

indicate existence of toxic or inhibitory materials in unbuf-

fered treated water

when used

as a diluent.

When

treated water

is

used as a

base for buffered diluents (and also when tap water must be used, which is
contrary to good laboratory practice because of variable composition and
progressive precipitation of mineral salts onto walls of dilution bottles), a

made for presence


ammonium, etc.).

periodic check should be


chlorine, copper,

To determine any

toxicity in prepared

the Interval Plating Procedure

"

of toxic substances (such as

phosphate buffered dilution water,

as described herein should be

done rou-

4.7

63

Dilution Water

weekly) along with the normal workload. The procedure,


in Figure 4:1, has not been subjected to collaborative
testing, but comparative studies have been made which indicate that the test
is sufficiently sensitive and satisfactory for use in laboratories that routinely
tinely (at least

shown schematically

add 1.25ml
stock phosphate
buffer

E. coli

(ATCC 25922)

from

slant

2-3ml Nutrient
Broth

60 minutes after 10 ^
dilution achieved

(2mj)(2mj)(^(2^
pour with

VRB

Incubate plates overnight at 32C. Count.

Compute average of five plates for each time.


Compute % change between zero and 60 minutes.

Figure 4:1. Interval Plating Procedure

agar

CULTURE MEDIA AND PREPARATION

64

Because the test is easy to do and because it is the most workable


system available, laboratory workers are urged to use this procedure.
The Interval Plating Procedure is a check on the dilution system as a
whole, not just the treated water which was used to prepare it. Detergent
residues which have persisted after washing bottles, phenolic or other residues in new caps, or chemical contaminants in reagents used to prepare
stock solutions may lead to a toxic dilution system, when treated water that
was used may be completely acceptable.
test milk.

1.

Procedure:
a.

The

treated water to be

examined

is

the procedure described in 4.7(B). This

amounts

into standard dilution bottles

made
is

by
99-ml

into dilution water

then dispensed

and autoclaved

at

in

121

for

15 minutes.
b. Escherichia coli (ATCC 25922) is used as the test organism. On the
day before the Interval Plating Procedure is to be done, 2-3 ml of Nutrient Broth is inoculated from a nutrient agar slant culture and incubated
at 32 C for 18 hours. One-tenth milliliter of the broth culture is then
added to 100 ml of Nutrient Broth in a flask and incubated for 4 hr at
32 C to obtain a culture in the mid-log phase of growth. The 4-hr culture
is then siluted serially to 10"** in three of the dilution blanks prepared as
indicated in a. The 10"^ dilution is immediately plated as the "zero"
time dilution by measuring 2 ml of the dilution into each of five plates
and mixing with Violet Red Bile Agar maintained in a water bath at
45 C. The culture is allowed to remain in the dilution water at room
temperature for 60 min, and then plated again using the same protocol
as for the "zero" time plating. The plates are then incubated at 32 C
for 18-24 hours. Colonies are enumerated and the following calculation
is made:

Mean Colony Count (60 min)

Mean Colony Count

Mean Colony Count


=

change

in

(0

(0

min)

min)

population between

and 60 minutes

The per cent change in colony count between zero and 60 min should
not exceed 15% to be considered acceptable.
4.8 Cleaning

Glassware and Testing

for Detergent

Residues

Modern detergents are very efl'ective for cleaning laboratory glassware.


Most of these are of the anionic type, usually with alkaline builders such as
silicates. Some detergents, especially the cationammonium compounds or "quats"), are highly bacteri-

phosphates, carbonates, or
ic

type (quaternary

and great care must be exercised to ensure their removal. Detergents


and soaps have a great affinity for all surfaces and because of this characteristic they displace dirt and allow it to be easily washed away. However,
because of this same characteristic, they are difficult to remove completely.

cidal

65

Culture Media Formulas

4.9

A. Cleaning glassware:

Most common detergents

in

laboratory use are satisfactory for general

purposes; however, occasionally deposits of "milk stone" or calcium salts


are encountered

which

resist

cleaning by ordinary means. These salts must

be removed by rinsing glassware several minutes in acid solutions before


eflFective cleansing can be achieved. A suitable solution for removing milkstone contains dichromate and sulfuric acid. This is prepared by dissolving
40 g of finely ground potassium dichromate in 150 ml of treated water. Place
in a pyrex vessel and add very slowly with continuous stirring 330 ml of
sulfuric acid such as used in the Babcock test for milkfat. During addition of
acid the vessel containing the dichromate solution should be cooled by placing

it

in a

cold water bath such as a stoppered sink.

The laboratory worker

when making the cleaning solution.


wash
is
The detergent
best done with hot water after preliminary rinsing
remove
most of the dirt. Soaking aids in removal of
with warm water to
stubborn residues. Glassware having residues which are not removed by the
detergent action should be immersed for 24 hr in the acid-dichromate cleanshould wear eye protection

and then rinsed thoroughly in tap and distilled water. Traces of


glassware
can be detected with pH indicator paper after the last
acid on
rinse. Six to 12 rinses with running tap water followed by several rinses with
ing solution

distilled

water are necessary for complete removal of detergent.

B. Detergent residues:

doubt remains as to the effectiveness of rinsing, especially if a quaterammonium compound has been used, the following procedure may be
used to detect bactericidal residues: glass petri dishes are prepared in three
ways: one set of three is washed and sterilized by the usual method; a secIf

nary

ond

set of three is

They

washed

in the

acid cleaning solution, then in castile soap.

are then rinsed four times in tap water and six times in distilled water

before sterilizing.

third set of petri dishes

is

dipped

in the

presently-used

detergent solution and sterilized without rinsing.

A sample of milk is plated in triplicate in these dishes and colonies are


counted after two days at 32 C. There should be no significant difference in
counts between the first and second sets of plates. A reduction in bacterial
count or diminished size of colonies may be apparent in plates of group three
if the detergent in use is bactericidal or bacteriostatic.
4.9

Formulas of Culture Media and Directions to Prepare Media


Use

for

This section contains formulas and preparation directions for two culture
media mentioned in this publication which may not be commercially available. The formula and specifications for Standard Methods Agar have been
retained since Standard Methods for the Examination of Dairy Products is
the original reference for this medium. In addition, comments on preparation
and use of a few of the commercially prepared media are included.

CULTURE MEDIA AND PREPARATION

66

A. Citrate Azide Agar:^^-

^'^

Yeast extract
Casitone or trypticase

(USP pancreatic

digest of casein)

Sodium citrate
Sodium azide

10

20

0.4

Ditetrazolium chloride (tetrazolium blue)

Agar

make

0.01 g
15

Microbiologically suitable water to

To

10

g
liter

prepare the basic medium, do not add sodium azide or tetrazolium

Heat to boiling to dissolve other ingredients. Dispense in 100-ml


amounts and autoclave at 121 C for 20 minutes.
Before adding final ingredients, temper medium to 48 C and to each batch
of 100 ml add
ml of 0.1 % sterile (121 C for 20 min) aqueous solution of
tetrazolium blue and
ml of 4 % sterile (121 C for 20 min) aqueous solution
of sodium azide. Final pH 7.0 0.2 (a 25 C.
Pour about 15 ml of the final prepared medium per plate. After solidification, stratify the plates with a thin layer of the same medium.
For enumeration of colonies, place a thin sheet of white paper (facial tissue) underneath a petri dish on the illuminated colony counter to enhance
color contrast between colonies and the background.
blue.

B. Eosin Methylene Blue Agar, Levine:^^

Note on preparation and use: Avoid overheating, as the agar is likely to


become soft and unsuitable for streaking. When it is to be used as a streaking
medium, melt agar in flowing steam or boiling water. Gently swirl contents
of flask to distribute flocculant precipitate and pour a thick layer into petri
plates. Set cover aside to leave about one-third of the plate exposed for
escape of moisture vapor and to dry surface of agar after solidification. A dry
surface promotes growth of distinct individual colonies and gives better type
diff"erentiation. Plates should be used within a week and can be stored inverted in the refrigerator. When stored, incubate non-inoculated plates as a

check for contamination.


C. Lactose Broth:

Note on preparation: When fermentation tubes or other containers are


prepared to examine 10-ml portions of samples, the lactose broth medium
must be of such strength that addition of that volume of sample to the medium will not reduce the concentration of ingredients below that in the standard medium. When 10-ml portions are inoculated into 10 ml of medium, the
medium must be formulated at double strength. When 10-ml portions are
inoculated into 20 ml of medium, the medium must be formulated at 1.5 times
the strength of the standard ingredients.

D.

MF-Endo

Broth:""

Note on use: For best

results,

medium should be prepared and used

the

67

Culture Media Formulas

4.9

same day; however,

it

may be

more than five days if kept


from bright Hght and refrigerated.

stored for not

tightly closed container, protected

Peptone
Glucose
Potassium dihydrogen phosphate (KH2PO4)

Magnesium

sulfate

(MgS04

10

g
g

THjO)

0.5

Agar

20

Distilled

chlortetracycline hydrochloride:.5

Rose Bengal Agar with

E.

in

water to make

g
g
liter

Suspend ingredients in water and heat to boihng to dissolve completely.


Dispense 100 ml of medium into screw-cap containers. Sterilize at 121 C for
15 minutes.

Prepare stock solution of


121

1% Rose Bengal

in distilled

water. Sterilize at

for 15 minutes. Store at 4 C.

Prepare stock solution of chlortetracycline hydrochloride by dissolving


mg in 100 ml of distilled water. Store at 4 C for no more than one week.
To 100 ml of melted and cooled agar, add 0.2 ml of the Rose Bengal and

100

ml of the chlortetracycline hydrochloride stock solutions just before dis-

tributing into appropriate tubes or plates. Final

pH

6.0

0.2

(qj

25 C.

Methods Agar:
Formulation of Standard Methods Agar shall conform to that for Standard
Methods Agar described in Standard Methods for the Examination of Dairy
Products, 13 ed.'^ This is the same formulation described for Plate Count
Agar, Dehydrated (Tryptone Glucose Yeast Agar) in Standard Methods for
the Examination of Water and Wastewater, 14th ed.^^ It is also the same
formulation as for Tryptone Glucose Yeast Agar of the Association of OffiF. Standard

Chemists.^ The formulation consists of the following

cial Analytical

gredients and quantities per

liter

Pancreatic digest of casein

5.0

Yeast extract

2.5

Glucose
Agar

1-0

15.0

make

Microbiologically suitable water to

Final

pH

in-

of medium:

should be 7.0 0.2

(a

25

after sterilization at

liter

121

for

15 minutes.

Ingredients used in formulation for Standards

Methods Agar

shall

con-

form to the following specifications:


1.

Pancreatic digest of casein

U.S. Pharmacopoeia XIX, p

Shall be of USP quality as given

in

the

744. 2"

Yeast extract Shall conform to specifications for yeast extract as


2"
in the U.S. Pharmacopoeia XIX, p 758.
Shall conform to specifications for glucose as given in the
3. Glucose
U.S. Pharmacopoeia XIX, p 128 '^'\ it shall be anhydrous.
2.

given

CULTURE MEDIA AND PREPARATION

68

Shall conform to the following specifications:

Agar

4.

General characteristics: The dry material shall be in granule or


flake form, cream-white to pale tan in color. An amount of 2.0 g should
be evenly suspendable in 100 ml of cold distilled water without lumping.
On application of heat, solution should be complete within 5 min at
a.

100 C.

After autoclaving the mixture at 121 C for 15 min, a hot 2.0% solution
clear or hazy, but should be without milkiness and free from

may be

extraneous matter or suspended particles which might be confused with


and cooling to room temperature,
the pH should be not less than 6.0.

bacterial colonies. After autoclaving

b.

Temperature of gelation and melting: A 1.5 % hot aqueous soluupon cooling, gel at not over 43 C, nor less than 33 C. After

tion should,

gelation, the solidified material should not melt at less than 70 C.


c.

Add

Viable spore count: The viable spore count should not exceed 50.
ml of sterile soybean casein digest broth USP, and

2 g of agar to 100

autoclave the mixture

at 121

for 5 minutes.

The mixture

obtain uniform distribution and, after cooling to 45

is

is

rotated to

poured into

three or four sterile petri plates. Incubate the plates for 48 hr at 32 to

C and

35

not be

G. Violet

count colonies. The

more than

Red

Bile

total

2,

should

Agar:

Note on preparation: Violet Red

Bile

Agar should not be autoclaved.^

In-

should be boiled for 2 min, then used immediately as a plating mediafter cooling to 45 C.

stead,

um

colony count, divided by

50.

it

4.10 Physical Standards for Standard


Standard Methods Agar medium
ments:
A.

It

shall

shall

Methods Agar

conform to the following require-

be prepared from ingredients which meet specifications for inMethods Agar medi-

gredients of, and according to the formula for, Standard

um

as described in 4.9(F) preceding.

be a uniform free-flowing beige or light bufif-colored powder.


have a moisture content of less than 5.0 %. Moisture is to be
determined using a moisture balance with a 125-W, 115- to 125-V infrared
industrial reflector bulb; a Powerstat transformer set at 100 is to be in the line
to prevent charring. The exposure shall be 15 minutes.
D. It shall be soluble in 2.35
solution in distilled water upon boiling for
1 to 2 min to yield a slightly opalescent-to-clear solution without precipitate.
E. It shall have a final reaction of pH 7.0 0.2 at 25 C after being autoB.

It

shall

C.

It

shall

claved at 121
F.

56

It

for 15 minutes.

shall not

develop an objectionable precipitate when held

at

50 to

for 2 hr after autoclaving that could interfere with the Standard Plate

Count.

4.1

69

Productivity Tests

4.11

Productivity Tests for Standard

Productivity of each

lot

Public Health Association

Methods Agar Medium

medium shall be compared with the American


(APHA) Reference Standard for Standard Meth-

of

ods Agar" with respect to a.) number of colonies and b.) size of the colonies.
This primary standard must be used in all testing to determine acceptability
of each

lot;

use of a secondary standard for

official testing is

not permissible.

This method was approved by the Coordinating Committee on Laboratory


Methods (APHA) and was adopted by the APHA in 1965 as recommended.
The method compares series of replicate plates for the test medium with a

on the Reference Standard, using four milk composites. An acceptmedium must yield counts within 10 9c of those obtained on the
Reference Standard. This protocol specifies that the testing laboratory
achieve results with no more than 10 % coefficient of variation and requires
20 replicate plates per milk composite for each medium. If the laboratory can
achieve a 5 9?^ coefficient of variation, only 10 replicate plates per composite
for each medium will be required [4.11(G)].
series

able test

A. Preparation of reference and test media:


1. Prepare each medium according to the manufacturer's directions on
the label of the container.
2.
3.

121

Dispense each medium in 15-ml amounts in tubes.


Sterilize according to the manufacturer's directions (generally

at

for 15 minutes.

B. Preparation of milk composites:


1
Prepare four composites of raw milk samples, using no less than five
well-mixed individual milk samples for each composite. Mix the individual
samples with a vortex mixer before each composite is made. Make composites as soon as possible (within 24 hr) after the samples are received in

the laboratory. Pasteurized milk composites


different
2.

Do a preliminary

dilution to use
If

may be prepared and

tested on

days from raw milk composites.


count,

that which

a preliminary count

is

if

desired, to determine the most satisfactory

will yield

approximately 200 colonies per plate.

not made, two dilutions will have to be plated for

the comparison to be described.


is made or if composites are to be held for
composites should be kept frozen and diluted to the appropriate dilution when ready for plating. Use composites within 72 hr after

3.

If

a preliminary count

testing, undiluted

preparation.

C. Dilution of milk composites:


When ready to begin plating and testing of media, dilute each composite in
10-fold dilutions, using sterile phosphate-buffered water, which is prepared
as described in 4.7(B).

'The Reference Standard

Eighteenth Street,

NW,

is

available from the

Washington,

DC

20036.

American Public Health Association, 1015

CULTURE MEDIA AND PREPARATION

70

D. Plating of diluted milk composites:


1. Melt each medium in a steam bath and hold in a water bath at 44 to
46 C for no longer than 24 hr, provided that a precipitate does not form. Do
not melt
2.

made

medium more

Plate

than once.

two consecutive

dilutions,

if

preliminary counts have not been

Use

dilutions of milk composites that


colonies
per
plate.
The allowable plate count
approximately
200
will yield
range is 100-300 colonies per plate, provided that no single plate count exceeds 300 and no average count is below 100.
3. Use a sterile 2-ml pipet (calibrated to deliver 2.0 0.02 ml) for all
plates for each dilution of each milk composite. Before sterilization, the pito establish a countable dilution.

pet should be thoroughly cleaned so that liquid does not adhere to

its

wall.

E. Plating pattern:
Plating shall be
1

done according

Number plates

to the following pattern:


according to the pipet-pour scheme given

Table

4:1.

COMPOSITE]

Two Milk Composite Schemes

in

Table

4:1.

4.11

71

Productivity Tests

3. Place 2 ml of the diluted milk composite in each plate, S-1 through


S-5 and T-1 through T-5, allowing milk to flon' freely from the pipet. Drain
for 5 seconds. Touch pipet tip briefly to a dry portion of the plate Pour one
tube of Reference Standard medium (15 ml) into each plate S-1 through S-5,
rotating each plate, as poured, to achieve adequate dispersal of inoculum.
Repeat with the test medium in plates T-1 through T-5.
4. Pipet the diluted milk to plates T-6 through T-10, then to S-6
through S-10. Pour appropriate medium into each plate in accordance with
Table 4:1. If 20 plates are required for each medium, continue this procedure with plates S-11 through S-15, then with T-11 through T-15, followed
by plates T-16 through T-20 and S-16 through S-20.
5. Reverse order for the second composite. Repeat for the other type of
milk composite.
.

6.

Invert

all

plates; incubate at 32

for 48 hours.

and colony size:


colony
counter
for enumeration. Productivity with respect to size
Use a
shall be considered comparable if the colonies on the test medium are generally similar in size to those obtained on the reference medium, using the
required number of samples as specified.
F. Enumeration

G. Statistical analysis and interpretation of results:


The method of comparison just described requires certain limits on coefficients of variation for the media involved. To calculate the coefficient of
variation for counts from each

X =
and

Calculate: the arithmetic


test

medium,

let

plate count obtained

= number of replicate plates.


mean (or average) plate count

for reference

and

media:

x=

^^
N

and the sample standard deviation for reference and

Then

test

media:

calculate the coefiicient of variation by:

c.v. =

(ijioo.

(This is usually expressed as a percentage.)


20 plates are used for each medium, the coefficient of variation cannot exceed 10%. If this value is exceeded for either medium, repeat replicates on both media until satisfactory precision is obtained. When 10 plates

When

CULTURE MEDIA AND PREPARATION

72

medium, the coeflficient of variation cannot exceed 5%. If


exceeded for either medium, repeat repHcates on both media

are used for each


this

value

is

until satisfactory precision is obtained.

Once satisfactory precision has been achieved, the average counts on the
two media are compared by calculating the corresponding Student's t-value.
This is done as follows:
let

Xr = average plate count for the reference medium


Xj = average plate count for the test medium
N = number of plates counted for each type of medium*
Sr = variance for the reference medium

and
St

variance for the test medium,

then calculate Student's

by

Xr

s|

Xi

s%

N
For each comparison, the t-value should be less than 2.70 when N = 20,
when N = 10. (A 1% level of significance is used for this
statistical test, corresponding values at the 5% level are 2.02 and 2.55, respectively.) If the calculated t-value for any milk comparison exceeds the
above values, the test medium should be rejected.
or less than 2.88

H. Statistical considerations for the number of replicates required:


The number of replicates required to detect a prescribed difference between average counts of test and reference media depends on the following
factors:'^

d = the difference to be detected between average counts of the test mediand the reference medium. This difference is of practical importance

um

*If diflferent

Let

This

is

plates are used for some reason, proceed as follows:


of plates counted for the reference medium
of plates counted for the test medium, then

numbers of

Nr = number
Nj = number

IXr

=
(N + Nt)

NrNt

distributed as Student's

priate significance levels can be

with

found

Xi

s^CNh -

+ s|(Nt +
Nt - 2
Nr
1)

Nr + Ni in

1)

2 degrees of freedom.

a table of t-values.

The appro-

4.11

73

Productivity Tests

when it is at least 10% of the average count of the reference medium, (i.e.,
d = .10 Xr)
a - standard deviation, a measure of the variability in counts. On the
basis of experimental tests, it may be assumed that the standard deviation is
the same for counts obtained on the reference medium and on the test medium. The magnitude of the standard deviation was found to be around 10% of
the average count. By requiring each comparative series of plates to exhibit
no greater than 10% coefficient of variation, an estimate may be established
for the standard deviation as 10% of the average count for the reference
medium (i.e., cr = .10 Xr). For more precise results, if each comparative
series of plates

is

required to exhibit no greater than

5%

coefficient of varia-

an estimate may be established for the standard deviation as 5% of the


average count for the reference medium, {i.e., a = .05 Xr).
a = level of significance, the probability of concluding that there is a difference between average counts obtained on the reference medium and on
the test medium when, in fact, there is none. This probability should be
tion,

small. Accordingly, a

1%

(a

.01) level of significance

has been used in the

following table.

no difference between the average counts


of the test medium and of the reference medium when, in fact, a difference
does exist. This probability should also be small. Accordingly, values of
0.05 and 0.01 were employed for ft in the following table.
To determine the number of replicates required by the above criteria, the
ft

the probability that there

is

"standardized difference'' D, where

D =

is

computed. This

ratio de-

er

pends on d (the magnitude of the difference to be detected) and on a (the


standard deviation). Three situations are presented below relating d and or:
1. Assume that the testing laboratories can achieve results with coefficients of variation of 10%, and that a 5% difference is to be detected between
average counts obtained on the test medium and the reference standard,
then

A
o-

2.

Assume

^S ^
=

.10

Xr

medium and

.50.

5%

and that a

5%

difference

difference, and

is

to be detected

between

the reference standard, then

Similar calculations can be used for a

10%

.10

that the testing laboratories can achieve results with coeffi-

cients of variation of

the test

10%

coefficient of variation

and a

X^ uofuev of D imt

ctte

mmTt?"

v?f

-sriicices -^smursu:

m eadt smtuhen vie-

;C&^i|irilM>lT4

*5

onr.irrc

"^'

..A.

"^r

T'

-^

-rencrilm

i.

it

-unr

"tai -tfw

rv

otuipcai uanini-

.-.,

>

^-:

-r:-" >.-. v;:er.TMrtium.Sir:lirvifiSB;-^

rwsr -~...
-

^u/"*.

Jvparaiwm jf cfteiitft.

Suu)ci{tnr. 4nic Warfant.

-n

vwater^MwiyMs.

luwi

'llism-

Canter Sir DisBeCartrti. ^ctiuna.

CHAPTER

STANDARD PLATE COUNT METHOD


W.S. Clark,

5.1

Jr., A.R. Brazis, J.L. Fowler,


C.K. Johns, and F.E. Nelson

Introduction

Historically, the

main

cultural procedure to determine viable bacterial

populations in dairy products has been an agar plate method, which

is

highly empirical technique because bacteria occur singly, in pairs, chains,

number of colonies that develop per


gram of sample frequently may be smaller than the actual
number of individual cells present. Additionally, some bacteria because of

clusters or packets. Consequently, the


milliliter

or per

requirements for special nutrients, reaction to oxygen, unfavorable


incubation temperatures or other factors are unable to form visible colonies under conditions employed in this procedure.

injury,

The Standard Plate Count, employing uniform standardization of equipment, materials and incubation, is of considerable value. The method is suitable for measuring bacterial populations in most types of dairy products. It is
the method specified in the Grade A Pasteurized Milk Ordinance ^^ to examine raw and pasteurized milk and milk products. It also is recommended for
industry application in detecting sources of contamination by testing line
samples taken at successive stages of processing. Cultured dairy products,
(e.g., cultured buttermilk, non-fermented acidophilus milk), or dairy products to which a bacterial culture has been added, ordinarily are not tested by
the Standard Plate Count.
While standardization of equipment, materials and incubation in doing the
Standard Plate Count has been of considerable value, questions continue to
be raised concerning the ability of this procedure to completely reflect sanitary practices used to produce and handle milk. As a result, procedures for
preliminary incubation of milk in conjunction with determining the Standard
Plate Count may be useful to provide additional information about the bactesample (see Appendix A). Additionally, a more commay be obtained by
incubation of additional plates, at other temperatures and for various periods of time.
riological quality of a

plete picture of the bacterial content of dairy products

ISC Liaison: W.S. Clark,

Jr.

77

STANDARD PLATE COUNT METHOD

78

5.2

Equipment and Supplies

A. Work area:
The work area should be a table or other rigid support, level, with ample
surface, in a clean, well-lighted (at least 50- and preferably 100-footcandles

and well-ventilated room reasonably free from


(bacteria, yeasts and molds)
in air in plating areas, as determined during plating by exposure of poured
plates, should not exceed 15 colonies per plate during a 15-min exposure
at

each working surface

dust and drafts.

^')

The number of microorganisms

[see A.8].

B. Storage space:

Cabinets, drawers or shelves should be clean and adequate for protection

of glassware, apparatus and other equipment from breakage.


C. Refrigerator:

and maintain temperature of samples between


may be used for storage of prepared media when desired. Record temperature daily.
refrigerator should cool

0-4.4

(32-40 F) until tested, and

D. Thermometers:

Thermometers of appropriate range, may be mercury-filled

(or having a
lower than - 1 C), or of an adjustable type, with a graduation interval not to exceed 1 C. Unless otherwise
specified, accuracy should be checked at least every two years with a thermometer certified by the National Bureau of Standards or one of equivalent
distinctively colored fluid with a freezing point

accuracy.

Where

a record

is

desired of temperature in refrigerators, auto-

claves, hot-air ovens, or incubators, automatic temperature-recording in-

may be used.
There are two general types of mercury thermometers in I4se in laboratories, those calibrated for total immersion and those designed to be partially
immersed. Partial-immersion thermometers have a line completely around
the stem of the thermometer at the point to which they should be immersed.
If this line is absent, the thermometer is designed for total immersion. As an
example, a partial-immersion thermometer should be used in an incubator
[5.2(P)] or refrigerator [5.2(C)] because only part of the thermometer is immersed in a control vial of water, the temperature of which is being measured. Conversely, a thermometer placed on the bottom shelf of a water bath
is totally immersed in the warm (or cold) environment and should be of the
struments

total-immersion type.

The

easiest

way

to

check the calibration of laboratory thermometers

put them in a water bath, either partially or totally

immersed

in the

is

to

water,

according to the way they will be used in the laboratory. Also place in the
water bath a thermometer certified by the National Bureau of Standards.
Most, but not all, of these thermometers are calibrated for total immersion.

Equipment and Supplies

5.2

Vigorous

stirring of the

79

water

in

the bath

is

essential to insure uniform tem-

perature during thermometer caHbration.

Check

the calibration by comparing the temperature reading on the certithermometer with that of the laboratory thermometer at or very near the
temperature the thermometer will be used to measure (e.g., an incubator
thermometer should be checked at 32 C because this is the temperature of
interest). If the thermometer is to be used for several purposes, it should be
checked at the different temperatures of use. If there is a difference in the
temperature reading between laboratory and certified thermometers after
the reading of the certified thermometer has been corrected as indicated by
the certificate, attach a tag to the laboratory thermometer to show the
amount of correction that should be applied to obtain an accurate measurement of temperature.
fied

E.

Transfer pipets {glass and plastic):^^'

'^'

'**

Pipets should be nontoxic, with walls straight, tips ground or fire-polished


(glass), calibrated for bacteriological use,

and conforming to

APHA

specifi-

Use only pipets with unbroken tips and having graduations distinctly marked to contrast sharply with the color of milk or diluted
milk. Discard pipets with damaged tips.
cations (Fig. 5:1).*

F. Pipet containers:
Stainless steel or

aluminum pipet containers are preferred. Copper con-

tainers shall not be used. Char-resistant, high-quality sulfite pulp kraft paper

may be

used.

G. Dilution bottles:
Dilution bottles should have a capacity of about 150 ml, be of borosilicate
glass, and closed with Escher- type stoppers or screw caps. Use friction-fit
liners in screw caps, as required, to make the closure leakproof. Be sure that
each batch of dilution blanks is properly filled [5.3(D)]. Dilution bottles
should be marked indelibly at 99 1 ml graduation level. Or, bottles or test
tubes of about 15-ml capacity. Plastic caps for bottles or tubes and plastic

closures for sample containers must be treated,


residues. This

to

remove

toxic

be accomplished by autoclaving them twice while they


water, or exposing them to two successive washings in

are submerged in
82-C (180 F) water containing a

suitable detergent.

H. Petri dishes or plates:


Dishes should have bottoms of
*

when new,

may

Volume delivered

in

4-sec

maximum,

last

at

least

85-mm

inside diameter,

drop of undiluted milk blown out, or

last

drop of

ml tolerance 0.025 ml. To allow for residual milk and milk dilutions
on walls and in the tip of the glass pipet under the specific technique hereinafter described for
rapid transfers, such pipets shall be graduated to deliver 1 .075 mi of water at 20 C (68 F). If the
pipet is of styrene plastic, it should be calibrated to deliver 1.055 ml of water at 20 C.
diluted milk touched out,

STANDARD PLATE COUNT METHOD

80

mm

deep, with interior and exterior surfaces of bottoms, free from bubbles, scratches or other defects. Petri dishes may be made of glass or plas12

tic.^-

^^

11 ml

1.1
1

ml
II

o
o
T
1(/)

<

-J

<

i_
E
E
in

(M
I-

<

<

ml

5.2

/.

Equipment and Supplies

81

Petri dish containers:

Containers of stainless steel or aluminum, with covers, preferred. Charpaper may be used. Singleservice petri dishes may be stored in their original containers.

resistant sacks of high-quality sulfite pulp kraft

Balance:
Balance should be sensitive to 0.1

J.

g,

with 200-g load.

single-pan balance

preferred.

K. Mechanical shaker:
A shaker as described in the 11th edition of this manual

may be

used.

Water bath:

L.

A water bath of appropriate size should be thermostatically controlled, to


hold melted medium at 44-46 C (111.2-114.8 F).
M. Colony counter:
Standard apparatus, Quebec model preferred,^" or one providing equivalent magnification

N.

and

visibility.

Tally:

mechanical counting device should be used.

O. Instrumental colony counter :^'^

An instrumental colony counter may be used when determined to give


counts equivalent to those obtained manually [5.11].
P. Incubator or incubator room:

An

incubator or incubator room must be properly constructed and conTemperature within the incubator must not vary more than 1 C

trolled.

from the 32-C

setting.^

tor or incubator

room

there will be at least

Determine temperature variations within the incubaat


in.

two or more locations. Load incubators so that


of space between adjacent stacks of plates and

between walls and stacks.


Incubator rooms must be well insulated, equipped with properly distributed heating units, and have forced-air circulation.
Keep incubators in rooms where temperatures are within the range of
16-27 C (60.8-80.6 F). Laboratory personnel are responsible 1.) for recording temperatures in the top and bottom of the incubator or incubator room
twice daily in the morning after the incubator has been closed all night, and
just before the end of the day's work; and 2.) for adjusting the thermoregulator. Place thermometers on top and bottom shelves, and in between as
needed. Thermometer bulbs must be submerged in water within small, tightly closed vials, as air temperature is an unreliable index of actual agar tem-

peratures.

Optionally use automatic devices of predetermined accuracy for con-

STANDARD PLATE COUNT METHOD

82

trolling

and recording temperatures

Check accuracy of these devices

in

incubators and/or incubator rooms.

periodically

by comparing readings with

those from standard thermometers.

When

standard thermometers are used, minimum- and maximum-registhermometers may be placed in each incubator to indicate undetected
gross temperature deviations and the degree of such deviations. Do not depend on readings from these special thermometers for daily records of temperatures. Minimum- and maximum-registering thermometers are unnecessary when automatic devices of predetermined accuracy for controlling and
recording temperatures are in continuous and proper operation.
tering

Q. Autoclave:
An autoclave shall be of a size sufficient to prevent crowding of the interior and be constructed to provide uniform temperatures within the chamber

up to and including the sterilizing temperature of 121 C. Additionally, it


must be equipped with an accurate mercury-filled thermometer or bimetallic
helix-dial thermometer properly located to register minimum temperature
within the sterilizing chamber (temperature-recording instrument optional),
pressure gauge and properly adjusted safety valve. Optionally provide temperature recorder-controller. Small 5- to 10-qt (4.73- to 9.46 -liters) pressure cookers, equipped with a thermometer for temperature control,
substituted for an autoclave

when proper temperature

is

may be

maintained and

re-

corded and satisfactory results are obtained.


R. Hot-air sterilizing oven:

The oven should be of a size sufficient to prevent crowding of the interior,


constructed to give uniform and adequate sterilizing temperatures, with
vents suitably located to insure prompt and uniform heat penetration, and
equipped with an indicating or recording thermometer suitably located to
register minimal temperatures. The thermometer should have a range broad
enough for the intended use [up to 220 C (428 F)].

5.3 Materials

A. Standard Methods Agar:

Standard Methods Agar should be prepared as described


B. Microhiologically Suitable

in

Chapter

4.

(MS) water:

MS

water should be prepared as described in 4.6. The term "water," as


used herein when describing preparation of media, reagents, dilution blanks,
etc., means only water that has been treated to free it from traces of dissolved metals and bactericidal and inhibitory compounds.
C. Dilution water:

Use only buffered

MS

water for dilutions

[4.7J.

Make

monthly to determine whether toxic substances are present

tests at least

[4.6].

Examination of Samples

5.5

83

D. Dilution water blanks:

MS water so that after sterilization each


Autoclave bottles of MS water at 121 C for 15 minthan 99-ml amounts are required (e.g.. 9 ml, 90 ml, etc.)

dilution bottles with buffered

Fill

will

contain 99

When

utes.

less

2 ml.

apply proportionally smaller deviation tolerances for amounts in dilution


blanks. Optionally use correctly calibrated automatic water-measuring devices.

When

is used, measure MS water directly into


and use prepared blanks promptly.

bulk-sterilized diluent

sterile dilution bottles

5.4 Sterilization
A. Steam:
[See 5.2(Q) and 4.4(A)]

Hot

B.

air:

[See 5.2(Q) and 4.4(B)]


C. Recording:

Where records of operations may be

desired or required for legal testimo-

ny, preferably record time at which oven or autoclave reaches sterilization

temperature,

each

lot

minimum temperature

used, and time of discontinuing heat for

of materials sterilized. Recording devices are useful for this pur-

pose.

Examination of Samples

5.5

A. Temperature:
After collection of samples, refrigerate and expeditiously transport to the
testing laboratory.

Upon

arrival at the laboratory,

determine the temper-

ature of each set of samples by inserting a precooled thermometer into a

separate pilot container treated exactly as the samples; record the temperature.

Do

not insert a thermometer into any sample intended for micro-

biological examination before removal of the test portion.

B. Collection, storage,

and examination times:

Standard methods to collect samples of milk and milk products are described in Chapter 3. Record time and dates of sample collection and receipt
at the

laboratory for each set of samples. At the laboratory, store samples at

until tested. Initiate testing of samples within 36 hr after collection


and record time of plating. Fluid milk samples that have been frozen should
not be tested microbiologically, as freezing causes a significant change in the
viable bacterial count in milk.*^

0-4.4

C.
If

Chemical

tests:

chemical tests are necessary,

analysis [5.6(B)].

first

remove portions

for microbiological

STANDARD PLATE COUNT METHOD

84

1.

METHOD OF EMPLOYING

ml

OF SAMPLE
10"

10'

DILUTION RATIO

NONE
1

ml

ml

99

SAMPLE

ml

0.1 ml

LABELING KEY

^^T^

VOL OF SAMPLE

1ml

\^T^

ml

0.1

ml

ml

0.1

4^
10~^ml

lO'^ml

10'''ml

\
<f

10"^ml

10"'*ml

PER DISH
2.

DILUTION RATIO

METHOD OF EMPLOYING

11 ml

OF SAMPLE

10"

NONE

10

11 ml

-3

ml

99

SAMPLE

ml

ml

0.1

ml

ml
I

0.1

ml

LABELING KEY

VOL OF SAMPLE

ml

10'''

ml

lO'^ml

lO'^ml

10"'^ml

PER DISH
Figure 5:2. Examples for preparing dilutions.

5.6 Preparing

Samples

A. Identifying plates:
Identify each plate with sample number, dilution, and other desired information before shaking samples and making dilutions.
B. Shaking samples

and

dilutions:

Before removal of the sample from its container, thoroughly and vigorously mix contents until assured that representative portions will be removed. Invert filled containers repeatedly until contents are homogeneous.
(If necessary to insure a homogeneous sample, aseptically pour the product
from the filled carton back and forth into a sterile container.) Before opening
a sample container,

may contaminate

remove from the closure

all

obvious materials which


unopened sample

the sample. Optionally wipe the tops of

5.7

Diluting

85

Samples

containers with a sterile cloth saturated with

70%

alcohol.

The

interval be-

tween mixing and removing the test aliquot shall not exceed 3 minutes.
Immediately before transferring test portion of milk or cream (except from
filled containers as noted above) and of dilutions thereof, shake container,
making 25 complete up-and-down (or back-and-forth) movements of about
one foot (30 cm) in 7 seconds. ^^' '^ Optionally, a mechanical shaker may be
used to shake the dilution blanks for 15 seconds.

Samples

5.7 Diluting

A. Selecting dilutions:

For Standard Plate Counts, select dilution(s) so that the total number of
^ on a plate will be between 30 and 300 (see Fig. 5:2). For example,
where a Standard Plate Count is expected to reach a number between 3,000
and 300,000, prepare plates containing 1 100 and 1 1000 dilutions. Dilutions
of 1 10 and 1 100 should be used for low-count samples.
colonies

B. Measuring sample portions:

Use a

and subsequent transfers from the same conIf the pipet does become
contaminated before completing transfers, replace it with a sterile pipet. Use
a separate sterile pipet for transfers from each different dilution. Add test
tainer

if

sterile pipet for initial

the pipet

is

not otherwise contaminated.

C (59-77 F).
CAUTION. Do not prepare or dispense dilutions or pour plates in direct
sunlight. When removing sterile pipets from the container, do not drag tips

portions of milk to dilution blanks adjusted to 15-25

over exposed exteriors of the pipets remaining in the case because exposed
ends of such pipets are subject to contamination. Do not wipe or drag pipet
across lips and necks of vials or dilution bottles.

Do

not insert pipets more

than one inch (2.5 cm) below the surface of the sample or dilution.

Draw

test

portions above the pipet graduation, then raise the pipet tip above the liquid

and adjust to the desired mark by allowing the lower side of the pipet
contact the inside of the containers (Fig. 5:3) in such a manner that
drainage is complete and excess liquid does not adhere * when pipets are
removed from sample or dilution bottles. Do not flame sterile pipets.
1. Milk and Milk Products: When measuring dairy products having a
viscosity similar to milk, complete each transfer by letting the column drain
from the graduation mark to the rest point of the liquid in the tip of the pipet
within 2-4 seconds. Promptly and gently blow the last drop out if transferring into a dilution blank. Make transfers carefully and do not rinse pipets in
dilution water. Plates can be prepared according to one of the methods

level,

tip to

shown

in Fig. 5:2.

Concentrated and Cultured Dairy Products: Where the solids consamples appreciably exceeds that of whole milk, prepare
the initial 1 100 (or 1 10) dilution by weighing a test portion into a warmed
2.

tent or viscosity of
:

STANDARD PLATE COUNT METHOD

86
dilution blank.

Weigh

g (or 11 g) aseptically into dilution bottles containing

99 ml of sterile, warmed (40 C) buflFered dilution water. Viscosities of these


dairy products vary at different temperatures, and differences occur in specific gravities, depending upon their composition. Furthermore, the amount
of trapped air will vary even in properly agitated samples.

Measuring dilutions:
measuring a diluted sample of dairy product, hold pipet at an angle
of about 45 with tip touching inside bottom of petri dish or inside neck of
dilution bottle. Lift cover of petri dish just high enough to insert pipet. Allow
2-4 sec for diluted milk or cream to drain from the graduation mark to the
rest point in the tip of the pipet; then, touch tip once against a dry spot on the
glass. Do not blow out into a petri dish. When 0.1-ml quantities are measured, hold pipet as directed and let diluted sample drain to the proper graduation point but do not retouch pipet to plate.
After depositing test portions in each series of plates, pour the medium

When

promptly [5.8(B)].

Figure 5:3. Measuring diluted sample or portions. Lower side of the pipet
touches the inside surface of the container.

87

Sterility Controls

5.9

5.8 Plating

A. Melting medium:
Melt required amount of medium quickly in boiling water or by exposure
steam in a partially closed container, but avoid prolonged ex-

to flowing

posure to unnecessarily high temperatures during and after melting. If the


medium is melted in two or more batches, use all of each batch in order of
melting, provided that contents of separate containers remain fully melted.
Discard melted Standard Methods Agar that develops a precipitate. Do not
medium. Freshly sterilized agar, after tempering to
45 C, also may be used.
resterilize the plating

Cool melted medium promptly to approximately 45 C and hold in a water


bath between 44 and 46 C, see 4.5(F).'*' Set a thermometer into an aqueous
solution of 1.5% agar in a separate container identical to that used for medi-

um;

this

temperature-control solution must have been exposed to the same

Do not depend upon the sense of touch


proper temperature of the medium when pouring agar.

heating and cooling as the medium.


to indicate the

and mixing agar:


number of samples

B. Pouring
Select the

to

be plated

in

any one series so

that not

more than 20 minutes (and preferably only 10 min) elapse between diluting
the first sample and pouring the last plate in the series .*- '^ (Should a continuous plating operation be done by a team, plan the work so that the time
between initial measurement of a test portion into diluent or directly into a
dish and pouring of the last plate for that sample is not more than 20 minutes.) Introduce 10 to 12 ml of liquefied medium at 44-46 C into each plate by
gently lifting the cover of the petri dish just high enough to pour the medium.
Carefully avoid spilling medium on outside of container or on the inside of
the plate lid when pouring. As each plate is poured, thoroughly mix the
medium with the test portions in the petri dish, taking care not to splash the
mixture over the edge, by rotating the dish first in one direction and then in
the opposite direction, by rotating and tilting the dish, or by using mechani-

Having thus spread the mixture evenly over the bottom of the
to solidify (within 10 min) on a level surface. After solidification, invert plates to prevent spreading colonies from developing [5.1 1(1)]
and promptly place them in the incubator.
cal rotators.

plate, allow

it

5.9 Sterility Controls of

Medium, Dilutions and Equipment

A. Material controls:
Check sterility of dilution waters and medium by pouring control plates
and, if desired,
for each sterilizaton lot of dilution blanks and medium used

for

each

lot

of petri dishes and pipets.

STANDARD PLATE COUNT METHOD

88
B. Additional controls:

number of samples in a series may vary considerably, prepare


one agar control plate for samples plated in the morning and another
agar control for samples plated in the afternoon. If colonies appear on agar
control plates, prepare additional control plates to determine if contamination came from the medium, water blanks, plates or pipets, or air [5.2(A)].
Since the

at least

5.10 Incubation
Incubate plates
Plates

at

32 1

for 48

hr for the Standard Plate Count. ^-

must reach the temperature of incubation within 2 hours. Incubation

times and temperatures for special bacterial groups, and procedural


ferences, are given in Chapters 6 and

Avoid excessive humidity

in the

trolling ventilation

15% of

its

and

dif-

7.

incubator to reduce the tendency for

spreader formation, but prevent excessive drying of the


than

^^

air circulation.

Agar

in plates

medium by con-

should not lose more

weight during 48 hr of incubation.

Counting Colonies on Plates and Recording Results

5.11

Count plates promptly after the incubation period [5.10]. If impossible to


count at once, store plates after the required incubation at approximately
4 C for not more than 24 hr,'^ but avoid this as a routine practice. When
counting colonies on plates, proceed according to directions given in
5.II(B-I). Also, for each lot of samples, record
ity tests

on materials

on the report

(dilution blanks, agar, etc.) used

results of steril-

when pouring

plates

[5.9(B)].

A. Counting of colonies:
1. Manual counting: Count colonies with the aid of magnification under
uniform and properly controlled artificial illumination, using a tally [5.2(N)].
Routinely use a colony counter - equipped with a guide plate ruled in square

centimeters [5.2(M)]. Plates should be examined in subdued

light.

Avoid

mistaking particles of undissolved medium, sample, or precipitated matter in


plates for pinpoint colonies.

Examine doubtful objects

carefully, using high-

from foreign matArrange schedules of laboratory analysts to prevent eye fatigue and the
inaccuracies that inevitably result from eyestrain.
2. Instrumental counting: Instrumental colony counters, when determined in individual laboratories to yield counts that 90% of the time are
within 10% of those obtained manually, may be used for counting agar
plates. When using colony counting instruments, exercise the following pre-

er magnification,

where required,

to distinguish colonies

ter.

cautions:'^

proper placement of
dish on colony counter surface
dishes
avoid "counting" stacking or of
petri

ribs

legs

plastic petri

89

Counting Colonies and Recording

5.11

do not count plates having unsmooth


agar surfaces
avoid plates having food
or
bubbles
the agar
do not count plates having spreaders or extremely
surface colonies
avoid scratched plates
wipe
and
dish bottoms before counting.
(stippled)

particles

air

in

large

fingerprints,

B.

One

films

off"

petri

plate with 30-300 colonies:^

Select a plate with 30-300 colonies unless excluded by 5.11(1).

Count

all

colonies, including those of pinpoint size, on the selected plate, record dilution used,

See Table

and report total colonies as a basis for the Standard Plate Count.
5:1, Sample Nos. 1001 and 1004.

C. Duplicate plates:
1.

Count

plates with 30-300 colonies and average the counts to obtain

Count (Sample No. 1011) except that if more than one


is prepared but only one plate yields 30-300 colonies, count both plates unless excluded by 5.11(1.1) or 5.11(1.2), include
these counts in the arithmetic average, and compute the count per milliliter
by multiplying the average number of colonies by the dilution used 15.7(A)].
See Table 5:1, Sample Nos. 1012 and 1013.
2. When both duplicate plates in one dilution but only one of the duplithe Standard Plate

plate of a given dilution

cate plates in the consecutive dilution yields 30-300 colonies, use only the
plates contained 30-300 colonies to compute Standard
See Table 5:1, Sample Nos. 1010 and 1015.
3. When both duplicate plates from consecutive decimal dilutions yield
30-300 colonies and are to be counted, compute the Standard Plate Count
per milliliter for each dilution and proceed as in 5.1 1(D) and 5.12. See Table
5:1, Sample No. 1014.

dilution

where both

Plate Count.

D. Consecutive dilutions (30-300 colonies):


If plates from two consecutive decimal dilutions yield 30-300 colonies
each, unless excluded by 5. 1 1(1), compute the Standard Plate Count per milliliter/or each dilution by multiplying the number of colonies per plate by the
dilution used [5.7(A)] and report the arithmetic average as the Standard Plate
Count per milliliter unless the higher computed count is more than twice the
lower one, in which case report the lower computed count as the Standard
Plate Count per milliliter or per gram, as applicable. See Table 5:1, Sample
Nos. 1002, 1003, 1014 and 1015.

E.

No

plate with 30-300 colonies:

no plate with 30-300 colonies and one or more plates have more
than 300 colonies, use plate(s) having a count nearest 300 colonies and count
If

there

is

STANDARD PLATE COUNT METHOD

90

plate(s) as in 5.11(D) and 5.11(H). Report as the Estimated Standard Plate


Count (ESPC). See Table 5:1, Sample No. 1009.

F. All plates with


If

from

plates

all

fewer than 30 colonies:

dilutions yield fewer than 30 colonies each, record actual

number of colonies on lowest dilution (unless excluded by 5.11(1) and report


count as Estimated Standard Plate Count per milliliter or per gram, as appliSee Table

cable.

5:1,

Sample No.

1007.

G. Plates with no colonies:


If plates

Table

5:1.

from

all

Examples

dilutions of

for

any sample of raw and/or heat-processed

Computing Standard

Gram
Sample

Plate

Count per

Milliliter or

per

91

Counting Colonies and Recording

5.11

dairy products have no colonies and inhibitory substances have not been
1
times the corresponding lowest
no colonies appear on the 1:100 dilution, report
count as "less than 100 (<100) Estimated Standard Plate Count" per milliliter or per gram, as applicable. See Table 5:1, Sample No. 1005.

detected, report count as less than (<)

For example,

dilution.

if

H. Crowded plates {more than 300 colonies):


If the

number of

colonies per plate exceeds 300, count colonies in those

portions of the plate that are representative of colony distribution and calculate the

Estimated Standard Plate Count from these counts.

If there are

few-

er than 10 colonies per square centimeter, count colonies in 13 squares, selecting,

if

representative, 7 consecutive squares horizontally across the plate

and 6 consecutive squares at right angles, being careful not to count a square
more than once. (The sum of colonies in 13 representative square centimeters multiplied by 5 yields the estimated colonies per plate when the area
of the plate

When

is

65 cm-.)

there are

more than

10 colonies per square centimeter, count colo-

nies in 4 such representative squares. Multiply the average

number found

per square centimeter by the appropriate factor to determine estimated number of colonies per plate. Because the average inside diameter of the bottom
of a standard glass petri dish

is

91

mm,

normally multiply the average num-

ber of colonies per square centimeter by 65.


plastic dishes with larger or smaller

If

pressed-glass or single-use

diameters than 91

mm

are used, labora-

tory personnel should determine the actual diameter and use a correction
factor for multiplication.

Do

not report counts on

crowded

plates

from the highest dilution as too

count (TNTC). Where bacterial counts on crowded plates are


greater than 100 colonies per square centimeter, report as greater than (>)
6,500 times the highest dilution plated. See Table 5:1, Sample No. 1006.
Report counts as "Estimated Standard Plate Count" per milliliter or per
gram, as applicable.
When all colonies on a plate are accurately counted and the number exceeds 300, report as "Estimated Standard Plate Count" per milliliter or per
gram, as applicable. See Table 5:1, Sample No. 1009.

numerous

/.

to

Spreaders:

Distribution: If spreaders occur on the plate(s) selected [5.11(1.2)],


count colonies on representative portions thereof [5.11(H)] only when a.)
colonies are well distributed in spreader-free areas, and b.) the area covered
by spreader(s), including the total repressed-growth area, if any, does not
1.

exceed one-half of the plate area. Where the repressed-growth area alone
exceeds one-quarter of the total area, report results of the test as directed
in 5.11(1.3).

Counting spreading colonies: When the counting of spreading colocannot be avoided, count each of three distinct types as one source. The
2.

nies

STANDARD PLATE COUNT METHOD

92

first

type

is

a chain of colonies, not too distinctly separated, that appears to

be caused by disintegration of a bacterial clump when the petri dish is rotated to mix the agar with the test material. If only one such chain exists,

count

it

as a single colony. If one or

more chains appear

separate sources, count each source as one colony.

Do

to originate

from

not count each indi-

vidual growth in such chain(s) as a separate colony.

The second type of spreading colony is that which develops in a film of


water between the agar and the bottom of the dish. The third type is that
which forms in a film of water at the edge or over the surface of the agar.
These two latter types develop largely because of an accumulation of moisture at the point from which the spreader originates. When dilution water is
uniformly distributed throughout the medium, bacteria rarely develop into
spreading colonies. Any laboratory with 5% of plates more than one-fourth
covered by spreaders should take immediate steps to eliminate this trouble.
Note the conditions
spreader(s) cover(s)

when

for reporting results as laboratory accidents

more than one-half of

the plate [5.11(1.3)].

Spreader growth, laboratory accidents, or bacterial growth inhibprepared from samples a.) have excessive spreader growth
[5.11(1.2)]; or b.) are known to be contaminated or are otherwise unsatisfactory, report as "Spreaders" (Spr) or "Laboratory Accident" (LA). See
Table 5:1, Sample No. 1008. If the test for inhibitory substances (Chapter 9)
3.

itors: If all plates

is

positive for a sample, report the Standard Plate

itors" (GI).

The analyst may be

Count

as

"Growth

Inhib-

inclined to suspect the presence of inhib-

sample being examined when plates have no growth


in lower dilutions; such developments
should not be interpreted as evidence of inhibition until the presence of inhibitory substances has been confirmed.
itory

substances

in the

or have proportionately less growth

5.12

Computing and Recording Counts

To compute

the Standard Plate Count, multiply the total

nies or the average

number

(if

duplicate plates of the

same

number of

colo-

dilution) per plate

by the reciprocal of the dilution used. Record dilutions used and number of
colonies on each plate counted or estimated.

When colonies on duplicate plates of consecutive dilutions are counted,


compute the Standard Plate Count for each dilution [5.1 1(D)] before averaging to record sample Standard Plate Count.

Avoid creating fictitious ideas of precision and accuracy when computing


Standard Plate Counts, by recording only the first 2 left-hand digits. Round
counts off to 2 significant figures only at the time of conversion to Standard
Plate Count by raising the second digit to the next highest number only when
the third digit from the left is 5, 6, 7, 8 or 9; use zeroes for each successive
digit toward the right from the second digit (see examples in Table 5:1).
Record results of sterility control tests on materials [5.9]. Record the temperature of the incubator during incubation of agar plates.

5.15

93

References

5.13 Reporting

and Interpreting Counts

Report counts as the Standard Plate Count or the Estimated Standard


Count per milliliter or per gram, as applicable. When instrumental
colony counters are used, report counts as Standard Plate Count (I) or ESPC

Plate

(I).

The matter of interpreting bacterial counts is beyond the scope of this


manual. Consult local regulations as well as the Grade A Pasteurized Milk
Ordinance.
5.14 Personal Errors

Avoid inaccuracies

in

counting caused by carelessness, impaired vision,

or failure to recognize colonies. Laboratory workers should strive to duplicate their

own

counts on the same plate within

5%

and the counts of other

analysts within 10%.^

5.15 References
1.

American Public Health Association.

Methods

1960. Standard

for the

Examination of

Dairy Products. 11th ed. American Public Health Association, N.Y.


2.

Archambault,

J., J.

CuROT, and M.H. McCrady.

1937.

The need of uniformity of conAmer. J. Pub.

ditions for counting plates (with suggestions for a standard colony counter).

Health 27:809-812.
3.

Babel,

F.J.,

Collins, E.B., Olson,

Jr., J.C.

Peters,

I.I.,

Watrous, G.H. and M.L.

Speck. 1955. The standard plate count of milk as affected by the temperature of incubation.
J. Dairy Sci. 38:499-503.
4.

Berry, J.M., McNeill, D.A., and


bacterial counts,

5.

J.

L. D.

Witter.

1969. EflFect of delays in pour plating

on

Dairy Sci. 52:1456-1457.

BoLAFFi, A., and W. Litsky. 1959. Studies on the use of plastic petri dishes for the cultiJ. Milk Food Technol. 22:67-70.
Breed, R.S., and W.D. Dotterer. 1916. The number of colonies allowable on satisfactory agar plates. Tech. Bull. 53, New York Agr. Exper. Sta., Geneva, N.Y.
Butterfield, C.T. 1932. The selection of a dilution water for bacteriological examinavation of bacteria.

6.

7.

tions. J. Bacteriol. 23:355-368;


8.

Courtney,

J.L. 1956.

The

Pub. Health Rep. 48:681-691.

relationship of average standard plate count ratio to

proficiency in plating dairy products.


9.

Donnnelly,
sis

J.

employee

Milk Food Technol. 19:336-344.

C.B., Harris, E.K., Black, L.A., and K.H. Lewis. 1960. Statistical analysplit with state laboratories. J. Milk Food Tech-

of standard plate counts of milk samples

nol. 23:315-319.
10.

11.

Felland, H.S., and J.W. Nadig.


from glass and

plastic pipettes. J.

Fowler,

Clark,

in

J.L.,

13.

W.S. Foster,

J.F.,

doing the standard plate count as described

Dairy Products.
12.

Jr.,

1965. A comparative study of the volumetric deliveries


Milk Food Technol. 28:124-126.

J.

Food

in

and A. Hopkins. 1978. Analyst variation


Standard Methods for the Examination of

Prot. 41:4-7.

Fruin, J.T., and W.S. Clark,


colony counter vs. a true count.

Jr.

1977. Plate count accuracy: analysts

and automatic

Food Prot. 40:552-554.


HuHTANEN, C.N., Brazis, A.R., Arledge, W.L., Cook, E.W., Donnelly, C.B., Ferrell, S.E., Ginn, R.E., Lembke, L., Pusch, D.J., Bredvold, E. Randolph, H.E.,
Sing, E.L., and D.I. Thompson. 1970. A comparison of horizontal versus vertical mixing
J.

procedures and plastic versus glass petri dishes for enumerating bacteria
Milk Food Technol. 33:400-404.

in

raw milk.

J.

STANDARD PLATE COUNT METHOD

94
14.

HuHTANEN, C.N., Brazis,


R.E., Jezeski,

holding dilutions
15.

A.R., Arledge, W.L., Cook, E.W., Donnelly, C.B., Ginn,


PuscH, D., Randolph, H.E., and E.L. Sing. 1972. EflFects of time of
on counts of bacteria from raw milk. J. Milk Food Technol. 35:126-130.

J.J.,

Huhtanen, C.N., Brazis, A.R., Arledge, W.L., Cook, E.W., Donnelly, C.B., Ginn,
R.E., Murphy, J.N., Randolph, H.E., Sing, E.L., and D.I. Thompson. 1970. Effect of
dilution bottle mixing

methods on

plate counts of raw-milk bacteria.

J.

Milk Food Technol.

33:269-273.
16.

Huhtanen, C.N., Brazis, A.R., Arledge, W.L., Donnelly, C.B., Ginn, R.E., Randolph, H.E., and E.J. Koch. 1975. Temperature equilibration times of plate count agar
and a comparison of 50 versus 45 C for recovery of raw milk bacteria. J. Milk Food Technol. 38:319-322.

17.

Marth, E.H., Proctor,

J.F., and R.V. Hussong. 1965. Effect of pipette type and sample
on plate count results. J. Milk Food Technol. 28:314-319.
Prickett, P.S. and N.J. Miller. 1931. A modified APHA milk dilution pipette. Amer. J.

size
18.

Pub. Health 21:1374-1375.


19.

20.

21.

22.

Jr., R.B., Bradshaw, J.G., and D.W. Francis. 1969. Effect of freezing raw milk
on standard plate count, J. Dairy Sci. 52:1720-1723.
Richards, O.W., and P.J. Heijn. 1945. An improved darkfield Quebec colony counter. J
Milk Technol. 8:253-256.
RiPPEN, A.L. 1961. Modem concepts of dairy plant engineering. J. Dairy Sci. 44:731-736.
U.S. Department of Health, Education and Welfare. 1965. Grade A Pasteurized
Milk Ordinance Recommendations of the Public Health Service. U.S. I^ublic Health
Service, Washington, D.C.

Read,

23.

Yale, M.W., and C.S. Pederson. 1936. Optimum temperature of incubation for standard
methods of milk analysis as influenced by the medium. Amer. J. Pub. Health 26:344-349.

CHAPTER

COLIFORM BACTERIA
P. A.

Hartman, W.L. Green, G.E. Huskey, and


A.C. Salinger

Introduction

6.1

The coliform group of bacteria comprises

all

aerobic and facultatively an-

aerobic, gram-negative, nonspore-forming rods able to ferment lactose with

production of acid and gas

organisms

is

at

32

within 48 hours.

One source

of these

the intestinal tract of

warm-blooded animals; certain bacteria of

members

of this group. Typically, these organisms

nonfecal origin also are

are classified in the genera. Escherichia Enterobacter {^ormevXy Aerobacter)


,

and Klebsiella.
ed

in

A few

lactose-fermenting species of other genera are includ-

the coliform group. In proportion to the

any of these types

numbers present, existence of

products is suggestive of unsanitary conditions or


practices during production, processing or storage.
Application of the test for coliforms is intended neither to detect fecal
in dairy

pollution specifically nor to identify Escherichia coli in dairy products, but

rather to measure the quality of the practices used to minimize bacterial


contamination of processed dairy products.^- ^^ Such tests are conducted

following pasteurization primarily to detect significant bacterial recontami-

cream and other processed dairy products. ^^


Results of tests on raw samples are to be interpreted differently from those
obtained by testing pasteurized milk.'^'^^ Coliforms in small numbers may
enter raw milk and cream under normal conditions of production and hannation of milk,

dling.'^

With increased use of farm bulk tanks and pipeline milkers, some laboranow making coliform counts on raw milk to determine the degree
of contamination during milk production. With both raw and pasteurized
samples, in the absence of storage-interval and temperature records for milk
or cream before testing, it is impossible to conclude from positive tests alone
to what degree bacterial growth following initial contamination may have
contributed to test results. With cultured products, some limitations on age
of sample before testing seems necessary because of the rapid and marked
reductions encountered in coliform counts within 48 hr of processing.*^
tories are

ISC Liaison: R.T. Marshall

95

COLIFORM BACTERIA

96

For information concerning coliforms

in

other dairy products, refer to dis-

10), butter, margaand related products (Chapter 11), cheese and other cultured products
(Chapter 12), and ice cream and related products (Chapter 13).

cussions of concentrated and dry milk products (Chapter


rine

6.2 Definitions

positive presumptive test for

cream

is

indicated when:

at least 0.5

Agar

C on

a solid plating

[6.8, 6. 10], or 2.)

Lactose Bile Broth


at 32

members of the coliform group

typical dark red colonies (normally

milk and
measuring

in

mm in diameter on uncrowded plates) appear within 24

incubation at 32
Bile

1.)

''-

'^' *^

medium

'-

'-

'^'

^'^

2 hr of

such as Violet Red

fermentation tubes containing

2%

show gas formation within 48

Brilliant
3 hr

Green

incubation

[6.9, 6.11].

should be made of doubtful colonies from Violet Red


by transferring each of five colonies (if available) to tubes of
2% Brilliant Green Lactose Bile Broth.
A completed test is made by streaking material, from typical colonies on
solid media [6.10] or from Brilliant Green Lactose Bile Broth tubes showing
gas production [6.11], onto plates of Eosin Methylene Blue Agar. Coliform
colonies on Eosin Methylene Blue Agar are dark or have dark centers and
colorless peripheries; coliform colonies on Violet Red Bile Agar are red.
Pure cultures so isolated should produce gas in fermentation tubes of Lactose Broth at 32 C within 48 hr and consist only of gram-negative, nonsporeforming rods.
Bile

confirmed

Agar

test

plates

6.3 General Interpretations


Results of coliform tests, especially results obtained by using the tube
method, have sometimes been interpreted qualitatively, with little emphasis
on their quantitative significance. Inadequate directions for interpretation
may be partially responsible for unsatisfactory terms in which test results
have been expressed, and for failure to specify limiting coliform densities in
some sanitary codes. Because small test quantities often will fail to yield
appreciable numbers of coliforms, negative results should be interpreted by
plant operators and by control officials with due consideration of the volume
of sample tested.

Since results of determinations of total number of bacteria in dairy products by the agar plate and direct microscopic
liliter
is

methods are reported per milwhen solid media are used

or per gram, the generally accepted practice

to report results of coliform determinations as

milliliter" (or

"Coliform colony count per

per gram). Control administrators, dairy plant operators and

laboratory personnel are usually familiar with reports based on such quan-

Workers in some areas prefer to use the fermentation tube technique,


is one standard procedure used in water and wastewater control laboratories, and to report results similarly
for example, as "Coliforms, Most
Probable Number/100 ml" (or /lOO g), or "Coliforms, MPN/100 ml"

tities.

which

(or /lOO g).

97

General Procedure

6.6

With the quantities ordinarily tested, any positive result on a pasteurized


product suggests recontamination and indicates the presence of defective
equipment or a need for improved plant sanitation. Regardless of the volume
basis for reporting results, proper emphasis should be placed on all positive
findings and the cause should be ascertained and eliminated.
When coliforms are enumerated in manufactured products, the question
always remains whether the counts actually reflect the numbers of viable
coliforms present. In products that contain bacterial cells which may have
been stressed either during processing or in storage, and where debilitation
or sublethal injury of coliforms may occur, both solid and liquid selective
media can be inhibitory. Thus, counts on some manufactured milk products
may be low and may not indicate the true populations of viable coliforms
present. Environmental stresses such as sublethal heat or chemicals, freezing, or dehydration may interfere with quantitative recovery of coliform organisms on selective media.'- -' This has been found true for fluid milk,
cheese and frozen dairy products.^- '^ Recovery may also be influenced by
the diluent or the

medium

used.**-* Progress

recovery of injured coliforms.^-

6.4

being

made

in increasing

Sampling

Sampling
6.5

is

'"

is

done

in

accordance with procedures given

in 3.13.

Equipment, Supplies and Media

Refer to Chapter 5 for standard equipment and supplies needed

[5.2],

except for special items as follows:


Inoculation loop (3-mm) and needle.
Fermentation tubes.
6.51

Media

The preparation of appropriate media is described in Chapter 4. Preferably


When media are prepared from component ingredients, the analyst must assume full responsibility for correct formulause dehydrated media.
tion.

A. Violet Red Bile Agar.


B. Brilliant Green Lactose Bile Broth, 2%.
C. Eosin Methylene Blue Agar, Levine.
D. Lactose Broth.
E.

6.6

Nutrient Agar.

General Procedure

fluid milk and cream by using either solid or liquid


Since organisms giving positive coliform tests are practically nonexistent in properly pasteurized milk collected directly from pasteurizers, presence of such organisms is usually interpreted as the result of con-

Examine samples of

media

[6.8, 6.9].

tamination introduced by equipment and/or processing downstream from the

COLIFORM BACTERIA

98
pasteurizer. Therefore, a completed test

and

official

ucts

is

generally not needed in routine

control of pasteurized fluid milk products or manufactured prod-

made from milk given

a heat treatment equivalent to or greater than that

of pasteurization. However, questionable colonies should be confirmed by


transfer to Brilliant

When

Green Lactose

Bile Broth.

fermentable substances other than lactose are present

in

samples,

confirm the presence of lactose fermenters by appropriate transfers of dark


red colonies on solid medium, or of broth from tubes showing gas, to

Green Lactose

Brilliant

Bile Broth [6. 10]; or proceed with the completed test

16.11].

6.7 Relative Value of Plate

and Tube Methods

Choice of method will depend largely upon limits for number of coliforms
which the analyst is interested. Other considerations are materials required and laboratory facilities and labor available for conveniently handling
in

volume of work involved.


five or more coliform colonies appear on single plates, counts are
usually more precise than are counts obtained by using five fermentation
tubes.** For routine examination of milk or cream with numbers of coliforms
the

When

usually greater than 5 per milliliter, or in preliminary surveys of pasteurizing


sufficient to use one or two plates, each containing 1 ml of
numbers of coliforms are routinely less than 5 per milliliter
normal achievement in well-operated pasteurizing plants, especially where
process samples are being examined tests of greater sensitivity may be required. Increased sensitivity can be achieved by using a larger sample;

plants,

it

sample.

is

If

4-ml portions in plates poured with 15-20 ml of medium are satisfactory.


When counts are still less than 5 coliform colonies per plate and accurate
results are desired, a multiple-tube procedure must be used so that larger
samples can be examined.
The multiple-tube method results in a most probable number that is an
estimate of the number of bacteria within limitations of the procedure. For
example, when five tubes of 10 ml each are inoculated, the 95% confidence
limits, based on coliforms per 100 ml, could vary as follows: between less
than 0.05 and 6.0 bacteria with all negative tubes (Table 6:11); between
0.05 and 13 bacteria with one positive tube; between 2 and 19 bacteria with
two positive tubes; and between 6 and 53 bacteria with four positive
tubes.

Because of the irregular distribution of bacteria, even when coliforms

average

per

milliliter, the probabilities

are that

of a sample will contain no coliforms; but


portions of

1%

if

some 37% of 1-ml portions


test is made with five

each

ml each, completely negative results

may

be expected less than

of the time.

The tube method, using five portions of 10 ml and five of 1 ml, permits
estimation of numbers of coliforms from 0.02 to 2.40+ per milliliter
(1.8 to 240+/100 ml) and, when the result is completely negative, furnishes
evidence that no coliform was cultivated from 55 ml of sample.

6.10

99

Confirmed Test

Caution should be exercised before drawing conclusions from the results


'^ The precision of

of single examinations by either plate or tube method. ^^results obtainable

is

low

inoculated with one or

a.) when few positive tubes appear among several


more sample volumes(s), or b.) when a very low

colony count from several plates

6.8

is

obtained.

Conform Test with a Solid Medium

1 ml of milk sample or decimal dilution thereof into sterile plates.


each plate 10-15 ml of Violet Red Bile Agar tempered to 44-46 C.
Pour an agar control for each flask of medium used.
To enhance sensitivity of tests without unduly increasing the number of
dishes used, plate portions up to 4 ml per dish and use 15-20 ml of medium.
To facilitate mixing of the sample and the agar medium when plating highsolids and viscous products such as cream, ice cream, and buttermilk, prepare initial dilutions of 1:2 or 1:10 and distribute 10 ml into three dishes,
using 15-20 ml of medium per dish. If high counts are consistently encountered, distribute 1 ml of a 1:10 dilution into one plate.
Mix contents of plates thoroughly by tilting and rotating each dish. Allow
the mixture to solidify (5-10 min) on a level surface, then distribute an additional 3-4 ml of the plating medium as an overlay, completely covering the
surface of the solidified medium to inhibit surface colony formation. Invert
and incubate plates for 24 2 hr at 32 C. Dark red colonies measuring
0.5 mm or more in diameter on uncrowded plates are counted and results are

Transfer

Add

to

recorded as directed

in 6.12.

On crowded plates, coliform colonies may show atypical characteristics


and may measure less than 0.5 mm in diameter; furthermore, strains of some
nonconforms and yeasts may produce colonies comparable in color and size
on crowded plates. ^^ In such instances it is necessary to pick representative colonies and confirm the presence of lactose fermenters [6.6, 6.10], or to subject them to the completed test [6.11], or both.
Results from plates having excessive numbers of colonies may be unreliable,
particularly for samples containing substantial numbers of organisms able to
grow at refrigeration temperatures. Avoid crowded plates, if possible, by
plating a sufficient number of dilutions with selected volumes of sample.
to colonies of coliforms

6.9 Coliform Test with a Liquid

Medium

Inoculate fermentation tubes of 2% Brilliant Green Lactose Bile Broth with


appropriate volumes (such as 10.0, 1.0, 0.1, 0.01 ml, etc.) of sample, using
five

portions of each test volume in separate tubes. Prepare decimal dilutions


one positive and one negative tube in each series inoculated

to obtain at least

for use in calculating the

at32C

for 48

6.10 Confirmed Test


If additional

most probable number present. Incubate the tubes

hr [6.12(B)].

From a

information

is

Solid

needed

Medium

to confirm a positive

presumptive test

COLIFORM BACTERIA

100

from Violet Red Bile Agar, promptly transfer typical and/or atypical colonies
to 2% Brilliant Green Lactose Bile Broth fermentation tubes and incubate for
48 hr at 32 C. Gas production indicates a positive confirmed test. Failure to
show gas production within 48 hr excludes members of the coliform group. If
growth on a solid medium is without clearly isolated typical colonies, streak
sample material on the surface of solidified Violet Red Bile Agar plates for
isolation. Incubate for 24 hr at 32 C and transfer the isolated growth to
2% Brilliant Green Lactose Bile Broth fermentation tubes. Incubate tubes
until gas appears, but no longer than 48 hr; proceed with the completed test
of positive tubes as in

6.

1 1

Completed Test from a Liquid Medium


When additional information is needed following

6.1

liquid

medium

[6.9, 6.10],

a positive test from a


promptly streak an Eosin Methylene Blue Agar

plate with a trace of the liquid culture so that discrete colonies are obtained
after the plate has been incubated for 24 hr at 32 C. From these, inoculate a
Nutrient Agar slant and a lactose fermentation tube from one typical colony

from two or more colonies that most


and the Lactose Broth tube(s) for 48 hr at 32 C. Gas formation in Lactose Broth and
presence of gram-negative, nonspore-forming, rod-shaped bacteria from the
slant constitute a positive completed test.
or, if only atypical colonies are present,

closely

conform

to coliform-Iike colonies. Incubate the slant(s)

6.12 Reporting Results

A. Tests with a solid medium:


lino coliforms appear on

plate(s), report "less

"n"

number

than n per milliliter" (or

would be reported if only


1 coliform colony had been secured from the total volume plated. For example, if 2 ml of undiluted milk have been plated, report results as "Coliform count less than 0.5 per milliliter".
When less than 15 coliform colonies develop on a plate(s) from a diluted
per gram), substituting for

the

that

Keep in mind,
however, that precision decreases as the number of colonies per plate decreases."* When less than 15 coliform colonies routinely develop on plate(s)
from diluted samples, lower dilutions should be plated. If less than 5 coliform colonies routinely develop on plate(s) from undiluted samples, ^^ use of
multiple-tube procedures should be considered. [See 6.7]
Preferably record results from only those plates that contain between
15 and 150 coliform colonies per plate. If plates from two consecutive decimal
dilutions yield colony counts of 15 to 150, compute the counts for each dilution by multiplying the number of colonies per plate by the dilution used and
report the arithmetical average as coliform count per milliliter or per gram,
unless the higher computed count is greater than twice the lower count, in
and/or undiluted sample(s), record the results as observed.

6.12

101

Reporting Results

which case report the lower computed count. Use only two significant figures followed by zeros.
If more than 150 colonies develop on the highest-dilution plate, estimate
numbers as described in Section 5.1 1(H). Record results as "Estimated coliform count" (ECC). Counts greater than 1500 per plate should be reported

>1500 times the reciprocal of the dilution.


Where multiple plates are used to distribute several milliliters of the same
dilution, sum counts for the plates and multiply by the appropriate factor.
as

B. Tests with a liquid medium:^^-

Record

results, indicating

each, and

number of

''''-"

number of tubes used, volume introduced

positive tubes obtained; for example,

if

into

three of five

tubes with 10-ml portions in each have gas and none of five tubes with

1-ml portions

in

each have gas, record as follows:


10 ml
3/5

1ml
0/5

The numerator indicates the number of positive tubes and the denominator
the number of tubes inoculated. Interpret the laboratory record from Table
6:1 showing most probable numbers 16.13] and report "Coliforms, MPN per
milliliter" (or per gram). The coliform count given in the example would be
8/100 ml or 0.08/ml.

Table 6:1 lists the most probable numbers of coliforms per 100 ml, corresponding to the frequency of positive fermentation tests, secured from a
variety of multiple-portion inoculation systems, beginning with 10-ml test

more than one organism may be responsible for each posimost probable number of organisms is a logarithmic function of

portions. Since
tive test, the

results

when

test portions are

volume inoculated

number

listed in

decimally related. Therefore,

if

the greatest

ml rather than 10 ml, multiply the most probable


Table 6:1 by 10. Thus for results of
is

1ml
3/5

0.1ml
1/5

most probable number, 0.11 per ml (11/100 ml), is multiby 10 to arrive at 1.1 per ml (110/100 ml) as the final most probable

the corresponding
plied

number of coliforms
oculated

is

in the

sample. Similarly,

0.01 ml, multiply the

if

the greatest

most probable number

volume

listed in the table

in-

by

1,000.

When more

than three different volumes are tested, use results from only
Use results of the smallest volume having a

three consecutive volumes.

reading of 5/5 and results obtained with the next two smaller volumes.
Significant results in the following examples are in boldface type; values

with asterisks are not to be used in the computations:

COLIFORM BACTERIA

102

Example
(a)

1.0

ml

0.1ml

0.01 ml

0.001 ml

6.13

MPN

Tables

103

104

COLIFORM BACTERIA

6:11. Selected MPN Estimates and 95% Confidence Limits of Estimates for Fermentation Tube Tests When Only Five Tubes With 10-ml (*)
or Five Tubes With 10-ml and One Tube Each With 1.0- and 0.1-ml

Table

Volumes Are Used

6.15

3.

105

References

BusTA, F.F.

1976. Practical implications of injured microorganisms in food.

J.

Milk Food

Technol. 39:138-145.
4.

CowELL, N.D. and M.D. Morisetti. 1969. Microbiological techniques some statistical
J. Sci. Food Agric. 20:573-579.
Delay, P.D. 1947. The significance of the coliform test in pasteurized milk. J. Milk Food

aspects.
5.

Technol. 10:297-299.
6.

DE Man,

The probability of most probable numbers. European J. Appl. Microand personal communication.
FouRNELLE, H.J. and H. Macy. 1950. A comparative study of presumptive media for the
J.C. 1975.

biol. 1:67-78,
7.

8.

9.

coliform group. Amer. J. Pub. Health 40:934-942.


GoEL, M.C., Kulshrestha D.C, Marth E.H., Francis D.W., Bradshaw J.G., and
R.B. Read, Jr. 1971. Fate of coliforms in yogurt, buttermilk, sour cream, and cottage
cheese during refrigerated storage. J. Milk Food Technol. 34:54-58.
Hall, H.E., Brown, D.F., and K.H. Lewis. 1967. Examination of market foods for coli-

form organisms. Appl. Microbiol. 15:1062-1069.


10.

Hartman,

p. a.,

Hartman.

P.S.

and

W.W. Lanz.

1975.

VRB-2

agar for stressed

coli-

forms. Appl. Microbiol. 29:537-539, 865.


1

1.

12.

HosKiNS, J.K. 1934. Most probable numbers for evaluation of coli-aerogenes


mentation tube method. Pub. Health Rep. 49:393-405.

Maxcy, R.B.

1970. Non-lethal injury

tests

by

fer-

and limitations of recovery of coliform organisms on

Food Technol. 33:445-448.


McCarthy, J. A., Thomas Jr., H.A., and J.E. Delaney. 1958. Evaluation of the reliability of coliform density tests. Amer. J. Pub. Health 48:1628-1635.
McCrady, M.H. 1915. The numerical interpretation of fermentation tube results. J. Infect.
selective media. J. Milk

13.

14.

Dis. 17:183-212.
15.

McCrady, M.H.
Health

16.

J.

1918. Tables for rapid interpretation of fermentation tube results. Pub.

(Toronto) 9:201-220.

McCrady, M.H. and

Archambault.

1934. Examining dairy products for members of


Amer. J. Pub. Health 24:122-128.
McCrady, M.H. and E.M. Langevin. 1932. The coli-aerogenes determination in pasteuriJ.

Ihe Escherichia-Aerohacler group.


17.

zation control.
18.

J.

Morris, R.L. and

Dairy Sci. 15:321-329.


J.

Cerny.

for coliforms in milk.


19.

J.

1954. Significant abnormalities in the violet red bile technique

Milk Food Technol. 17:185-187.

Nelson, F.E. and M.P. Baker.

1953.

The

influence of time and temperature of plate in-

cubation upon bacterial counts of market milk and related products, particularly after hold-

20.

21.

22.

23.

ing under refrigeration. J. Milk Food Technol. 17:95-100.


Oblinger, J.L. and J. A. Koburger. 1975. Understanding and teaching the most probable
number technique. J. Milk Food Technol. 38:540-545 and 39:78.
Ordal, Z.J. 1970. Current developments in detection of microorganisms in foods: Influence of environmental factors on detection methods. J. Milk Food Technol. 33:1-5.
Reinbold, G.W. 1971. Bacteriological testing of milk for regulatory purposes usefulness
of current procedures and recommendations for change. 111. Raw milk quality where do
we go from here? J. Milk Food Technol. 34:260-263.
U.S. Department of Health, Education & Welfare. 1965. Grade A pasteurized milk
ordinance Recommendations of the Public Health Service. USPHS, Washington, D.C.
Weiler, W.A. and S.E. Hartsell. 1969. Diluent composition and the recovery of Esche-

24.

richia coli. Appl. Microbiol. 18:956-957.

How

25.

Woodward,

26.

Works Assoc. 49:1060-1068.


Yale, M.W. 1936. Escherichia-Aerobacter group

R.L. 1957.

probable

is

the most probable

in ice

cream,

number?

J.

Amer. Water

(b) Its significance

portance, p 17-30. Proc 36 Ann. Conv. International Association of Ice

and im-

Cream Manufac-

turers.
27.

Yale, M.W.

1937.

Comparison of

solid with liquid

presence of lactose fermenting bacteria


569.

in

media as a means of determining the

pasteurized milk. Amer.

J.

Pub. Health 27:564-

CHAPTER

THERMODURIC, THERMOPHILIC, AND


PSYCHROTROPHIC BACTERIA
E.R. Vedamuthu, Lester Nankin, Z.J. Ordal, and
Carl Vanderzant

Thermoduric Bacteria

7.1

7.11

Introduction

Thermoduric bacteria can survive exposure to temperatures considerably


above their maximal temperature for growth. In the dairy industry, the term
is applied to those organisms which survive, but do not grow, at pasteurization temperature. They usually include species of Micrococcus, Microbacterium, Streptococcus, Lactobacillus, the coryneform group of bacteria ^, Bacillus, and, occasionally, gram-negative rods.^^ The principal
sources of contamination are poorly cleaned and sanitized utensils and
equipment on farms and in processing plants. Control of thermoduric bacteria is an operational problem involving both dairy farms and processing
''* ^**
plants.**' ^These bacteria may contribute significantly to high Standard
Plate Counts on pasteurized milk and other milk products. Hence, the
thermoduric count has been used in the dairy industry primarily as a test of
care employed in utensil sanitation and as a means of detecting sources of
organisms responsible for high counts in pasteurized products. Present-day
methods of milk storage on the farm, transportation to the processing plant,
pasteurization, and post-pasteurization handling and marketing have introduced new dimensions to the dairy trade. Under these circumstances,
thermoduric psychrotrophs (especially Bacillus species) may constitute a
major concern in the shelf life of pasteurized milk.'^- ^^
To identify the degree of contamination with heat-resistant bacteria, bacterial counts on raw milks subjected to laboratory pasteurization can be
made.
7.12 Collection of Samples

To

detect bacteria that survive pasteurization, normally take samples of

producers' milk as collected (Chapter

3)

essing plant.

ISC Liaison: G.W. Reinbold

107

or finished products from the proc-

THERMODURIC, THERMOPHILIC, PSYCROTROPHIC BACTERIA

108

7.13 Equipment

A. Test tubes,

sterile:

Screw-capped, 20x125

mm;

cap with rubber or

plastic liner.

B. Pipets, sterile: Graduated, 5-ml or 11-ml delivery (TD).

C. Thermometers: Thermometers used in the water bath and in the pilot


tube with milk should cover the critical range of temperature during pasteurization, should have divisions of 0.1 C, and should be checked at least bien-

with a NBS certified thermometer.


D. Water bath: Electrically heated, thermostatically controlled at
62.8 0.5 C, equipped with stirrer and thermometer. The volume of water
nially

should be sufficient to absorb the cooling effect of tubes placed


in temperature of more than 0.5 C.

in the

bath

without a drop

E. Metal, plastic or wire rack: For holding test tubes.

7.14 Procedure

Transfer 5 ml of a thoroughly mixed milk sample to a test tube, being


and upper portions of the tube with

careful to avoid contaminating the lip

While assembling a series of tubes for laboratory pasteurization, hold


to 4.4 C. Place a pilot tube containing 5 ml of milk,
in ice water at
with thermometer inserted therein, in the rack during the preliminary holdmilk.

tubes

ing period in the ice bath, as well as during pasteurization. Place rack of

tubes

in

or they

pasteurizing bath at 62.8 C. Tubes should be immersed completely

may be immersed

so that the water line

the level of milk in the tubes.


least 62.8

short-time

When

within 5 minutes.

(HTST)

is

approximately

The temperature of

Do

V2 in.

above

the milk should reach at

not attempt to simulate high temperature

pasteurization.

the temperature in the pilot tube reaches 62.8 C, start timing the

holding period. At the end of the 30-min holding period, immediately place
rack of tubes in an ice water bath and cool tube and contents to 10 C or
in the pilot tube. Determine the
Laboratory Pasteurization Count (LPC) by the Standard Plate Count (Chapter 5). Simplified viable count methods (Chapter 21) may be used.

below, as indicated by the thermometer

7.15 Interpretation

Presence

in individual

producers' samples of appreciable numbers of bac-

teria that survive laboratory pasteurization indicates

contamination from

poorly sanitized farm utensils. Poorly sanitized processing equipment in


milk plants also may be a source of contamination with thermoduric bacteria.

7.16 Reporting

Report results as "Laboratory Pasteurization Count per milliliter"


Where one of the simplified methods is used, add to the reported
count the appropriate designation to identify the method.
(LPC/ml).

7.2

109

Thermophilic Bacteria

7.17 Limitations

Laboratory pasteurization called for


ature long-time

(LTLT)

tions obtainable in

in the

method simulates low-temper-

pasteurization and does not correspond with condi-

modem HTST

pasteurizers. Suitable laboratory tech-

niques to simulate modern pasteurization are only in developmental


stages *' " and have to be widely tested and evaluated before adopting for
routine usage.

The
cover

plating
all

medium and

culturing conditions used in the test

heat-injured but viable cells, and

may

may

not re-

not reflect the recovery pat-

For current application of the LPC


equipment sanitation on the farm and in the processing

tern in fluid milk or other dairy products.

as an indicator of

however, this method is adequate.


Recent studies have shown that the LPC can be influenced by temperature
and time of plate incubation, pH of the plating medium, and type of peptone
used in the plating medium. These factors influence repair and recovery of
plant,

injured cells after laboratory pasteurization treatment. ^^'

^^'

'^'^

Thermophilic Bacteria

7.2
7.21

Introduction

In the dairy industry, the term thermophilic bacteria applies particularly to

bacteria

which grow

in

milk held at elevated temperatures (55

including pasteurization

(LTLT), 62.8

or higher),

C.^'**

Thermophilic bacteria usually are species of Bacillus which enter milk


from various sources on the farm, or from poorly cleaned equipment in the
processing plant. When milk is held at high temperatures for long periods,
these bacteria rapidly increase in numbers and may cause flavor defects or

problems with respect to bacterial standards. Counts of thermophilic bacteria are obtained by the Standard Plate Count method (Chapter 5), with
plate incubation at 55 1 C. Numerous large, rod-shaped bacteria that are
strongly stained in milk films prepared from pasteurized milk according to
the procedure for the Direct Microscope Count (Chapter 14) are probably
thermophilic bacteria.

7.22 Procedure

Prepare dilution and plates as described for the Standard Plate Count
5), except use 15 to 18 ml of agar per plate, and incubate for 48 hr at

(Chapter
55

C.

7.23 Reporting
After determining the colony count, report as "Thermophilic Bacterial
count per milliliter" (or per gram); or TBC/ml (TBC/g).

THERMODURIC, THERMOPHILIC, PSYCROTROPHIC BACTERIA

110

7.3
7.31

Psychrotrophic Bacteria
Introduction

Microorganisms which play a significant role in biological processes in


low-temperature environments have usually been called psychrophilic (coldloving). This term seems to imply optimum growth at low temperatures.
Relatively few of the "psychrophiles" isolated from dairy products have
optimum growth temperatures below 20 C.*^ It has been suggested that the
term "psychrotrophic" (to increase, to develop in the cold) be applied to
those organisms able to grow relatively rapidly at 7 C and below without
reference to optimum temperature for growth. ^^' ^^ In the dairy industry, the
term "psychrotrophic" indicates organisms capable of appreciable growth
in milk and milk products at commercial refrigeration temperatures, 2 to 7 C,
irrespective of their optimum growth temperature.^^' ^^* ^^ Species of
Pseudomonas, Flavobacterium, Alcaligenes, Acinetobacter, and Bacillus
are often encountered among psychrotropic bacteria. Certain species of
Streptococcus belonging to groups III and IV as listed in the 8th edition of
Bergey's Manual
and yeasts and molds found in dairy products also can
'^,

be included

From

in the

psychrotrophic group.

a quality control point of view, psychrotrophic bacteria are prob-

ably the single most important group of organisms which

may be

present

in

The importance of psychrotrophic bacteria has greatly increased with extended storage of raw and pasteurized milk and other dairy

dairy products.

products. These bacteria are generally non-pathogens, but in dairy products

they can cause a variety of oflF-flavors, including fruity, stale, bitter, putrid
and rancid flavors, as well as physical defects.^- " ^'*' ^^ They are also involved in loss of flavor in cultured dairy products.^" The influence of the
psychrotrophic flora of pooled raw milk intended for manufacturing purposes, on yield, process eflficiency and final quality (body, texture, and flavor) of the end product is being recognized.'^ Most of these bacteria are
gram-negative rods, although some gram-positive, spore-forming, thermoduric rods belonging to the genera Bacillus and Clostridium are lately coming into prominence as thermoduric psychrotrophs.'"- ^^ Psychrotrophic
bacteria are rarely present in the udder. The numbers of psychrotrophic bacteria in raw milk depend upon sanitary conditions prevailing during production and upon time and temperature of milk storage before processing. The
influence of psychrotrophic bacteria on the shelf life of pasteurized milk will
depend mainly upon the number present after packaging, the rate of growth,
the storage period, and the biochemical activity of the organisms. Even if no
detectable changes occur, an increase in numbers of psychrotrophs may
cause problems in meeting bacterial standards.
Although most psychrotrophs are killed by pasteurization, a few, usually
Bacillus spores, may survive pasteurization and cause spoilage of milk during extended storage.**- '** '" However, the presence of psychrotrophic orga-

7.3

111

Procedure

nisms

in

pasteurized products usually can be attributed to post-pasteuriza-

tion contamination.

The Standard

Plate Count method with low-temperature incubation of


used to enumerate psychrotrophic organisms. In the interest of uniformity of method, a single combination of temperature, 7 C, and time,
10 days, for incubation has been selected to enumerate this group of bacplates

is

teria.'

Because extended refrigerated storage favors growth of this group of bacit is important to determine their presence as quickly as possible so
that if the product is heavily contaminated the source of contamination can
be ascertained and remedied.
Several new techniques have been reported, but tests which may be applicable to raw milk may not be adequate for pasteurized samples and vice
versa. Also tests for milk may not be applicable to cottage cheese. None of
these new techniques have been subjected to collaborative study. The more
promising techniques involve selective media for gram-negative bacteria,
preliminary incubation of samples or plates, use of SPC agar in conjunction
with the oxidase test, and enumeration of microcolonies by optical or electronic counting procedures.'^' '' '^' ^^ These tests, however, do not discriminate between spoilage organisms and inert types.
Preliminary incubation technique (Appendix A) may give more reliable
counts, but this method does not solve the problem of the relatively lengthy
interval needed for obtaining results.^' '^' ^' Also, it does not provide information on the relationship between psychrotrophs and keeping quality of
milk. Use of selective media on a routine basis could signal potential psychrotroph-related problems before they become explosive; further, this
method allows manipulation of incubation temperature (for poured plates) to
teria,

allow shorter time interval before results are available.^


in

'^

Use of SPC agar

conjunction with the oxidase test'' does not involve any change

tory procedures,

and both the

SPC and

in labora-

potential psychrotrophic count can

be obtained on the same plate. The keeping quality test of Moseley (Appenif the currently recommended

dix A) can probably be greatly improved

C can be shortened with


incubation
temperatures.
media
and
higher
the combined
Method
status, these
accorded
Standard
Although unofficial, and not yet
quality
on
milk
as aff'ected
methods offer tools by which specific information
rapidly.
There
is,
however,
relatively
determined
by psychrotrophs can be
widely
varying
under
the
conditions
for
use
no single, all-inclusive method
encountered in the dairy industry.

5- or 7-day

initial

incubation of the sample at 7

use of selective

7.32 Procedure

Prepare dilutions of product and then plate as described for the Standard
Count (Chapter 5) except incubate plates at 7 1 C for 10 days. Take

Plate

special care that melted

is cooled to 44 46 C before pouring


some psychrotrophic bacteria. ^"^ At temper-

medium

plates to prevent destruction of

THERMODURIC, THERMOPHILIC, PSYCROTROPHIC BACTERIA

112

may grow,

atures above 7 C, other organisms

possibly yielding misleading

results.

To

evaluate

/7/flA7r

sanitation for effectiveness in eliminating psychrotrophs

from pasteurized products, the 5- or 7-day keeping quality


dix A) is most useful.

test (see

Appen-

7.33 Reporting
After determining the colony count, report as "Psychrotophic Bacterial

Count per

Baumann,
organisms

2.

G.W. Reinbold. 1963. The enumeration


Milk Food Technol. 26:351-356.

D.P., and

Buchanan,

of psychrophilic micro-

R.E., and N.E. Gibbons, 1974. Bergey's Manual of Determinative Bacteriol-

The Williams

Desai, M.N., and T.J.

&

Wilkins Co., Baltimore, Maryland.

Claydon.

1964. Preliminary incubation of

aid in evaluating bacteriological quality.


4.

PBC/ml (PBC/g).

in dairy products. J.

ogy. 8th Edition.


3.

per gram); or

References

7.4
1.

milliliter'' (or

DiCKERSON,

Jr.,

R.W., and R.B. Read,

J.

raw milk samples as an

Milk Food Technol. 27:333-448.

Jr. 1968.

Instrument for study of microbial thermal

inactivation. Appl. Microbiol. 16:991-997.


5.

Elliott,

A., Emmons, D.B., and A.R. Yates. 1974. The influence of the bacterial qualion the properties of dairy products. A review. Can. Inst. Food Sci. Technol. J.

J.

ty of milk

7:32-39.
6.

EM., Nelson,

Foster,

F.E., Speck, M.L.,

Doetsch, R.M., and

Dairy Microbiology. Prentice-Hall, Inc., Englewood


7.

Cliffs,

Freeman, T.R., Nanavati, N.V., and W.E. Glenn.


chrophilic bacteria in pasteurized milk.

J.C.

Olson,

Jr. 1957.

N.J.

1964. Faster enumeration of psy-

preliminary report.

J.

Milk Food Technol.

27:304-307.
8.

Grosskopf, J.C, and W.J. Harper.


milk.

9.

Hammer, B.W., and


Inc.,

10.

11.

12.

13.

New

Hankin,
nisms

in

F.J.

1957. Dairy Bacteriology, 4th ed.

L., and

W.F. Dillman.

1968.

pasteurized dairy products.

Hii

EMAN,

J.

John Wiley

&

Sons,

rapid test to find potentially psychrophilic orga-

Milk Food Technol. 31:141-145.

in

der Anlieferungsmilch. Milchwissenschaft 24:721-726.

J.L. 1940. Thermoduric bacteria in pasteurized milk.

review of the literature.

Dairy Sci. 23:1143-1160.

International Dairy Federation.


clusions. Int. Dairy Fed.

16.

Babel.

Hankin, L., Pernice, A., and W.F. Dillman. 1968. Quality control of milk production by
means of the cytochrome oxidase test. J. Milk Food Technol. 31:165-170.
Hartley, J.C, Reinbold, G.W., and E.R. Vedamuthu. 1969. Effects of medium and
incubation temperature on recovery of microorganisms from manufacturing-grade. Grade
A and pasteurized milk. J. Milk Food Technol. 32:90-93.
Heeschen, W., Reichmuth, J., Tolle, A., and H. Zolder. 1969. Zur Bestimmung der

J.

15.

life

York.

psychrotrophen Keimflora
14.

1969. Role of psychrophilic spore-formers in long

Dairy Sci. 52:897 (Abstr.)

J.

Johns,

CK.

Annual

1969.

Seminar on psychrotrophic bacteria

Con-

Bull. Part 1:46-50.

1960. Preliminary incubation for

raw milk samples.

J.

Milk Food Technol.

23:1375141.
17.

MossEL, D.A.A., and H. Zwart.


types

18.

among Enterohacteridceae

Olson,
(Abstr).

H.C

1960.

The

isolated

1963. Spoilage of milk

rapid tentative recognition of psychrotrophic

from food.

J.

Appl. Bacteriol. 23:185-188.

by thermoduric psychrophiles.

J.

Dairy Sci. 46:362

7.4

19.

20.

113

References

OvERCASE, W.W., and K. Atmaram. 1974. The


fluid milk. J. Milk Food Technol. 34:233-236.
Parker, R.B., and P.R. Elliker.

role of Bacillus cereus in sweet curdling of

1953. Effect of spoilage bacteria

on diacetyl content and

flavor of cottage cheese. J. Dairy Sci. 36:843-849.


21.

22.

Reinbold, G.W., Johns, C.K., and W.S. Clark, Jr. 1969. Modification of the preliminary
incubation treatment for raw milk samples. J. Milk Food Technol. 32:42-43.
Shehata, T.E., and E.B. Collins. 1971. Isolation and identification of psychrophihc spe-

23.

from milk. Appl Microbiol. 21:466-469.


Stroup, W.H., Dickerson, Jr., R.W., and R.B. Read,

24.

exchanger for microbial thermal inactivation research. Appl. Microbiol. 18:889-892.


Thomas, S.B. 1948. Psychrophilic microorganisms in milk and dairy products. Dairy

cies of Bacillus

Jr. 1969.

Two

phase slug flow heat


Sci.

Abstr. 20:358, 369, 448^68.


25.

Thomas,

S.B. 1969. Methods of assessing the psychrotrophic bacterial count of milk.

J.

Appl Bacteriol. 32:269-296.


26.

Thomas,

S.B.,

Druce, R.G., Peters,

G.J.,

and D.G. Griffiths. 1967. Incidence and


A reappraisal and review. J.

significance of thermoduric bacteria in farm milk supplies:

Appl Bacteriol., 30:265-298.


27.

Thomas, W.R., Reinbold, G.W., and F.E. Nelson.

1963. Efl"ect of temperature

of plate incubation on the enumeration of pasteurization-resistant bacteria

and time
Milk

in milk. J.

Food Technol. 26:357-363.


28.

Thomas, W.R., Reinbold, G.W., and F.E. Nelson.


on enumeration of pasteurization-resistant bacteria

1966. Effect of

in milk. J.

pH

of plating

medium

Milk Food Technol. 28:156-

160.

29.

30.

31.

Thomas, W.R.. Reinbold, G.W., and F.E. Nelson. 1966. Effect of the type of bacteriological peptone in the plating medium upon the enumeration of pasteurization-resistant bacteria in milk. J. Milk Food Technol. 29:182-196.
Vanderzant, C, and A.W. Matthys. 1965. EfiFect of temperature of plating medium on
the viable count of psychrophilic bacteria. J. Milk Food Technol. 28:383-388.
Witter, L.D.

1961. Psychrophilic

bacteria A review.

J.

Dairy Sci. 44:983-1015.

CHAPTER

SCREENING AND CONFIRMATORY METHODS FOR


THE DETECTION OF ABNORMAL MILK*
William W. Ullmann, A. Richard Brazis,
William L. Ariedge, W.D. Schultze,

and W.C. Lawton

Introduction

8.1

Regulations in most progressive areas of the country require that milk

be from one or more healthy cows; that

shall

colostrum; and that milk otherwise abnormal

it

shall

may

be practically free of

not be offered for sale.

This includes milk from cows with mastitis, milk from cows treated with
medicinal agents which

may be

secreted in the milk, and milk containing

pesticides, herbicides, or other agents

which are judged

to be prejudicial to

the well-being of the milk consumer.

Concentrations of leucocytes
ally

in

excess of 500,000 per

milliliter are

considered indicative of mastitis or other abnormality.'"

rion,

By

gener-

this crite-

in the very early or very late lactation or cows with a low-grade or


udder infection are likely to produce milk having an excessive number

cows

latent

of leucocytes.

Screening and confirmatory tests described herein estimate the number of


somatic cells (including leucocytes)

in milk.

Although polymorphonuclear

leucocytes are principally associated with inflammation of

mammary glands,

enumeration of all nucleated somatic cells has been generally found more
feasible in determining normality or abnormality of milk. The tests are not
intended for diagnosis of mastitis.

8.2

Sampling (See

3.13)

8.3 Screening Tests


8.31

Modified Whiteside Test

The amount and opacity of a precipitate, which is formed when milk and
sodium hydroxide are mixed together ^^ in the prescribed manner, roughly
parallels the somatic cell count of the milk. The modified Whiteside test
ISC Liaison: W.W. Ullmann
115

DETECTION OF ABNORMAL MILK

116

(MWT) may

be applied both to quarter ^' ^^ and to blended herd milk.'^^ Assignment of a grade to the test reaction is subjective. A picture guide is
available for grading this reaction [8.31(A.6)]. Equipment and supplies are
easily obtainable and the method is quick, simple and inexpensive.
A. Equipment and supplies:

Smooth, dark surface, such as blank bakelite sheets or window glass


enameled on one side; areas of about 4 cm^ may be
marked by lines etched on the surface.
2. Sodium hydroxide (NaOH), 4%: Weigh out 4 g of NaOH pellets (do
not use pellets that have fused together from moisture) and make up to
1.

(not highly polished)

1(X)

ml with

distilled

water; store

in flint glass

or suitable screw-capped polyeth-

ylene or polypropylene bottles. Discard the solution

if it

becomes cloudy.
size. Use

Medicine droppers: Of good quality, with uniform bore


separate droppers for milk and for NaOH.
4. Applicator sticks: 6 in. long, for mixing milk and NaOH.
3.

5.

Sample containers:

6.

Picture guide:* For scoring test reactions.

Sterile [3.11(C)].

B. Procedure:
All samples

must be stored

at

0-4.4

C from

the time of collection until

examination. Testing must be completed within 36 hr after collection.


1.

Place 5 drops of cold, properly agitated milk [5.6(B)] on one spot on

the test plate. Rinse the dropper in cold water.

drops of 4% NaOH.
and with an applicator stick held at a low angle, beat the
first, and then, with a circular motion, spread it over an area of no

2.

Add

3.

In 20-24 sec

mixture at
more than 4 cm^ of the test plate.
4. Score the reaction (Fig. 8:1). The
permit clear observation of the reaction.
5.

Complete one

test

light

source should be sufficient to

before starting the next. In each succeeding

sample, rinse the dropper to remove any traces of the previous sample or
any moisture. The same dropper may be used throughout, unless the tests
are to be repeated.
C. Grading reactions:
Negative (-). The mixture is opaque and milky in appearance, and entirely free from precipitate throughout. The somatic cell count is usually under
0.5 million per milliliter.

Trace

(T).

The mixture

opaque and milky, but fine particles of coagmay be few or many and are easily overexamined closely in good light. The somatic cell

is still

ulated material are present. These

looked unless the

test is

^Available from photographer C. Hadley Smith, Carey Building, Ithaca, N.Y. 14850 ($1.50).

8.31

117

Modified Whiteside Test

Figure 8:1. Appearance of scoring reactions in the modified Whiteside

test

under magnification.

count

is

may be

usually between 0.5 and 1.0 million per milliliter but a few counts
slightly higher.

Positive (1+).

The background

is

less

opaque but

still

somewhat milky.

Slightly larger particles of coagulated material are present and quite thickly
scattered throughout the area. These reactions are easily discernible when

compared

to a trace reaction, although only very slight

observed. The somatic


milliliter.

cell

count

is

usually between

clumping will be
and 2 million per

DETECTION OF ABNORMAL MILK

118

The background is slightly watery in appearance, with deficlumps of coagulated material that are easily differentiated from those of
the 1+ reaction. Fine strings or threads may form instead of clumping, if
stirring has been very rapid. The somatic cell count is usually 1.5 to 2 million
per milliliter, although some counts may be higher.
Positive (3+). The background is definitely watery, with larger clumps of
coagulated material than those observed in the 2+ reactions. The somatic
Positive (2+).

nite

count is usually in excess of 3 million per milliliter.


Milk having a high somatic cell count and which produces strong reactions
usually thickens shortly after stirring is started and has a tendency to follow
cell

the applicator stick in a

breaks up into what

is

somewhat gummy mass. As stirring


2+ or 3+ reaction.

is

continued,

it

typical for a

D. Precautions:
1. Milk must be thoroughly agitated before sampling and testing. No
cream plug or sedimentation should occur in individual samples.
2. The temperature of each sample must be in the range of 0-4.4 C and
the storage period between sampling and testing must not exceed 36 hours.
3.

Good

lighting

E. Recording

is

essential

and reporting

when reading

the test [5.2(A)].

results:

Record results in reaction categories (i.e., negative, trace, 1 + 2+ or 3 + ).


For guidance, refer to the grading chart. Do not report numbers of somatic
,

cells in place

of test reaction categories.

8.32 California Mastitis Test

The

California Mastitis Test

(CMT) was developed to test


^^ The test may be

vidual quarters at the side of the cow.^^-

milk from indiapplied to bulk

tank milk and other blended supplies.

The

CMT

purple).

reagent consists of a detergent plus a

The reaction between

acid) of cell nuclei

is

the detergent and

a measure of the

pH

indicator (bromcresol

DNA

number of somatic

(deoxyribonucleic

cells in the milk.

At

a concentration of 150,000-200,000 cells per milliliter, a precipitate begins to

form, thickening into a gel as number of cells increases. The grading system
is based on the amount of gel formation, but assignment of test scores is
essentially subjective. This test has the advantages of simplicity, rapidity,

low cost and convenience.


A. Equipment and supplies:
For programmed screening of tank samples, it is faster and possibly more
accurate to dispense milk and reagent with automatic syringes, which can be
set to deliver 2-ml quantities. For cowside, bucket, and occasional tank testings, a polyethylene squeeze bottle may conveniently replace the automatic
syringe for dispensing the reagent.
1.

2.

Cornwall continuous-pipetting
Cornwall 2-ml syringe.

outfit.

8.32 California Mastitis Test

119

DETECTION OF ABNORMAL MILK

120

Cornwall metal pipet holder.


Laboratory cannula, 4-in. (10 cm.)

3.

4.
5.

Special plastic paddle.

6.

CMT

7.

Sink and cold water faucet or bucket, for waste and rinse water.

reagent,'

produced under

license.'^

B. Procedure: (Fig. 8:2).


Collect and store sample(s) as specified in 3.13. Test sample(s) within

1.

36 hr after sampling.
2.

Mix sample(s)

3.

Place 2 ml of milk into a cup or paddle.

as specified in 5.6(B).

Add 2 ml of CMT reagent.


Gently rotate paddle (or cup) in a circular pattern for 10 sec so that
milk and reagent mix thoroughly.
6. Immediately score reaction.
7. Rinse the paddle and shake off excess moisture. The paddle need not
be dried.
C. Recording and reporting results:
Record results in reaction categories as indicated in Table 8:1. Cell count
4.

5.

ranges for the various reaction scores are


ues.'^'

^'^*-

^^'

Do

'"

in

accordance with published

val-

not report numbers of somatic cells in place of test reac-

tion categories.

D. Precautions:
If

1.

milk

is

kept refrigerated, reliable readings can be obtained up to

36 hours. Thereafter, milk tends to lose

its reactivity. Unrefrigerated milk


cannot be tested accurately after about 12 hours.
2. Somatic cells tend to migrate with milkfat. Therefore, thorough mixing of milk just before testing is essential to assure a representative sample.
3. The CMT reaction must be scored between 10 and 15 sec after mix-

Weaker

ing starts.

reactions fade thereafter.

Because of the

4.

critical

time factor

in

reading the

or other devices containing more than four cups


not possible to

make

is

CMT,

use of paddles

not recommended.

It is

accurate readings on more than four cups within

5 seconds.
5.

It is

important to use

CMT reagent produced

under license to be sure

of obtaining a standardized reagent.


6.

It

is

important that lighting be sufficient to permit clear observation

of reactions.
8.33 Wisconsin Mastitis Test

The

principle of

samples was

first

raw milk
the California Mastitis Test. Subsequently, methods

measuring viscosity after adding detergent

used

in

^Available from Dairy Research Products, Inc.


Inc., Fort

Atkinson, Wise. 53538.

Box

288,

to

Yarmouth, Me; or from

NASCO,

121

8.32 California Mastitis Test

Table

Symbol

8:1.

Grading and Interpretation of Milk, California Mastitis Test


Interpretation

Suggested

Visible Reaction

Meaning
Negative

(cells

per ml)

Mixture remains liquid with no evidence

0-200,000

of precipitate formation

Trace

slight precipitate

forms, seen to best ad-

150,000-500,000

vantage by tipping paddle back and forth

Weak
positive

and observing mixture as it flows over bottom of cup. Trace reactions tend to disappear with continued movement of fluid.
A distinct precipitate forms, but with no 400,000-1,500,000
tendency toward gel formation. In some
milks, reaction

is

reversible with contin-

ued movement of the paddle and the precipitate

Distinct
positive

may

disappear.

Mixture thickens immediately with some 800,000-5,000,000

Upon swirlmove toward the

suggestion of gel formation.


ing,

mixture tends to

center, leaving bottom of outer edge of

Strong
positive

cup exposed. When the motion is stopped,


mixture levels out, once again covering
bottom of cup.
A gel is formed which causes surface of
mixture to become convex. Usually there
is a central peak which remains projected

Generally

>

5,000,000

above the main mass after motion of


paddle has been stopped. Viscosity is
mass tends
bottom of cup.
This symbol should be added to the CMT An alkaline reacscore whenever the reaction is distinctly tion reflects dealkaline, as indicated by a contrasting pression of secre-

greatly increased, so that the


to adhere to

Alkaline

milk

deeper purple color.

tory activity, oc-

curring as a result
either

of

mation

inflam-

or

of

drying-off of the
gland.

Acid milk

acid
Bromcresol purple is distinctly yellow at Distinctly
pH 5.2. This symbol should be added to milk in the udder
is rare. When enthe score when mixture is yellow.
countered, it indicates

fermenta-

tion of lactose

bacteria.

by

DETECTION OF ABNORMAL MILK

122

quantitatively measuring the viscosity of a detergent-milk mixture


were developed.^- ''^ One of these, the Wisconsin Mastitis Test (WMT),^^ is
is rapid, simple and inwidely used in the United States. The
expensive. It provides an objective and sensitive means of grading the reaction obtained. Reaction will diminish significantly in samples that have not
been continuously refrigerated at 0-4.4 C ^' or that have not been tested

for

WMT

within 36 hr after collection.

A. Equipment, supplies and reagents:


1.

Test tubes:* Clear plastic, flexible, measuring 12.1 x 130

with an air vent having an approximate diameter of 3

mm

mm

ID,

located about

mm

from the outside bottom of the tube.


Metal caps:* With an orifice in the center 1.09-1.10 mm in diameter;
made of 25-gauge (0.5-mm) brass.
3. Tube racks:* Aluminum, to hold firmly ten 12.1 x 130 mm plastic

65

2.

WMT tubes.
4. Automatic syringe: Capacity 2-ml, for pipetting milk samples [assembled from Becton Dickinson (B-D) syringe No. 1250S, or equivalent].
The syringe should be adjusted to deliver 2 ml of milk and the accuracy
checked by collecting delivered volumes in a graduated cylinder.
5. Metal syringe holder: B-D No. 1250 MH, or equivalent.
6. Laboratory cannula: 50-100 mm long.
7. Continuous-pipetting outfit: 2-ml size, for pipetting reagent, B-D
No. 1251, or equivalent. The syringe should be adjusted to deliver 1.0 ml and
accuracy checked by collecting delivered volumes in a graduated cylinder.
8. Laboratory cannulas (2): 14-gauge, 100 mm long, B-D No. 1250 NR,
or equivalent. (These may be cut to the desired length with a file or hacksaw.) One cannula is connected to the 2-ml syringe and the other, approximately 82 mm long, is connected to the continuous-pipetting outfit just described. This 82-mm cannula should extend at least 6 mm below the level of
the surface of the milk sample when the pipetting outfit is resting on the two-

way

valve.
9.

over

Timer clock: With

dial

circular dial

and sweep second hand extending

markings.

Measuring square:^ Calibrated in 1-mm graduations.


reagent concentrate or
Reagent:^ Standardized commercial
equivalent. Prepare each day a supply of reagent diluted for use in the
as directed by the manufacturer. CAUTION: Old solutions of ready-to-use
10.

WMT

11.

WMT

may

reagent
12.

contain microbial populations sufficiently high to affect the

Water bath: To maintain reagent temperature

at 35

test.

(95 F) during

the test.
^Available from Dr. Z. D. Roundy, 725 South. 600 West, Orem. Utah 84057; or Dairy Re-

search Products, Inc.,

Box

288,

Yarmouth, Me. 040%

SAvailable under Catalog No. 5638-01930 from Belico Glass, Inc, Vineland, NJ, 08360; or

from Dairy Research Products, Inc, Box 288, Yarmouth,

Me

04096

8.33 Wisconsin Mastitis Test

123

WMT nozzle gauge.

13.

B. Care of samples:
Samples must be held continuously at 0-4.4 C. Samples should be tested

promptly after their removal from refrigeration the first time. Reliable results
will not be obtained on samples that have been warmed above 4.4 C and/or
have been cooled slowly in a convection-type refrigerator. All samples must
be tested within 36 hr after collection.
C. Procedure:
1. Rinse tubes and shake to remove excess water before use (or before
calibration). Rinse syringe in water and well-mixed sample [5.6(B)]. Dispense 2 ml of well-mixed sample into each tube, running it down the side of
the tube to avoid excessive foam. (Keep racks in ice water when adding
sample.)
2.

Quickly add two 1-ml portions of reagent warmed to 35 C to each of


good initial mixing of milk and reagent.

the tubes in one rack to provide

Deliver reagent below the surface of the milk (Fig. 8:3).

DETECTION OF ABNORMAL MILK

124

Figure 8:4. Mixing reagent and milk in the Wisconsin mastitis

Promptly cap tubes with caps previously arranged

3.

test.

right side

up

in

front of the rack.


Start mixing action 15 sec after adding reagent to last tube in the

4.

rack.

Mix by

placing a 10-ml

Mohr

pipet on the table and gently rocking

tubes back and forth on the pipet, permitting the liquid to run forward to

caps 10 times. Avoid banging the tubes. The ten excursions should be made
within 8-10 seconds. The temperature of the milk-reagent mixture at time of

must be 242 C (75+2 F).


1. Make any needed adjustment, such as straightening caps on
tubes, after the mixing action (at this stage, the viscosity is relatively stable.)
inverting

NOTE
5.

Invert rack immediately after the mixing action. Before inverting,

in a horizontal position while waiting for the sweep second hand


on the timer to reach a convenient starting point. Then invert rack rapidly
but smoothly and hold it in a vertical position throughout a 15-sec outflow
(Fig. 8:5). Return tubes to an upright position, remove caps, and let stand for
at least 1 min to allow liquid to drain down before reading.
6. Use measuring square [8.33(A.10)] to measure the fluid column remaining in each tube (Fig. 8:6). Measure to top of meniscus and record read-

hold tubes

ings in millimeters.

125

8.4 Confirmatory Tests

Figure 8:5. Timing inversion with the sweep second hand.

NOTE 2. Clean caps by placing them in a small container of warm water


and shaking them in two or three changes of water. Rinse tubes two or three
times in warm tap water [not over 45 C (113 F)] after each use. Remove
water by shaking the rack before each use.
D. Precautions:
nozzle gauge
1.
For calibration of caps, use the special

WMT

[8.33{A.13)] or equivalent. Correct size of the orifice

is

indicated

when

the

nozzle gauge wire can be inserted into the orifice 1.5 to 2.5 mm
end of the
without pressure.
tubes change shape. Frequent checking
2. With continuous use,
tubes have correct inside dimenof calibration is therefore necessary.
sions when the combined height of the column of 2 ml of milk plus 2 mi of
reagent reaches 37 mm. Approved glass pipets must be used to check cali-

WMT

WMT

WMT

bration.

Tubes not meeting

this

standard must be discarded.

8.4 Confirmatory Tests


Microscopic Procedures
Developers of the first microscopic procedure for examination of milk
the direct microfilms, Prescott and Breed, *^ identified two procedures
8.41

DETECTION OF ABNORMAL MILK

126

Figure 8:6. Measuring the remaining fluid in the


scopic count

(DMC) and

enumerate bacterial
sequently, Levowitz

test tube.

the direct microscopic leucocyte count

(DMLC)

to

and clumps, and leucocytes, respectively. Subpublished a "strip count" modification of the DMC
also applicable to the DMLC. Work completed by numerous investigators
has indicated that the term "somatic cells" is more indicative of those body
cells in milk generally associated with inflammation of the cow's udder than
the more specific term "leucocyte." Accordingly, the direct microscopic
somatic

cell

count

cells
*^

(DMSCC)

discussed herein includes somatic cell-count-

ing procedures that have evolved


'"' ^^- ^^' ^"

through studies of several investiga-

between analysts and by the same


procedures and one screening
DMSCC counting procedure described herein have been reported.^"
A. Advantages:
1. Milk films can be prepared quickly, stained and examined immedi-

tors.''-

**

analyst

among

Variations

the

three

in results

confirmatory

ately or later.

may be

stored as a relatively permanent record.

2.

Stained films

3.

Tentative identification of bacterial flora

may be made

nation of the milk film.


B. Disadvantages:
1.

Expensive equipment

is

required

(i.e.,

microscope).

during exami-

8.41

127

Microscopic Procedures

2.

Sample volumes are very small

(0.01 ml)

and great care must be

taken to minimize errors of measurement. Milk foam may cause inaccurate


measurement. The syringe may add metal filings to the sample.
3.

Preparation of stain and staining of slides calls for great care. Proper

performance of the

test requires

more extensive analyst

training than that for

indirect screening tests.


4.

Fatigue reduces precision of test performance and hence of test re-

sults.

Equipment, materials and solutions:


1.

2.
3.

Sample containers [3.11(C)].


Sample case and/or ice bath [3.11(D)] and [3.11(E)].
Level surface, in a well-lighted room reasonably free from dust and

drafts.
4.

Microscope

slides: (clean),

with circular areas of

cm^ marked on

the upper surface.


5.

Instruments for transferring 0.01 -ml quantities consisting of metal

syringe* with predetermined accuracy [14.9(B.l)], and pipet, calibrated ac-

cording to
6.

of

APHA

specifications, to deliver 0.01 ml of milk.

Bent-point needle:

Of 30-gauge

7.

Drying surface: Level, dust-free, with a thermostatically controlled

heat source for drying films at 40-45

and

wire, for spreading milk over an area

cm^.

(104-113 F).

8.

Forceps: For dipping and holding slides.

9.

Trays or jars: Equipped with tight-fitting covers, for holding solvents


submerging slides.

stains and/or for


10.

Slide-storage boxes.

11.

Compound

12.

microscope: Binocular preferred.


Oil-immersion objective: (93-lOOx magnification),

1.8

mm.

High-dry objective.
14. Substage, moved by rack and pinion, carrying an Abbe condenser
with iris diaphragm.
15. Mechanical stage.
16. Oculars: Huygenian or wide-field, not less than lOx magnification.
17. Microscope lamp: Separate, or connected to the microscope.
18. Stage micrometer slide: Ruled in 0.1- and 0.01-mm divisions.
^^
19. Ocular reticles:^ For use in strip reticle counting procedures
[8.41(E.2)], having two parallel lines, centrally located, with different spacing between the lines for various somatic cell concentrations and for use in
13.

'Bellco Glass Inc., Vineland,

NJ 08360, Catalog #5638-01930; Dairy Products,

Inc.,

Box

288,

Yarmouth, Me., 04096.


HDairy Research Products, Inc, Box 288, Yarmouth, Me., 04096 and Applied Research Institute, 90 Brighton Avenue, Perth Amboy, NJ 08861.
'American Optical Company Catalog No. K20I3, for 1.5 million cells per milliliter; Catalog
No. K2022, for 1.0 million cells per milliliter; Catalog No. K2023. for 0.75 million cells per
milliliter. Also available from Dairy Products Research, Inc, Box 288, Yarmouth, Me., 040%.

DETECTION OF ABNORMAL MILK

128

counting procedure [8.41(E.2)]. Preferably use a reticle with


and vertical lines, centrally located and perpendicular to

single-Strip

single horizontal

each other. Thus, each microscopic field


enumeration of somatic cells.

is

divided into four quadrants to

facilitate

Hand tally: Double tally preferred.


Newman-Lampert staining solution, Levowitz-Weber
[14.17] CAUTION: This stain should be prepared under a

20.

modifica-

21.
tion:

hood or

in

ventilated

a well-ventilated atmosphere.

22.

Immersion

23.

Staining solution (14.17).

oil:

Refractive index 1.51-1.52 at 20

(68 F).

D. Preparation of milk film(s):


1
Shake sample 25 times [5.6(B)] and allow it to stand a few minutes so
foam disperses. Rinse the 0.01-ml metal syringe or glass pipet several times
in clean tepid water and then several times in the milk sample. Withdraw test
portion of sample and remove excess milk from exterior of the tip with a
clean paper tissue or cloth. Holding the instrument in near-vertical position,
expel the test portion onto a slide near the center of the area intended for the
film

and

Make

touch-oflF.

sure that entire test portion

bent-point needle promptly spread milk over

marked

Legibly identify each film and slide of films as


2.

After spreading films,

level surface

fix

them by drying

it

is

at

is

delivered. With the

area.

Wipe needle

40-45

within 5 min on a

reasonably free from dust, insects, and drafts [8.41(C.7)].

TION: Too-rapid drying can cause

dry.

prepared.

CAU-

from the

slide

slide with fixed milk films in staining solution for 2

min-

films to crack and/or peel

during later treatments.


3.

utes.

Submerge

Remove

slide

and stand

it

in near-vertical position

on absorbent paper,

permitting excess stain to drain onto the paper. Forced air

may be used

to

dry stained films thoroughly. Rinse slides in three changes of tap water at
37-45 C (98.6-113 F), then drain and air-dry films (avoid heat or drafts).

Examine

NOTE.

films using a microscope.


It

is

advisable to conduct

graph, except water-washing and

final

all

procedures

in

the preceding para-

drying, in a ventilated

fume hood or

in

well-ventilated atmosphere.

Precautions to be observed: Metal syringes must be periodically checked


volume if the tip has

for proper calibration. Glass pipets will not deliver full

been chipped through to the bore. For use with the strip reticle counting
procedure [8.41(E.3)], duplicate milk films must be made from each sample.
E. Examination ofmilkfilm(s):
Counts are to be made using the same oil-immersion objective and eyepieces with which the microscopic factor (MF) was determined. After slides
have been placed on the mechanical stage, high-dry objective may be used
for preliminary location of the portion of milk film to be examined. Place one
drop of immersion oil on the film, move the oil-immersion objective into
alignment, and proceed with the count according to one of the approved

methods detailed

in

the following sections.

NOTE:

Regardless of the method

8.41

Microscopic Procedures

129

selected [8.41(E.l) or (E.3)], each somatic cell with an identifiable stained

nucleus should be counted.

stained nucleus should be identified as fol-

lows: For polymorphonucleated cells, count as a cell unit those cells having

two or more discernible nuclear lobes;

for other somatic cells, count as a cell

be essentially intact. When


doubt about a "cell" which may be only a fragment, do not count it.
1. Field counting procedure.
a. Adjustment and calibration of microscope: When determining
numbers of somatic cells per milliliter of milk, the microscopic field area
is fundamentally important because it determines the amount of milk
which can be examined in any one field. Field diameters providing microscopic factors from 300,000 to 600,000 are recommended. Microscopic factors are values by which the average number of somatic cells
per field is to be multiplied to determine the estimated number of cells

unit those cells having a nucleus that appears to


in

per

milliliter.

Manufacturers of binocular microscopes normally have adjusted tube


length and paired oculars for use at the most common interpupillary

mm)

distance (65
are to be

made by an
from

ies substantially

When

or especially for a given individual.


individual
that for

whose

estimates

best interpupillary distance var-

which the microscope was

set up,

use

paired oculars with diaphragms especially adjusted for the individual;

otherwise correct the counts obtained according to the diameter of the


field actually

observed.

Presently available standard equipment features binocular and

nocular microscope bodies of constant tube length. The resultant


brated

To

and resultant magnification remain constant regardless of

field

changes

tri-

cali-

in interpupillary setting.

direct light through the milk preparation, adjust

to provide

maximal

optical resolution [14.11].

microscope lamp

To determine

the micro-

scopic factor, replace milk preparation on the stage of the microscope


with a stage micrometer slide, ruled in 0.1- and 0.01-mm divisions and

proceed as follows:

Measure the

(1)

e.g.,

0.146

(2)

field

diameter

millimeters to the third decimal,

in

mm.

To determine

the area of field, square the radius

(r

Vi the field

diameter) and multiply by 3.1416.


(3)

To convert

area in
(4)

mm- by

the area of one field in mm'- to cm-, divide the field

100.

To determine

the

number of such

fields in

cm-, divide

cm^ by

the area of one field in terms of cm-.


(5)

Since only 0.01 ml of milk was spread over a 1-cm^ area, multiply

the

number of

per

milliliter

The
in

final

fields

by 100 to determine number of microscopic

fields

of milk.

value obtained

each instance

is

is

called the "microscopic factor"

dependent on a

definite tube length

(MF) and

and a

definite

DETECTION OF ABNORMAL MILK

130

combination of objective and ocular(s) used. The reciprocal of the MF


represents the fraction of 1 ml of milk actually seen in one microscopic
field. MF should be expressed in two significant figures (i.e., 480,000).
Use of the following condensed formula, in which the radius is expressed in millimeters, obviates the need to convert the area into square
centimeters, to determine the number of fields per square centimeter,
and to multiply by 100 to express results in cells per milliliter of milk.
'0,000

MF
Working

b.

When

factor (WF):

MF

stant, divide the

3.1416 X
the

r^

number of fields examined

by the number of

fields

counted

to

is condetermine the

WF.
Performance of

c.

count: Select successive rows, each per-

field

midway on
any side and two or three fields in from the edge. Proceed to count,
accumulating totals of somatic cells and of fields examined in accordance with Table 8:11 and the example below.
d. Selection of number of fields to be examined: Table 8:11 gives suggested microscopic factors (MF) with corresponding (1) field diameters
using lOx or greater oculars with 1.8-mm oil-immersion objectives, (2)
numbers of fields to be examined, and (3) calculated working factors
(WF), when making direct microscopic somatic cell counts per milliliter
pendicular to the previous row, across the film. Start about

(DMSCC/ml).

EXAMPLE.
the
cell

If

the technician

minimum number
concentration

is

using a microscope with a

of fields to be examined

is

10. If

MF of 500,000,

the estimated somatic

must

less than 3,000,000 per milliliter, additional fields

be counted. This estimate


10 fields

is

is

made by averaging

the cell count in the

first

examined and multiplying this average by the MF.


-^ 10 x 500,000 would yield an estimated somatic
If

40 cells were so

counted, for example, 40


cell

count of 2,000,000 per

milliliter.

Since this estimate

lies in

the 300,000-

3,000,000 range, an additional 10 fields (for a total of 20) must be counted.


After the

full

count per

number of required

milliliter is

fields

have been examined, the somatic

computed according

cell

to either of the following equa-

tions:

r^,r>^^
DM
sec

2.

off the

answer

total

to

two

total

a.

field as

the strip as

MF

fields

somatic cells x

WF

significant figures.

Single-strip counting procedure

croscopic

somatic cells x

No. of
or

Round

per ml

it

'.

This procedure uses the entire mi-

traverses the diameter of the milk film.

Calculation of the single-strip factor (SSF): Measure the diameter

of the microscopic

field for

the specific eyepiece-objective combination

8.41

Microscopic Procedures

Table

131

MF with corresponding field diameters,* numbers of


examined, and calculated WF for DMSCC per milliliter'

Suggested

8:11.

be

fields to

Microscopic Factors

Dependent values
Field diameters

Count

(mm)

300,000

400,000

500,000

600,000

0.206

0.178

0.160

0.146

limits per ml:

30,000-300,000

No. of fields
Working factor

30

40

50

60

10,000

10,000

10,000

10,000

300,000-3,000,000

No. of fields
Working factor

20

20

20

30

15,000

20,000

25,000

20,000

Over 3.000,000
No. of fields
Working factor

10

10

10

20

30,000

40,000

50,000

30,000

*Using lOx or greater oculars with 1.8-mm oil-immersion objectives.


magnification of at least 900x is necessary.

tA

used for counting. The MF of the microscope varies with the field diameter. A lOx ocular with a 1.8-mm oil-immersion objective is preferred.
Calculate the strip area.

The area of

ameter (D) of the microscope


(11.28

field

the single strip

is

equal to the di-

times the length of the milk film

mm).
Area of

single strip

Determine the number of single


ing 100

mm^

11.28

mm

(in

mm)

strips in the 0.01-ml milk film

by divid-

(area of 0.01-ml milk film) by the strip area.

No. of

single strips

100

mm^ ^

area of single strip

Convert the number of single strips in 0.01 ml of milk to a 1.0-ml basis


by multiplying the number of single strips in 0.01 ml by 100.

SSF = No.

of strips in 0.01 ml x 100

EXAMPLE:
Diameter of microscopic field = 0.142 mm
Length of strip (diam of 1 cm^ circle) = 11.28 mm
Area of single strip: 11.28 x 0.142 = 1.6 mm
No. of single strips in area of milk film
(0.01 ml of milk): 100 ^ 1.6 = 62.5

SSF =

62.5 X 100

6,250

DETECTION OF ABNORMAL MILK

132

Performance of the

b.

Focus on the

single-strip count:

film

edge

in

the oil-immersion field that appears to be at the maximum horizontal or


vertical excursion. Traverse the entire diameter of the milk film, counting those cells in the strip which touch one edge of the strip. Do not
count cells which touch the other edge. During scanning of the strip,
make continual fine focusing adjustments. The somatic cell count is

computed

as follows:

T^,o^^
DMSCC

per ml

Total No. of somatic cells in single strip

X single-strip factor (SSF)

EXAMPLE.

If

an analyst counts 200 somatic

assuming

computed

off the

answer

per ml

to

two

1,300,000 somatic cells per


3.

count

is

as follows:

DMSCC
Round

cells in a field-wide strip,

a single-strip factor [8.41(E.2)] of 6,250, the somatic cell

= 200 x

6,250

1,250,000

significant figures for a reported count of

milliliter.

Strip reticle counting procedure.^- ^

The

unit area

examined

is

strip of milk film, defined in length by the fixed diameter of the circular milk

width by the parallel lines on one of three special


eyepiece reticles [8.41(C.19)]. Selection of strip width according to maximum acceptable somatic cell concentration locates the region of optimum

film [8.41(C.4)]

and

in

count precision at this


a.

critical

concentration.

Calibration of microscope: Insert the appropriate eyepiece reticle

nonadjustable eyepiece of the binocular microscope. Align the


oil-immersion objective. Using the stage micrometer, measure the disin the

tance in millimeters between the parallel lines seen in the visual field,
estimating the third decimal place. This is the strip width. Strip length is
fixed at 11.28

mm

(diameter of

cm^

circle).

Determine area of

strip

in square millimeters. Divide film area by strip


(strip
area to get the number of such strips in the milk film. Multiply the result
by 100 to get the number of strips in 1 ml of milk. The computations are

width x 11.28)

expressed

in the

following equation:

Strip
^ reticle factor

(SRF) = r^rx

The SRF must be determined

-r^

rr-r-^

11.28 X strip width

for each microscope

and

m mm
reticle

combina-

tion.

Performance of strip reticle count: The horizontal diameter is located by observing the left or right edge of the milk film through highb.

dry or oil-immersion magnification while manipulating the vertical travel of the mechanical stage. Follow film edge to its widest horizontal
point. Rotate the eyepiece containing the eyepiece reticle until the par-

8.41

Microscopic Procedures

are horizontally oriented. Place the oil-immersion objective in

allel lines

alignment

not already done. Using the horizontal mechanical-stage

if

move

control,

133

the milk film completely across to

far edge, counting

its

somatic cells [8.41(E)]. Similarly, locate the vertical diameter of the


milk film by observing the top or bottom edge, and count cells found in
the vertical strip. When counting films with boundary lines, observe the
all

following rules:

Count somatic

(1)

field to

Count
Count

(2)

(3)

cells as

they pass through the center of the visual

lessen the need for fine focusing.


all

somatic cells entirely within both boundaries.

all

somatic cells which touch one of the boundaries; do not

count those somatic cells which touch the other boundary. Note that a
cell which just touches one boundary line from either the outside or the
inside is counted; a cell which touches the other boundary from either
side

is

not counted.

Computation of results:
a. Four strip procedure: When all four strips have been examined,
compute the average somatic cell count per strip (to two decimal places)
and multiply this value by the SRF. The resulting value is the direct

4.

microscopic somatic

^^,^^^
DMSCC
where

cell

count per
,

per ml

Hi +

milliliter:

Vi + H2 + V2 X

SRF

= count obtained on a horizontal strip, V = count obtained on


numbered and 2.

a vertical strip, and films are

EXAMPLE.

Using a microscope and reticle yielding an


the following counts are made: 72, 80, 78, 69.

DMSCC/ml =
Round

off to

two

72

+ 80 + 78 + 69

^^

SRF

of 15,160,

1,130,000

significant figures; thus, the reported

DMSCC

would be

1,100,000 per milliliter.


b.

Single-strip procedure:^^ Since precision increases with

number of

counted, as a count approaches the control limit the more strips


must be counted to determine compliance. The following procedure
strips

permits a single strip count for those milks falling into the extreme high
or low ranges but provides for counting of additional strips to confirm

those values which are close to the control limit. This

is

a modification

of the strip reticle counting procedure [8.41(E.3)], which permits rapid

examination of samples by examining only one strip. Milk samples are


processed exactly as in the strip-reticle counting procedure [8.41(E.3)]
but only the horizontal strip of the first film is counted. The number of
somatic cells found is compared with a low control value (C^) and a high
control value (C^), specific for the microscope and reticle used. If the

DETECTION OF ABNORMAL MILK

134

sample should be graded acceptable. If


may be considered as confirmed
(see example below) and the milk sample is graded unacceptable. If the
count is within the range C^ to C, the count must be confirmed. To
accomplish this, count the additional three strips required. Values of C^
and C^^ for the microscope and reticle are selected from Table 8:111.
They depend on the strip equivalent (SE), which is computed as folcount

is

less than Cl- the milk

the count

is

greater than C^. the count

lows:

_ Maximum
SE

EXAMPLE.
matic cells per

In

reticle

The SRF
is

cell

concentration

SRF

most areas, the

milliliter.

proper eyepiece

acceptable somatic

legal
is in

maximum number

is

1.5 million so-

the neighborhood of 15,000

when

the

used [8.41(C.19)]. Thus


1.500,000

^^ =

15,000

= '""

Cl and Ch from the table are, respectively, 73 and 137. The categories defined for a milk sample by the values Cl and Ch may be accepted with at least
as

much confidence as results


table of 95% confidence

attained using the confirmatory procedure.

The

limits designed for interpretation of conbased on the assumption that a coefficient of variation (CV) of 12% will describe the usual laboratory performance; in this
screening method an average CV of 19% is assumed.^" Therefore, any milk
sample which yields a count greater than Ch has more than 95% probability

firmatory counts

of containing
5.

sion

oil

^- ^" is

more somatic

than the legal

cells

limit.

Storage of slides. After slides have been examined, remove immer-

on films by dipping each

slide in

xylene for 15-20 sec and allowing

films to air-dry. Store slides with films in dust-free slide storage

future observations. Avoid accumulation of pencil


their presence will interfere with staining

marks on

boxes for

slides, since

procedures or with future re-exam-

ination of milk films.

8.42 Electronic Somatic Cell Counting Procedure

Somatic cells in milk have been counted electronically by use of several


published procedures which can be divided into a.) those employing a centri'''^'
and b.) those in which chemical disfuge to remove the fat globules
'

"

'*'-

The centrifugation procedure


Standard Methods."^^ The chemical procedure is standardized by the
Milk Marketing Board in the United Kingdom, and by the International
Dairy Federation in Europe.' Both procedures use the same electronic cell
counting device.
originated the chemical procedure and obtained a high degree of
Tolle
correlation between results of the chemical procedure and microscope counts;

persion of fat
is in

'''

is utilized.'-

^' ^'

'2-

''^'

'''

8.42 Electronic Somatic Cell Counting Procedure

Table

Gl

8:111.

135

Values of Cl and Cjj Appropriate for a Range of Strip Equivalents


(SE)*

DETECTION OF ABNORMAL MILK

136

Electronic scanning for cell counting provides high statistical accura-

4.

cy.
5. The electronic device can give data on cell size distribution as well as
count data.
B. Disadvantages:
1. Instrument cost is high but can be offset by rapid amortization.
2. Good techniques of cleanliness in the laboratory are required for the
instrument and glassware.
3. Calibration and standardization of the instrument are necessary.

C. Equipment

and reagents:

Coulter Counter:** (Model ZB, ZBI, ZF, FN, or equivalent)

1.

with 100 or 140 /xm aperture tube and

100-/>tl

fitted

manometer.

2. Automatic Diluter: 0.1-sampling and 9.9-ml. dispensing capacity,


and tolerant to ethanol.
3. Water bath(s): Either two water baths maintained at 80 and 60 C
( 1 C) or one quick-recovery bath capable of meeting both temperature

requirements.

Glass medicine

4.

ter, thin glass,

vials:^^ 7

dram, 66

mm

height, 25

mm outside diame-

snap-cap.

5.

Accuvettes:** 20 ml. capped plastic

6.
7.

Timer: 10 minutes.
Vortex Jr. Mixer.

8.

Racks

9.

Pipets: 10-ml, wide-tipped (for dispensing milk sample).

(2):

To accept medicine

vials

vials.

and accuvettes, 24-sample capac-

ity.

10.
in

10% formalin (40% w/v formaldehyde)


Twenty milligrams of eosine may be added to 100 ml of

Cell fixing solution: Filtered

deionized water.

the diluted formalin to provide a visual indication of fixing. Warning: the

and concentration of formaldehyde


alternate solution is Somafix.**
11.

in

formalin will change with time.

pH
An

Fat dissolving electrolyte solution.

volumes of 0.9% sodium chloride/water.


volumes of ethanol.
2.0 volumes of Triton X-100"
1.0 volume of formalin (409? w/v formaldehyde)
(hydroxymethyl)amino
Buffer this solution with 0.5 molar tris
methane primary std. to pH 7.0. Filter this solution.
An alternate solution is Somaton.**

84.5

12.5

12.

Calibration spheres: 5.0- or 3.4-fxm diameter monosized latex.**

Obtainable from Coulter

Electronics, Inc., 590

W. 20

St.,

Hialeah, Florida 33010.

^^Demuth Glass Works, Div. of Brockway Glass Co., Inc., Parkersburg, W.Va. 26101.
"Obtainable from Rohm & Haas, Philadelphia, Pa. 19104.

137

8.42 Electronic Somatic Cell Counting Procedure

D. Procedure:
1. Fixing milk samples.
a. Hold all milk samples

refrigerated at 4

before fixing.

Put 3 drops of Somafix into each medicine vial.


Before sub-sampling, invert milk samples 25 times.

b.
c.

Pipet approximately 10 ml of milk sample into medicine vials.


Immediately replace cap and mix samples on the Vortex Jr. Mixer (Steps d
and e must be undertaken as indicated to avoid coagulation of milk by Somafix).
Follow steps b, c, d, and e until all samples are thus treated.
f.
g. Rack sub samples and heat in water bath for 6 V2 min at 60 C (1 Vi min heat
d.

e.

up time and

min hold

time).

Time

this step with

Remove rack and cool samples in ice water


remove to room temperature (20 C) or refrigerate
2. Diluting and counting fixed samples.
h.

appropriate timing device.


(0 to 4

for 2

min and then

for later counting.

Calibrate the Coulter Counter according to instructions which follow and

a.

set the

lower threshold

Somaton solution do

at

4.4-/xm diameter (44.6 ^tm^). Using an accuvette and

five or six

blank counts on the Coulter Counter. The averIf it is not, clean the glassware used,

age of these counts should be below 20.


refilter the

must be

at

Somaton solution, or check electrical


room temperature for diluting.

interference. Fixed samples

b.

Mix each

c.

Invert each sample 2-3 times just before dilution.

d.

Dilute the fixed samples as follows:

(1)
(2)

fixed sample (consecutively 3-5 sec

on the Vortex

Jr.

Draw sample from the medicine vial into automatic diluter.


As medicine vial is taken away from diluter pipet, touch off (with

mixer).

the edge

of the vial) the milk drop remaining on the diluter pipet.


(3)

Expel milk and diluent from diluter into an accuvette, being careful to

prevent foaming. This can be done by directing flow

down

the side of the accu-

from making contact with


project under the surface of the solu-

vette at an appropriate angle, but keeping the pipet tip

the accuvette and never allowing the tip to


tion.

Do

not mix

in

accuvette.

place in rack. Dilute and rack all samples before going to


Also insert a capped Somaton blank and carry through the remaining
steps to serve as background count.
f.
Place rack of capped accuvettes in water bath at 80 C for 10 minutes. Be
certain that the top of the mixture in the accuvette is )4 in. below the water
e.

step

Cap accuvette and

f.

surface.
g.

Time

this step with appropriate device.

Cool samples

in ice

water

at

to 4

for 3

min and remove

to

room temper-

ature (20 C).


h. Invert each sample four times, allow bubbles to disperse (about 10 sec),
and count on the Coulter Counter. At this stage all samples should be analyzed
within one hour. The lower threshold of the Coulter Counter should be set at 4.4
/Am-diameter (44.6 /ixm'') and the manometer should be set for 100 /xl (0.1 ml)
of sample volume. The count readout minus blank counts multiplied by 1000
will be the cell count per ml of milk sample.

E. Calibration:
1.

Calibration will establish relationship between the threshold setting

on the Coulter Counter and spherical

particles of

known

size

3.40 or 5.0 jxm

DETECTION OF ABNORMAL MILK

138
in

diameter. The Coulter threshold

is

linear with particle size values in

cubic micrometers.
2. Make a dilute suspension of calibration particles in an accuvette with
Somaton. Place the accuvette in the counting position of the Coulter Counter and activate the counting cycle. A pulse pattern will appear on the Coulter oscilloscope. Pulses generated from monosized spheres will have uniform amplitudes. Adjust these pulse heights to between V3 and V2 the height
of the oscilloscope screen. Adjust the lower threshold shadow line to coincide with these pulse peaks. (Disable the upper threshold by rotating it full
counter clockwise). Note the lower threshold value. Repeat the lower
threshold adjustment three times and average the values. Rotate the lower

threshold clockwise until

it is set to a value just Vi of the average value just


determined. Take four or five counts and average them. They should be

between 1000 and 4000.

If not, dilute

or add calibration particles as required.

Calibration spheres will slowly settle out so occasional stirring

may be

re-

quired.

Adjust the lower threshold counter clockwise to a value of 1 V2 times the


averaged volume just determined. Take 4 or 5 counts and average them. Add
the averages from the Vi and 1 V2 positions and divide by two. This is the
V2 count. Return the lower threshold to the average value and take several
counts. These should be within 7% of the V2 count just determined. If they
are not, adjust the lower threshold slightly until they are. Record this lower
threshold value. Using this value and the cubic micrometer value of the calibration spheres, compute the appropriate threshold, aperture current, and
amplification settings for counting at 4.4-)U,m diameter (44.6 microns'^). Use
the computation in the Coulter Instruction Manual. A change of aperture
tubes requires a recalibration.

routine calibration check once a

week

is

sufficient.

F. Precautions:

Before fixing, samples must be stored in a refrigerator or ice bath.


Threshold calibration must be done with stable near-monosized
spheres of known size. They should be dispersed with an ultrasonic bath
1.

2.

before use.
3.

Samples should be

at

room temperature when

diluted with the fat-

dissolving solution.
4.

Containers receiving the sample milk must be clean and nearly parall stages. The same is required for the aperture tube and the

ticle-free at

outside electrode. Well-filtered fat-dispersion solution

may be used

for

flushing.
5. Blank samples of fat-dispersing solution alone should show a total
background count of less than 20 per 0.1 ml.
6. The outside electrode which extends into the sample should be kept
clean (use nitric acid); otherwise gas bubbles can collect on the electrode

surface.
7.

Check

the actual volumes of the

manometer and

diluter used.

8.5

References

8.5
1.

CuLLEN, G.A.
milk. Vet.

2.

139

References

1965.

The use of electronic counters

for determining the

number of

cells in

Record 77:858.

DiJKMAN, A.J., ScHiPPER. C.J.. BooY, C.J., and G. Postehummus. 1969. The estimation
number of cells in farm milk. Neth. Milk Dairy J. 23:168-181.
GiNN, R.E., Thompson, D.R., and V.S. Packard. 1976. A collaborative study of electronic somatic cell counting by the chemical method in raw milk. J. Food Prot. 40:456-458.
International Dairy Federation. 1971. Electronic counting of somatic cells in milk: A
recommended procedure for milk sample preparation and cell counting with a Coulter

of the
3.

4.

Counter. Annual Bulletin, Part


5.

11,

Square Vergote 41, 1040 Bruxelles, Belgique.


to the diagnosis of mastitis in cattle in connection

Jaartsveld, H.J. 1%2. Contribution

with mastitis control. Neth. Milk Dairy


6.

Levowitz, D.

J.

16:260-264.

1944. Reproducible data by microscopic method, p. 198. Bull. Int. Assoc.

Milk Dealers.
7.

8.

Macaulay, D.W., GiNN, R.E., and V.S. Packard. 1976. Experience in adoption and
comparative evaluation of the Coulter counter and DMSCC method for determining somatic
cell counts in milk. J. Milk Food Technol. 39:250-252.
Murphy, J.M., and J.J. Hanson. 1941. A modified Whiteside test for detection of chronic
bovine mastitis. Cornell Vet. 31:47.

9.

10.

National Mastitis Council. Subcommittee on Screening Tests. 1968. Direct microscopic somatic cell count in milk. J. Milk Food Technol. 31:350-354.
National Mastitis Council. Writing Committee, 1965. Current Concepts of Bovine
Mastitis.

NMC,

910 Seventeenth Street, NW, Washington, D.C.


1975. A technique for dififerential somatic cell counts

Seminar

11.

Newbould, F.H.S.

12.

on Mastitis Control I.D.F.


Pearson, J.K.L., Wright, C.L., Greer, D.O., Phipps, L.W., and J.M. Booth. 1970.
Electronic counting of somatic ceils in milk. A recommended procedure. U.K. Govern-

13.

ment. Northern Ireland Min. Agr., Stormont, Belfast, Northern Ireland.


Pearson, J.K.L., Wright, C.L., Greet, D.O., Phipps, L.W., and J.M. Booth. 1970.
A study of methods for estimating the cell content of bulk milk. J. Dairy Res. 37:467-480.

in

milk.

14.

15.

16.

Philpot, W.M., and J.W. Pankey. 1973. Comparison of four methods for enumerating
somatic cells in milk with an electronic counter. J. Milk Food Technol. 36:94-100.
Phipps, L.W., and F.H.S. Newbould. 1965. Isolation and electronic counting of leukocytes in cow's milk. Vet Res. 77:1377-1379.
Phipps, L.W., and F.H.S. Newbould. 1966. Determinations of leucocyte concentrations
cow's milk with a Coulter counter. J. Dairy Res. 33:51.
PosTLE, D.S., and H. Blobel. 1%5. Studies of bulk milk screening procedures for mast-

in
17.

itis.

18.

19.

tion
20.

Amer.

J.

Vet. Res. 26:90.

Prescott, S.C, and R.S. Breed. 1910. The determination of the number of body cells in
milk by a direct method. J. Infect. Dis. 7:362.
Read, Jr., R.B., Bradshaw, J.G., and A.R. Brazis. 1969. Influence of milk sample agita-

on abnormal milk scores.

Read,

Jr., R.B.,

Bradshaw,

J.

Dairy Sci. 52:1682.

J.G.,

and J.T. Peeler. 1971. Collaborative study of conin milk. J. Milk Food Technol. 34:285-288.

firmatory testing procedures for somatic cells


21.

Read,

Jr., R.B.,

Reyes. A.L., Bradshaw, J.G., and

ing of somatic cells in milk.

J.

J.T.

Peeler. 1967. Electronic count-

Dairy Sci. 50:669-674.

Jr., R.B., Reyes, A.L., Bradshaw, J.G., and J.T. Peeler. 1969. Evaluation of
seven procedures for detection of abnormal milk due to mastitis. J. Dairy Sci. 52:1359-

22.

Read,

23.

Regents.

1367.

University

of California.

1956.

California

mastitis

reagent.

U.S.

Patent

21,998,392.
24.

ScHALM, O.W., and D.O. Noorlander.


(October). Unpaginated.

1956.

The

California mastitis test. California Vet.

DETECTION OF ABNORMAL MILK

140
25.

26.

ScHALM, O.W., and D.O. Noorlander. 1957. Experiments and observations leading to
development of the California mastitis test. J. Amer. Vet. Med. Assoc. 130:199.
Schneider, R.. Jasper, D.E., and R.N. Hide. 1%6. The relationship between bulk tank
microscopic cell counts and the individual cow California mastitis test reactions. Amer. J.
Vet. Res. 27:1169.

27.

28.

29.

30.

31.

32.

33.

34.

ScHULTZE, W.D. 1968. Design of eyepiece reticles for use in the DMSCC method. J. Milk
FoodTechnol. 31:344-349.
ScHULTZE, W.D., and J.W. Smith. 1966. Comparison of the CMT and microscopic count
for estimating cell concentrations in quarter samples. J. Milk Food Technol. 29:126-129.
ScHULTZE, W.D., Smith, J.W., Jasper, D.E., Klastrup, O., Newbould, F.H.S.,
PosTLE, D.S., and W.W. Ullmann. 1971. The direct microscopic somatic cell count as a
screening test for control of abnormal milk. J. Milk Food Technol. 34:76-77.
Smith, J.W. 1%9. Development and evaluation of the direct microscopic somatic cell
count (DMSCC) in milk. J. Milk Food Technol. 32:434-441.
Smith, J.W., and W.D. Schultze. 1966. An evaluation of the California mastitis test as a
method of estimating the number of cells in milk. J. Milk Food Technol. 29:84-87.
Standard Methods for the Examination of Dairy Products, pp. 127-132. 13th ed.
1972. American Public Health Association, Washington, D.C.
Temple, H.C, and C.J. Haller. 1960. Quality tests on bulk milk to determine presence of
mastitis secretion. Annual Report, New York State Milk Sanitarians.
Thompson, D.R., Packard, V.S., and R.E. Ginn. 1976. Evaluation of the Coulter Counter
chemical method, DMSCC, WMT, tests for mastitis. J. Milk Food Technol. 39:854-

858.
35.

Thompson,

D.I.,

and D.S. Postle. 1964. The Wisconsin mastitis

test. J.

Milk Food Tech-

nol. 27:271.

36.

37.

ToLLE, A., Zeidler, H., and W. Heeschen. 1%6. Ein Verfahren zur electronischen
Zahlung von Milchzellen. Milchwissenschaft. 21:93-98.
Whiteside, W.H. 1939. Observations on a new test for presence of mastitis in milk. Can.
Pub. Health

J.

30:44.

CHAPTER

DETECTION OF ANTIBIOTIC RESIDUES IN MILK


AND DAIRY PRODUCTS
J.W. Messer, L.L. Claypool,
G.A. Houghtby, E.M. Mikolajcik, and E.L. Sing

9.1

Introduction

Widespread use of

antibiotics (e.g., penicillin

and tetracycline) and drugs

sulfonamides) has significantly contributed to control of diseases in


dairy cattle. Antibiotics and drugs are administered by infusion, by injection, and orally. Unfortunately, all three routes can permit the antibiotic or
drug to reach the milk supply, thus creating problems for the producer, proc(e.g.,

essor and consumer.

The need for qualitative and quantitative tests to detect antibiotic residues
and drugs in milk and dairy products has long been recognized. The cylinder
plate assay method was described in 1941.' The filter paper disc method was
developed in 1944-1945.'^- ^' Concurrent but independent work by many
investigators led to application of both methods to milk in 1950-1955. The
^^
principle of the reverse phase disc assay technique developed in 1960
^"
and
served as the basis of the agar diflfusion technique recently described
successfully used in

some

laboratories.'^'

'^

have been developed to assay commercial antibiotic preparations. They have not been widely used to assay milk and dairy products
since most will not detect the small amounts of antibiotics or drugs found in
milk and dairy products. In addition, to do the tests is difficult and time
consuming. If rapid, easy, sensitive chemical assay methods could be developed, they would readily be accepted and used.
The disc assay method '^ with modifications ^' ^- ^- ^' ^' '^' '*' '^ has been
used more extensively to determine the presence in fluid milk of residual
Chemical

tests

antibiotics, including penicillin, than the cylinder assay method.^-

Use

of standardized

materials will

reduce variability

procedures. With adequate care and suitable controls,


made.

ISC Liaison: E.H. Marth


141

in results

of

'' '^- ^^

assay
can be

all

reliable assays

ANTIBIOTIC RESIDUES

142

9.2 Bacillus subtilis Disc

Assay

Equipment and Supplies


A. Seed agar: Antibiotic Medium No. 1 ^' (Chapter 4).
B. Petri dishes: Flat-bottom pressed glass or plastic dishes.
C. Spore suspension: Use a standardized suspension of spores of Bacillus
subtilis (ATCC 6633). Store the spore suspension at 0-4.4 C.
9.21

Each lot of spores (from commercial sources or produced in the laboratomust be tested to determine the amount of inoculum for use in Methods
A, B, and C 19.22(A,B,C)]. To determine the number of spores per ml of
stock suspension, prepare dilutions of 10"^, 10"\ 10", and 10" of the stock
ry)

'^

spore suspension with sterile phosphate buffered water. Plate 1.0 ml of each
of the four dilutions in triplicate using Antibiotic Medium No. 1. Incubate

2 hr at 32 C. Average the count of the three plates


which has 30-300 colonies per plate. Multiply by the appro-

plates inverted for 24


for the dilution

priate dilution factor to obtain the

number of spores per ml

of the stock

suspension. Dilute the stock suspension as necessary to obtain the required


number of spores per ml of ''seeded" agar for Methods A, B, and C
[9.22(A,B,C)]. Preparation of a proper dilution of spore suspension for seed-

development and easy recognition of zones when the


sample contains the equivalent of, or less than, 0.05 unit of penicillin.'
D. Filter paper discs, penicillin-impregnated: Diameter Vi inch, (1.25 cm)
0.05 unit, with high absorbance, for use as reference standards. Refrigerate
discs as directed by manufacturer. Additional reference discs which contain
0.01, 0.025, 0.1, 0.25, 0.5 and 1.0 unit may be used if the concentration of
penicillin in samples is high and more exact information on the level of antibiotic present is needed. Since variability has been reported for commercial
filter discs impregnated with penicillin, new lots of discs must be checked
ing agar will permit

with

known

standards.

E. Standardized crystalline penicillin G, sodium or potassium: Store


at

-10 C. Do not use beyond


F. Filter

paper

expiration date.

discs, nonsterile blanks:

Diameter

Vi inch (1.25

cm), non-

toxic to B. subtilis, high absorbance, for sample application. Sterile blank

discs

may be used

if

they give results equivalent to those obtained with non-

sterile discs.

G. Filter paper discs, penicillinase-impregnated: Diameter Vi inch (1.25


cm), high absorbance, to identify pencillin.

NOTE.

All discs

used

in testing

must be of the same

size

and absorbance to

insure comparable results.

H. Penicillinase cone: Available from most


/.

Forceps: Dissecting, with

biological supply houses.

fine points.

Water baths: Thermostatically controlled.


K. Incubator: 32 1 C; 35 1 C, 37
C.

J.

143

9.22 Procedure

9.22 Procedure

A. Method
1.

(32

Prepare and

dium No.

1.

C OVERNIGHT INCUBATION):
sterilize

Cool the

(Chapter

sterile

4) a suitable

medium

to 50-55

amount of antibiotic MeC and prepare "seed"

agar suspension [9.21(C)]. Inoculated agar should contain approximately


1.0 X 10'^ spores per milliliter.'-^ Mix well but avoid incorporating air bubbles into the agar.
9:1

To

petri dishes

and allow the agar to

Use of

the

solidify

amount of medium

add the amount of agar specified


on a level surface.

in

Table

specified in the table will insure an agar

layer of uniform thickness regardless of the kind of petri dish employed.


larger and clearer, and lower concentrations of inhibitory
substances can be detected with a thin layer of agar than with a thicker layer
of medium.^' '^ Special care must be exercised to maintain uniformity of

Zones are usually

inoculum and depth of medium.


2. Divide the outside bottom of the petri dish with a marking pencil into
four or more approximately equal sections so the distance between discs will
be at least 20 mm (on centers) and label each with an identifying mark. With
a clean, dry forceps remove a paper disc from a vial or other suitable container and touch the edge into a well-mixed [5.6(B)] sample of raw milk. Allow milk to wet the disc by capillary action; avoid an excess of milk on the
disc. Immediately place disc, flat side down, on the agar surface near the

marked section. Place a 0.5-unit penicillin reference standard disc


on each plate. Vary the location of the reference disc so that in a
series of samples the reference disc will be placed in representative portions
of the plate. Do not wet reference discs, as freshly poured plates have
enough moisture on the agar surface to saturate the discs. Invert the plates
and incubate at 32 C for 14-24 hr until growth becomes apparent. Examine
plates and interpret results as in 9.23. Report as in 9.24.

center of a
[9.21(D)]

Table

9:1.

Quantity of

Medium Required

per Petri Dish to Insure Uniform

Thickness of Agar Layer*


.

Petri

^.

Diameter of

Dish

Size

Material and

Type

Layer

(mm)
100 X 22

Area or
Agar Layer
,

.,/

Amount

of

^'.
Required
,

(ml)

ANTIBIOTIC RESIDUES

144

Samples of raw milk which give a positive test must be heated to 82 C for
2-3 min,*^ cooled, and retested as in 9.23(A). This heat treatment inactivates
naturally occurring inhibitory substances in milk and eliminates the possibility of a false-positive test result. Alternatively, all samples can be heated
before testing, but this increases the amount of work, since less than 1% of
the samples are likely to give a positive result.

B.

Method B

(35

C SHORT INCUBATION):

Prepare seed agar and plates as in 9.22(A) except inoculated agar should
contain approximately 1.0 x 10*^ spores per milliliter. Invert plates with
discs in place and incubate at 35 C for 5-7 hours. Examine plates and interpret results as in 9.23. Report as in 9.24.

C.

Method C

(37

C SHORT INCUBATION):

Melt Antibiotic Medium No. 1, cool to 70 C and inoculate with a predetermined quantity of spore suspension [9.21(C)]. Inoculated agar should
contain approximately 5.0 x 10*^ spores of B. subtilis per milliliter.'^ Rotate

min in a thermostatically con70 C, mix the agar again, and prepare plates as in

the agar flask to distribute spores, hold 15


trolled

water bath

at

9.22(A).

Invert plates with discs in place and incubate at 37

comes apparent; normally observations can be made

until

growth beOp-

after 3-4 hours.

poured plates at 37 C for 1 hr before "spotting" with discs


wetted with milk.^ Examine plates and interpret results as in 9.23. Report as

tionally, hold

in 9.24.

9.23 Interpretation

Examine

plates for clear zones of inhibition.

Any

clear zone surrounding a

disc wetted with a heated sample is a positive test. To determine if the clear
zone resulted from penicillin or another inhibitor or both, proceed as in

9.23(A).

A. Tests for identification of the inhibitor:


1. To determine whether inhibition resulted from penicillin, place a
penicillinase-impregnated disc [9.21(G)] and a blank disc [9.21(F)] wetted
with the heated positive test sample and a 0.05-unit penicillin control disc on

add 0.05 ml of penicillinase cone [9.21(H)] to 5


Wet one blank disc [9.21(F)]
blank disc [9.2 IF] with a
one
sample
and
with the penicillinase-treated
Place
both discs and a 0.5-unit
sample.
of
the
same
aliquot
heated untreated

the

same

plate. Alternatively

ml of the heated milk sample and shake well.

on the same plate. Incubate as in 9.22(A), (B) or (C). If


no clear zone appears around the penicillinase disc, but one surrounds the
disc containing the test sample, the test demonstrates that penicillin is present. If a clear zone of equal size surrounds both discs, the test demonstrates
that penicillin is not present but another inhibitor is present. If the clear zone
surrounding the penicillinase disc is 5 mm or more smaller than the zone
surrounding the blank disc, the test demonstrates that both penicillin and
penicillin control disc

145

Equipment and Supplies

9.31

another inhibitor are present. If the clear zone surrounding the penicillinase
smaller than the zone surrounding the blank disc, the
disc is less than 5
test demonstrates only that an inhibitor other than penicillin is present.

mm

2.

Alternatively, place a disc wetted with the heated milk and one wet-

ted with penicillinase (or one commercially prepared with penicillinase) near

each other (edges 2-3 mm apart) on a plate. Place a 0.05-unit penicillin conon the plate. Incubate as in 9.22(A), (B) or (C). If penicillin is in the
milk, a characteristic neutralization zone appears at the juncture. If penicillin is not the inhibitor, a neutralization zone will not appear. This test
trol disc

demonstrates only the presence of penicillin or another inhibitor.


demonstrate both inhibitors simultaneously.

It

does not

9.24 Reporting

Report the presence of inhibitor only

in

heated milk samples.

9.23(A) were done, report the results demonstrated.


not done, report as positive for inhibitor.

To

If tests in

If tests in

9.23(A) were

estimate the concentration of

use reference discs [9.21(D)] as a guide. Report absence of inhibas Not Found or Negative.

penicillin,
itor

9.3 Modified Sarcina lutea Cylinder Plate


Penicillin in Non-fat Dry Milk 3. ^^
9.31

Method

for Detection of

Equipment and Supplies


Medium No. (Penassay Seed Agar) and Medium No. 4 (Yeast

A. Agar:

Beef Agar).

mm,

B. Petri dishes: Glass, 20 x 100

equipped with porcelain covers,


filter pads capable of absorbing water of syneresis. Comparable plastic dishes and lids may be used if the
lids are raised slightly to allow the escape of water.
C. Cell suspension: Use Sarcina lutea (ATCC 9341) which is maintained
as a stock culture on agar slants of Medium No. 1 and transferred to a fresh
slant every two weeks. Prepare the suspension as follows: Streak an agar

glazed on the outside, or glass cover

lids

with

slant heavily with the test organism and incubate for 18-24 hr at 32 C. Wash
growth from the slant with 1-2 ml of sterile physiological saline solution and
transfer to the dry surface of a Roux bottle containing 300 ml of Medium
No. 1. Spread the suspension evenly over the entire surface of the medium
with the aid of sterile glass beads. Incubate 18-24 hr at 32 C. Wash growth
from the agar surface with 50 ml of saline solution. NOTE: Before the actual

assay, determine by
to be

added

trial

plates the

stock suspension at 0-4.4

D.

1% phosphate

and dilute to

buffer,

tassium phosphate and


ter

optimum amount of

to seed agar to obtain the best

liter

for

<

the bulk suspension


zones of inhibition. Store this

weeks.

pH 6.0

0.1: Dissolve 8.0 g of

monobasic powa-

2.0 g of dibasic potassium phosphate in distilled

with distilled water.

ANTIBIOTIC RESIDUES

146

E. Cylinders: stainless steel with an outside diameter of 8

mm

diameter of 6

mm

0.1
and
an inside
F. Dispenser: For placing cylinders on the plate.
G. Water hath: Thermostatically controlled.

H. Incubator: 32

C; 30

0.1

C; and 37

0.1

mm

and

long.

C.

Roux hot tie.

/.

Glass heads: Sterile, for spreading suspension over the agar surface

J.

the

10

Roux

USP Sodium

K.

in

bottle.

Penicillin

reference standard: Follow the label direc-

by dissolving an

tions for storage of the standard. Prepare a stock solution

accurately weighed portion of the penicillin standard in buffer [9.31(D)] to


obtain a solution containing 1000 units/ml. Store at 0-4.4 C. This stock solution

may be used

for 2 days.

L. Non-fat dry milk: Antibiotic-free.

M.

Penicillinase, cone: Available from biological supply houses. Store at

0-4.4 C.

9.32 Procedure

A. Assay plate:
Prepare a suitable amount of Antibiotic
(Chapter

4),

and cool

petri dish. Distribute the

Add
Medium No.

surface.

Medium No.
and 4, sterilize
Add 10.0 ml of Medium No. to each sterile
medium evenly and allow to harden on a flat, level
1

to 48 C.

the appropriate

amount of culture [9.31(C)] to each 100 ml of


Mix well, taking care to avoid incorporating

4 cooled to 48 C.

any bubbles into the agar. Add 4.0 ml of this inoculated agar to each of the
plates which previously received 10.0 ml of the base layer. Distribute the
agar evenly by tilting the plate from side to side in a circular motion. Allow
agar to harden and use the plates on the same day they are prepared.
B. Standard response line:
1. Prepare this line each day samples are confirmed for penicillin. Prepare a control diluent from antibiotic-free non-fat dry milk [9.3 1(L)] and
phosphate buffer [9.31(D)J by combining the two materials at the rate of 9 ml
of buffer for every 1.0 g of non-fat dry milk. Use this diluent to dilute the
penicillin stock [9.3 1(K)] to obtain concentrations of 0.00625, 0.0125, 0.025,

0.05, 0.1,

and 0.2 unit/ml. The reference concentration

is

0.05 unit/ml.

plates [9.32(A)], place six cylinders [9.31(E)] equally

2. In 15 assay
spaced (60 intervals on a 2.8 cm radius) on the inoculated agar surface. Fill
three alternate cylinders on each of the 15 assay plates with the 0.05-unit/ml
concentration. Fill three alternate cylinders on three assay plates with each
of the other concentrations. The three plates containing the lowest concen-

tration of the standard are intended to

produce negative

results.

The other

12

plates are used to construct the standard response line. This will give fortyfive 0.05 unit/ml

er points

on the

determinations and nine determinations for each of the othline.

9.32 Procedure

3.

147

Incubate the 15 plates

at

30

for 16-18 hours. After incubation,

remove cylinders and measure the diameter of each zone of

invert plates to

inhibition as accurately as possible.


4.

Average the readings of the 0.05 unit/ml concentration and the nine

readings for each of the other reference concentrations tested on each set of
three plates. Also average the 45 readings of the 0.05 unit/ml concentration.

This average

is

the correction point for the other concentration points on the

curve. Correct the average value obtained for each point (0.00625, 0.0125,
0.025, 0.1 and 0.2 unit/ml concentration) to the figure

it

would be

if

the aver-

age for the nine 0.05 unit/ml readings for the specific concentration was the
same as the correction point. Thus, if in correcting the 0.025 unit/ml concentration, the

mm, and
plates

is

average of the 45 readings of the 0.05 unit/ml concentration

is

20

the average of the 0.05 unit/ml concentration of this set of three


19.8

mm,

the correction

is

mm.

mm.

+0.02

same

0.025 unit/ml concentration of these

If the

plates

average reading of the


is

17.0

mm,

the cor-

and the average of the


0.05 unit/ml concentration on two-cycle, semi-log graph paper, placing the
concentration in units/ml on the logarithmic scale and the diameter of the
zone of inhibition on the arithmetic scale. Construct the best straight line
through these points, either by inspection or by means of the following equarected value

is

17.2

Plot the five corrected values

tions:

3a

L
Where L and

+ 2b +

-e

..
H _

3e

+ 2d~ +

-a

calculated zone diameters for the lowest and highest

(0.0125 and 0.2 unit/ml) concentrations for the standard response line;
c

= average zone diameter

of the 45 zone diameters for the reference

concentration of 0.05 unit/ml; a,b,d,e

corrected average zone diam-

eters for each of the other concentrations used for the standard re-

sponse

line.

Controls:

In using this assay, the lowest standard concentration (0.00625 unit/ml)

intended to be a control which

may produce

is

negative results. This lowest

concentration represents control diluent to which penicillin has been added


at a level that is

normally below the

limit of detectability.

lowest concentration (0.00625 unit/ml)

will

On

occasion, the

produce a measurable zone

of inhibition. The next highest concentration (0.0125 unit/ml) should


always produce positive results. The sensitivity of this assay is normally
0.01 unit/ml. The non-fat dry milk plus phosphate buflFer [9.32(B)] should
always produce negative results.
D. Sample examination:
1. Screening procedure: Accurately weigh 10.0 g of non-fat dry milk

and add 90 ml of buffer [9.31(D)]. Mix thoroughly. If the concentration of penicillin is expected to be greater than 0.2 unit/ml in this mixture (2.0 units/g of

ANTIBIOTIC RESIDUES

148

original non-fat dry milk), dilute

an aliquot of the reconstituted milk with

control diluent [9.32(B)] to an estimated 0.05 unit/ml.


Place six cylinders [9.31(E)] on each assay plate [9.32(A)] at 60 intervals

on a 2.8
per

test.

cm radius. Use three plates per test. Two samples can be examined
On each of the three plates, fill two cylinders [9.31(E)] with the

0.05 unit/ml penicillin reference standard and

two cylinders with each of the

two untreated samples. Incubate plates for 16-18 hr at 30 1 C. After incubation, invert plates to remove cylinders and measure diameter of clear
zones of inhibition produced. Average six control readings for the reference
concentration and six readings for each sample. Any test sample producing
an average zone of inhibition of 9. 1 mm or greater is a presumptive positive
test and must be confirmed as in [9.32(D.2)].
2. Confirmatory procedure: To identify the activity as penicillin, add
penicillinase cone [9.3 1(M)] at the rate of 0.5 ml per 10 ml of sample. Mix,
and incubate for 30 min at 37 C. Use three assay plates [9.32(A)] per test
sample. On each of the three plates, fill two cylinders [9.31(E)] with the
0.05 unit/ml penicillin reference standard, two cylinders with the untreated
sample, and two cylinders with the penicillinase-treated sample. Incubate the
plates for 16-18 hr at 30 1 C. After incubation, invert plates to remove
the cylinders.

Measure the diameter of each zone of inhibition as accurately as possible.


Average the zone readings of the standard, untreated sample and pensample on the three plates.
zone of inhibition with the untreated sample and no
zone with the penicillinase-treated portion of the sample is a positive test for
penicillin. If the average zone of inhibition of the penicillinase-treated portion of the sample is the same size as the average zone produced by the
untreated portion of the same sample, penicillin is not present. If the average
icillinase-treated portion of the
3.

Interpretation:

zone of inhibition produced by the penicillinase-treated portion of the


sample is 0.1 mm less than the average zone of inhibition produced by the
untreated portion of the same sample, penicillin plus another nonspecific
is present.
Calculation of penicillin concentration:

inhibitor
4.

To

calculate the antibiotic

content of a sample, average the zone readings of the six 0.05 unit/ml control
zone readings and the six zone readings of the sample on the three plates. If
the sample gives a larger average zone size than the average of the control,
add the difference between them to the zone size of the 0.05 unit/ml reference standard of the response line [9.32(B)] to obtain the concentration of
the sample from the response line [9.32(B)]. If the average sample value is
lower than the average of the 0.05 unit/ml control, subtract the difference
between them from the 0.05 unit/ml reference standard of the response line
[9.32(B)] to obtain the concentration of the sample from the response line
[9.32(B)]. Multiply the concentration in units/ml obtained from the response
line [9.32(B)]

by the dilution factor of 10 x to obtain the

final

concentration

in

149

References

9.5

terms of units/gram.

If additional dilution

of the sample has been made, the

appropriate dilution factor must be taken into account in calculation of

final

potency.
9.33 Reporting
penicillin. Report the absence of
Samples found positive for nonspecific
must be assayed by other methods before reporting.

Report only samples found positive for


penicillin as negative for penicillin.

inhibitors

9.4 Specific Antimicrobial Cylinder

Methods

Methods to assay milk and milk products (cheese, buttermilk, ice cream,
dry milk and evaporated milk) for specific antibiotics (bacitracin, chlortetracycline, oxytetracycline, tetracycline, streptomycin, dihydrostreptomycin,

neomycin,

been described

penicillin,

this publication

if

polymyxin, erythromycin, and novobiocin) have

identification of the antibiotic present

is

necessary,

should be consulted.

References

9.5
1.

and

'^

Abraham,

E.P.,

Chain,

E.,

Fletcher, CM., Florey, H.W., Gardner, A.D., Healt-

LEY, N.G., and M. A. Jennings. 1941. Further observations on penicillin. Lancet 241:177.
2.

3.

Arret, B. and A. Kirshbaum. 1959. A rapid disc assay method for detecting penicillin in
milk. J Milk Food Techno! 22:329-331.
Carter, G.G. 1974. Detection of penicillin in dry powdered milk by the Sarcina lutea
cylinder plate method. National Center for Antibiotic Analysis, FDA, USDHEW, Washington, D.C.

4.

and R.L. Morris. 1955. A modified disc assay method for detecting antibotics
Milk Food Technol. 18:281-283.
Grove, D.C. and W.A. Randall. 1955. Assay Methods of Antibiotics. Medical Encyclo-

Cerny,

J.

milk.

J.

in
5.

N.Y.

pedia, Inc.
6.

Baughman, R.W., Nelson, F.E., and P. A. Hartman. 1961. Rapid antimethods usingBacillus stearothermophilus J. Milk Food Technol. 24:143-146.
Johns, C.K. 1960. Further observations on testing milk for penicillin. J. Milk Food TechIgarashi, R.T.,

biotic assay

7.

nol. 23:266-268.
8.

9.

Johns, C.K., and I. Berzins. 1956. Observations on the determination of antibiotics in


milk. J. Milk Food Technol. 19:14-17.
KosiKOWSKi, F.V., Henningson, R.W., and G.F. Silverman. 1952. The incidence of
antibiotics, sulfa drugs and quaternary ammonium compounds in the fluid milk supply of

New York
10.

11.

State.

J.

Dairy Sci. 35:533-539.

KosiKOWSKi, F.V., and R.A. Ledford. 1960. A reverse-phase disc-assay test for antibiotics in milk. J. Am. Vet. Med. Assoc. 136:297-301.
Kramer, J., Carter, G.G., Arret, B., Wilner, J., Wright, W.W., and A. Kirshbaum.
1%8. Antibiotic residues
Antibiotic

12.

&

in milk,

dairy products, and animal tissues. National Center for

FDA, USDHEW, Washington, D.C.


Thornberry, H.H., Ehrlich, J., McGuire,

Insulin Analysis.

Loo, Y.H., Skell, P.S.,


G.M., and J.C. Sylvester.

1945.

Assay of streptomycin by the paper

J.M., Savage,

plate

method.

J.

Bacteriol. 50:701-709.
13.

Marth, E.H., Alexander,

F.J.,

ods for detection of antibiotics

and R.V. Hussong. 1963. Studies on disc assay meth-

in milk. J.

Milk Food Technol. 26:150-154.

ANTIBIOTIC RESIDUES

150

14.

Methods of Analysis.

1975. Twelfth Edition. Section 16.124. Association of


Chemists, P.O. Box 540, Benjamin Franklin Station. Washington, D.C.
Packard, V.S., Tatini, S., and R.E. Ginn. 1975. An evaluation of methods for detecting
and comparative incidence of penicillin residues in different types of raw milk. J. Milk Food

Official

Official Analytical

15.

Technol. 38:601-603.
16.

17.

Parks, O.W., and F.J. Doan. 1959. Sensitives of the disc assay and triphenyltetrazolium methods for antibiotics in milk. J. Milk Food Technol. 22:74-76.
Pater. B. 1976. A collaborative study of the Delvotest-P method to detect low penicillin
J. Food Prot. 40:23-24.
SuNO, F.A., Czarnecki, R.B. and W.K. Harris.

concentrations in milk.
18.

19.

20.

21.

Vincent,

J.G., and

H.W. Vincent.

penicillin determination. Proc. Soc.


22.

1958.

The incidence of

penicillin in the

market milk supply of a local New England area. J. Milk Food Technol. 21:211-214.
Silverman, G.F., and F.V. Kosikowski. 1952. Systematic testing of inhibitory substances in milk. J. Milk Food Technol. 15:120-124, 137.
Van Os, J.L., Lameris, S.A., Doodewaard, T., and J.G. Oostendorp. 1975. Diffusion
test for determination of antibiotic residues in milk. Neth. Milk Dairy J. 29:16-34.
1944. Filter paper disc modification of the

Oxford cup

Exper. Biol. Med. 55:162-164.

Wright, W.W. 1962. Overnight microbial


Anal. Chem. 45:301-306.

plate assay for penicillin in milk.

J.

Assoc.

OflF.

CHAPTER

10

MICROBIOLOGICAL METHODS FOR


CONCENTRATED AND DRY MILK PRODUCTS
G.W. Reinbold, W.S.

Clark,

Jr., J.L.

Dizikes,

and C.K.Johns

10.1

Evaporated Milk

Evaporated milk is obtained by removing a portion of the water from milk.


Following homogenization, evaporated milk is further processed by heat (either before or after sealing in a can) to prevent spoilage.

The

resulting prod-

commercially sterile or micro! 'iologically stable. When conducting a


microbiological examination of canned evaporated milk, if the bacterial content is a pure culture, highly heat-resistant spores usually are found. Mixed
cultures indicate gross under-processing, contamination after heat processuct

is

ing, or

contamination during culturing.

When

cans are swollen, inoculation of product into recovery broths and


is useful in establishing microbiological contamination. When testing of flat cans is indicated, preincubation [10.1(E)] is
direct plating [10.1(B)]

called for before examination.

A. Collection and preparation of samples:


Collect samples, using apparatus and procedures as given in 3.21. The
same sampling procedures apply to both field and laboratory sampling of
sealed containers. Measure representative initial test portions from samples

and proceed with dilutions 10.1(B). Bacteriological results obtained on previously opened containers should be interpreted with caution.
B. Diluting, plating and incubating plates:
For direct plating, measure representative 0.5-ml portions of sample (or
5 ml of 1
10 dilution) into each of two petri dishes. Where necessary to
make a 1 10 dilution, transfer 11 ml of sample into a dilution bottle contain:

ing 99 ml of microbiologically suitable

pare higher dilutions from the

ISC Liaison: W.S. Clark,

(MS) water

[4.6].

10 dilution. If the

Jr.

151

Where needed,

sample

is

pre-

coagulated or

MICROBIOLOGICAL METHODS: CONCENTRATED AND DRY MILK PRODUCTS

152

Otherwise abnormal, optionally use

MS

water to which has been added

1.25% sodium citrate as the first dilution blank to dissolve the milk.
Prepare four sets of plates in duplicate for each dilution, using Standard

Methods Agar

[4.9(F)]. Incubate

two

sets of plates at 32

for 48'^

one aerobically and one anaerobically.

48

3 hr.

Incubate sets similarly at 55


Use from 15 to 18 ml of agar in plates incubated at 55 C.

3 hr

for

C. Counting plates:

Count plates

When the number of colonies per plate is fewer than


number found. Also record the number of colonies on

[5.11].

30, record the actual

control plates. Verify the identity of doubtful objects resembling colonies.

D. Reporting:
Report counts as Standard Plate Count (SPC) per milliliter if plates were
incubated at 32 C and as Thermophilic Bacterial Count (TBC) if incubated at
55 C. Counts of fewer than 30 colonies per milliliter are reported as estimated counts and should be interpreted with caution. Counts from plates
incubated anaerobically should be appropriately designated.

unopened containers:
Commercial sterility of product can be determined by incubation of unopened cans at 32 C and 55 C for one week followed by culturing. If the
contents have an abnormal appearance after incubation, large numbers of
E. Incubation of

organisms usually are present. Presence of nonsporeforming organisms usually indicates post-sterilization contamination, frequently caused by imperfect cans. Cans containing milk which appears normal after incubation and
which yields no colonies on plates may be assumed to be commercially sterile.

10.2 Concentrated
Concentrated milk

and Sweetened Condensed Milks

is

obtained by removing a portion of the water from

is not processed by heat to prevent spoilage.


Sweetened condensed milk is obtained by removing a portion of the water
from a mixture of milk and safe and suitable nutritive sweeteners. The quantity of nutritive sweetener used is sufficient to prevent spoilage. Both of
these products may be homogenized.
Although sweetened condensed milk (whole or skim), with or without
added sugar, commonly is sold at retail in hermetically sealed cans, large
quantities of sweetened and unsweetened products are stored temporarily in

milk.

It

is

pasteurized, but

1), barrels, tank trucks or tank cars,


be incorporated into other foods relatively soon after manufacture.
Proper heat processing destroys all coliform bacteria in condensed products. Since bulk concentrated milk usually is exposed to contamination more
often than canned milk, and since canned milk, once opened, is subject to

bulk containers such as cans (10 gal) (38


to

10.3 Dry Dairy Products

153

recontamination, the coliform test

may be used

to indicate possible recon-

tamination after processing or after exposure.

Yeasts and molds are the most common cause of spoilage of sweetened
condensed milk. Presence of yeast or mold indicates lack of sanitary care.
A. Collection and preparation of samples:
Collect samples, using apparatus and procedures given in 3.21 except as
follows: When necessary to insure uniformity of contents immediately before removal of test portions

usually contains about

45%

from sample jars (sweetened condensed milk


sugar), heat in water bath to a temperature

which does not exceed 45 C, mix contents thoroughly and transfer promptly. Prepare the initial dilution by weighing
g of sample directly into a
bottle containing 99 ml of MS water, or weigh into a wide-mouth bottle and
add MS water.
1 1

B. Diluting, plating, incubating,

When
10.1(B).

and counting

plates:

analyzing coagulated or otherwise abnormal samples, proceed as

For

plating, incubating

and counting plates, proceed as

in

in 10.1(B)

and 10.1(C).
C. Reporting results:

Report results as Standard Plate Count per gram of concentrated/sweetened condensed milk. When the plates have fewer than 30 colonies, report
the actual number of colonies as the Estimated Standard Plate Count per
gram. Also record the number of colonies on control plates [5.9].

D. Test for conforms:


Using
10 dilutions of bulk condensed or condensed milk from previously opened, hermetically sealed containers, proceed and report results
as directed in Chapter 6.
1

E.

Yeast and

mold count:

Using appropriate dilutions, proceed as in 11.5. Report results as Yeast


and Mold Count (YMC) per gram of concentrated or sweetened condensed
milk.

10.3 Dry Dairy Products

Dry dairy products

in tote

bins or in 50- or 100-lb (22.5-45 kg) bags fre-

quently are incorporated into ice cream mix to provide the required milk
solids. They also are used in cottage cheese, cheese food preparations, and
for reinforcing low-fat milk

constituted in the

home

and yogurt. These products also may be

re-

for beverage or culinary use.

The coliform group, when present in dry dairy products, has the same
when present in pasteurized milk [6.1]'' Food poisoning out-

significance as

154

MICROBIOLOGICAL METHODS: CONCENTRATED AND DRY MILK PRODUCTS

breaks caused by coagulase-positive staphylococci have been associated


with dry milk and special foods prepared therefrom.-- Although not all co'

agulase-positive cocci in foods are toxigenic, their presence in large

numbers

milk suggests a need for additional tests to determine their toxicity


[2.2(N)]. Within recent years, occurrence of Salmonella in dry milk and re-

in

lated products has

dairy products

is

been the object of intensive study."' Mold content of dry


influenced by exposure and handling during and after

drying.

Standards for dry milks include Standard Plate Count, coliform and direct
These methods generally are applicable to all types
microscopic counts.^of dry milk products, including those sold for infant feeding and for special
''^

diets.

processed by special methods which result in a


product with improved reliquefication characteristics. Nonfat dry milk may
be classified by heat treatment based on use of the whey protein nitrogen
test.' This is of practical importance in indicating the suitability of sprayprocess nonfat dry milk for specific purposes.
Instant-type dry milk

is

A. Collection and preparation of samples:


Collect samples, using apparatus and procedures as noted

in 3.21.

Equipment and supplies:


Weighing scoop, sterile: Aluminum scoop and counterweight. Use
sterile scoop [3.12]; or immediately before each use, dip scoop in alcohol
B.

1.

and flame.
2.

Spatula or stainless steel spoon: Sterile.

3.

Aluminum

into pieces
4.

and

foil

or paper: Standard

foil

or hard-surfaced paper, cut

sterilized.

Dilution blanks: See 4.7(A,B) and 5.3(D) or use 1.25% sodium citrate

blanks for relatively insoluble dry milks that otherwise will not go into solution readily.

The pH of

the resulting suspension should be less than 8.0.

C. Dilutions:

Spray dried milk, when freshly prepared, is much more soluble than is
Spray dried milk is hygroscopic and becomes less soluble
if its moisture content increases during storage. Samples should be protected
from moisture absorption. Most dry milks can be dissolved in MS water
[4.6]. Dissolve the more insoluble samples in 1.25% sodium citrate dilution
blanks [10.3(B.4)]. Do not use alkalinized blanks unless tests show that their
roller-dried milk.

use

is

necessary.'^

Preferably examine samples soon after manufacture.

Whenever

possible,

the date of manufacture should be recorded. Before opening a container for

make the contents homogeneous by shaking or by


and inverting the sample containers; or mix the contents carefully by

transferring a test portion,


rolling

10.3 Dry Dairy Products

155

using the sterile spatula or spoon. For


tion bottle of

MS

initial dilutions,

adjust blanks to 45

g of dry milk directly into a 99-ml wide mouth diluwater; or using the sterile spatula or spoon, weigh 11 g of

and promptly weigh

1 1

dry milk onto sterile aluminum

foil

or hard-surfaced paper and immediately

transfer contents aseptically to the initial dilution blank. Dilutions are easily

made

in

wide-mouth 8-oz (240 ml) leakproof containers. In preparing

initial

observe special precautions to dissolve dry milk completely to


obtain a homogeneous mixture.'' Agitate the dilution blank mildly to wet the
sample completely. Allow to soak 2 min; shake dilution bottle, making
25 complete up-and-down movements of about one ft in 7 seconds. Prepare
dilutions,

appropriate dilutions.

D. Plating, incubating and counting plates:


Incubate plates at 32 C for 48 3 hours. Count colonies in accordance
with 5.1 1. Since samples may contain appreciable numbers of aerobic spore
formers, plates

may

colonies accurately.
the

number and

count. ^

If

contain spreading colonies that

An

make

overlay of 3-5 ml of sterile agar

is

it

difficult to

count

reported to reduce

size of spreaders without significantly reducing the plate

may be confused with colonies,


under low power of the microscope.

undissolved particles of dry milk

verify identity of doubtful colonies

E. Reporting results:

Report results as Standard Plate Count per gram of dry dairy product.
Since determinations by the agar plate method do not reveal all the sanitary conditions of production, processing
ples using the direct microscopic

analysis

^-'^

[10.3(G,H,I)]

may be

method

and storage, examination of sam^ and other microbiological

[14.4]

of value.

F. Direct microscopic count (see Chapter 14):

Because of the progressively lethal effect of processing and storage on


microorganisms in dry milk, determination of the number of viable bacteria
may not reliably indicate its previous sanitary quality or the sanitary handling of raw milk before drying. Direct microscopic examination of stained
preparations of dry milks will give valuable additional information [14.4].^
1. Preparing and staining films: With readily soluble samples, follow
10.3(G) (11 g of milk in a 99-ml water blank). Use a binocular microscope,
500,000-600,000 '- factor preferred [14.12], to count sufficient fields [14.14,
14. 18] and insure proper illumination [14. 1 1]. To avoid undissolved particles
of casein in films prepared from less readily soluble samples, use 1.25% so-

dium

citrate

blanks for the

solution stain [14.17].

Do

10 dilution.

Apply the Levowitz-Weber

not refrigerate stain, and discard

it

when

single-

precipi-

tate or foreign matter appears.

Make
tion of
if

only direct microscopic clump counts [14.6], following the definiin 14.18. Some cells stain poorly but must be counted

"clump" given

they are identifiable as microorganisms.

It

must be recognized

that in

MICROBIOLOGICAL METHODS: CONCENTRATED AND DRY MILK PRODUCTS

156

single-sample comparisons, count differences between laboratories


rather large.
2.

may be

'^

Reporting results: Using an appropriate microscopic factor [14.12],

multiply the count by 10 (to compensate for the

10 dilution), correct

if

necessary for the film area used, and observe precautions [14.16]. Report
results as Direct Microscopic Count per gram of dry milk.

G. Test for coliforms:

Weigh
ceed as

11

in

g of milk into a 99-ml wide-mouth dilution blank at 45 C, pro6, and report results (per gram) as directed.

Chapter

H. Yeast and mold count:


Using appropriate dilution [10.3(C)], proceed as in 11.4. Report the computed result as Yeast and Mold Count per gram of dry milk.
Thermoduric, thermophilic and psychrotrophic bacteria:
Using appropriate dilutions, proceed as in Chapter 7. Results should be
reported as the count of the specific type of organism per gram of dry milk.
/.

10.4 References
1.

2.

3.

American Dry Milk Institute,

Inc. 1971. Standards for grades of dry milks, including

methods of analysis. ADMI Bull. 916. 130 North Franklin Street. Chicago, 111.
Anderson, P.H.R. and D.M. Stone. 1955 Staphylococcal food poisoning associated
with spray-dried milk. J. Hyg. 53:387-397.
Armijo, R., Henderson, D.A., Timothee, R., and H.B. Robinson. 1957. Food poisoning outbreaks associated with spray-dried milk
An epidemiological study. Amer. J Pub

Health 47:1093-1100.
4.

Clark, W.S.,

Jr.,

Reinbold, G.W., Dizikes,

J.L.,

Johns, C.K., and D. Hotchkiss.

Evaluation of plate count for dry milk. (In preparation)


5.

6.

Cone, J.F., and U.S. Ashworth. 1949. Comparison of methods of reconstituting milk
powder for the plate count, with an analysis of variance. Food Res. 14:165-176.
George, Jr., E. Olson, Jr., J.C. Jezeski, J.J., and S.T. Coulter. 1959. The growth of
staphylococci in condensed skim milk.

7.

8.

Heinemann,

milk.
9.

J.

Dairy Sci. 42:816-823.

Growth and thermal destruction of Micrococcus pyogenes var.


aureus in heated and raw milk. J. Dairy Sci. 40:1585-1589.
Heinemann, B. 1960. Factors affecting the direct microscopic clump count of nonfat dry
J.

B.

1957.

Dairy Sci. 43:317-328.

Macy, H.

1928.

Some

observations on the bacterial content of dried milk.

J.

Dairy Sci.

11:516-526.
10.

Marth, E.H.
J.

11.

Miller,

J.J.

richia coli.
12.

1969. Salmonellae and salmonellosis associated with milk

and milk products.

Dairy Sci. 52:283-315.

J.

and P.S. Prickett. 1938. Note on

violet red bile agar for detection of

Esche-

Dairy Sci. 21:559-560.

Pedraja, R., Choi, R. Mengelis, A. and E. Small. 1963. Joint collaborative study of the
clump count method in dry milks and its statistical consideration. J.

direct microscopic

Dairy Sci. 46:810-818.


13.

U.S. Public Health Service. 1969. Grade A dry milk products. Supplement 1 to the Milk
Ordinance and Code 1965. Recommendations of the Public Health Service. U.S. Public
Health Service, Washington, DC.

14.

Willis, A.T., 1969. Techniques for the study of anaerobic spore-forming bacteria. In,
Methods in Microbiology, pp. 80-115. (J.R. Norris and D.W. Robbins, Eds), Vol 3B. Aca-

demic Press, N.Y.

CHAPTER

11

MICROBIOLOGICAL METHODS FOR BUTTER,


MARGARINE AND RELATED PRODUCTS
Claude Harper,

Jr.,

Jim

L.

Dizikes,

and

Vernal S. Packard

11.1

Introduction

Methods to measure the microbiological quality of butter or similar foods


must take into account their peculiar properties, such as composition and
physical structure. The water content is relatively low (16-17%) and, in wellworked butter or margarine, is distributed in numerous microscopic droplets
If salt is added, all of it is dissolved in water, thus
producing a relatively high chloride concentration in the aqueous phase.
This, together with the thorough moisture distribution, provides significant
protection against microbial spoilage of butter or margarine.
On the other hand, if butter has been contaminated in the manufacturing

throughout the product.

process and

if

conditions, such as poor dispersion of water and high temper-

ature, favor microbial growth, spoilage

may

occur. Psychrotrophic bacteria

molds ^ and coliforms ^ do not survive pasteurization and are rarely found in butter from
well-conducted operations. When present, they indicate faulty sanitation.
Estimates of numbers of these organisms in samples of cream or butter taken
are prominent in this type of deterioration. Yeasts and

at various stages

tamination.

of processing are useful in detecting the sources of conPlate Count can be used as an important measure-

The Standard

ment of processing sanitation since viable cultures of lactic acid bacteria are
seldom used during processing of commercial butter or margarine.
Microbiological methods described here for butter are also applicable to
margarine and other similar-type spreads (butter-margarine combinations,
low-fat vegetable fat spreads, 60% vegetable fat spreads).

11.2 Microbiological

Among

Methods

methods applicable to butter are: a.) cultural methods for determining Standard Plate Count, coliforms, yeasts and molds, psythe

ISC Liaison: D.W. Mather


157

MICROBIOLOGICAL METHODS: BUTTER, MARGARINE AND RELATED PRODUCTS

158

chrotrophic and proteolytic bacteria and b.) the microscopic method for

esti-

mating mold filaments as an indication of the quality of the cream used.'


In a comparative study ^ it was demonstrated that the enterococcus count
(Appendix A) is a useful index of sanitary quality of butter and that enterococci are more salt-tolerant and survive low-temperature storage better than

do coliform bacteria.
Plating methods to detect proteolytic and psychrotrophic bacteria are useful in

evaluating the keeping quality of butter.

The method for yeast and mold count prescribes use of Acidified Potato
Glucose Agar as the growth medium. Recently Chlortetracycline-Rose Bengal Agar (Appendix A) has been proposed because it oflFers certain advantages such as easy detection of yeast and mold colonies.^This medium
"^

has not yet been tested

11.3

a collaborative study.

in

Sampling Procedure

Follow instructions for sampling butter

11.4 Bacterial
A. Procedure:
Before plating,

[3.23].

Counts

warm

dilution blanks

and pipets to 40 C. Rotate contain-

more than 50% of capacity with

ers, filled to not

a water bath at not over 40

butter or similar product, in

(usually less than 15 min) until product

is fluid

enough to pipet. Avoid separation of fat and serum fractions. Wet the pipet
by drawing into it 11 ml of sterile, warm dilution water and discharging the
contents (the warm dilution blanks may be used for this purpose). With a

minimum

of product adhering to the exterior of the pipet, immediately trans-

fer 11 g of

thoroughly mixed, melted sample into 99 g of

warm

(40 C) dilution

water. Shake dilution bottle [5.6(B)].

Divide 5 ml of
1

2 dilution).

For

10 dilution equally into

10,

two

petri dishes (equivalent to

100 and higher dilutions, follow the procedure

for milk [5.7(B)]. Plating of undiluted butter or similar product


tory,

because

it

is

unsatisfac-

prevents breakup of clumps of cells and uniform dispersal of

fat in the plates.

B. Standard Plate Count (for lactic culture-free butter or margarine):

Prepare 1
100 and other suitable dilutions. Proceed as in 5.7(B.2).
Report as Standard Plate Count (SPC)/gram of butter, margarine, or
:

re-

lated product.

C. Conforms:

Prepare 1 2 and other dilutions. Proceed as in 6.8.


Report as Coliform Count (CC)/gram of butter, margarine, or related prod:

uct.

159

11.6 References

D. Proteolytic count:
2 or other suitable dilutions. Pour plates with Standard MethPrepare 1
ods Agar to which 10% of sterile skim milk is added just before pouring.
Incubate plates at 21 2 C for 72 hours. Flood plates with \% HCI or
10% acetic acid solution for 1 minute. Pour off excess acid solution. Count
colonies surrounded by clear zones produced by proteolysis.
Report as Proteolytic Count (PC)/gram of butter, margarine, or related
:

product.
E. Psychrotrophic count:
1
2 or other suitable dilutions. Proceed as in 7.32.
Report as Psychrotrophic Bacterial Count (PBC)/gram of butter, marga-

Prepare

rine, or related product.

11.5 Yeast

and Mold Counts

Prepare Potato Glucose Agar, preferably from dehydrated base, according


to manufacturer's directions. Before pouring plates, provide for suppression
of bacterial growth by adjusting the medium to pH 3.5 0.1 (see formula
instructions) with sterile (filtered or autoclaved)

only to the

amount of agar needed

for

10%

tartaric acid.

Add

immediate use, because remelting

acid
will

destroy the solidifying properties of the acidified agar.

Pour plates

[5.8]

with 15-18 ml of Acidified Potato Glucose Agar and allow

to solidify. Invert plates

and incubate

at 21

2 C.

merous, count plates on the

third

Normally, count plates

mold colonies are nuday and count again on the fifth day, if

after 5 days of incubation, using a colony counter.

If

possible.

Report as Yeast and Mold/gram of butter, margarine, or related product.

11.6 References
1.

Association of Official Analytical Chemists.

1975. Official

Methods of Analysis.

12th ed. Association of Official Analytical Chemists. Washington, D.C.


2.

BucHBiNDER,

and E.C. Alff. 1947. Studies of coliform organisms

in dairy

products.

3.

Mycopathol. Mycol. Appl. 35:281-289.


Cooke, W.B., and A.R. Brazis. 1967. Occurrence of molds and yeasts

in dairy

products.

4.

5.

6.

L.,

Mycopathol. Mycol. Appl. 35:281-289.


Macy, H., Coulter, S.T., and W.B. Combs. 1932. Observations on the quantitative
changes in the microflora during the manufacture and storage of butter. Minn. Agr. Exper.
Sta. Tech. Bull. 82. University of Minnesota, St. Paul.
Overcast, W.W., and D.J. Weakley. 1969. An aureomycin-rose bengal agar for enumeration of yeast and mold in cottage cheese. J. Milk Food Technol. 32:442-445.
Saraswat, D.S.. Reinbold, G.W., and W.S. Clark, Jr. 1965. The relationship between
enterococcus, coliform, and yeast and mold counts on butter. J. Milk Food Technol.
28:245-249.

7.

Shadwick, G.W.,

Jr.

1938.

study of comparative methods and media used in microI. Yeast and mold counts. Food Res. 3:287-298.

biological examination of creamery butter.

CHAPTER

12

MICROBIOLOGICAL METHODS FOR CHEESE AND

OTHER CULTURED PRODUCTS


Norman

12.1

F.

Olson, Robert

F.

Anderson, and
Robert Sellars

Introduction

Cheese making generally depends on the natural presence


addition to milk or curd, of specific types of microorganisms.

in milk,

or the

The sequence

of changes that usually occurs during the manufacturing process can be predicted within biological limitations; however, growth and activity of micro-

organisms are influenced by time, temperature, salt, pH, nutrient requirements and other factors during the ripening process. Different groups of
bacteria may develop sequentially. One group often enhances growth of others, but the metabolic by-products of most microbial species eventually may
inhibit their growth partially or completely. Some of these changes are essential for development of characteristic flavor in certain types of cheese.
The microbial flora varies among different types of cheese and even between
different cheeses of the

same

type.

Numbers

of bacteria in natural or proc-

essed cheese are not generally related to public health or safety. For example, the presence or absence of coliforms should not be used as an index
of sanitation because growth and death rates of coliforms are unpredictable
in cheese during ripening. Although comparatively few cheese-borne disease
outbreaks have been reported, some have occurred in recent years.^-*^'*~"
The pattern of microbial growth in other cultured products is less complex
than in ripened cheese. In cottage cheese, cultured milks, sour cream, and

similar products, the specific organisms producing lactic acid initially are

may provide

to some degree an index of culture


bacteria and yeasts and
such
coliform
as
development, while organisms
as indices of sanitary
and
may
be
used
contaminants
molds are present as
conditions. The microbiology of these latter products is discussed in stan-

present in large numbers and

dard

tests. ^'

ISC Liaison: G.W. Reinbold


161

162

MICROBIOLOGICAL METHODS: CHEESE AND OTHER CULTURED PRODUCTS

12.2 Collection

and Preparation

of

Samples

A. Procedure for sampling:


Collect samples, using apparatus and procedures as given in Chapter

The sample should be representative of the cheese

3.

to be tested. Five approx-

imately equal portions, each consisting of not less than 5 g taken from

dif-

ferent parts of a single piece of cheese, are adequate as a representative

sample when combined.


B. Apparatus:

Provide equipment and supplies as needed, in accordance with specificaChapter 3. The following additional apparatus is recommended:

tions in
1.

Spatula, sterile.

Blender, mechanical.
Container for blender, sterile: Stainless steel or other nontoxic metal
or glass, with leakproof blending blade assembly. The unit may be autoclaved or sanitized with a 200-ppm chlorine solution for 5 minutes. Rinse
chlorine solution from container with an ample single rinse of sterile
0.1% aqueous sodium thiosulfate solution. The blender should be selected
on its ability to produce rapid and complete emulsification of cheese samples.
2.

3.

4.

Balance: The capacity and pan of the balance should be sufficient to


the sample, container and container cover. The balance

accommodate

should have a precision of

0.1 g

and readability of

0.1

gram.

C. Procedure for cheese other than cottage cheese:

Using aseptic technique, thoroughly comminute or mix each sample

until

representative portions can be removed. Heat 99-ml dilution blanks of sterile, freshly prepared (less than 7 days old), aqueous 2% sodium citrate to
40 C. Aseptically transfer 11 g of cheese to a sterile blender container previously warmed at 40 C and add the warmed sodium citrate blank. Mix for
2 min at a speed sufficient to emulsify the sample properly, invert the con-

from the interior walls, and remix for approximately


C from friction in agitation may occur with some mechanical blenders. This should be
determined before use of the blender so corrective action can be taken, if
needed. If heating is unavoidable with equipment available, mixing periods
of less than 2 min may be used provided that complete emulsification is obtainer to rinse particles

10 seconds. Inadvertent heating to temperatures in excess of 40

tained.

The

10 dilution should be plated or further diluted immediately,

great are being taken to avoid air bubbles or foam.

As an

alternative method,

sterilized

transfer

it

*NASCO.

177-ml

(6 oz)

to a flat surface

Fort Atkinson,

10

mg

Wl

is

rapidly weighed into a pre-

equivalent.'"^ Close the bag,


and macerate the contents into a fine paste by

Whirl-Pak bag* or

53538

its

12.3 Microbiological

Analyses

X 125-mm

rolling a 15

163

test

tube or similar cylindrical object over the bag.

The sample should not be forced into the corners or the tied seal area of the
bag. Open the bag and add 9 ml of 1% sodium citrate at 40 C. Reclose the
bag and roll the contents, as described above, to form a fine emulsion and
proceed with plating immediately. Enumeration of bacterial species, such as
lactic streptococci, that form chains may not be feasible with this method.''

However,

problem was not evident in a collaborative study involving


Cheddar and Romano cheeses in nine laboratories in the United

this

analysis of
States. 12

D. Procedure for cottage cheese:


Place the sterile blender container on a proper balance and tare. With a
mix contents of the cottage cheese container; or if in a tightly

sterile spatula,

closed plastic sample pouch, gently knead and mix the enclosed curd.
Aseptically

remove

the cover of the blender and place

balance beside the container. Weigh


container.

Add

99 ml of

warmed

it

upside

down on

the

g of cottage cheese into the sterile


(40 C) sterile 2% sodium citrate solution as
11

described in 12.2(C) to disperse and dilute the cottage cheese curd. Proceed

and plate appropriate dilutions immediately. The alternative


bag method described in 12.2(C) may also be used.
as in 12.2(C)

E. Procedure for cultured milk, cultured cream, yogurt, acidophilus


milk, Bulgarian buttermilk,

and

similar cultured or acidified

semifluid products:

After thoroughly mixing the sample, weigh

wide-mouth container, add 99 ml of


shake

until a

amounts of

homogeneous dispersion

11

g of product into a sterile

sterile buffered distilled


is

water (40 C),

obtained, and withdraw appropriate

The sample may


mechanical blender as described in

this 1:10 dilution for plating or further dilution.

be dispersed

in

2% sodium

citrate with a

12.2(D).

12.3 Microbiological Analyses


A. Yeast and mold count:
Pipet appropriate amounts of the 1:10 dilution of product from the blender
container or a shaken 1:10 dilution of product [12.2(C,D,E)], into sterile pet-

and add Acidified Potato Glucose Agar. Where the count is expected
ml evenly among three plates and count the total
colonies appearing on the three plates, if higher dilutions prove to be too
sparsely populated. Report as Yeast and Mold Count per gram of product.
ri

plates

to be low, distribute 10

B. Test for coliforms:


Pipet appropriate

amounts of the

1:10 dilution of product

from the blender

container or a shaken dilution tl2.2(C,D,E)], into sterile petri plates and add

MICROBIOLOGICAL METHODS: CHEESE AND OTHER CULTURED PRODUCTS

164

Violet

Red

Bile Agar.

Samples of cottage cheese, sour cream, and cultured

milk must be plated within 24 hr after manufacture to obtain meaningful


results.^ Coliform-like colonies in Violet Red Bile Agar should be confirmed

and other products containing fruit or added sugar. Report the


Count per gram of product. Alternately, inoculate a series
Coliform
count as
Brilliant
Green Lactose Bile (2%) Broth with appropriate volof
of tubes
sample and proceed as in 6.9. Report results as
prepared
of
the
umes
for yogurt

'^

MPN

Coliform

per gram or other appropriate quantity.

Psychrotrophic bacterial count:


Using appropriate dilutions, proceed as

in 7.32.

12.4 References
1.

Allen. V.D.. and W.D. Stovall.

2.

cream.
3.

1960. Laboratory aspects of staphylococcal food poi-

Milk Food Technol. 23:271-274.


Barber, F.A., and H. Pram. 1955. The problem of false coliform counts on

soning from Colby cheese.

J.

J.

Foster, E.M., Nelson, F.E., Speck. M.L., Doetsch, R.N.. and J.C. Olson,
Dairy Microbiology. Prentice-Hall, Englewood

4.

5.

6.

7.

9.

Cliffs.

Jr. 1957.

N.J.

Zealand

J.

Dairy Sci. Technol. 7:7-11.

Menzies, D.B. 1944. An outbreak of typhoid fever in Alberta traceable to infected Cheddar
cheese. Canad. J. Pub. Health 35:431-438.
Minor. T.E., and E.H. Marth. 1972. Staphylococcus aureus and staphylococcal food
intoxications.

10.

ice

GoEL. M.C.. KuLSHRESTHA, D.C., Marth, E.H., Francis, D.W., Bradshaw, J.G., and
R.B. Read, Jr. 1971. Pate of coliforms in yogurt, buttermilk, sour cream and cottage
cheese during refrigerated storage. J. Milk Food Technol. 34:54-58.
Hammer, B.W., and F.J. Babel. 1957. Dairy Bacteriology, 4th ed. John Wiley and Sons.
N.Y.
Hendricks, S.L.. Belknap. R.A., and W.J. Hausler. Jr. 1959. Staphylococcal food intoxication due to Cheddar cheese. I. Epidemiology. J. Milk Food Technol. 22:313-317.
Martley, F.G. 1972. The effect of cell numbers in streptococcal chains on plate-counting.

New
8.

fruit

Milk Food Technol. 18:88-90.

III.

Staphylococci

in

dairy foods.

A review.

J.

Milk Food Technol. 35:77-82.

Park, H.S., Marth, E.H., and N.F. Olson. 1973. Fate of enteropathogenic strains of
Escherichia coli during manufacture and ripening of Camembert cheese. J. Milk Food
Technol. 36:543-546.

11.

Stiles,

G.W.

1945. Brucellosis in goats; recovery of Brwc^-Z/a w^//7e.s/i'

from cheese manu-

Rocky Mountain Med. J. 42:18-25.


Wilkinson, R.F., and G.H. Richardson. 1975. Comparative and collaborative

factured from unpasteurized goat's milk.


12.

studies on

the preparation of hard cheese samples for microbiological assay using a plastic

technique.

J.

Milk Food Technol. 38:583-585.

pouch

CHAPTER

13

MICROBIOLOGICAL METHODS FOR ICE CREAM


AND RELATED FOOD PRODUCTS
G.A. Muck, J.H. Martin, and R.L. Winslow

13.1

Introduction

Methods

to determine the sanitary quahty of frozen dairy foods are similar


appUcation and Hmitations to those used for fluid milk and cream. Directions to determine the bacteriological quality of certain product ingredients
in

are given in other chapters as follows: evaporated milk, condensed milk, dry

milk in Chapter 10; butter in Chapter

1 1

and

fluid

milk and cream in Chapter

5.

Microbiological methods described here are also applicable to frozen dairy foods in

which vegetable

fats

have been substituted for milkfat such as

Mellorine and Parevine and to water ices, pops, and non-dairy creamers.

Standard Plate Counts of bacteria in frozen dairy foods and ingredients, as


other dairy products, are indices of plant sanitation and of processing and
handling conditions. The method may be applied to all types of samples
taken at successive stages of manufacture to detect bacterial contamination
or growth or both.
in

Coloring materials, extracts and flavors,


confections

fruits, nuts,

cakes, pastries and

ingredients frequently added to pasteurized cream mix or


frozen product may be important sources of bacterial conice

to the partially

tamination of frozen dairy foods.


Ingredients added to frozen dessert mixes before pasteurization normally
do not contribute to microbial contamination of the pasteurized mix unless
these ingredients contain bacterial spores; however, bacteriological analysis

may be
late,
fiers

important

in establishing the quality

of ingredients such as choco-

cocoa, eggs, milk, cream, other dairy ingredients, stabilizers, emulsiand other food additives.^ The Standard Plate Count, coliform count,

yeast and mold count, and spore counts are useful tests to determine the

With coliforms, the problem of false counts in


cream should be recognized.^ There can be organisms other than

quality of such ingredients.


fruit ice

ISC Liaison: D.W. Mather


165

166

MICROBIOLOGICAL METHODS: ICE CREAM AND RELATED FOOD PRODUCTS

coliforms present in fruit ice cream which give positive results with the coH-

form

test

and, therefore, confirmed tests and caution are required

in inter-

pretation of such tests.

few instances, certain foodborne disease epidemics and other forms

In a

of illness have been traced to frozen dairy foods.

Any

ingredient of a frozen

dairy food could be a source of appreciable microbiological contamination.

The

extent of such contamination depends upon conditions of manufacture,

length and temperature of storage,

methods of defrosting or dissolving, and

contact with sanitary equipment.

Equipment and Supplies

13.2

A. Grinding equipment:
Grinding of coarse gelatin (6-10 mesh), agar-agar, Irish moss, and other
emulsifiers

mixtures

and

stabilizers to

in dilution

40-mesh

size or smaller ensures

more uniform

water. Grinding equipment must be sterile before use.

B. Dilution blanks:

Preferably prepare wide-mouth bottles to facilitate sample introduction.

tablespoonful of sterile glass beads

in

each dilution blank

may

facilitate dis-

persion of samples of emulsifiers and stabilizers.


C. Sterile spatula {or similar instrument):
Sterile spatulas or similar instruments should be of stainless steel for

transfer of granular materials to previously weighed dilution bottle.

13.3

Sampling Procedures

See 3.25 for procedures applicable to sampling of

ice

cream and

related

frozen products [3.25(B)].

Samples for

13.4 Preparation of

Plating

A. Unmelted samples:

When
dition,

representative samples are received

at

the laboratory in frozen con-

keep them frozen and preferably make

aseptically weighing an

a 1:10 dilution thereof by

1-g test portion of frozen product directly into a

MS water [5.3(B)]. This


samples
procedure obviates the inconvenience of melting
and the need for
extra equipment.
dilution blank containing 99 ml of sterile buffered

B. Melted samples:
If
sis,

the sample

hold

it

at

is

partially melted but

room temperature

still

the product temperature does not get

quite viscous at the time of analy-

more than 15 minutes. Be sure that


above 4.4 C during this time. Refrig-

for not

13.5 Standard Plate

Count

167

erate perishable unfrozen samples at 0-4.4

until

time of analysis, but nev-

er hold longer than 36 hr from the time of collection. Aseptically weigh


(or

of sterile

C. Coloring, flavoring,

Where necessary
not more than 40 C
to 40

1 1

well-mixed sample directly into a dilution bottle containing 99 ml


buffered distilled water [5.3(B)].

g) of

and other added materials:

homogeneity, warm samples in a water bath at


no more than 15 minutes. Use water blanks adjusted

to ensure

for

for the initial 1:10 dilution.

Vigorously shake samples of coloring solutions, measure out appropriate


amounts, and plate suitable dilutions. Weigh 1 g of well-mixed dry colors
into 99 ml of sterile diluent and plate, usually measuring 1-ml and 0.1-ml
amounts of the resulting 1:100 dilution into the plates. Vigorously shake extracts and flavors and plate 1-g and 0.1-g (or 1-ml and 0.1-ml) amounts.
Owing to the bactericidal properties of some solvents used for extracts and
flavors, (frequently diluted alcohol), direct plating of 1-g (or 1-ml) amounts
may result in plates without colonies, whereas corresponding 1:10 dilutions
may show abundant growth of spore-forming types.
Weigh representative 1-g portions of nuts or fruits directly into a dilution
bottle containing 99 ml of sterile diluent. Allow the mixture to soak for 5 min,
shake vigorously, and plate 1- and 0.1-ml portions of the resulting
1

1:10 dilution.

Weigh
blanks

1 1

g (or

g)

of cake, pastry or confection into 99-ml sterile diluent

when preparing

initial

dilutions.

D. Stabilizers and emulsifiers:


Mix contents of the sample jar thoroughly by stirring or shaking.

Where

necessary, aseptically grind or pulverize coarsely ground material so that


will

pass through a sterile flour

the dilution at not

sifter (not less

than 40-mesh). Weigh

above room temperature. To


powder. Shake vigorously

tents of the bottle while introducing the

then allow the stabilizer to rehydrate at

than 20 min;

if

a longer period

is

it

g into
prevent lumping, swirl con-

room temperature

more convenient, hold

for 15 sec,

for not

more

dilutions at 0-4.4

(32-40 F). Transfer the primary 1:100 dilution to a water bath at not

more

than 40 C, swirling the contents at intervals to hasten dispersion.

Where

the stabilizer

is

not completely dispersed within a heating interval

of 20 min, plate representative portions of the suspended material. Prepare

no more than four samples

at a

time before plating. Plate samples within

20 min after preparing dilution blanks.

13.5 Standard Plate

Count

Proceed with general instructions for diluting and plating samples and for
incubating and counting plates [5.6-5.14].

168

MICROBIOLOGICAL METHODS: ICE CREAM AND RELATED FOOD PRODUCTS

Because

it

takes longer to prepare

ordinarily limit the

initial

number of samples

dilutions of frozen dairy foods,

in a single series

of plating (six to

exposure of mixtures to temperatures which might permit bacgrowth will not exceed 20 minutes.

eight) so that
terial

13.6 Test for Coliforms [6.8]

Because of the presence of sucrose in ice cream, coliform-like colonies on


Red Bile Agar should be confirmed.

Violet

13.7 Psychrotrophic

Count

Using appropriate dilutions, evaluate ice cream mixes, milk shake mixes,
and other mixes intended for freezing or semifreezing, and sold through refrigerated distribution channels [7.32].

13.8 Yeast

and Mold Count

Using appropriate dilutions [13.5], proceed as in 11.5. Report the result as


"Yeast and Mold Count" (YMC)/gram of product examined.
Where molds may be present in products like frozen, crushed, and pureed
fruit, prepare a homogeneous mixture of pulp from a representative portion
of sample and examine microscopically for mold filaments according to
methods outlined in the AOAC's Official Methods of Analysis.^

13.9 References
1.

Association of Official Analytical Chemists.

1970. Official

Methods of Analysis,

Washington, D.C.
Barber, F.W., and H. Fram. 1955. The problem of false coliform counts on
cream. J. Milk Food Techno!. 18:88-90.
11th ed. Association of Official Analytical Chemists.

2.

3.

Hall, H.E.

1971.

The

Technol. 25:230, 232.

fruit ice

significance of Escherichia coli associated with nutmeats.

Food

CHAPTER

14

DIRECT MICROSCOPIC METHOD FOR BACTERIA


D.I.

14.1

Thompson,

Lyie Eckberg

and George Sherman

Introduction

The direct microscopic method consists of examining under a compound


microscope stained films of a measured volume of milk or milk product dried
on glass slides [14.8-14.18]. Staining permits recognition of distinctive
shapes and arrangements of bacteria and somatic cells in films. This method
makes possible rapid examination of films to determine whether quality
problems exist. Actual counts of clumps of bacteria and individual somatic
cells are reported.

The accuracy and reproducibility of results by this method depend largely


on effective training of qualified people in the performance and uses of the
method.
14.2 Applications to

Raw

The microscopic method

Milk to be Pasteurized

ofters a rapid technique for determining the ex-

in raw milk or cream samples taken directly


from the producer or from pooled supplies in tankers or storage tanks. Individual samples may be examined within 10-15 minutes. The arrangement
and morphology of bacterial cells in the stained film may suggest a possible
cause of a high count, while excessive numbers of somatic cells point to
abnormal udder conditions, especially mastitis. Such interpretations always
should be made cautiously, even by well-trained analysts. When actual
counts are to be reported, when litigation may be involved, or when other
exacting situations may exist, actual numerical estimates may be made.

tent of bacterial

contamination

14.3 Applications to Pasteurized Milk

The microscopic method may be used


contamination

in

to determine the extent of bacterial

pasteurized fluid milk and cream. Because dead cells lose

their stainability to

some degree and

generally only very small numbers of

viable bacteria occur in pasteurized products, this

ISC Liaison: J.L. Dizikes


169

method should not be

DIRECT MICROSCOPIC METHOD

170

used to determine compliance with finished-product standards but should be


used only as an aid in troubleshooting. Bacteria in large numbers in stained
films prepared from uncultured pasteurized products regardless of type or
viability indicate undesirable conditions.

14.4 Applications to Dry Milk

Most dry milks contain only

relatively small

numbers of viable

bacteria.

examination of films prepared from reconstituted samples [10.3(F)] can provide information on the previous history of the product
and may be useful in quality control programs.** Direct microscopic clump
counts have been incorporated into government ^^-'^^ and industry '-^ standards. Arguments have been presented against their use ^ and evidence has
appeared indicating that wide variations on the same samples might be expected between different laboratories.'^ Also, the relationship between direct microscopic counts of skim milks and of corresponding dry products
made from them was found to vary widely.^ Finally, effects of heat on stainability of bacteria in dry milk ^^ should be recognized.
Microscopic

14.5

Sources of Error

As with other methods

in

Method

for assessing the bacterial content of milk, results

by the direct microscopic method are estimates only. Training and experience of the analyst are prime factors in determining accuracy, reproducibility and significance of results obtained.** Even with the most exacting
technique, replicate estimates may vary appreciably. Among factors responsible for variation are:

inaccuracy

in

measuring 0.01-ml quantities;


and staining of slides;

faulty preparation

failure of bacteria to stain;

the minute

amount of milk

actually

examined

in counting;'"^

irregular distribution of bacteria in the films;

count a sufficient number of fields;


poor microscopy inadequate or excessive illumination of the microscope, poor focusing, improper use of colored filters;
drying films on a nonlevel surface;
eye fatigue, and
mistakes in observation and calculation.
failure to

14.6 Bacterial Estimates

When

large numbers of bacteria are present and are uniformly distributed,


from poor-quality milk will have numerous clumps per field examined,
whereas films from high-quality milk contain few or no bacteria in many
fields. Thus, examination of only several fields often will indicate the general
quality characteristics of the sample. Since presence or absence of bacteria
may be determined readily, both good-quality and poor-quality samples
films

may

be recognized easily.

14.7 Bacterial

Counts or Grades

171

Table 14:1. Suggested Microscopic Factors with Corresponding (1) Field


Diameters,* (2) Numbers of Fields To Be Examined, and (3) Calculated
Working Factors (WF),^ When Making Direct Microscopic (Clump) Counts
per

Dependent values,

MF

milliliter

DIRECT MICROSCOPIC METHOD

172

Figure 14:1. Slide with circular square-centimeter areas.

two or three quality

classes, with limits far

enough apart,

if

more than two

classes are used, to represent appreciable differences in sanitary quality.

14.8 Procedure for Collecting

Take samples
preservatives

'^'

Samples

in sterile, preferably
'"

only as needed

precooled, containers, optionally using

in the

absence of refrigeration; or, immediml of well-mixed

ately after taking the sample, aseptically transfer 0.01

sample to a

14.9

slide [14.9(A)]

and prepare the

film prornptly [14.15-14.17].

Equipment and Supplies

Except for specialized items required for these

tests, refer to

Chapter

5 for

other equipment needed.

A. Microscope slides:

Clean microscope slides for milk and cream films must be used. Optional
X 3 in., (2.5 x 7.50 cm)
1
in., (5.0 x 7.50 cm) or 2 x 4.5 in.
(5.0 cm X 11 .25 cm) with clear, circular (diameter approximately 1 1 .28 mm)
1-cm^ area ^'^ delineated on each, and the remaining area etched (Fig. 14:1).
Slides described in 8.41(C.4) may also be used. Slides may be cleaned

2x3

sizes

for repeated use.

Use

of clear glass slides with a guide

is

not acceptable.

Several procedures are reported to be satisfactory for cleaning used

They are often cleaned by submerging in hot detergent solutions until


from residue, rinsed thoroughly in flowing tap water, dried, and proper-

slides.

free
ly

stored for reuse.

New

may be soaked

an effective cleaning solution (e.g., dichroin flowing tap water, and


then in distilled water. Slides are dried with minimal exposure to dust, insects and other contaminants. Stainless steel or glass holders or other suitable equipment may be used for transferring slides from one cleaning step to
slides

in

mate-sulfuric acid), rinsed thoroughly (10-15 sec)

14.9

Equipment and Supplies

173

another to avoid fingerprints and other residues on areas where films are
Used slides may be soaked in a hot chemical detergent or wetting

placed.

agent until

all

residues are removed, then rinsed and treated as just de-

and dry
immediately before use.
Slides, new or thoroughly washed, may be stored with a surface coating of
Bon Ami or other comparable detergent. Shortly before use, the detergent
film is wiped off with a clean, dry cloth. Some workers flame and cool slides
scribed. After cleaning, optionally store slides in alcohol. Drain
slides so treated

'"*

immediately before use; an alternative method


hol and flame them just before use.

is

to store slides in

95%

alco-

B. Transfer instruments for 0.01 -ml quantities:


1.

tities

Metal syringe:* For rapid and convenient transfer of 0.01-ml quan-

of milk or cream, having a semiautomatic spring-actuated plunger and

a stainless steel piston sliding in a close-fitting stainless steel measuring


tube, and with an adjustable setscrew to permit presetting the instrument for

repeated accurate measurements of 0.01-ml quantities (Fig. 14:2).


Accuracy of syringe adjustment should be determined before use and periodically thereafter as necessary.

Where

certification

is

required before use,

appropriate regulatory agencies should verify the accuracy of the setting


before applying the certification symbol over the setscrew. This provides the

necessary assurance that the setscrew cannot be readjusted accidentally or


otherwise without visible evidence thereof.

NOTE: The mechanical principle of the syringe makes possible positive


measurement of 0.01-ml portions without necessitating manual adjustment
of the milk column. It also makes possible accurate measurement of test
portions of cold heavy-fluid creams, which cannot be done with 0.01-ml
pipets.

Trouble with artefacts (metallic oxide particles)

may be encountered

with metal syringes.

To determine

the weight (and indirectly the volume) of milk delivered by a

determine the specific gravity of a homogetest charge [14.15(A) or (B)],


wiping the exterior of the tip of the instrument as usual. Weigh the charged
instrument on an analytical balance. Expel the charge as usual near the center of the 1-cm^ area and if a syringe is being used, spread the charge evenly
over the area with the tip of the piston or needle. Weigh the discharged
instrument. The difference in weight between the instrument charged and
discharged should average (over 5 to 10 weighings) 0.0103 gram.
Do not allow residues to dry on the instrument. Immediately after use,
disassemble and clean the measuring tube and piston. Do not interchange
0.01-ml syringe

'*^

nized milk sample.

or pipet,^

first

Withdraw a representative

parts from one syringe with parts of another. Handle parts carefully

*Available from Applied Research Institute, 90 Brighton Avenue, Perth


or from Dairy Research Products, Inc..

Box

288,

Yarmouth, Me. 04096.

Amboy,

when

N.J. 08861,

DIRECT MICROSCOPIC METHOD

174

'^^^-:::^

APPROX20mm

6-7mm

PLUNGER

(P^

r^
BARREL

SPRING
PISTON

MEASURING
TUBE

0.01ml

14.9

Equipment and Supplies

pH

7.0.

first

175

Observe the extra precaution of cleaning new instruments before

use.

Before assembling the syringe,


residues have been

make

removed from

certain that both detergent and milk

the instrument.

To

reassemble, slide the

removed) over the piston and plunger, carefully insert the piston
into the measuring tube at the countersunk opening, and tighten the threaded
joint. Be sure threaded joints are always tightened to identical positions.
Ail syringes, whether or not officially branded, which after repeated use
are tested and found not to conform with milk deliveries as specified above,
should be returned to the manufacturer with instructions to recondition
spring

(if

them. Ordinarily syringes should be tested for accuracy biennially, or more


if needed. WARNING: Except in emergencies, do not loosen the
setscrew for adjusting the plunger stroke to readjust the stroke.

frequently

2.

Pipet:

For bacteriological use only, calibrated to deliver 0.01-ml

quantities of milk according to

APHA

specifications."* Straight, thick-walled

capillary tubing with a bore of such diameter that a single graduation

mm

40-60

from the pipet

tip.

The

tip is

mark

is

blunt and formed so as to discharge

the milk cleanly (Fig. 14:2). Calibrated to contain 0.1395 g (approximately


140 mg) of mercury, it will discharge 0.01 ml of milk at 20 C (68 F), or cali-

brate with milk as in 14.9(B.l).

NOTE: When

not

in use,

keep pipets submerged

in

and the bore

suitable soapless detergent or strong cleaning solution.

When

filled

with

preparing for

use, rinse the bore and exterior thoroughly in clean water until free of the

detergent or cleaning solution.

C
A

Bent-point needle:
bent-point needle

is

used for spreading milk or cream from a 0.01-ml

pipet over a square-centimeter area.

D. Diying surface:

clean, dust-free, level surface should be provided with suitable heat

source for drying films

at

40-45

(104-113 F).

thermostatic control

is

box (metal, wood or composition frame) over an electric bulb or substage microscope lamp, or use a
commercially available drying box (Fig. 14:3) for this purpose. Use of a
cross test level is suggested, to be certain that the surface on which films are
made and dried is perfectly level.

desirable. Optionally use the top of a cabinet or

E. Forceps

and partitioned slide holders:

Forceps and partitioned

slide holders are required for dipping

and holding

slides.

F. Trays or jars:

Trays or jars should be equipped with tight-fitting covers, and be of convenient size for holding solvents and stains and/or submerging slides held in
partitioned racks.

DIRECT MICROSCOPIC METHOD

176

Figure 14:3. Film-drying box, closed type.

G. Slide storage boxes, cases or files:


Slide storage boxes, cases or files

must be clean, dust-free and insect-

proof, for storage [14.18].

H. Compound microscope:

A
tive,

binocular microscope

is

preferred, with

1.8-mm oil-immersion objecAbbe condenser

substage actuated by a rack and pinion carrying an

having a numerical aperture (NA) of 1.25 or higher. It should be equipped


with an iris diaphragm, a flat mirror if the light is not an integral part of the
microscope assembly or mounted on the base, a mechanical stage, and oculars as follows: single for

monocular or paired

for binocular, 10 x magnifica-

tion recommended (12.5-15x permitted). For both enumerative and grading


purposes, the ocular(s) should be calibrated to provide a microscopic factor
of 300,000 to 600,000 [I4.12J. Wide-field oculars with high eyepoints are ad-

vantageous, especially for those


/.

who wear

eyeglasses.

Ocular disc subdivided into quadrants:

To use

the disc (see Fig. 14:4), place it in the ocular so that it rests on the
edge of the reducing diaphragm. Face the etched surface of the disc accord-

177

Equipment and Supplies

14.9

Figure 14:4. Ocular disc, showing general arrangement of guide

ing to directions provided

lines.

by the microscope manufacturer. Only one disc

is

required for a binocular microscope. Before using such a disc, determine the

microscopic factor corresponding to the area to be examined [14.13].

Microscope lamp:
microscope lamp is necessary where the light is not a part of, or attached to, the microscope frame. It should be of an advanced or professional
type, with a light source equivalent to 100-watt illumination, having a reflector, condensing lens system, and iris diaphragm to provide Kohler-type illumination. Any simple lamp system of proper intensity that provides critical
J.

illumination

is

also satisfactory.

Modern "on base" or attached

illuminators equipped with an aspheric

condenser lens, reflector, and 110-volt, 15-watt clear bulb are acceptable if
light quality and intensity are adequate. Choose a type specifically designed
for a monocular or binocular microscope.
The ordinary separate substage illuminator lacking a reflector and an aspheric condenser lens

is

not acceptable for use in estimating bacterial num-

bers.

K. Mechanical stage:
A regular mechanical stage as supplied with the microscope, or a special
stage for slides 2 x 4.5 in. (5.0 x 11.25 cm) may be used.

L. Stage micrometer slide:

stage micrometer slide ruled in 0.1- and

M. Hand tally:
See 5.2(N) (double hand

tally preferred).

0.01-mm

divisions.

DIRECT MICROSCOPIC METHOD

178

KOHLER ILLUMINATION

Figure 14:5. Diagram showing adjustment of illumination source and microscope condenser to obtain Kohler-type illumination. (Redrawn from original
of the American Optical

Company. Instruments

Division, Buffalo, N.Y.)

14.10 Materials
A. Chemical reagents (as required):

Chemical reagents
fied.

shall

be of the highest-purity, unless otherwise speci-

Keep containers of stock reagents

tightly closed.

B. Methylene blue chloride, certified:

Dry

stain certified

by the Biological Stain Commission as being

satisfac-

tory for the purpose intended; or, liquid stains prepared from dry stains so
certified shall

be used.

C. Immersion

oil:

Immersion

with a refractive index 1.51-1.52 at 20

type

is

14.11

used.

oil

Type B (medium

viscosity)

is

C and

of a nondrying

preferred.

Illumination Adjustment

When

an "in base" or "on base" illuminator

to secure

maximum

brightness of field.

Use

is

used, adjust the condenser

the condenser diaphragm only to

reduce glare.

To obtain maximum optical resolution when a separate lamp is used, it is


necessary to adjust both the illumination source and the microscope condenser properly to obtain Kohler-type illumination (Fig

14:5).

However, mi-

14.11 Illumination

Adjustment

179

"CRITICAL" ILLUMINATION

LD

Figure 14:6. Diagram showing microscope and lamp adjustment to obtain


critical illumination.

(Redrawn from

original of the

American Optical Com-

pany, Instruments Division, Buffalo, N.Y.)

croscope and lamp adjustment to provide


is

critical illumination (Fig 14:6) also

acceptable.'^

To

obtain Kohler illumination with a binocular microscope, proceed as

follows:

A. Position microscope near edge of table to provide maximum ease of


Have body of microscope in a vertical position. Place illuminator in
front of microscope, approximately 6-8 in. away, depending on focal length
use.

of the lamp condensing system.


B. Turn on the lamp, close lamp diaphragm to provide a small-diameter

opening {Vs-Va in.) (3-6 mm) and tilt lamp so that the light beam is about
centered on the flat microscope mirror and is uniformly reflected into the
microscope condenser. Focus lamp condenser so that filament image is in
sharp focus on diaphragm leaves of the microscope condenser. This can be
determined by looking into the microscope mirror at the proper angle to see
the reflection of the microscope condenser diaphragm.
C. Place a stained smear on the stage. Turn the low-power or high-dry

Rack the microscope substage condenser up as far


go and open the microscope condenser diaphragm wide. Focus on
the specimen. Adjust the binocular eyepiece to see one merged field comfortably. Also, adjust the individual eyepieces to compensate for refraction
differences. (If the microscope is equipped with only an oil-immersion lens,
follow A, C, D, E and F, being sure that the initial lamp diaphragm opening
objective lens into place.
as

is

it

will

very small.)

DIRECT MICROSCOPIC METHOD

180

D. Place the specimen

sharp focus and, disregarding rainbow effects

in

Hghting, slowly rack the microscope condenser

down

until a solid

in

many-

sided spot of white light (image of the lamp diaphragm opening) appears.
Adjust position and tilt of illuminator, focus of microscope condenser and,
if

necessary, angle of mirror

to obtain

spot of

even

field

of view and

focus, slowly open the lamp condenser

field in

until the spot of light just

Remove

F.

spot in the

over the entire field with sharpest possible edges to the


not for any reason subsequently change height of micro-

scope condenser.
E. While looking at the

diaphragm

light

light

Do

light.

to center the

fills

the field of view completely.

down open drawtube. If light is too bright,


over open end of drawtube. One should see clearly

ocular and look

left

place a clear glass

filter

an image of the lamp filament. If this is visible, Kohler illumination has been
achieved. If not, move the lamp either closer or farther away to obtain maximum definition of the filament image.
G. Replace ocular, raise microscope body, change to oil-immersion objective lens, apply immersion oil to slide, and bring smear into sharp focus.
H. While looking into the microscope, slowly close illuminator diaphragm
until the field of view is just filled with light. Remove left ocular once again
and, while looking

close microscope condenser diaphragm


Replace ocular and microscope is ready

down drawtube,

until its closing is just detectable.

for use.
If light is

/.

or

too intense for comfort, it


clear daylight

more neutral-density or

scope, usually

in

the

filter

insertion of one
between the lamp and micro-

may be reduced by
filters

holder of the lamp. Positioning the

a greater distance from the microscope

may be somewhat

light

source at

less desirable in

reducing light intensity. Do not change adjustment of lamp, microscope condenser diaphragm or microscope condenser height to change intensity of
light. If light is inadequate for good visibility after proper illumination adjust-

ment, use a

light

source of greater intensity.

14.12 Adjustment and Calibration of Microscope

When

determining numbers of bacterial clumps per milliliter of milk


is fundamentally important because it de-

[14.1], the microscopic field area

termines the amount of milk which can be examined in any one field. Field
diameters providing microscopic factors from 300,000 to 600,000 are recommended. Microscopic factors are values by which the average number of
bacterial

clumps per

ber of clumps per

field is to

be multiplied to determine the estimated num-

milliliter.

Manufacturers of binocular microscopes normally have adjusted tube


length and paired oculars for use at the most
(65

mm)

made by an
from

common

or especially for a certain individual.


individual

whose

interpupillary distance

When

estimates are to be

interpupillary distance varies substantially

that for which the microscope

was

set up, use paired oculars with dia-

181

14.14 Working Factor

phragms

especially adjusted for that individual; otherwise, correct the


counts obtained according to the diameter of the field actually observed.
FYesently available standard equipment includes binocular and trinocular

microscope bodies of constant tube length. With this feature, the resultant
calibrated field and the resultant magnification remain constant regardless of
changes in interpupillary settings.

14.13 Calculation of Microscopic Factor

To direct light through a milk preparation, adjust the microscope lamp to


provide maximal optical resolution [14.11]. Verify the manufacturer's indicated microscopic factor [14.12, 14.14]. To determine the microscopic factor, replace

milk preparation on the stage of the microscope with a stage

micrometer

slide ruled in 0.1-

and 0.01-mm divisions and proceed as

fol-

lows:

A. Measure

diameter

field

To determine

B.

area of

in millimeters to the third

square radius

field,

(r

decimal, as 0.146

mm.

Vz field diameter)

and

multiply by 3.1416.
C.

To convert

ters, divide field

area of one

field in

square millimeters to square centime-

area in square millimeters by 100.

D. To determine the number of such

fields in

cm^

divide

cm^ by the

area of one field in terms of millimeters by 100.

Since only 0.01 ml of milk or cream was spread over a 1-cm^ area,

E.

multiply

per

number of

milliliter

by 100

fields

to

determine number of microscopic

fields

of milk.

determined by the ocular diaphragm. The final value


(MF) and in each instance is dependent on a definite tube length and a definite combination of objective and
ocular(s) used; the distance from the eye to the ocular must remain constant.
The reciprocal of the MF represents the fraction of one ml of milk actually
seen in one microscopic field. MF should be expressed in two significant

The

field

obtained

is

viewed

is

called the microscopic factor

figures (e.g.. 480.000).

Use of
is in

the following condensed formulas in which the radius or diameter

millimeters, obviates the need for conversion of area into square cen-

timeters, determination of
plication

by 100

number of fields per square centimeter, and

to convert the result to

MF

10,000

,,^

3.1416 X

or

clumps per

^
MF

r^

milliliter

multi-

of milk.

40,000
3.1416 x d^

14.14 Derivationof Working Factor


For convenience in routine work, where number of fields examined is conworking factor (WF) may be used. This factor is obtained by dividing

stant, a

DIRECT MICROSCOPIC METHOD

182

MF

To determine the
determined by number of fields
examined. b\ number of clumps found. Table 14:1 shous typical MPs. their
corresponding field diameters and indicates the W'F as gov erned b\' the magnitude of the count. (WFs are also expressed in two significant figures.)
the

number

usually by a constant

estimated count, multiply the proper

of fields counted.

\\"F. as

14.15 Using Transfer Instruments and Preparing Films

Employing careful technique, keep instruments and glassware scrupulousclean and free of foreign materials. Cleanliness of surfaces coming in contact with milk or cream is especially important, although sterilit\ is unnecessary". Before measuring the test portion, shake sample vigorously 25 times
[5.6iB)]. Guard against foam in measurmg test portions. As 0.01-ml test portions are spread. legibK' and indelibh identify each film. Use a number or
other suitable symbol on the etched margin of the slide, on the chart, or on
1\

both to provide unmistakable identification of each film.

To avoid

bacterial

growth uhen making films, work rapidly but carefulK


dr>' them at 40-45 C within 5 min on a level surface
.

After spreading films.

protected from dust [14.9iDi]. To prevent films from cracking and or peeling
from the slide during later treatments, do not heat too rapidly. Protect films
and slides from undue e.xposure to dirt. dust, insects, and other damage from
the time of film preparation until all counting is completed [14.16-14.18].
After mixmg the sample thorough!) [5.6\ B i]. measure 0.01-ml portions b\
procedures A or B as follows:
'^'^

A. Use of a 0.01-ml syringe.


Before use and between successive samples, rinse measuring tube

m clean

water (25-35 C) by dipping tip slightly beneath the surface and repeatedly
drawing in and expelling water. Before transferring test portions to the slide,
dip tip of tube not more than one cm below the surface (excluding foam) of
well-mixed milk or cream, and repeated!) rmse tube b\ drauing in and expelling portions. Holding tip beneath the surface. ful!\ release plunger and
withdraw a test portion. With clean paper tissue or cloth remove excess milk
or cream from exterior of

tip.

(Slides with circular

mit spreading of the film with a rotary motion

1-cm- areas readily per-

when

a syringe

is

used.'-)

Holding the instrument in nearl\ vertical position, place tip near center of
area for the film and expel the 0.01-ml test portion. \V ith plunger fullv depressed and syringe nearly vertical, spread test portion with tip of extended
piston rod over an exact square-centimeter area. Do not release plunger until
after its removal from film. Optionally use a bent needle to spread the film
[14.9(C)].

B. Use of 0.01-ml pipet:


Before use and between samples, rinse graduated portion of pipet

water (25-35 C) by dipping the

tip slightly

in

clean

beneath the surface and repeat-

14.17 Preparation and Use of Stain

183

and expelling water. Before measuring test portion, rinse


which test portion is to be
removed. W ithdrav^ portions of well-mixed samples slightly above the graduation mark. Take precautions that saliva does not get into pipet to contaminate milk sample. \Mpe exterior of pipet with a clean, dry paper tissue
edly drav^ing

traces of residual uater from bore in sample from

tip. adjust length of column to the


near center of area to be covered and expel a

or towel and. b\ absorbing liquid at the

graduation mark. Place

tip

test portion. With a clean bent-point needle, promptly spread


sample o\ er an exact square-centimeter area. Wipe needle between samples
on a clean. dr\ tissue or tov^el.
If cream samples are tempered at 30-35 C (86-95 Fi for not more than
10 min before v^ithdrawal of a test portion, more uniform deli\eries uith the

0.01 -ml

pipe:

ma\ be expected.

14.16 Handling of Slides During Staining


For handlmg
steel, or

slides containing dried tilms. optionally

use glass, stainless

other holders partitioned to permit multiple transfers of slides. Such

holders must be sealable and maintained free from residues that might subsequentl) contaminate the staining solution

in

uhich

slides are dipped.

14.17 Preparation and Use of Stain

Use

Make

the Le\owitz-\\ eber modification of the

Newman-Lampert

staining solution in accordance with directions given herein.

stain.

^^

Avoid

use of liquid reagents containing suspended foreign matter. After stain has

been formulated, handle it in a manner that minimizes e\aporation of


solvents and consequent formation of precipitate that might introduce artefacts into stained films. Sohent loss must be minimized at all times to allow
proper staining.
.4.

Preparation of staining solution:


stains under a fume hood to avoid toxic fumes. Slowly

CAUTION Prepare

meth\lene blue chloride to 52 ml of 95^f ethyl alcohol


in a 200-ml flask. Swirl to dissolve.
4-".
12-24
4.
filter
through fine filter paper (Whatman
hr at
2 C.
stand for
4
ml
of
glacial acetic acid and store in a
42 or equivalent), then add
clean. tightK closed bottle with a closure that is not affected by stain readd
and
Uet
No.

0.6 g of certified

44 ml of tetrachloreihane (technical)

agents.

B. Staining procedures:
Slides should be immiersed in a container that can be covered to prevent

evaporation while slides are

in stain solution.

Repeated use of

stain in

an

uncovered \essel may result in formation of precipitate.


Submerge slides containing fixed dried films singh or in multiples [14.16]
into stain for two mmutes. Dram off' excess stain by resting edge of slide on

DIRECT MICROSCOPIC METHOD

184

absorbent paper. Dry thoroughly by forced air if available. Rinse dried,


stained shdes in three changes of tap water at 35-45 C, then drain and air-dry
before examining the films microscopically.

Proceed as above

in

preparing and staining cream films.

When small numbers of raw milk films are to be stained, flooding of slides
may be more practical than submersion. Care must be taken to limit flooding
exposure so that evaporation does not progress to the point where precipitation of dye occurs. Use adequate ventilation.
C. Storage of staining solution:

Discard used stain whenever the solution becomes contaminated or otherWhen not in use, keep containers of staining solution tightly

wise unsuitable.

closed to prevent evaporation. This applies to surplus supplies as well as to


"working batches" of stain between successive operations. Avoid use of
containers or closures which

may

taminate staining solutions. Store

dissolve or disintegrate and thus con-

in a relatively

dark cool place but do not

refrigerate.

14.18 Examining Films

To obtain estimates of the clump count per milliliter, examine film with
oil-immersion objective after placing one drop of immersion oil on each film.
Count as separate clumps any cell or group of cells (apparently of the same
type) separated by a distance equal to or greater than twice the smallest
diameter of the two cells nearest each other. Regardless of proximity to each
other of cells of different types, count each type as a separate unit. Count
diplococcal forms as single clumps.

Two procedures are favored by examining representative portions:


A. Select successive rows at right angles to each other across the slide,
starting about midway on any side. Proceed by passing over the first two or
three fields to within two or three fields of the opposite edge. If it is necessary to examine more fields, start midway at the top or bottom of the film
and continue until the correct number of fields have been counted as shown
in 14.15

(Table

B. Count

all

14:1).

clumps seen

in

one or more field-wide

strips

running com-

pletely across the film. This procedure requires less time than the

examine a

When

it

sufficient area of the film


is

and

is

first

to

less fatiguing.^*

anticipated that the quality of milk

is

such that the

maximum

have to be examined, count clumps in 10-15 fields


to establish an approximate number of clumps per field, and thus tentatively
establish the count limit within which the sample will fall. From this, the
actual number of fields to be counted can be determined for the microscopic
factor being used. After examining the number of fields required on the basis
of a preliminary count, verify that the clump count falls within limits prescribed for number of fields to be counted [14. 14]. Clean objectives with lens
papers only, and store in a vertical position.

number of

fields will not

185

14.20 References

Preserve preparations for a reasonable interval after rendering reports, to


if results are contested. Arrange periodically

permit reexamination of slides

for checking results of laboratory workers, especially

may be based upon such

After examination of films and removal of

may be preserved

solvent, slides

when

punitive action

reports.
oil

by use of xylene or other

fat

indefinitely.

14.19 Standards
Standards of raw milk proposed by the USDA include three classes of
milk differentiated by the direct microscopic clump count. '^ Moreover,
[14.16] are being applied.
USDA standards for dry milk based on the

DMC

14.20 References
1.

2.

3.

American Dry Milk Institute, Inc. 1970. Recommended sanitary/quality standards


code for the dry milk industry. Bull. 915. ADMl, Chicago, III.
American Dry Milk Institute, Inc. 1971. Standards for grades of dry milks, including
methods of analysis. Bull. 916. ADMI, Chicago, 111.
Breed, R.S. 1911. The determination of the number of bacteria in milk by direct microscopic examination. Centralb.

4.

5.

6.

f.

Bakt. Part

II

30:337-340.

Breed, R.S., and J.D. Brew. 1916. Counting bacteria by means of the microscope. Tech.
Bull. 49, N.Y. Agr. Exp. Sta. Albany, N.Y.
Brew, J.D. 1914. A comparison of the microscopical method and the plate method of
counting bacteria in milk. Bull. 373, N.Y. Agr. Exp. Sta. Albany, N.Y.
Claiborne, F.B., and K.E. Cox. 1951. The direct microscopic count on preserved milk
samples: An effective measure for uniform state-wide control. J. Milk Food Technol.
14:105-108.

7.
8.

9.

Coulter, S.T.

milk.
10.

1957. Quality considerations.

J.

Dairy Sci. 40:1012-1015.

Forest, H.L., and E. Small. 1959. The direct microscopic clump count as a measure of
quality of nonfat dry milk. Proc. 15 Int. Dairy Congr. 3:1883-1889.
Heinemann, B. 1960. Factors affecting the direct microscopic clump count of nonfat dry
J.

Dairy Sci. 43:317-328.

Levine, B.S. 1950. Effect of formaldehyde on the direct microscopic count of raw milk.
Pub. Health Rep. 65:931-938.

11.

Levowitz, D. 1944. Reproducible data by microscopic method,

p.

193, Bull. International

Association of Milk Dealers.


12.

Levowitz, D.
scopic count.

13.

Levowitz,

An

1957.

J.

inquiry into the usefulness of the standard

methods

direct micro-

Milk Food Technol. 20:288-293.

D., and

M. Weber.

1956. .An effective "single solution" stain.

J.

Milk Food

Technol. 19:121, 128.


14.

1952. Retention of milk and milk-product films on glass slides during stainand rinsing for microscopic examination. J. Milk Food Technol. 15:195.
Moats, W.A. 1961. Chemical changes in bacteria heated in milk as related to loss of staina-

MiLONE, N.A.
ing, defatting

15.

bility. J.

16.

Dairy Sci. 44:1431-1439.

Newman, R.W.

1952.

The Smith 0.01-ml syringe

in the

microscopic grading of milk.

J.

Milk Food Technol. 15:101-103.


17.

Pedraja, R., Chol, R., Mengelis, A., and E. Small. 1963. Joint collaborative study of
clump count method in dry milks and its statistical considerations. J.

the direct microscopic

Dairy Sci. 46:810-818.


18.

Richards, O. 1956. The effective use and care of the microscope. American Optical
pany, Buffalo, N.Y.

Com-

DIRECT MICROSCOPIC METHOD

186

19.

United States Department of Agriculture. 1963. Agricultural Marketing Service,


Minimum standards for milk for manufacturing purposes and its production
and processing recommended for adoption by state regulatory agencies. Fed. Register
Dairy Division.

(June 26).
20.

United States Department of Agriculture.

1969.

Consumer and Marketing

Service,

Dairy Division. United States Standards for grades of nonfat dry milk. Fed. Register

(March

12).

CHAPTER

15

REDUCTION METHODS
J.C. Flake, R.

15.1

Dabbah, and D.B. Weddle

Methylene Blue Reduction Method

A. Introduction:

The methylene blue reduction method

indirectly estimates the

number of

bacteria in milk in terms of the time interval required, after starting in-

cubation, for a dye-milk mixture with a characteristic blue color to

become

white [15.1(L)]. Different "methylene blue reduction time'' intervals permit


rapid grouping of samples into classes or grades.
ability of bacteria in milk,

oxygen dissolved

in the

when incubation

mixture, which

in

is

The method depends on the


grow and to utilize

started, to

turn lowers the oxidation-reduc-

To measure this
oxygen depletion,

dem-

tion potential of the mixture.^

bacterial activity and to

onstrate visibly the rate of

a solution of methylene blue

thiocyanate,-" an oxidation-reduction indicator which produces initially a

blue color in the mixture (concentration about

250,000),

is

added. The

and
at
1 C (97
hence depletion of oxygen and to shorten the required period of observation.
Certified tablets of proper strength to prepare dye solutions are available
commercially [15.1(H.8)].
mixture

is

incubated

36

In general, reduction time

tent of the sample.

is

2 F) to hasten bacterial activity

inversely related to the

Numerous comparisons

initial bacterial

con-

of reduction times with estimates

''^ and with other


have demonstrated this general relationship. This general relationship has also been confirmed in an extensive study ^ in which a pertinent
statistical basis for setting equivalent classes among the reduction tests and

of bacterial populations by the agar plate method


tests ^^'^^

total

counting methods was used.

raw milk:
The method is used to grade raw milk, especially for manufacturing purposes. The small amount of equipment and materials and the simplicity of
B. Application to

ISC Liaison: J.L. Dizikes


187

"

REDUCTION METHODS

188

method permit simultaneous testing of several hundred samples. Since it


combines speed and adaptability, this test may be used in milk plants, receiving stations, condensing plants, cheese factories, and similar establishments to rapidly grade incoming milk supplies.
Because decolorization of methylene blue is influenced by metabolic activities of each bacterium, and because a relatively large amount of milk is
used in the test, reduction time is less affected by uneven distribution of

the

bacteria than are numerical estimates of bacterial colonies or clumps. Fur-

may be reduced
depending on the prevailing growth phase of bacteria at
time of testing and on types of bacteria present.
Objections have been raised to application of the methylene blue reducthermore, milks containing equivalent numbers of bacteria
at different rates,

The modified test presently in use


some of these objections by maintaining the
more uniform distribution throughout the mix-

tion test in grading milks of high quality.

[15.1(A-L)] attempts to meet


bacteria and fat globules in
ture.''

^' '^' '^-

Nevertheless, where good sanitary control

bacterial content

is

is

maintained and

low, the reduction test becomes progressively less appli-

cable, and results obtained by other

methods are more meaningful.

Lack of correlation with counting methods:


The lack of correlation between reduction times and numerical estimates
C.

of bacterial populations
1.

sample

may

result

from any of the following

Differences in reducing activities of bacterial


is

incubated

at

36

when

the

C. For example, most thermoduric bacteria

are less actively reducing than are

and thus are

factors:

species

less readily detected

many

other

by reduction

common
tests. ^^

milk contaminants

This

is

also true for

psychrotrophic bacteria.'"2.

Failure of

medium and
3.

some

at the

bacteria that reduce dye to develop colonies on the

incubation temperature used.

Variations in proportion of bacteria carried into the cream layer by

rising fat globules.'^


4. Reducing activity of bacterial cells is not diminished by clumping,
whereas plate and direct microscopic counts are lowered by clumping.
5. Presence in the sample of inhibitory substances, such as residues of
'''^^
antibiotics from mastitis therapy.'**'

D. Interpreting and reporting reduction times:


1.) classification of samples
two or more major groups, according to appreciable differences in bacterial quality, and 2.) use of descriptive grading terms to indicate whether or

Application of the grading principle permits

into

not the product

is

"acceptable."

When

reduction times are shorter than

those permitted by regulations or industry standards, appropriate grading

terms clearly indicating lack of compliance should be used. Under no

cumstances report results as number of bacteria.

cir-

Methylene Blue Reduction Method

15.1

189

Although colostrum, mastitic milk, and milk from cows far advanced in
may have shorter reduction times than normally would be expected
from their bacterial content, the influence of such abnormal milk in mixed
herd milk is virtually eliminated by dilution with normal milk. Because reduction times of poor quality milk may be lengthened materially by incorporation of oxygen, caution should be exercised in differentiating between
milks reducing in less than one hour.^*^ For routine control work, record
results at hourly intervals. Assign class designation of samples according to
the grading system used.
lactation

E. Standards:

Some state regulations provide for grading of manufacturing milk by the


methylene blue test. The sanitary codes of two dairy product associations
have uniform standards for this test.'^*' USDA recommended standards for
manufacturing milk ~'^ do not include the methylene blue reduction test because of its low correlation with the other bacterial tests.
Equipment and supplies for collecting samples:
Provide equipment and supplies as in 3.32(A).

F.

G. Procedure for collecting samples:

Proceed as

in 3.32(B).

H. Equipment and supplies:


1. Dipper or pipet: Delivery 10 ml.
2. Buret or pipet: With 1-ml graduations.
3. Tubes, vials, or suitable nontoxic plastic bags: See 3.32(A.l).

Tube

4.

tubes; or

flat

closures:

Rubber or other

suitable stoppers for individual

(metal) plates, slightly larger than tube rack tops, having one

surface covered with soft, impervious material; or other suitable

means

to

allow inversion of tubes in racks without loss of contents and without contamination of adjacent tubes.

Incubator for samples: Water bath of sufiicient size designed

5.

a.)

thermostatically controlled heat source to maintain samples at 36


(97

36

2 F); b.) with

volume of water

with

sufficient to bring contents to at least

within 10 min after placing samples therein; c.) to maintain water level

slightly

above

level of contents in tubes;

and

d.) to protect

samples from

action of light during incubation.

Sheet metal or wire racks: See 3.32(A.2).


Cooling bath: To hold sample at 0-4.4 C (32-40 F) until tested.
8. Methylene blue thiocyanate tablets: Certified by the Biological Stain
Commission; dye content per tablet, approximately 8.8 mg.
6.
7.

9.

Light-resistant flask, stoppered:

Amber

(zero transmission at wavelengths less than 380

or low-actinic glass bottle

mm)

with screw-cap closure

or other container off"ering similar protection, capacity 250 ml [15.1 (J)].


10.

Interval timer.

REDUCTION METHODS

190
/.

Precautions:

Glassware must be chemically clean.


ical

compounds

Do

not use chlorine or other chem-

as bactericidal agents on glassware, dippers or closures.

Avoid recontamination of that portion of the sterilized stopper which is inserted into the sample tube. Place tubes in racks. Protect sterilized equipment from dust and from contamination by unnecessary handling.
Preparing dye solution:
Autoclave or boil 200 ml of

J.

so that, after

Add

distilled

water

in a light-resistant

stoppered

more than 200 ml of distilled water to the flask


cooling, contents will measure 200 2 milliliters. Do not sub-

flask [15.1(H.9)].

slightly

steam pipeline condensate or special demineralized waters for diswater [5.3(B)]. Do not adjust volume of heated water by adding nonsterile water. With clean, dry forceps, or using some other procedure that
will minimize contamination of the tablet, promptly transfer one methylene
blue tablet to the flask of hot water. Allow the tablet to dissolve completely
stitute

tilled

before cooling the mixture.

Use

solution in proportion of

sterile, light-resistant glass

solutions weekly.

Mark

ml to 10 ml of sample. Keep dye solution

in

containers in a cool, dark place. Prepare fresh

date of preparation on containers.

K. Testing samples:

With buret or pipet, transfer 1 ml of methylene blue thiocyanate solueach tube either shortly before (preferred) or shortly after placing
10 ml of sample therein. After adding dye either to empty tubes or to milk in
tubes, avoid prolonged exposure of tubes to light, especially direct sunlight.
Immediately before or after transferring milk to tubes, identify each tube
legibly and indelibly with the producer's name or number 1.) by labeling
each tube, 2.) by making an appropriate chart for each rack of tubes, or 3.) in
some other unmistakable manner. Cover or stopper tubes loosely as soon as
the sample has been added and place them in a cooling bath. To monitor the
temperature adequately, place one tube containing 10 ml of milk and a thermometer [5.2(D)] in the rack with other sample tubes.
When each rack or batch of samples is complete, either 1.) start incubation promptly or 2.) transfer rack(s) of tubes or vials from cooling bath
to suitable temporary storage at 0-4.4 C to await a more convenient time for
tion to

incubation.

When

ready to incubate, bring temperature of the

test portions

Check heating rate by means of the


thermometer in the temperature-monitor tube. Loosen stoppers slightly. To
distribute cream when the temperature reaches 36 C, tighten stoppers, slowto 36

within 10 minutes [15.1(H.5)].

each rack of tubes three times, and record the time as the beginning
Do not substitute shaking for inversion.
If the number of samples or the temperature of contents, when samples
are removed from the cooling bath and placed in the incubating bath, are
ly invert

of incubation.

15.2 Resazurin Reduction

Method

191

such as to require tempering for longer than 10 min, preheat samples in an


auxiliary water bath controlled at a temperature not to exceed 40 C (104 F);
or place tubes in bath in smaller numbers and at successive periods. When
necessary to increase the rate of heat transfer, use more heated water or
forced agitation of water, or both. Determine temperature of samples by
placing a thermometer in the pilot tube containing 10 ml of milk; otherwise,
treat the pilot

tube as test samples in the batch are treated.

L. Recording

and reading

tests:

During incubation, observe color changes as follows:


1. If any sample is decolorized after incubation for 30 min, record

methylene blue reduction time as MBRT 30 minutes.


2. Following the initial 30-min reading, make all subsequent readings

at

successive hourly intervals.

Record such readings as reduction times in whole hours between


inversion of tubes [15.1(K)] and readings when decolorization is
noted. For example, if color disappears between 0.5- and 1.5-hr readings,
record the result as MBRT 1 hr; similarly, if between 1.5- and 2.5-hr readings, as MBRT 2 hr; and so on.
4. Immediately after each reading, remove and record all decolorized
samples and then slowly make one complete inversion of the remaining
3.

initial triple

tubes.
If readings are made at hourly intervals only, and reduction times are determined on that basis, samples may become decolorized shortly after a
reading. If such samples are credited with reduction times corresponding in
hours to time of reading when samples were found to be decolorized, recorded reduction time may in an extreme instance be almost one hr longer

than the actual reduction time. Such erroneous lengthening of recorded reduction times invariably implies that samples are of better bacterial quality
than the true result would indicate. Reading of samples as prescribed, there30 minutes.

fore, limits potential errors in recorded reduction time to

Because of uneven disappearance of blue color that may be noted as retest, record at each reading any sample as

duction progresses during the


decolorized

when

four-fifths of the visible portion of contents

is

white.

remind the observer when successive examinations should be made. To interpret reduction times, see 15. 1(D). Assign class
designation to samples according to grading system used.

Use an

interval timer to

15.2 Resazurin Reduction

Method

A. Introduction:
Resazurin, another oxidation-reduction indicator, is used in the resazurin
test. Resazurin has a high tinctorial value, imparting to fresh milk

reduction

a characteristic blue color. Reduction of dye

is

indicated by a gradual color

REDUCTION METHODS

192

change through various shades of purple and mauve to

full

pink, at which

point the resazurin apparently has been completely changed to resorufin. In


milk, this stage of reduction is irreversible. The second stage, consisting of
further reduction of resorufin to dihydroresorufin and characterized by a

fading of the pink color,

is

reversible. ^^ Reduction of

dye from the

original

more independent of both oxygen content and Eh


(oxidation-reduction potential) than does the change from pink to
blue to pink appears to be
^hjte9.16.20

Two

tests

have come into use. The "triple reading test"'^ measures time

required (up to 3 hr) to obtain dye reduction to a prescribed color end point.

"one-hour test,"^* the degree of color change

In the
1

is

determined after

hr of incubation.

raw milk:
The resazurin reduction test requires relatively little equipment and can be
done by reasonably careful workers who have normal color vision. In addiB. Application to

tion to

promptly furnishing indirect information concerning bacterial content

and/or activity, resazurin


associated substances.^'

is

^^-

sensitive to the reducing action of leucocytes or


'^-^"^^

This sensitivity

is

regarded as advanta-

geous by most workers. Inherent limitations of this method when applied


routinely to rnilk supplies are similar to those noted for the methylene blue
reduction test [15.1(C)], although results of this test seem to be less affected
by antibiotic residues in milk "^- ^' than are those of the methylene blue test.

Interpreting and reporting reduction times:


Application of the grading principle permits 1.) classification of samples
into two or more major groups, according to appreciable differences in bac-

use of descriptive grading terms to indicate whether or


"acceptable." When reduction times are shorter than
those permitted by regulations or industry standards, appropriate grading
terms clearly indicating lack of compliance should be used. Under no circumstances report resuhs as numbers of bacteria.
The triple reading test, because of its longer incubation period, has advantages over the one-hour test. Some properly cooled milks containing many
terial quality,

and

not the product

2.)

is

and other milks containing large numbers of leucocytes


may show little color change during the first hour of incubation.
Such milks may be missed by the one-hour test but often are detected by

dormant bacteria
**

''*

'*'

^^

'^

further incubation.

'^^'*

comparable to the methylene blue reduction


test in its agreement with the Standard Plate Count,^- '^ but the end point is
reached in about half the time required for reduction of methylene blue.*^

The

triple

reading test

*^

is

D. Standards:

The

provided for observing color and inthe end of three successive hourly intervals and classifica-

original triple reading test

verting tubes at

'''

15.2 Resazurin Reduction

193

Method

by results of the three hourly readings. Other schemes to


'^^'^
raw milk are based on somewhat different reading times.-tion of milk

USDA
standards
in

recommended standards

one for milk handled

in

for manufacturing milk

-^

classify

provide dual

farm bulk tanks and one for milk handled

cans. These time intervals represent the time required to reach the color

P 7/4 [15.2(G.3)].
The one-hour test is recognized

standard of 5

in the sanitary codes of two dairy product


Three classes of milk are established on the basis of purple,
lavender and pink end points [15.2(G.3)] after one hr of incubation. A recent
evaluation of the one-hour test ^ indicates that use of dual standards one
would
for milk handled in cans and one for milk handled in farm bulk tanks
be conducive to more uniform classification of milk supplies.

associations.-*'

Equipment and supplies for collecting samples:

E.

Provide equipment as

in 3.32(A).

E. Procedure for collecting samples:

Proceed as

in 3.32(B).

G. Equipment and supplies:

Provide materials as in 15.1(H). substituting certified resazurin tablets for


methylene blue thiocyanate tablets.
1. Resazurin tablets*: Certified by the Biological Stain Commission;
dye content per tablet approximately 11 mg.
2. Light source and filter: Essential in color comparisons tl8.4(A.7)].
3.

Color standards': For triple reading test, use a single color standard,
For the one-hour test, use a color grader consisting of four

7/4 (purple).

color standards: 5

and 10 P

PB

PB 7/5.5 (bluish purple); 5 P 7/4 (purple);


To prevent fading, avoid exposure of color

7/4 (blue); 10

7/8 (pinkish purple).

standards to strong lighting conditions.

H. Precautions:
Proceed as
/.

in 15.1(1).

Preparing dye solution:

Prepare solutions as

in 15.1 (J), substituting certified

methylene blue thiocyanate


J.

resazurin tablets for

tablets.

Testing samples:

except substitute resazurin solution for methylene


blue thiocyanate solution. When tube contents reach 36 C, slowly invert (do

Proceed as

in 15.1(K).

Available from Allied Chemical. P.O. Box 431. Morristown. NJ 07960.


^Color standards are available from Munsel Color Company. Inc.. 2441 North Calvert Street,
Baltimore.

MD

21218.


REDUCTION METHODS

194

not shake) each rack of tubes three times and then incubate [15.1(H.5)].

Avoid making

which can cause colors to change very

tests in bright sunlight,

rapidly.^

K. Reading the test:


Because color comparisons

may be

whether by daylight

or by

artificial light

unsatisfactory, examine tubes under a fluorescent tubular daylight

lamp against

background

a neutral gray

^- '^

who can make

of tubes only to persons

[18.4(A.7)]. Assign examination

the necessary color comparisons

accurately.
L. Resazurin triple reading test:^^

Proceed as

Adhere

in

15.2(E-K).

procedure during the incubation period:


for 1 hour. Record color at an appropriate time
before 1 hr has elapsed or at the end of 1 hr, in accordance with the grading
system used. Compare each tube with single color standard 5 P 7/4
1.

to the following

Incubate

at

36

[15.2(G.3)].
2.

After the

initial

incubation period of

hr, invert all tubes

once,

whether or not a color comparison has been made. Then continue incubation

C for an additional hour.


During or at the end of the second hour of incubation, make color
comparisons at appropriate time stipulated by grading system followed.
4. After the second hour of incubation, invert tubes once and continue
36

at

3.

incubation for a third hour.


5.

Make

final

color comparison at appropriate time during third hour of

incubation or at end of third hour, as dictated by grading system used.

M. Resazurin one-hour

test:

Proceed as in 15.2(E-K). After incubation at 36 1 C for 1 hr, compare


each tube with color standards under standard lighting conditions
[18.4(A.7)]. Assign class designation to samples according to grading system
used.

15.3 References
1.

Abele, C.A.

1945.

The methylene-blue reduction

content of milk, to determine

its

test as a

means of estimating

the bacterial

suitability for pasteurization or as a basis for grading. J.

Milk lechnol. 8:67-79.


2.

American Dry Milk Institute,


code for the dry milk industry.

3.

Dabbah,

R.,

Inc.

ADMI

Moats, W.A., Tatini,

1970.

Recommended

Bull. 915. Chicago,

S.R., and J.C.

sanitary/quality standards

III.

Olson,

Jr.

1969. Evaluation of the

resazurin reduction one-hour test for grading milk intended for manufacturing purposes.

J.

Milk lood Technol. 32:44-48.


4.

Dahhah,

R.,

Tatini, S.R., and J.C. Olson, Jr. 1967. Comparison of methods for grading
J. Milk Food Technol. 30:71-76.
The use of the resazurin comparator

milk intended for manufacturing purposes.


5.

Davis, J.G., and L.G.


light.

Newland.

Dairy Ind. 8:555-563.

1943.

in artificial

195

15.3 References

6.

7.

8.

Evaporated Milk Association. 1970. Evaporated milk industry sanitary standards code
and interpretations. Evaporated Milk Association, Washington, D.C.
Prayer, J.M. 1937. Dyes and methods as they afifect the methylene blue test. Bull. 424.
Vermont Agr. Exp. Sta., Burlington, Vt.
FR.AYER, J.M. 1938. The resazurin test

A preliminary study. Bull. 435. Vermont Agr. E.xp.

Sta., Burlington. Vt.

11.

Prayer, J.M. 1942. Dye reduction in milk related to Eh, pH and dissolved gases. Bull. 498.
Vermont Agr. Exp. Sta., Burlington, Vt.
Garvie. E.I., and A. Rowlands. 1952. The role of microorganisms in dye-reduction and
keeping-quality tests. 11. The effect of microorganisms when added to milk in pure and
mixed culture. J Dairy Res. 19:263-274.
Greene, V.W., and R.M. Jamison. 1959. Influence of bacterial interaction on resazurin

12.

Johns, C.K. 1938. Concerning the accuracy of the methylene blue reduction

9.

10.

reduction times.

J.

Dairy Sci. 42:1099-1100.


test. J.

Dairy

Sci. 21:227-237.
13.

Johns. C.K. 1939. Pactors influencing the reduction of methylene blue

in

milk. Sci. Agr.

19:435-457.

16.

Johns. C.K. 1939. Place of the methylene blue and resazurin reduction tests in a milk
control program. Amer. J. Pub. Health 29:239-247.
Johns, C.K. 1941. Some aspects of the resazurin test. p. 173. 15th Annual Report, New
York State Association of Dairy and Milk Inspectors.
Johns, C.K. 1942. Recent Canadian research on the resazurin test. Bull, 34th Year, Inter-

17.

Johns, C.K. 1954. Relation between reduction times and plate counts of milk before and

14.

15.

national Association of Milk Dealers 11:256-265.

after pasteurization.
18.

19.

20.

22.

Milk Pood Technol. 17:369-371.


penicillin in milk

Johns, C.K. and R.K. Howson. 1940. A modified resazurin


estimation of milk quality. J. Milk Technol. 3:320-325.

test for the

on the dye

more accurate

Johns, C.K. and R.K. Howson. 1940. Potentiometric studies with resazurin and methylene blue

21.

J.

Johns. C.K. and J.G. Desmarais. 1953. The effect of residual


reduction tests for quality. Canad. J. Agr. Sci. 33:91-97.

in milk. J.

Dairy Sci. 23:295-302.

Johns, C.K., and H. Katznelson. 1949. Penicillin and dye reduction tests for milk quality. J. Milk Technol. 12:133-136.
Little, L. 1940. Comparative studies upon the methylene blue and resazurin tests. J. Milk
Technol. 3:274-279.

23.

24.

Nelson, P.P.. and V.D. Poltz. 1938. Studies on resazurin as an indicator of the quality of
milk. p. 89. Report. 1936-38, Kansas Agr. Exp. Sta.
Ramsdell, G.A., Johnson, W.T.. Jr.. and P.R. Evans. 1935. Investigation of resazurin
as an indicator of the sanitary condition of milk.

J.

Dairy Sci. 18:705-717.

A. 1951. The effect of penicillin on the methylene blue reduction time and

25.

Rowlands,

26.

Thornton, H.R.

keeping quality of milk. Proc. Soc. Appl. Bacteriol. 14:7-14.


1933. The use of the methylene blue reduction

test.

Canad.

J.

Pub. Health

24:192-196.
27.

Thornton, H.R.. and R.B. Sandin.

1935. Standardization of the

methylene blue reduc-

by the use of methylene blue thiocyanate. Amer. J. Pub. Health 25:1114-1117.


28. Thornton. H.R., Strynadka. N.J., Wood, P.W., and C. Ellinger. 1934. Milk contamination and the methylene blue reduction test. Canad. J. Pub. Health 25:284-294.
29. United States Department of Agriculture, Consumer and Marketing Service. 1972.
Milk for manufacturing purposes and its production and processing. Requirements recommended for adoption by state regulatory agencies. Ped. Register 37:7046-7066 (April 7).
30. Wilson, G.S. 1935. The bacteriological grading of milk. Special Report Series 206, Medical
tion test

Research Council (England).

CHAPTER

16

MICROBIOLOGICAL TESTS FOR EQUIPMENT,


CONTAINERS, WATER AND AIR
Bengsch and
Donald Vesley

R.Y. Cannon, C.E. Beckelheimer, Harold

Introduction

16.1

Good sanitation practices require that contamination of dairy products


from equipment, containers, water, and air be eliminated. Following
pasteurization of the product, contamination with pathogenic microorganisms has a direct public health significance. In addition, contamination
with psychrotrophic organisms will have a direct bearing on the shelf life of
refrigerated products. Only one psychrotrophic organism per package of
fluid milk products is significant to maintenance of quality.*^

Sample Collection (Chapter 3)

16.2

16.3 Tests for Sanitation of


1

Equipment

Rinse Solution Method


Effectiveness of cleaning and sanitizing containers and equipment can be

6.31

determined by repeated flushing of a measured volume of sterile, buff"ered


sanitizer-neutralizing solution or nutrient broth over the product contact surface and then determining the bacterial population of the rinse solution by
plating or

membrane

filter

techniques.'

Large equipment assemblies can be checked by circulating water that has


been chlorinated and neutralized, or sterilized by membrane filtration '^ and
determining the increase in bacterial population by membrane filter.' Sanitation of assemblies may also be checked by sampling milk progressively at
^^
points of access along processing lines.

A. Equipment and supplies:


1. Transfer pipets, sterile.
2.

3.
4.

Petri dishes, sterile.

Standard Methods Agar, VRB Agar, MF Endo Broth


Stock phosphate buffer solution [4.7(A.l)].

ISC Liaison: R.T. Marshall


197

[4.1].

MICROBIOLOGICAL TESTS: EQUIPMENT, CONTAINERS, AIR AND WATER

198

sterile: Add 1.25 ml of stock phosphate bufml of 10% aqueous sodium thiosulfate, 4 g of Asolectin* and
10 g of Tween 20^ to microbiologically suitable (MS) water and make up to
1 liter. Adjust to pH 7.2 and dispense sufficient quantities into screwcapped
vials made up to contain 20 ml, 100 ml or other needed volumes, following
sterilization. Asolectin is hygroscopic and should be stored in a desiccator.
Weigh the powder and rapidly dissolve by heating over boiling water. (Neutralizing Buffer, Difco Laboratories, Detroit, is available in dehydrated
form. A solution similar to the above rinse solution is produced on rehydra5.

Buffered rinse solution,

fer solution, 5

tion.)
6. Rinse water for large volume rinsing of "cleaned in place" (CIP)
equipment: Large volume of water may be treated or sanitized by chlorinating to a residual concentration of 25 mg per liter, holding for 10 min, and then
neutralizing by adding an excess of 10% sodium thiosulfate solution.
Tap water may also be sterilized by membrane filtration followed by addi-

tion of

sodium thiosulfate

Sodium

7.

to inactivate residual disinfectant.'^

thiosulfate solution, 10%: Dissolve

MS

water and make up to 1


place, or in an amber bottle.

liter; filter;

8.

Hypodermic syringe and needles,

9.

Ethyl alcohol, 70%.

lOOg of Na2S203-5H20

in

store in the refrigerator in a dark

sterile.

B. Rinsing containers:

Sample containers, obtained as directed

in

3.13(D) will be handled as

fol-

lows:

Firm-walled capped items: Remove containers from the conveyor

1.

line

without touching

valves.

lips

or interiors before containers reach the

Aseptically introduce 20 ml

of sterile,

[16.31(A.5)] into each container, then insert the containers into the

beyond the

lines

larger than
that

filler

filler

buffered rinse solution

conveyor

valves, to be capped mechanically. For container

gal, introduce 100

mechanical cappers are

in

ml of rinse solution. Should inspection show


an unsanitary condition, apply laboratorytaken from the conveyor before

sterilized closures to three other containers

filler valves. Proceed as in 16.3 1(B. 3).


Firm-walled uncapped items: Paper, laminate or plastic containers
that are formed, filled and sealed in a single device must be obtained after
sanitization but before the product to be packaged is introduced. Swab a

reaching the
2.

portion of the side surface of containers with


sterile

inject

70%

alcohol, aseptically

fill

hypodermic syringe with 20 ml of sterile, buffered rinse solution, and


into the container. For a container larger than
gal, introduce 100 ml
1

of rinse solution. Aseptically apply a piece of cellophane tape to cover the


area punctured by the needle. Proceed as

in 16.3 1(B. 3).

'Associated Concentrates, 32-60 Sixty-first Street. Woodside,


^Atlas Chemical Industries, Inc., Wilmington,

DE

19839.

NY

11377.

199

16.31 Rinse Solution Metliod

Shaking firm-walled containers: Grasp the container firmly with its


in horizontal position. Shake vigorously 10 times, each shake being
a "to-and-return'' thrust of about 20 centimeters. Turn the container 90
and repeat the horizontal shaking treatment; turn the container 90 twice
more and repeat the horizontal shaking. Swirl the container vigorously
3.

long axis

20 times

in

a small circle with the long axis in vertical position, then invert

and repeat.
4. Flexible-walled items: Swab all caps on fill tubes of collapsed liners
with a soft paper towel moistened with 70% alcohol, then remove and introduce, aseptically, 20 ml of buffered rinse solution [16.3 1(A. 5)] with a sterile

pipet.

70%

gal,

introduce 100 ml of rinse solution.

swab an area of

the tube adjacent to the liner with

For containers larger than

Where tubes

are sealed,

alcohol and introduce rinse with a sterile hypodermic syringe; apply

cellophane tape to the puncture area.

on a smooth, clean, firm horizontal surface as


flat as its construction permits. With hands or a roller, "work" the rinse
solution back and forth 10 times, contacting all interior surfaces completely.
With sterile scissors, cut an end of uncapped containers to obtain contents.
Lift fill tube of flexible liner before removing closure (or cutting tube with
sterile scissors) to avoid losing rinse solution. Then lower tube to permit
Arrange the collapsed

liner

aseptic transfer of rinse to sterile container.

method may be employed


on hookups designed for cleaning in
place. A sufficient volume of treated rinse water [16.31(A.6)] is added to a
storage vat or tank at the upstream end of the assembly, then pumped or
allowed to flow by gravity through the assembly. A control sample (approximately
liter) of treated rinse water is obtained before rinsing. Samples are
collected at the point of discharge from the first, middle and final portions of
rinse water. Samples may also be collected at various points throughout the
5.

Equipment assemblies: The

rinse solution

on individual items of equipment or

assembly.

and counting plates:


Container samples: Distribute 10 ml of the 20-ml or 100-ml rinse
solution equally among three sterile petri dishes; seed two other petri dishes
with 1-ml portions of the rinse. Pour dishes which received 10 ml of rinse
with 15-18 ml of Standard Methods Agar; pour others in the usual manner.
Plating rinse solutions

1.

Incubate plates at 32 C for 48 3 hr and count colonies. Results are recorded as Residual Bacterial Count (RBC) per specified container capacity in ml
as follows:
a.

When

containers are rinsed with 20 ml (or 100 ml) of rinse solution


total number of colonies on plates by

and 10 ml are plated, multiply


2 (or 10) to record

present, record as
b.

When

numbers of colonies per container.


<2 (or <10) per container.

If

no colonies are

containers are rinsed with 20 ml of rinse solution and 2 ml


number of colonies on plates and multiply by 10.

are plated, count total

MICROBIOLOGICAL TESTS: EQUIPMENT, CONTAINERS, AIR AND WATER

200

Numbers

of coliforms in rinse solutions are determined by dividing 5 ml of


between two plates and culturing as directed in 6.8. Number of

rinse solution

colonies on the two plates multiplied by 4 when 20 ml of rinse solution are


used, or by 20 with 100 ml of rinse solution, is recorded as coliforms per
specified container capacity.

Membrane

filter

procedures

may

also be used with the rinse solutions.

Yeasts, molds, proteolytic bacteria, or other specific microorganisms may


be determined similarly by use of appropriate differential media and in-

cubation temperatures and times.


2. Equipment samples: Rinse solutions obtained from cleaned-in-place
(CIP) systems or processing assemblies and control samples require use of
the

membrane

samples taken
yield

(if

filter

at the

procedure

'

for analysis.

Average the yields of rinse

beginning, middle and end of drainage and subtract the

any) of control samples. Calculate ratio of sample volume to rinse


yield to obtain an indication of numbers of

volume and multiply by corrected

organisms present in the entire system. Presence of specific types of organisms can be determined by employing appropriate differential media and
incubation conditions, e.g., MF-Endo Broth would be used for coliform
counts.

Milk samples obtained from processing lines (as

in

Chapter

3)

may be

plated directly to determine where significant change in total counts occur.


Samples may be laboratory-pasteurized (Chapter 7) and then plated to de-

termine presence of thermoduric bacteria. Presence of psychrotrophic bacteria may be determined by incubating samples at 7 C for 5 days and then
replating. A substantial increase in count indicates presence of psychrotrophs. Points of entry of other types of microorganisms can be determined by plating with appropriate differential media.

6.32 Surface Contact Methods


Microbiological examination of surfaces requires selection of the proper

method. RODAC (Replicate Organism Direct Agar Contact) plates and the
swab procedure are usually the methods of choice. The swab technique
should be used for areas such as cracks, corners or crevices, i.e., areas of
such dimension that the swab will be most effective in recovering organisms
from them. The RODAC procedure is better used on flat surfaces such as
walls, floors, ceilings and equipment surfaces. Selection of the proper technique
A.

is

essential for meaningful results.'*-

Swah

''^

contact method:

The swab technique

swab moistened in an
The swab is then rinsed

consists of brushing a sterile

appropriate solution over a measured surface area.

in a measured amount of sterile solution and the liquid examined


microbiologically. Select areas for swabbing which appear to be unclean or

thoroughly

difficult to clean.

Equipment surfaces which are

regular, smooth, ground,

polished, large or accessible are easier to clean than those which are irregu-

16.32 Surface Contact

lar,

ly

Methods

201

rough-seamed, ridged, small, rounded on small

reached.

If

radii, or less

convenient-

these latter areas are not properly cleaned, residues will accu-

mulate, and surfaces will not be sanitized

when chemical

solutions are

applied.
1.

Equipment and

See Chapter

supplies:

5,

16.31(A), and the following

modifications:
a. Screw-capped
screw-capped vials

vials

Dispense rinse solution [16.31(A.5)]

(7 to 10

cm

in small

long) prepared to contain 5 ml of solu-

swabs with wood applicator sticks are autois no change in pH).


Sterile nonabsorbent cotton, firmly twisted to apb. Cotton swabs
proximately 5 mm in diam by 2 cm long over one end of a wood applicator stick 12 to 15 cm long. Package in individual or multiple convetion after autoclaving. (If

claved

in

the rinse solution, ascertain that there

nient, protective containers (glassine envelopes, test tubes or vials),

with swab heads


cotton swabs

MS

is

away from

the closure, and autoclave. If toxicity of

suspected, autoclave a representative sample of swabs

ml per swab) at 121 C for 15 min and do the Interval


MS water (Chapter 4) to show the absence or
presence of toxicity. Dacron and rayon swabs may also be used.'^
Swabs made of calcium alginate fic. Calcium alginate swabs
bers 2.5,8,17,22 Qpg soluble in aqueous solutions (rinse, culture media,
in

water

(5

Plating Procedure for

etc) containing

1%

of sodium hexametaphosphate (or sodium glycero-

phosphate, or sodium citrate, or 1% of any mixture of these). All organisms dislodged from swabbed surfaces are thus liberated. When using
calcium alginate swabs,* prepare rinse solution vials to contain 4.5 ml
(rather than 5.0 ml) after sterilization.

swab Prepare a 10% sosodium hexametaphosphate or other solubilizing compound,


package in appropriate vials and autoclave.
e. Mechanical shaker (optional).
2. Procedure:^' ^' Open the sterile swab container; grasp the end of a
stick, being careful not to touch any portion which might be inserted into a
vial, and remove aseptically. Open a vial of bufi^ered rinse solution; moisten
the swab head and press out excess solution against the interior wall of the
vial with a rotating motion. Hold the swab handle to make a 30 angle with
the surface. Rub the swab slowly and thoroughly over approximately 50 cm^
of surface three times, reversing direction between successive strokes.
Move swab in path 2 cm wide by 25 cm long or other dimensions to cover
equivalent area. Return the swab head to the solution vial; rinse briefly in
solution, then press out the excess. Swab four more 50 cm- areas of sanitized
surface, as above, rinsing the swab in solution after each swabbing; remove
excess liquid. After the fifth area has been swabbed, position the swab head
d.

Solubilizing solution for calcium alginate

lution of

*Inolex Biomedical Division,

Glenwood, IL 60425.

MICROBIOLOGICAL TESTS: EQUIPMENT, CONTAINERS, AIR AND WATER

202

in the vial and break or cut it with sterile scissors leaving the swab head in
the vial. Replace screw cap; put vial in a waterproof container packed in

cracked ice and deliver to the laboratory. With calcium alginate swabs follow the standard procedure, but after swabbing the final (fifth) 50 cm'^ area
and depositing the swab head in the rinse vial add 0.5 ml of sterile solubilizing solution.
3.

remove

Preparing and plating rinse solutions from swabs: At the laboratory,


vial from refrigerated storage. Shake vigorously, making 50 com-

plete cycles of 15

cm

in 10 sec, striking the

palm of the other hand

at the

end

manually; or

may be put into appropriate racks and shaken


mechanical shaking may be employed for a time interval which

treats cotton

swab heads

of each cycle.

Groups of

vials

similarly to the

manual procedure. Plate

2-, 1- and/

or 0.1-ml portions of rinse solutions. Plate additional decimal dilutions, if


plates with Standard Methods Agar. Incubate

deemed necessary. Pour

plates, count colonies, and then calculate the number of colonies recovered
from 50 cm- (equivalent to 1 ml of rinse). When other than "total counts"
are sought, plate with appropriate selective or differential media and in-

cubate as required.
^^''^^
B. RODAC Plate {Agar Contact) Method:^'^The RODAC plate method provides a simple agar contact tool for sampling surfaces. It is of value in estimating sanitary quality of surfaces en-

countered

in

dairy operations, food-processing plants, and hospitals. Col-

laborative studies

'^

have indicated that the mean per cent recovery of

was slightly
50%, both for the swab (47%) and RODAC plate (41%) methods.
However, results from the RODAC plate method were more reproducible
than those from the swab technique. The RODAC plate method is particularly recommended when quantitative data are sought from flat, impervious

aerosolized Bacillus subtilis spores from stainless steel surfaces


less than

It should not be used for pervious, creviced or irregular surfaces.


Product contact surfaces amenable to RODAC sampling necessarily will
be flat and impervious, and thus relatively easy to clean and disinfect. Other
environmental surfaces, such as floors, more likely will be heavily contaminated. In either event, a sufficient number of spots should be sampled to

surfaces.

obtain representative data.


1. Apparatus and materials: Disposable plastic RODAC plates* may be
purchased prefilled with test medium, or they may be filled in the laboratory.
They should be prepared with 15.5-16.5 ml of Standard Methods Agar, and
the meniscus of the agar should rise above the rim of the plate. Where surfaces have been subjected to germicide treatment, incorporate appropriate
neutralizers in the medium. Use 0.5% polysorbate (Tween) 80 to neutralize
substituted phenolic disinfectants and 0.07% soy lecithin to neutralize qua-

ternary

ammonium compounds.

^Falcon Plastics, 550 West 83rd Street, l.os Angeles, CA. 90052.

203

16.4 Tests for Water Supplies

Dey-Engley medium" may be used.

It

contains a variety of ingredients able

to neutralize germicides likely to be used on surfaces.^'


If qualitative

may be

data are sought, differential media

used, or prefera-

bly colonies should be transferred to differential media from the original

broad-spectrum medium. Following preparation, plates should be incubated


C for 18 to 24 hr as a sterility check. They should then be used within
succeeding
12 hr unless appropriately wrapped and refrigerated to slow
the
at 32

dehydration.
2.

Procedure:

Remove plastic cover and carefully press agar surface to


Make certain that entire agar meniscus contacts the

surface being sampled.

surface, using a rolling uniform pressure on the back of the plate to effect

contact. Replace the cover and incubate plate in an inverted position for

24-48 hr at 32 C. When heavy contamination is present, incubate 24 hr to


prevent overgrowth. When contamination is light, 48 hr should be allowed
for growth of colonies. Count colonies and record as either number of colonies per

RODAC

plate or

number of colonies per cm^.

16.4 Standard Tests for Water Supplies

Water

for milkhouse

and milking operations

shall

be from a supply proper-

protected and operated, and shall be easily accessible, adequate


for the purpose, and of a safe, sanitary quality.'
ly located,

To be

suitable for use, water must, in addition to

complying with sanitary

requirements, be practically free of microorganisms that could

initiate

prod-

uct spoilage. Spoilage of refrigerated milk and milk products has frequently

been caused by inoculation with water-borne organisms (primarily psychrotrophs).-^ This contamination has occurred either directly, through
product contact with the water itself (e.g., by milk traversing a surface that
was freshly rinsed with water, or by cheese curd or butter washed in water);
or indirectly, by microbes metabolizing nutrient residues on incompletely
cleaned equipment surfaces. Since most of the microorganisms of water are
destroyed by chlorination or heat, in many plants all water is treated to keep
spoilage at a

minimum.

Procedures to sample and test water supplies to determine conformance


with requirements for coliform concentration by both standard most probable number (MPN) and membrane filter methods are completely detailed in
the most recent edition of Standard Methods for the Examination of Water
and Wastewater. Although limitations for total bacterial counts are not included in drinking water specifications, information on numbers of organisms developing at various incubation temperatures is helpful both in
classifying untreated waters and in assaying the efficiency of treatment procedures. The membrane filter technique is particularly useful to determine

"Difco, Inc., Detroit, Mich. 48233.

MICROBIOLOGICAL TESTS: EQUIPMENT, CONTAINERS, AIR AND WATER

204

numbers of organisms in supplies containing so few


procedures do not yield meaningful results.

that the usual plating

16.5 Tests for Microbiological Quality of Air (See Appendix)

The demand for improved shelf life of dairy products has put increased
emphasis on the microbiological quality of air in dairy environments. Entrance of a very few viable contaminants from air into pasteurized and cultured dairy products can cause spoilage.^ Entry of any microorganisms into
sterilized dairy products is undesirable. The requirement that dry milk products be free of Salmonella emphasizes the need to reduce the microbial content of air used for cooling and/or instantizing of dry milks.
Sources of airborne contamination in dairy plants include people, various
dusts, drains, insects, rodents, and products and materials received into the
plant.

'^'-'^

Basic methods to determine viable particulate matter in air include such


techniques as sedimentation, impingement in liquids, impaction on solid surfaces, centrifugation, and filtration.'"

16.6 References
1.

2.

3.

American Public Health Association.

1975. Standard Methods for the Examination of


Water and Wastewater, 14th ed. American Public Health Association, Washington, D.C.
Angelotti, R., Foter, M.J., and K.H. Lewis. 1958. A comparative evaluation of methods for determining the bacterial contamination of surfaces. Food Res. 23:175-185.
Angelotti, R., Wilson, J.L., Litsky, W., and W.G. Walter. 1964. Comparative evalu-

ation of the cotton

swab and

contamination from stainless


4.

Baldock.
terial

5.

RODAC

methods

steel surfaces.

for the recovery of Bacillus subtilis spore

Health Lab. Sci. 1:289-296.

J.D. 1974. Microbiological monitoring of the food plant:

contamination of surfaces.

Barnes, J.M.

1952.

J.

Methods

to assess bac-

Milk Food Technol. 37:361-368.

The removal of bacteria from

glass surfaces with calcium alginate,

gauze, and absorbent cotton-wool swabs. Proc. Soc. Appl. Bacteriol. 15:34.
6.

Bond, R.G., Halbert, M.M., Keenan, K.M., Putnam, H.D., Ruschmeyer, O.R., and
D. Vesley. 1963. Development of a method for microbial sampling of surfaces, with special
reference to reliability. Final report under contract PH-86-62-182, Division of Hospital and

Medical

Facilities.

Bureau of State Services, Public Health Service, U.S. Department of

Health, Education and Welfare, Washington, D.C.


7.

BucHBiNDER,

L.,

BucK,

Jr.,

1947. Investigations of the

8.

T.C, Phelps, P.M., Stone, R.V., and W.D. Tiedeman.

swab

rinse technic for

nation of cleansed eating utensils. Canad.


9.

10.

J.

alginate soluble

utensils.

wool for the exami-

Pub. Health 44:464-467.

Cannon, R.Y., and K.K. Reddy. 1970. Contamination of milk with air-borne microorganisms through the vacuum defoamer. J. Milk Food Technol. 33:197-201.
DiMMicK. R.L.. and A.B. Akers. 1969. An Introduction to Experimental Aerobiology.
John Wiley

11.

examining eating and drinking

Amer. J. Pub. Health 37:373-378.


Cain, R.M. and H. Steele. 1953. The use of calcium

&

Sons, Inc., N.Y.

Engley, F.B., and B.P. Dey.

1970.

universal neutralizing

medium

for antimicrobial

chemicals. Chemical Specialities Manufacturer's Association, Proceedings of the 56th

Meeting.

205

16.6 References

12.

13.

Favero, M.S.. McDade, J.J.. Robertson, LA.. Hoffman, R.K., and R.W. Edwards.
1%8. Microbiological sampling of surfaces. J. Appl. Bacteriol. 31:336-343.
Greene, W.W., Vesley, D.. Bond, R.G., and G.S. Michaelsen. 1962. Microbiological
contamination of hospital

14.

faces
15.

air.

1.

Quantitative studies. Appl. Microbiol. 10:561-566.

Hall, L.B., and M.J. Hartnett.


in hospitals.

1964.

Measurement of

on

bacterial contamination

sur-

Pub. Health Rep. 79:1021-1024.

Harper, W.J., and C.W. Hall.

Technology and Engineering,

1976. Dairy

p. 545.

The Avi

Publishing Co., Inc., Westport, Ct.


16.

Heldman, D.R.
plants.

17.

18.

J.

HiGGiNS, M. 1950. A comparison of the recovery of organisms from cotton-wool and calcium alginate wool swabs. Ministry of Health and Public Health Laboratory Service (Gt.
Brit.) Monthly Bull. 9:50-51.
HoLLiNCiER. N.F., and L.H. Lindberg. 1958. Delayed recovery of streptococci from

Amer.

throat swabs.
19.

1967. Significance and control of air-borne contamination in milk and food

Milk Food Technol. 30:13-17.

Marshall,

J.

Pub. Health 48:1162-1169.

plants as determined by use of the rinse


20.

filter

method.

Tiedeman, W.D.,
utensils, pp. 68-70.

22.

J.

Sing, E.L., Elliker, P.R., Christensen, L.J., and W.E. Sandine. 1967. EflTective
ing procedures for evaluating plant sanitation.

21.

in twelve fluid milk processing


Milk Food Technol. 36:237-241.

R.T., and R. Appel. 1973. Sanitary conditions

N.Y.
Tredinnick,

1948.

J.

test-

Milk Food Technol. 30:103-111.

Chairman. Technic for the bacteriological examination of food


report. Amer. J. Pub. Health Yearbook, 1947-48, Part 2,

Committee

J.E., and

J.

Tucker.

1951.

The use of calcium

alginate wool for

swabbing

dairy equipment. Proc. Soc. Appl. Bacteriol. 14:85.


23.

Walter, W.G., and


flat

24.

surfaces.

J.

Witter, L.D.

J.

Potter. 1963. Bacteriological

field

studies on eating utensils and

Environ. Health 26:187-197.


1961. Psychrophilic

bacteria A review.

J.

Dairy Sci. 44:983-1015.

CHAPTER

SEDIMENT
E.O. Wright,

W.L

IN

17

MILK

Arledge, W.S. Clark,

Jr.,

M.H. Roman, and R.W. Webber

17.1

Introduction

A sediment test consists of filtering a specific volume of milk through a


white sediment disc and observing type and amount of residue. Appearance
and quantity of residue roughly serve to measure visible insoluble matter in
samples. Discs are compared in appearance with several discs prepared by
filtering

milk with

known amounts

of standard sediment mixtures, which

permits classifying milk according to sediment content. Measured portions


fine sediment mixtures filtered directly onto a set of

from either coarse or

official photographs of standard discs are used


comparisons with tests on raw milk samples.''''^'*' Discs prepared from a
fine sediment mixture are preferred for comparison with tests on bulk tank,
processed and consumer package samples. Special procedures, often involving chemical treatment and heating of samples, may be required to make
cream and other fluid dairy products filterable.'^
Sediment found in milk in different regions and at different seasons of the
year may vary in composition, specific gravity, color and other physical
characteristics, so that a quantity of sediment in one locality may appear

standard reference discs, or

for

on sediment discs from equivalent amounts obtained elsewhere.


on dairy farms has reduced the
value of sediment tests on milk as delivered to receiving plants. Although
presence of sediment in milk indicates unsanitary methods of production or
handling, its absence does not prove that sanitary conditions have prevailed.
When most sediment has been removed from milk before delivery, farm

different

Efficient use of single-service strainers

inspection during milking or plant inspection during processing

may

reveal

times or have been cleaned.^

whether products have been kept clean at all


Likewise, even when appreciable sediment is present in milk when received
by the processing plant, efficient use of filters and clarifiers at the plant
serves to reduce the value of sediment tests on retail products. Despite these
limitations, these tests are simple and permit rapid measurement of sediment
introduced by careless practices on farms or in processing plants.

ISC Liaison: W.S. Clark.

Jr.

207

SEDIMENT

208

Sediment testing of milk

at

IN

MILK

milking time before filtering will assist in deter-

mining the degree of cleanliness. This procedure can be used to aid the
dairyman in those instances when unacceptable sediment tests continually
result.

17.2 General

Two

Methods

methods are used to determine sediment in milk: the


"mixed-sample" method and the "off-bottom" method.
The mixed-sample method generally uses standard discs prepared from
fine sediment mixtures for comparisons. This method commonly is used to
general

determine sediment in milk from farm bulk tanks, transportation tanks and
storage tanks, during processing, and in the consumer package.
The ojf-bottom method uses discs prepared from coarse or fine sediment
mixtures for comparisons (choice depends on type of sediment for which

samples are being tested). This method was developed to measure amount of
sediment which collects on bottoms of cans of unstirred milk as oflFered by
dairymen. The off-bottom method eflfectively demonstrates carelessness in
production to dairymen and offers evidence of unsanitary methods to control
officials.

Off-bottom sediment

because

a.)

pended

fine

some

sediment;

ing; c.) surface

tests

may

not always reveal true conditions,

unstirred milk as delivered contains considerable susb.)

milk

may have been

agitated shortly before test-

contours of some can bottoms vary; or

d.) the

operating the sediment gun (for example, air-operated guns

technique of

may work

allow enough time to draw the gun across the bottom of the can)

fast to

too

may

vary.

The mixed-sample method is recommended for milk in farm bulk tanks.


shown no uniformity among tanks as to dimensions,
amount of milk, or location of sediment.** It has been been demonstrated that
for practical purposes sediment recovered from a
gal (3.79 liters) of mixed
Field experience has

sample

is

equivalent to the sediment from a

pt (0.47 liter) oflf-bottom

sample.**-^

Methods

for determining extraneous matter in dairy products, exclusive

of sediment in milk, are laboratory procedures, whereas methods for detecting sediment in raw milk are classed as platform tests. Procedures to detect
and determine extraneous matter in other dairy products have been described elsewhere in this book.

17.3

Equipment and Supplies

A. Tester:*

The

tester shall be simply constructed, easily cleaned,

and quickly adjust-

able between testing of samples to permit sanitary removal of the used disc

*Available from Sediment Testing Supply

Company,

Company,

1512

West Jarvis Avenue, Chicago,

III.

430 East Main Street, Box 157, Greenwood, Ind.


46142; NASCO, Ft. Atkinson, Wise. 53538; and MOG Division. New York Laboratories, 901
E. New York Ave., Brooklyn, NY 11203.
60626; Clark Dairy Supply

Inc..

17.3

Equipment and Supplies

209

and replacement with a clean

NOTE

disc.

Before using, check tester for reproduc-

sediment must not bypass filter


impeded in its flow. Select type of tester suited to method of
sampling and sample size.
For proper sampling procedure, see Section 3.34.
ibility

of results (see

17.3(C)). Milk or

disc or be

Mixed-sample method, pressure or vacuum-type tester:


Equip a single-unit off'-bottom type of tester, or other suitable tester, with
a special head having appropriate filtering diameter for sample size.
B.

C. Ojf-hottom tester:

The off-bottom tester may be a single-unit type, for intake of one pt (0.47 1)
of milk on upstroke of plunger and discharge through disc on downstroke,
or a two-unit type containing one unit for removal of one pt (0.47 1) of milk
from bottom of can and another for filtering the sample. Filtering diameter
should be IVs

NOTE:

in.

(2.86 cm).

Directions for use of standard sediment material

in

checking sedi-

ment testing devices for reproducibility have been published.'-^ There are
two basic requirements; .) measure quantity of milk delivered to assure that
appropriate sample size is withdrawn and passes through disc; and 2.) be
sure the entire sample passes through disc and that no portion of sample or
1

sediment bypasses disc.

D. Sediment discs

:^

Standard sediment discs, Wa in. (3.18 cm) in diameter, for use over the flat
in the tester, to expose a filtration diameter of Ws in. (2.86 cm),

screen
0.40

in.

(1.02 cm), 0.20 in. (0.51 cm), 0.14 in. (0.35

as applicable.

The

cm) or 0.10

in.

(0.25

cm)

disc must not contain phenolic resins or other chemicals

may contaminate milk.


NOTE: To test sediment discs,

that

filter 12

mg

of fine standard sediment mix-

ture (60-ml aliquot) through the disc, using a clean flask to catch the
trate.'-^

Transfer the

filtrate to

fil-

a beaker. Rinse flask three times with water

and add rinsings to the beaker. Refilter the filtrate through a 7- or 9-cm
S & S No. 589 White Ribbon paper, or equivalent, that has been washed
with about 200 ml of water, dried to constant weight at 100 C, and cooled in
a covered dish in a desiccator before weighing. Rinse beaker and paper thoroughly with water. Dry paper to constant weight as above. Test not fewer
than three discs. Average weight of sediment per disc passing through three
or more discs should not exceed 2.8 mg. A standard disc prepared from a
fine sediment mixture should not appear to have sediment buried beneath
the surface.

^Kendall

Company, Fiber Products

Eaton, Ohio 45320.

Division, Walpole, Mass. 02081 and

Food

Filters Corp.,

SEDIMENT

210

IN

MILK

White cardboard or special cards:


White cardboard or special cards for mounting discs with a semi-gloss to

E.

dull finish are preferred.

17.4 Preparation

and Use

of Standard

Sediment Discs

Directions to prepare coarse and fine sediment standard discs have been
published,^ and laboratories wishing to prepare standard discs

these directions.

However,

it

may be more

may

refer to

practical to use photographs of

standard discs.

The grading method


or less than that on the

is

as follows:

first

When

disc, read

it

sediment

in

a sample

as the value of the

first

is

equal to

disc. If the

sediment exceeds that shown on a standard disc or photograph, read it to the


next higher classification and report sedimentation as "not exceeding
mg." Report
mg," or in the event of a rejection, report it as "exceeds

whether a mixed or an off-bottom sample was used. Rapid screening for


sediment content may be accomplished by comparing sample sediment disc
to nearest standard disc. The screening method should not be used for milk
exceeding the highest standard sediment disc on recognized official charts.^
A. Photographs of standards:
Photographs of standard discs may be used as a guide in grading sediment
tests. Do not use photographs that have become faded, stained, soiled or
otherwise damaged. Photographs of standard discs with diameters of P/s in.
(2.86 cm), 0.40 in. (1.02 cm), 0.20 in. (0.51 cm), 0. 14 in. (0.35 cm) and 0.10 in.
(0.25 cm), have been prepared by the U.S. Department of Agriculture* in
cooperation with the U.S. Food and Drug Administration and the American
Public Health Association.

17.5

Procedure

A. Mixed samples:'''

'"

Pass the sample through a disc [17.3(D)], held in correct position in the
Temper one pt (0.47 liter) of milk to 35-38 C (95-100 F) and filter

tester.

in. (1.02 cm) in diameter [17.3(A)]. If a single-unit offbottom tester with a special head is used, temper a sample larger than one pt
(0.47 liter) to 35-38 C, withdrawing one pt (0.47 liter) with tester while stirring; or, draw one pt (0.47 liter) into tester and warm milk by holding tester
under running hot water before discharging milk through discs. (Milk varies
in its rate of flow through discs. Pasteurized milk may be more difficult to
filter than raw milk. Other factors influencing rate of flow are temperature,
fat content, freezing, high acidity, degree of clumping of fat globules, stage
of lactation, presence of abnormal milk, and amount of sediment in sample.)

through a disc 0.40

*These grading charts may be purchased from the Standardization Branch, Dairy Division,
Department of Agriculture. Washington, D.C. 20250.

Agricultural Marketing Service, U.S.

211

17.6 References

Remove

disc

from

tester

and mount on white cardboard, a special card, or

sized paper [17.3(E)]; or, store in an individual transparent


If disc is

placed on paper while

still

waxed envelope.

moist, milk acts as an adhesive. Grade

by comparison with standard discs [17.4(A)] and indicate on report whether


was graded wet or dry. Character of sediment may be determined by
microscopic examination.
To prevent decomposition on storage, spray used discs with an alcoholic solution containing 2.5 g each of menthol and thymol in 100 ml, or
disc

40%

formalin.

Do

tached, moisten

it

not use glue to affix disc to paper. If disc becomes dewith a few drops of water and remount. Protect from con-

tamination.
B. Off-bottom samples:

Take sample from bottom of can while moving sampling device diametrically across bottom of can, or around circumference if can has a high-center
bottom. Synchronize withdrawal of pint sample with movement of tester
head as follows: Pull plunger up in tube as head of tester is moved once
across bottom of can, or around circumference.
If the tester is of the single-unit type, discharge milk with a complete
downstroke of the plunger. After tester is removed from can, draw plunger

up 6 or 8 in. (15-20 cm) and quickly push plunger to


disc from tester and proceed as in 17.5(A).

its full

length.

Remove

C. Equivalent diameter disc methods:

0.20-, 0.14- or 0.10-in. (0.51-, 0.35-, or 0.25-cm) diameter sediment

cm) diameter disc in the mixed


head having appropriate filtering diameter, is substituted for the tester having the 0.40-in. (1.02 cm)
diameter filtering disc. Suitable equipment is available as indicated in 17.3.
The following mixed milk sample sizes are used with the appropriate filter-

disc can be substituted for the 0.40-in. (1.02

sample method.

suitable tester, with special

ing diameter:

cm)
0.20-in.
0.14-in. (0.35 cm)
0.10-in. (0.25 cm)

4-oz (120 ml) sample


2-oz (60 ml) sample
1-oz (30 ml) sample

Follow procedures described

(0.51

filtering

filtering
filtering

in 17.

5A

diameter

diameter
diameter

for doing the test.

17.6 References
Methods

for the Examination of

1.

American Public Health Association.

2.

Dairy Products, pp. 150-155, 12th ed. American Public Health Association, New York.
Association of Official Analytical Chemists. 1976. Official Methods of Analysis, pp.

3.

866-869. 12th ed. Association of Official Analytical Chemists, Washington, D.C.


Clarke, J.O. 1947. Determination of filth and extraneous matter in dairy products. Amer.
J.

4.

1967. Standard

Pub. Health 37:728-732.

General Specifications for Dairy Plants Approved for USDA Inspection and
Grading Service. October 10, 1975. Federal Register, Vol. 40, No. 198, pp. 47910-47940,
Washington, D.C.

SEDIMENT

212

5.

6.

IN

MILK

Joiner, C.R. 1952. Report on sediment tests


35:99-100, 340-344.

in

milk and cream.

J.

Assoc. Off. Anal. Chem.

Joiner, C.R. 1953. Report on sediment tests

in

milk and cream.

J.

Assoc. Off. Anal. Chem.

36:87-88, 310-315.
7.

KiHLSTRUM, E.E. and R.W. Delhey.

1960.

Farm

bulk tank sediment testing.

J.

Milk Food

Techno!. 23: 108-112.


8.

LiSKA, B.J. and H.E. Calbert. 1954.


testing using off-the-bottom standards

holding tanks.
9.

10.

Watson, N.E.
Watson, N.E.

J.

A study of mixed milk sample methods of sediment


and equipment, for possible use with farm bulk milk

Milk Food Technol. 17:82-85.

1952.

sediment

pump

for farm tanks.

J.

Milk Food Technol. 15:43.

1956. Determination of sediment in milk in farm tanks and/or plant storage

tanks and tank trucks.

J.

Milk Food Technol. 19:228-229.

CHAPTER

18

PHOSPHATASE METHODS
F.J. Babel, T.L.

18.1

Eddleman, D.H. Kleyn and


G.K. Murthy

Introduction

The phosphatase test is applied to dairy products to determine whether


was done properly and also to detect the possible addition of
raw milk to pasteurized milk. Studies ^ have indicated that all raw milk contains the enzyme phosphatase and that its thermal resistance is greater than
that of nonsporeforming pathogens which could possibly be present in milk.
The phosphatase test detects improper pasteurization on the basis of residupasteurization

phosphatase activity.
negative phosphatase reaction cannot be interpreted as an absolute index of proper pasteurization. The test is based on inactivation of phosphatase to a certain level of activity and it is possible that a blend of overheated
and underheated milk, or a blend of overheated milk and a very minute
amount of raw milk might yield a negative phosphatase test.
al

Though other
valves, sealed

controls, such as temperature recorders, flow diversion

pumps

of

known

to assure proper pasteurization,

delivery capacity, etc., have been designed


it

is

advisable for plant operators and regu-

latory agencies to use the phosphatase test as a routine control procedure.

The

test is

simple and

is

very sensitive to exposures less than those estab-

lished for pasteurization.

based on the principle


liberates phenol
from a disodium phenyl phosphate substrate or phenolphthalein from a phenolphthalein monophosphate substrate when the tests are conducted at a
suitable temperature and pH. The amount of phenol or phenolphthalein
liberated from the substrate is proportional to the activity of the enzyme.

The phosphatase

tests described in this chapter are

that the alkaline phosphatase

enzyme present

in

raw milk

Phenol is measured colorimetrically after its reaction with 2,6-dichloroquinonechloroimide (CQC) to form indophenol blue. Phenolphthalein is detected by addition of sodium hydroxide.
Since publication of the original Kay-Graham '^ procedure for estimation
of phosphatase activity, many changes have been made in the method. The
ISC Liaison: D.W. Mather
213

PHOSPHATASE METHODS

214

test

has been simplified and made more rapid, more sensitive, and more
'^' '** "^
Several substrates, other than disodium phenyl phos-

quantitative.^'

suggested for estimating phosphatase activiOnly the substrate specified in a particular method should
the phosphatase methods outlined in this chapter.

have

phate,

2. 12, 13, 17,

ty

be used

in

been

21,22

18.2 General Precautions

Because of the extreme sensitivity of phosphatase tests employing disodium phenyl phosphate as substrate to minute amounts of phenol and phenolic compounds, it is essential that all pipets, glassware, stoppers, etc., and
all

reagents be phenol-free to avoid false positive tests.

A. Glassware:
All glassware should be rinsed with warm water, washed with a detergent
which contains no phenolic compounds, rinsed in tap water and then distilled water or other suitable reagent grade treated water, air- or oven-dried,
and protected from contamination during storage. It is desirable to follow
this procedure immediately after use.
Representative pieces can be tested to determine whether they are free of

phenol.

B. Closures:

Use

of plastic closures should be avoided since phenolic contamination

from them has been encountered.

Gum

rubber stoppers generally are free of

phenolic compounds, but they should not be used

in

containers of butyl

alcohol.

C. Reagents:

Keep

all

reagents tightly stoppered to prevent deterioration and con-

tamination.

Presence of free phenol in buffered substrate containing disodium phenyl


phosphate frequently is a cause of blue color in control tests. This color is
caused by deterioration of the buffer substrate or by disodium phenyl phosphate that is not phenol-free. Deterioration of buffer substrate [I8.6(B.I)]
can be delayed by heating to 85 C for 2 minutes.'" Phenol-free disodium
phenyl phosphate should be used, if possible. To test for phenol in buffered
substrate, add 2 drops of CQC to jO ml of buffered substrate and incubate for
5 min at room temperature. If any color develops, the buffered substrate
should be discarded and a fresh solution prepared.
If the disodium phenyl phosphate is not phenol-free, a satisfactory buffered substrate can be made as follows: In a separatory funnel, dissolve 1.1 g
of disodium phenyl phosphate in 50 ml of buffer [18.6(C.2)], using the Cor-

215

18.3 Controls

or 0.5 g of disodium phenyl phosphate in 25 ml of buflFer [18.4(B.l)],


using the Scharer tests. Add 2 drops of CQC, mix well, and allow 10 min for

nell test;

color development. Extract the indophenol blue color by shaking vigorously

with 10 ml of /2-butyl alcohol [18.4(B.6)]. Allow to stand until the alcohol


layer separates completely.
solution to

liter

Remove

the alcohol layer and dilute the aqueous

with buffer [18.6(C.2)], using the Cornell

500 ml with water, using the Scharer

test;

or dilute to

tests.

Phos-Phax tablets* are used in the Scharer rapid test [18.4(B.3)], disPhos-Phax tablet in 5 ml of water. Add 2 drops of CQC, shake well,
and allow 5 min for color development. Extract color with 2 ml of //-butyl
alcohol. Allow to stand until the alcohol separates, remove the alcohol with
a pipet, and discard. Dilute the aqueous solution to 50 ml with water. These
If

solve

solutions should be prepared shortly before use, as they


age. Store refrigerated no longer than a

CQC

powder should be stored

preparing

CQC

decompose with

week.

refrigerated in tightly closed bottles.

solutions, bring the

powder

to

When

room temperature before

opening the bottle to avoid moisture condensation on the reagent. RefrigCQC solution and discard it when it deteriorates (turns brown).

erate the

18.3 Controls Applicable to All

Phosphatase Procedures

Controls are used to detect deterioration or contamination of reagents; to


detect presence of interfering substances such as flavoring materials having
a phenolic structure; to account for variations in color of the indophenol blue
that results

from

and fats, particularly where


been done; and to check for possible

interfering coloring materials

direct extraction with butyl alcohol has

presence of microbial phosphatase,

A.

Negative control:
negative control should be used with each type of dairy product being

approximately 5 ml or 5 g of product in a test tube containing a


at 90 C for one min followed by rapid cooling. Stir
during heating to insure complete inactivation of phosphatase. Test the
heated sample for phosphatase. Any color developed in the negative control
is not due to residual phosphatase, but indicates contamination of reagents
tested. Place

thermometer and heat

or presence of interfering coloring materials or both. Ideally, the negative


control should

show no

color.

B. Positive control:

As

a check on the proper functioning of reagents, a single positive control

will sufifice for

each series of samples tested. Add

0.1

ml of fresh raw mixed

^Supplied by Applied Research Institute, 90 Brighton Ave.. Perth

Amboy.

N.J.. 08861

PHOSPHATASE METHODS

216

herd milk to 100 ml of raw milk which has been heated


followed by rapid cooling to room temperature.
is

done on

this

When

at

90

for

one min,

the phosphatase test

sample, a positive result should be obtained.

C. Interfering substance control:

Since vanillin and other

compounds with phenolic

structures

may be

pres-

ent in chocolate-flavored milk, pressurized creams, toppings and ice cream,


false-positive tests

may be

obtained

when

the substrate

is

disodium phenyl

phosphate. Sterile milk products processed by the high-temperature short-

may contain substances that react in the phosphatase test to


produce false-positive results. When positive results are obtained on samples, the test should be repeated using the same amount of sample but subtime procedure

stituting for buffered substrate the

same amount of

buffer solution.

In the absence of disodium phenyl phosphate or phenolphthalein

mono-

phosphate any color in this control is caused by interfering substances and


not by residual phosphatase. However, if the color of the test using buflFered
substrate
trol,

is

greater than the color obtained

in the

interfering-substances con-

underpasteurization and/or contamination with raw product

is

in-

dicated.

D. Microbial phosphatase:
The possibility of phosphatase production in dairy products by microorganisms, particularly in cream, has been demonstrated by Hammer and
Olson ^ and Barber and Frazier.'^ Microbial phosphatases are considerably
more heat resistant than is alkaline milk phosphatase \ so differentiation is
possible with the following technique:

phosphatase test is positive, repasteurize a 5-ml portion of


C for 30 min in a test tube. Completely immerse the tube
or immerse so that the water line is approximately 4.0 cm above the level of
the milk in the tube. The temperature of the milk should reach at least 62.3 C
within 5 minutes. Keep a thermometer immersed in the sample or in a suitable control. Cool and do phosphatase tests on the repasteurized sample, the
original sample, and the negative control. If the repasteurized sample does
not show a significant reduction in color, the original result was due to microbial phosphatase. It should therefore be classified as false-positive and
If the original

the sample at 62.8

reported as negative.
E. Collection of samples (Chapter 3):

samples should be placed in clean containers and refrigerated until


It is desirable to take samples for phosphatase testing before samples
are admixed with nuts, fruits, candy and other such additives. However, if
flavored products are to be tested, it is recommended that such additives be
removed by straining before testing.
All

tested.

18.4 Scharer Rapid

217

Phosphatase Test

18.4 Scharer Rapid

Phosphatase Test "-27

This method should be limited to preliminary screening only.

A. Equipment:
1. Test tubes: Must be of uniform composition, wall thickness, and
calibrated at 5-. 5.5- and
bore. A convenient size for the test is 12 x 144
8.5-ml to top of meniscus; for preparation of cheese samples, use 18 x 150

mm

mm

test tubes.

test tubes.

2.

Test tube stoppers: Phenol-free, to

3.

graduated in 0.1-ml divisions.


Water bath: Thermostatically controlled to 40 1 C.
to 110 C, accuracy checked with an NBS-certified
Thermometer:

4.
5.

Pipets:

ml and

fit

5 ml,

thermometer.
6. Dropping bottles: Amber glass, size V4 oz (7.5 ml), with dropper calibrated to deliver approximately 50 drops of CQC solution per milliliter.
7. Light source and filter: Such as dental X-ray viewer consisting of a
single 14- to 22-watt daylight-type fluorescent light and a plastic or glass
light filter approximately 10 cm high by 30 cm wide; or other suitable equivalent equipment.
8. Color standards: Prepare phenol standards [18.4(C.3)] to contain 1,
2 and 5 fxg of phenol per 5 ml of solution.
9. Hand homogenizer or mechanical blender.
B. Reagents:
1.

BuflFer:

Dissolve

(NaHC03-Na2C03-2H20)

100 g of sodium sesquicarbonate dihydrate


or 46.89 g of sodium carbonate plus 37.17 g of so-

water and make up to one liter.


ml of the above buffer to 500 ml. Alternateby Applied Research Institute) tablet in
(supplied
1
Buf-Fax
dissolve
ly,
50 ml of water. For determinations on cheese, sour cream, and buttermilk
prepare a double-strength bulfer control by diluting 25 ml of buffer to 250 ml;

dium bicarbonate
2.

in

Bufi'er control: Dilute 25

Buf-Fax

ml of water.
phenol-free disodium
phenyl phosphate crystals in water, add 25 ml of buffer, and make up to
500 ml with distilled water. Optionally prepare solution by dissolving 1 Phosalternately, dissolve
3.

tablet in 25

Buflfer substrate: Preferably dissolve 0.5 g of

tablet in 50 ml of water. For determinations on cheese, buttermilk, and


sour cream, prepare a double-strength buffer substrate and make solution up
to only 250 ml; if Phos-Phax is used for the double-strength buffer substrate,

Phax

dissolve
4.

Phos-Phax

CQC

tablet in 25

reagent: Dissolve 30

ml of water.

mg

of crystalline

CQC

in 10

ml of methyl

or ethyl alcohol. Optionally prepare the solution by dissolving 1 Indo-Phax


tablet in 5 ml of methyl alcohol. Store the solution under refrigeration and

discard after one week, or

if it

turns brown.

PHOSPHATASE METHODS

218

5.

Catalyst: Dissolve 200

mg of copper sulfate (CUSO4

SHoO) in 100 ml

of water. Where Indo-Phax

tablets are used, the catalyst

since the tablets contain the

copper

can be omitted

CQC.

alcohol (neutralized): Neutralize the free acid in the alcohol

6. A2-Butyl

as follows:

salt as well as the

Add

0.1

N NaOH

to alcohol until a small portion tested with

bromthymol blue gives a green or

light

blue color.

To

test,

shake a sample of

the alcohol with an equal quantity of neutral distilled water, allow to sepa-

and add a few drops of indicator solution to the water layer. Alternateadd 50 ml of diluted buflfer (10 ml of buffer tl8.4(B.l)] plus 40 ml of
distilled water) to each gallon (3.785 liters) of alcohol, shake well, and store
rate,
ly,

in the refrigerator.
7.

-Butyl alcohol, 7.5%: Add 7.5 ml of neutralized A?-butyl alcohol to

92.5 ml of distilled water.

Standards:

Stock phenol solution: Weigh 1.000 g of reagent-grade anhydrous


liter volumetric flask, make up to the mark with 0.1
HCl, and mix (1 ml contains 1 mg of phenol). This stock solution remains
1.

phenol, transfer to a
stable for several
2.
1

liter

months

in the refrigerator.

Dilute phenol solutions: a.) Dilute 10 ml of stock phenol solution to

with water; each

milliliter contains \Q fxgof phenol, b.) Dilute 10 ml of


described to 50 ml with water. Prepare fresh daily; each

Solution

a. just

milliliter

contains 2 (xg of phenol.

Phenol standards: To a series of test tubes similar to those which will


be used for the test procedure, add dilute phenol and water in the amounts
specified in Table 18:1. PREPARE FRESH DAILY.
For the Scharer rapid method, prepare only phenol standards containing
1, 2, and 5 fxg of phenol per 5 ml of solution. Add 0.5 ml of buffer tl8.4(B.l)],
mix, and add 6 drops of CQC reagent 118.4(B.4)] containing copper catalyst,
or 2 drops of catalyst [18.4(B.5)] and 6 drops of CQC to each. Mix immediately and incubate for 5 min at 40 C in a water bath.
3.

Table
Phenol Solution

18:1.

Preparation of Phenol Standards

Phosphatase Test

18.4 Scharer Rapid

4.

219

Butyl alcohol-extracted standards: Extract each of the standards

[18.4(C.3)] with 3 ml of butyl alcohol. After the butyl alcohol has separated,

draw

ume

ofiFa

portion of the butyl alcohol extract and dilute with an equal vol-

of butyl alcohol.

Test-samples which have been extracted but not diluted should be compared directly with these extracted and diluted standards.

D. Controls:
1. Chocolate milk, cheese, ice cream, or other flavored milk products:
Do an interfering substance control [18.3(D)] on each flavored product.
2. Cream, butter and cheese: Do a microbial phosphatase control
[18.3(D)] on each positive sample.
3. Positive and negative controls: Do at least one positive control
[18.3(B)] for each series of samples and one negative control [18.3(A)] for
each kind of product.
E. Procedure:
1.

Pipet 0.5-ml portions of samples and controls into suitable test

tubes.
a.

Buttermilk, sour cream, and creamed cottage cheese are best pre-

pared for sampling by homogenizing in a mechanical blender.


To prepare for sampling, transfer 5 g
b. Cheese or dry cottage cheese
x
containing
2 ml of 7.5% neutralized -butyl
150
mm)
to a test tube (18
grind
with
glass
rod.
Add
an additional 18 ml of 7.5% neualcohol and
a
macerate further if necessary,
-butyl
alcohol.
Shake
well,
tralized
allow
for
30
minutes.
A 0.5-ml portion for testing is
and
to stand
about
middle portion.
withdrawn
from
the
relatively
clear
then
prepare
samples,
melt
butter
at 40 C, mix, and pipet a
Butter
To
c.

0.5-ml portion into a

test tube.

Reconstitute product with


d. Concentrated and dry milk products
water to the original concentration of milk solids and test in the manner
specified for the original product.
2.

When

testing milk, cream, ice cream, butter, chocolate milk or re-

constituted milk products, add 5 ml of buffer substrate [18.4(B.3)].

When

cream, cheese, or cottage cheese, add 5 ml of buffer


substrate prepared with double strength buffer as stated in 18.4(B.3). For
reliable results, the pH of the mixture should be tested and adjusted, if necessary, to pH 9.5 to 10 with solid buffer [18.4(B.l)]. Samples should be kept
testing buttermilk, sour

cold before testing.


3.

4.

40

Stopper tubes and mix by inverting several times.


Place tubes in a water bath, warm to 40 1 C, and then incubate at

C for 15
Remove

minutes.

tubes from the water bath, add 6 drops of CQC reagent containing catalyst (add 2 drops of catalyst if CQC does not contain copper
sulfate) to each tube, stopper, mix, and reincubate for five minutes.
5.

PHOSPHATASE METHODS

220

6. Remove tubes from the water bath, cool in an ice water bath, add
ml of neutralized cold -butyl alcohol, restopper, and extract indophenol
blue by gently inverting the tubes four times through a half circle. (Take
about 2 sec to invert tubes, pause about 2 sec, take another 2 sec to return
tubes upright; pause 2 sec and then repeat). Lay tubes on their sides on a flat

surface for 2 min to permit separation of the butyl alcohol, then repeat the
mixing and separation steps.
7. If all the butyl alcohol has been emulsified and no clear butyl alcohol
layer remains, cool tubes for 5 min in ice water and then centrifuge them for
5 minutes.
8.

Stand tubes erect and compare colors of the butyl alcohol layers with
Comparison of colors shall be made using a standard light

standards.

[18.4(A.7)].

F. Interpretation:

Only 0.5 ml of milk or milk product was tested, but dilution of the standards 1:1 is equivalent to multiplying by 2 or converting to 1 ml; therefore
the value corresponds to micrograms of phenol per milliliter of product. A
value of

fxg

or

more of phenol per

milliliter

tuted milk product or cheese extract

is

of milk, milk product, reconsti-

indicative of improper pasteurization

or of contamination with unpasteurized products.

18.5 Modified Spectrophotometric


This test

is

Phosphatase

IVIethod

^^

^^

a modification of the Scharer rapid test, and samples of various

products should be prepared for testing as described in Section 18.4(E).

A. Apparatus:
1.

mm

Test tubes: Borosilicate glass, 16 x 125

or 16 x 150

mm

glass-

stoppered.
2.

Stoppers: Phenol-free rubber; or glass stoppers.

3.

Pipets:

4.

6.

Water bath: Same as 18.4(A.4).


Thermometer: Same as 18.4(A.5).
Dropping bottle: Same as 18.4(A.6).

7.

Centrifuge: Safety head, or equivalent, that will hold six 15-ml tubes

5.

at

Same

as 18.4(A.3).

a fixed angle; or any centrifuge to

accommodate

16

x 150-mm glass-stop-

pered centrifuge tubes.


8.

Spectrophotometer or colorimeter: Coleman Model 6A, or equiva-

9.

Cuvettes: Selected, round, 12 x 75

lent.

mm.

B. Reagents:

See section 18.4(B) for reagents used

in this

method.

18.5 Modified Spectrophotometric

Phsophatase Method

221

C. Standards:

Stock phenol solution: Same as 18.4(C.l).


Dilute phenol solution: Same as 18.4(C.2).
3. Phenol standards: Same as 18.4(C.3).
For construction of the standard curve, prepare only standards containing
0, 2, 5, 10, 15, 20 and 25 /Ag of phenol per 5 ml of solution. Add 0.5 ml of
buflFer [18.4(B.l)], mix, and add 6 drops of CQC reagent [18.4(B.4)] containing copper catalyst or 2 drops of catalyst [18.4(B.5)] and 6 drops of CQC
reagent to each standard. Mix immediately and incubate for 5 min at 40 C in
a water bath.
4. Extraction with A?-butyl alcohol: Add 5 ml of //-butyl alcohol to each
of the above standards [18.5(C.3)]. Extract the indophenol blue by the procedure given in [18.4(E.5)], and centrifuge for 5 minutes.
5. Construction of standard curve.
a. Withdraw 2 ml of the butyl alcohol layer from each extracted standard [18.5(C.4)] and dilute with an equal volume of butyl alcohol in
matched cuvettes and mix.
phenol standard at absorbb. Zero the spectrophotometer on the
ance (100% transmission) with the wavelength dial at 650 nm and read
the absorbance value of each standard.
c. Plot absorbance readings versus micrograms of phenol on standard coordinate paper and draw a best straight line through the points of
2, 5, 10, 15, 20 and 25 /xg of phenol per 5 ml of prepared standards
1.

2.

[18.4(C.3)].

D. Controls:
See, section 18.4(D) for controls applicable to this

method.

E. Procedure:

Samples for testing various products should be prepared as indicated

in

Section 18.4(E).
1.

Pipet 0.5 ml of well-mixed sample into 5 ml of buflFer substrate in a

16 X 125

mm

test tube.

Stopper with a phenol-free

gum

rubber stopper and

mix by inversion.
2.

Incubate the

test mixture, together

tive control [18.3(A)] of the

with a similarly prepared nega-

same kind of sample,

at

40

for

hr in a

water bath.
3.

Remove samples from

the water bath, add 6 drops of

CQC

reagent

(containing catalyst) to each, mix, and reincubate for 5 minutes.

Remove samples from the water bath, cool in ice water for 5 min,
ml of cold butyl alcohol, and mix by inversion [18.5(C.4)].
5. Centrifuge samples [18.5(C.4)] for 5 min and pipet about 3 ml of the
butyl alcohol layer from each sample into a cuvette.
4.

add

PHOSPHATASE METHODS

222

6.

Zero the instrument on the negative control and read the absorbance

value of the test sample at a wavelength of 650 nm.


F. Interpretation:

Determine the concentration of phenol

in the test

sample by comparing

the absorbance reading on the sample to the standard curve. Results thus

obtained give the micrograms of phenol per milliliter directly i.e., only
ml of sample was used in the test but the standards were diluted 1:1 and

0.5

the net result

of sample.

equivalent to multiplying by 2 or converting to one milliliter


value greater than 2.3 /xg of phenol per milliliter is indicative

is

Any

of improper pasteurization.

18.6 Cornell

Phosphatase Test

^^-^

A. Apparatus^:
1.

2.

3.

Test tubes: Borosilicate glass, 16 x 150 mm.


Pipets: 1 ml and 5 ml (or 10 ml) graduated in 0.1-ml divisions.
Weighing balance: Accuracy, 5 mg.

Water bath: Temperature thermostatically controlled at 37 1 C.


Thermometer:
to 110 C, certified against an NBS standard thermometer.
6. Funnels: Size suitable for 11-cm filter paper.
7. Filter paper: Whatman No. 42, 11 cm.
8. Dropping bottles: Amber, 25- to 50-ml capacity; dropper to deliver
4.

5.

approximately 43 drops per

milliliter.

10.

Same as 18.4(A.7) (optional).


Color standards: Same as 18.6(C).

11.

Spectrophotometer or colorimeter: Bausch

9.

Light source:

& Lomb

Spectronic 20

or equivalent.
B. Reagents:
1. Carbonate buffer substrate: Dissolve 11.5 g of anhydrous sodium
carbonate (NagCOa), 10.2 g of anhydrous sodium bicarbonate (NaHCOg),
and 1.1 g of disodium phenyl phosphate in distilled water and make up to
1

liter.

2.

Trichloroacetic-hydrochloric acid precipitant: Dissolve 25 g of triHCl (approxi-

chloroacetic acid crystals in 50 ml of water, add 50 ml of cone

mately 36%), and mix thoroughly.


3. Sodium carbonate solution, 8%: Dissolve 80 g of anhydrous sodium
carbonate in distilled water and make up to 1 liter.

^Materials and reagents, including permanent color standards


Scientific

Company,

711 Forbes Avenue, Pittsburg, Pa. 15219.

may be

obtained from Fisher

18.6 Cornell

223

Phosphatase Test

Copper sulfate-sodium hexametaphosphate solution: Dissolve 0.5 g


(CUSO4 5 H2O) and 50 g of sodium hexametaphosphate

4.

of copper sulfate

crystals in distilled water and

CQC

5.

make up

to

liter.

solution: Dissolve 0.2 g of crystalline

CQC

in

25 ml of absolute

methyl or ethyl alcohol.


//-Butyl alcohol:

6.

Bp

117.5 C;

do not

neutralize.

C. Phenol color standards:

Stock phenol solution: Dissolve

1.

make up

to

liter.

Keep

g of phenol crystals in water and


and after use if not prepared

refrigerated before

fresh.
2. Buffer solution: Dissolve 11.5 g of anhydrous sodium carbonate
(NaaCOs), 10.2 g of anhydrous sodium bicarbonate (NaHCO;,), and 0.1 g of
liter.
copper sulfate (CUSO4 5 H2O) in distilled water and make up to
3. Diluted phenol solution: To 2 ml of stock phenol solution [18.6(C.l)]
add enough buffer solution [18.6(C.2)] to make 500 ml. This solution con1

tains 4

)u,g

of phenol per milliliter of solution.

Make

Proportioning solutions, for standards and

fresh daily.

development: Pipet
x 150 mm) portions in the ratios listed in the first two
columns of Table 18:11. With one exception, later adjusted, all mixtures are
10-ml volumes before color development.
Add to each of the above 10-ml volumes exactly 2 drops of CQC with
quick stirring and invert the test tube once. Incubate at 37 C for 5 min for
color development. Add 5 ml of /?-butyl alcohol to each test tube. Invert the
tubes five times to extract the color. Seal tops with a proper closure and
4.

final

into test tubes (16

store in the refrigerator or in an ice-water bath.

Table

18:11.

Phenol Color Standards*

Buffer Solution

Diluted Phenol Solution

[18.6(C.2)]

[18.6(C.3]

(ml)

(ml)

yug

Phenol

per Tube

10

15.6

0.4+

9.5

0.5

12

16

20
40

10

Mixtures in the first two columns give the respective phenol concentrations.
^Remove 6 mi of the mixture and discard. Develop color on the remaining 10 ml of

solution.

PHOSPHATASE METHODS

224

5.

Construction of standard curve:

Remove approximately

ml of alco-

hol from the extracted standards [18.6(C.4)] with a pipet and place in clean,

small cuvettes or into colorimeter tubes.

ed from the

Read

against butyl alcohol extract-

phenol standard solution using a colorimeter or spectrophotometer set at absorbance (100% transmission) and at 650 nm. On standard
coordinate paper plot the absorbance or optical-density values against the
phenol concentrations used. Draw a line through the points and use as the
standard curve.

D. Controls:

Do

controls as indicated in 18.3.

vored milks, high values


fering substances.

in

On

chocolate milk, ice cream, and

negative controls

may

fla-

indicate presence of inter-

Such values are subtracted before

interpretation.

E. Procedure:

Sample preparation: For milk, cream, chocolate milk, buttermilk,


cream mix, and condensed milk, pipet ml of product into a test
tube, add 10 ml of warm (37 C) carbonate bufl"er substrate, and mix. For
ripened cheese, cottage or cream cheese, butter, and dry milk, transfer 0.5 g
1.

whey,

ice

Add 1 ml of warm (37 C) carbonate buflFer substrate


and macerate the solid into a paste or into many small particles with a glass
rod. Then add an additional 9 ml of carbonate buff"er substrate and stir.
2. Incubation: Place test tubes in a water bath maintained at 37 C. Allow 5 min for tubes to attain 37 C and then incubate for one hour.
3. Precipitation: Following incubation, remove test tubes and carefully
pipet down the side 1 ml of trichloroacetic-hydrochloric acid precipitant.
Filter after a few seconds into a clean test tube (16 x 150 mm), preferably of

of product to a test tube.

a type calibrated at 5-ml intervals.


4. Color development and measurement: To 5 ml of clear filtrate add
ml of the copper sulfate-sodium hexametaphosphate solution. Pipet into
the tube 5 ml of warm sodium carbonate solution (8%) and mix. Add exactly
2 drops of CQC solution and without delay invert the tube once. Permit color
to develop at 37 C for exactly 5 minutes. Add 5 ml of Aj-butyl alcohol, invert
the tube five times, and let stand for one minute. Compare colors visually
and read against alcohol standards.
Optionally, in place of visual reading, remove approximately 3 ml of ex1

tracted butyl alcohol solution from each test tube with a clean pipet into a

small clean cuvette or colorimeter test tube 12

mm

in

diameter, place in the

colorimeter, and measure absorbance or optical density at 650

nm

against an

from a suitable negative control set at 100% transmittance (18.3(A) and 18.6(D)). Determine the phenol concentration by reference to a standard curve as prepared under Section 18.6(C.5).
alcohol-extracted

filtrate

F. Interpretation:

Express values directly, without factor multiplication of any kind.

Any

value over 1.0 fxg of phenol per 0.5 ml of milk or other fluid dairy product, or

18.7 Rutgers

225

Phosphatase Test

per 0.25 g of cheese or other solid dairy product indicates underpasteurization, or recontamination with raw milk, or both.
Milk and cheese quantities listed in the above interpretation are derived
from the filtrate volume analyzed, which represents approximately half of
the original sample volume or weight.

Phosphatase Test

18.7 Rutgers

12

Collaborative studies ^' have indicated that this test is satisfactory as a


screening test for estimating phosphatase activity in skim milk, milk and
light cream. The principle of the test is that phosphatase can hydrolyze phenolphthalein monophosphate; phenolphthalein

ed by addition of

alkali.

The color

is

compared

is

released and can be detect-

visually with a standard pre-

pared from the same milk.


A. Equipment:
1.

bore.

Test tubes: Must be of uniform composition, wall thickness, and

Tubes

15

x 100 mm are recommended.


ml, graduated or volumetric.

2.

Pipets:

3.

Water bath: Thermostatically controlled

to 37

C.

B. Reagents^:
1. Substrate concentrate: Dissolve 3.9 g of dicyclohexylamine salt of
phenolphthalein monophosphate and 73.2 g of 2-amino-2-methyl-l-propanol

in

21.9 ml of HCl.

The pH

is

10.15 at 25 C.

Standard concentrate: Dissolve 10 mg of phenolphthalein, 40 mg of


tartrazine and 73.2 g of 2-amino-2-methyl-l-propanol in 21.9 ml of HCl.
2.

The pH
3.

is

10.15 at 25 C.

Color developer: Dissolve 10 g of

NaOH

in

water and dilute to

100 ml.
4.

//-Butyl alcohol, 7.5%: Adjust 75 ml of /?-butyl alcohol to

addition of approximately

N NaOH

All reagents are stable indefinitely

and make up to

if

pH

by

liter.

refrigerated.

C. Procedure:

The procedure
the

AOAC

is

currently limited to skim milk, milk and light cream by

(Official, Final Action).

Although

unofficial, the

method

is

appli-

cable to low-fat milk, half and half, heavy cream, plain ice cream mix,
cheese, butter, and concentrated and dry milk. Also, the method can be used

between residual and reactivated alkaline phosphatase.


two test tubes.
Place 5.0 g of cheese in a test tube (18 x 150 mm) and
a. Cheese
add 20 ml of 7.5% neutralized /7-butyl alcohol. Grind sample with a

to differentiate
1.

Pipet 1.0-ml portions of samples into

^Reagents

may be purchased from Applied Research

boy, N.J. 08861.

Institute,

90 Brighton Ave., Perth

Am-

PHOSPHATASE METHODS

226
glass rod for 2

paper.
b.

min and

immediately through

filter

Use 1.0-ml portions of

Melt

sample

Butter

at

Concentrated and dry milk

nal concentration of solids.

Reconstitute with water to the

Mix and use 1.0-ml portions


warm to 37 1 C.

2.

Place tubes in water bath and

3.

Add

filter

40 C, mix, and use 1.0-ml portions for

testing.
c.

Whatman No.

filtrate for testing.

origi-

for testing.

drop (0.04 ml) of substrate concentrate to one tube and

drop

(0.04 ml) of standard concentrate to the other tube.


4. Mix and incubate at 37 1 C for 30 minutes.
5.

Add

drop (0.04 ml) of color developer to each tube, mix and com-

pare visually.

D. Interpretation:
If

the tube containing substrate concentrate

shows

less pink color than the

tube containing standard concentrate, the sample can be considered proper-

The standard contains 4

of phenolphthalein per test which

ly

pasteurized.

is

equal to the amount of phenolphthalein liberated by

/xg

ml of pasteurized

milk contaminated with 0.1% raw milk in 30 minutes. Preparation of a stan-

dard

in the

which
18.8

is

same milk as

very important

the sample results in colors having the

in visual

same hue

comparisons.

Phosphatase Reactivation in Dairy Products Heated by


High-heat Short-time and Ultra-pasteurization Methods and
Ultra High-temperature Methods

The phosphatase

test

has been used successfully to detect inadequate pas-

teurization of milk and milk products processed by conventional methods at


62.8 C for 30 min, or 71.7 C for 15 seconds. Dairy products heated higher

than the conventional temperature (71.7 C) by high-temperature short-time

(HTST) or by high-heat short-time (HHST) methods

(88.3

for

sec to

(UP) 138 C for 2 sec) may give


C
heating and cooling, and when
immediately
after
a negative phosphatase test
However,
positive
test may be observed in
refrigerated (4.4 C or below).
a
products not adequately stored, particularly in those held at or above 10 C
'**~'"
for extended periods.**- ^'
Development of restored phosphatase activity
has been termed "reactivation." Absence of a high bacterial population in
these products eliminates the possibility of bacterial phosphatase.
Reactivation may occur in products processed at temperatures above
100 C. Reactivation is greatest in products processed at 104.4 C, incubated
at 34 C, and adjusted to pH 6.5. If magnesium (Mg) salts are added to
HHST- or UP-processed products after heat treatment but before incubation or storage, they catalyze reactivation. Products with a high fat content show increased reactivation due to high initial concentration of phosphatase, but an increase in holding time during heating will reduce
reactivation. Homogenization of products before heat processing decreases
100

for 0.01 sec) or by ultra-pasteurization

227

Phosphatase

18.8 Reactivated vs Residual

reactivation. Phosphatase reactivation

is

low or does not occur

in

products

with titratable acidities of 0.40 to 0.45% (calculated as lactic acid) or more.


Cow's milk shows seasonal variations in phosphatase concentration and in
its

reactivation behavior.^''

The differentiation of reactivated phosphatase from residual phosphatase


(in raw or under-pasteurized product) is based upon the fact that the enzyme
is capable of reactivation when stored with Mg salts at 34 C, and will show a
four- to ten-fold increase in phosphatase activity over the same product
stored without added

Mg

salts.

:^''^^
A. Differentiation of reactivated from residual phosphatase
results
phosphatase,
compare
To differentiate reactivated from residual

obtained on testing a

5 dilution of milk or milk

product containing mag-

nesium acetate with the results of tests of an undiluted portion of the same
sample without Mg(C2H302)2 after storage at 34 C for one hour.
Reagents

1.

a.

Magnesium

acetate solution: Dissolve 35.4 g of Mg(C2H302)2 4

Warm

in 25 ml of water.

slightly to aid solution.

H2O

Pour the solution

into a 100-ml volumetric flask, rinse the original container several times

with 5-ml portions of water and add the rinsings to the volumetric flask.
Allow the solution to cool and make up to 100 ml. This solution contains
of Mg per milliliter.
Control and diluent: Place 10 ml of each sample to be tested in a
boiling water bath for 1 min after the temperature of the sample has

40.1

mg

b.

reached 95 C. Cool the sample and use a portion for dilutions as

re-

quired, for a boiled control.

B. Procedure:

Place a 5-ml aliquot of the sample to be tested in a screw-cap (pheand add 0.1 ml of water. To a second 5-ml aliquot of the

1.

nol-free) test tube

same sample
0.1-ml
2.

while

Mohr

in

an identical tube, add 0.1 ml of Mg(C2H302)2 solution with a

pipet and mix well.

Incubate both aliquots for


in

hr at 34 C; mix samples occasionally

the water bath.

3. Remove samples from the water bath and cool in ice water. Dilute
ml of the sample containing Mg with 5 ml of the corresponding boiled milk
or milk product control (1-1-5 dilution).
4. Using the modified spectrophotometric [18.5] or the Cornell [18.6]
phosphatase procedure, test the undiluted sample without Mg and the
1
5 diluted sample with Mg added for phosphatase activity.
1

-I-

C. Interpretation:
If the

-(-

5 diluted

sample containing

Mg

has equal or greater activity

than the undiluted sample without Mg, the sample is considered negative for
residual phosphatase and indicative of reactivation. If the diluted sample has

PHOSPHATASE METHODS

228

sample, and

less activity than the undiluted

phatase test result was positive, the sample

the initial conventional phosconsidered positive for residu-

if

is

phosphatase.

al

D. Precautions:

A false-positive

phosphatase

may be obtained when a sample containbeen allowed to stand longer than 4 hr at

test

ing reactivatable phosphatase has

about 16 C or 2 hr at about 20 C.
Reactivated phosphatase in an unrefrigerated product
al phosphatase.

may be

positive test

obtained with a sterile milk product because of

However,

reactivated phosphatase.

will test like residu-

if

the sterile product

is

allowed to stand

room temperature for extended periods (2 hr at 20 C) the differential test


may no longer be applicable. To confirm presence or absence of alkaline

at

phosphatase, follow the procedure for microbial phosphatase [18.3(D)] and


use appropriate controls [18.3]. If the phosphatase test is positive, it may
indicate presence of microbial phosphatase. Then,

it

may be

necessary to

resort to microbiological tests to assess the quality of the product.

18.9 References
1.

Association of Official Analytical Chemists.


12th ed.

2.

1975. OflRcial

Methods of Analysis,

Washington, D.C.

Greeley. 1967. New substrate for alkaline phosphatase in milk.


Chem. 50: 555-557.
Barber F.W. and W.C. Frazier. 1943. The development of a positive phosphatase test

Babson A.L. and


Assoc.

3.

AOAC,

Offic.

S.J.

J.

Agr.

in

refrigerated, pasteurized cream. J Dairy Sci 26:343-352.


4.

BuRGWALD, L.H.

1939.

The phosphatase

test.

review of the literature on

for detecting irregularities in the pasteurization of milk and dairy products.

its

application

J.

Dairy Sci.

22:853-873.
5.

Campbell,

McFarren.

J.E. and E.F.

1961. Collaborative study of a differential test for

reactivated and residual phosphatase in dairy products.

J.

Assoc.

Offic. Agr.

Chem. 44:444-

448.
6.

Eddleman, T.L. and

F.J.

Babel.

1958. Phosphatase reactivation in dairy products. J.

Milk Food Technol. 21:126-130.


7.

Fram, H.
cream.

8.

J.

1957.

Phosphatase reactivation

in

high-temperature, short-time pasteurized

Dairy Sci. 40:1649.

Gilcreas, F.W. and W.S. Davis. 1936. An investigation of the amylase and phosphatase
tests as an indication of pasteurization. Annual Report, International Association of Milk
Sanitarians 25:15-39.

9.

Hammer, B.W. and H.C. Olson.


croorganisms.

10.

J.

Henningson, R.W.
bicarbonate buffer.

11.

J.

1960.

13.

Chemical

Kay, H.D. and W.R. Graham,


J.

in milk. J.

Assoc.

Kleyn, D.H. and W. Yen.


ty in milk. J.

Assoc.

stability

of disodium phenyl phosphate in carbonate-

Jr. 1933.

Phosphorus compounds of milk. VI. The

effect of

Dairy Res. 5:63-74.

Kleyn, D.H. and S.H.C. Lin.


assay system

by mi-

Dairy Sci. 43:769-772.

heat on milk phosphatase.


12.

1941. Phosphatase production in dairy products

Milk Technol. 4:83-85.

1968. Collaborative study of a


Offic. Anal.

new

alkaline phosphatase

Chem. 51:802-807.

1970. Spectrophotometric determination of phosphatase activi-

Offic. Anal.

Chem. 53:869-871.

18.9 References

14.

229

KosiKOWSKi, F.V.

1949.

simple universal dairy products phosphatase

Science

test.

110:480-481.
15.

KosiKowsKi, F.V.

1951.

The

effectiveness of the Cornell phosphatase test for dairy prod-

ucts. J. Dairy Sci. 34:1151-1158.


16.

17.

KosiKOWSKi, F.V. 1964. Evolutionary changes in the Cornell phosphatase test. J. Milk
Food Technol. 27:268-270.
Lin, S.H.C. and D.H. Kleyn. 1967. New alkaline phosphatase activity assay system as
applied to milk.

18.

McFarren.

J.

Dairy Sci. 50:942.

Black, L.A. and J.E. Campbell. 1963. Modified spectrophotometric


J. Milk Food Technol. 26:395-396.
McFarren, E.F., Thomas, R.C. Black, L.A. and J.E. Campbell. 1960. Differentiation
E.F.,

phosphatase method.
19.

of reactivated from residual phosphatase in high-temperature short-time pasteurized milk

and cream.
20.

J.

Assoc.

Offic. Agr.

MuRTHY, G.K., Cox,


ultra

Chem. 43:414-426.

and L. Kaylor. 1976. Reactivation of alkaline phosphatase in


high-temperature short-time processed liquid milk products. J. Dairy Sci. 59:1699S.

1710.
21.
22.

23.

J. 1966. Automated test for phosphatase. J. Dairy Sci. 49:1482-1487.


Reynolds, R.G. and W.J. P. Telford. 1967. Application of an automated procedure to the
milk phosphatase test. J. Milk Food Technol. 30:21-25.
Scharer, H. 1938. A rapid phosphomonoesterase test for control of dairy pasteurization. J.

O'Brien,

Dairy Sci. 21:21-34.


24.

Scharer, H.

1938.

Improvements

pasteurization of milk and


25.

Scharer, H.

its

in the rapid

products.

J.

phosphatase

test for detection of

improper

Milk Technol. 1:35-37.

1939. Application of the rapid phosphatase test to dairy by-products.

J.

Milk

Technol. 2:16-20.
26.

Scharer, H.

1945. Application of the phosphatase test to cheese.

Amer.

J.

Pub. Health

35:358-360.
27.

Scharer, H.

1953.

The Scharer modified phosphatase

test. J.

Milk Food Technol. 16:86-

88.
28.

Wright, R.C. and


ment.

29.

J.

Tramer.

1953. Reactivation of milk phosphatase following heat treat-

Dairy Res. 20:177-188.

II. J.

III. J.

J.

Tramer.

1953. Reactivation of milk

phosphatase following heat

treat-

phosphatase following heat

treat-

phosphatase following heat

treat-

Dairy Res. 20:258-273.

Wright, R.C. and


ment.

31.

J.

Wright, R.C. and


ment.

30.

I.

J.

Tramer.

1954. Reactivation of milk

Dairy Res. 21:37-49.

Wright, R.C. and

J.

Tramer.

1956. Reactivation of milk

ment. IV. Influence of certain metallic ions.

J.

Dairy Res. 23:248-256.

CHAPTER

19

CHEMICAL METHODS
L.J.

J.A. Burke,

19.1

Bianco, G.H. Richardson, J.W. Sherbon,


R. Ginn, and D. Liden.

W. Roth, D.A. Biggs,

Introduction

This chapter has been enlarged to provide broader coverage of chemical


methods used in testing dairy foods. In general, selected chemical methods

considered to be important from public health and economic viewpoints are


presented. Chemical methods considered only for screening are included in

Appendix B.
19.2

Added Water

19.21

Milk

in

Thermistor Cryoscope

In 1956 Shipe

-^

reported that thermistors could be advantageously used


on milk. He also presented a tech-

for critical freezing point determinations

Associate Referee of Dairy Products,


he described a thermistor cryoscope method. Tests made with the
thermistor cryoscope, using 2 ml of milk require only one to two minutes.
More recent reports have confirmed the reliability of the thermistor cryonique for their use and

in 1958'-** as

AOAC,

scope.

**

'^

Solutes, or materials dissolved in a liquid solvent, generally lower the


Such lowering usually is proportional to the

freezing point of the solvent.

concentration of solutes

in

the solvent.

As applied

to milk

when water

is

added, the concentration of salts dissolved in the serum is diluted. Dilution


of milk progressively gives a freezing point more nearly approaching that of
water.

Use of the freezing point in surveillance of milk for presence of added


water should be approached with caution and with an understanding of
causes for variations found in the freezing point.
Lactose and chlorides account for about 75% of the freezing point depression and the one tends to compensate for the changes in concentration of the
ISC Liaison: G.H. Richardson
231

CHEMICAL METHODS

232

Most of the variation has been attributed


changes in the nonchloride ash fractions of the milk. The freezing point
upper Hmit value of -0.525 H or -.507 C has been established as official,''
and milk at that value or below is presumed to be free of added water. However, determination of the freezing points of authentic raw and processed
milk samples is essential for establishing evidence of the addition of water.
Henningson reported that evidence exists that the freezing point varies with
such factors as season of the year, feed, ambient temperature, breed of the
animal, time of milking, the animal's access to water, weather, morning and
evening milk, and possibly other factors. Many of these factors apparently
Other, according to Henningson.'^
to

are interrelated.

affected also by vacuum treatment of milk, steriand freezing and holding samples before determining the
freezing point. Addition of milk solids will markedly lower the freezing

The freezing point can be

lization of milk,

point.

A. Apparatus:
The assembled cryoscope consists of:
1. Cooling bath, to permit rapid adjustment of sample to near-freezing
temperatures.
2.

Stirrer.

Freezing mechanism.
Thermistor probe, to permit interpretation of temperature readings
on a scale with a range of 0.001 to - 1 C and successive 0.001- C graduations
at 1.2-mm intervals. Calibrate the instrument as per the manufacturer's di3.

4.

rections.
is needed:
x 100 mm (some models

In addition, the following apparatus


5.

Sample tubes, without

tubes 16 X 100
6.

tion or
7.

mm

with

lip,

16

may

require

lip).

Rubber stopper or other closure for each tube, to prevent evaporacontamination of contents.
Test tube rack.

(5.0-7.5 cm) deep, to hold ice water and cracked ice.


and dry, 2-ml capacity, may be disposable pipets.
B. Standardization of cryoscope:
8.

Bath, 2 to 3

9.

Pipets, clean

Use

in.

either of the following standards:

Sucrose solutions, 7% and 10%: 7 g and 10 g of sucrose, analytical


made up to 100 ml of water; or
2. Sucrose solution equivalents, 7% and 10%: 0.6892 g (-.408 C or
-.422 H) and 1.0206 g (-.600 C or -.621 H) of NaCl, analytical grade,
each made up to 100 ml of water.
Calibrate each instrument following directions in manufacturer's operating manual.
Calibration should be checked weekly or more often, depending on the
1.

grade, each

number of samples

tested.

19.22

233

Vapor Pressure Osmometer

Determine freezing point of the standards, using the same


unknowns.

procedure

test

as for

C. Precautions:
First determinations should establish the freezing point range. Normally,
second and third replicates are needed to establish most accurate freezing
points. Samples with large amounts of added water should be tested for fat
and protein to confirm a problem.
Periodic checks include:
1.

Calibration against standard solutions.

2.

Measurement of bath temperature (normal range -4

to

-7 C) and

level of solution.
3.

Position of thermistor (black speck at tip should be near center of

2-ml sample).
4. Adjustment of
D. Procedure:
It is

tests,

stirrer

essential to use the

and freezer-vibrator.

same technique with each instrument throughout

both on standard solutions and on milk samples. Instruments

may

have different required manual steps and readout capabilities. Use thoroughly mixed samples of milk having a titratable acidity not over 0.18%.
1. Transfer 2 ml of sample to a test tube using a 2-ml pipet.
2. Cool and freeze sample according to manufacturer's instrument instructions.

E. Calculation:

Calculate added water as follows:

milk about T/100.

By common

1%

Per cent added water

Where T = base

of water raises the freezing point of

usage,
-^

100

(T - T')

freezing point of authenticated samples, and T'

true

freezing point of test sample.

For example, if the authenticated sample freezes at -0.542 C.T. or -0.525


and unknown milk freezes at -0.520 C, t', then
jj ^
Per cent added water

-0.542 - (-0.520)

ro"^7

,^^
, ^^
or =
^ ^^^ " 4.06

4%

Vapor Pressure Osmometer ^e. 27


An osmometer has been developed which measures vapor pressure as a
function of dewpoint depression. A small thermocouple detector, located in
the sample chamber headspace, senses temperature at vapor pressure equilibration in the chamber. Only 5-7 /xhteT samples are used. The readout is in
milliosmols (mOs) per kilogram of water. Added water in milk has been
The same precautions associated with
quantitated with this instrument.
19.22

^*^

CHEMICAL METHODS

234

Ref 18, AOAC 1975 ed, 16.093) are


required with the vapor pressure osmometer when passing judgment upon
milk sample "added H2O" status.
thermistor methodology

(SMEDP

13,

A. Apparatus and reagents:


1. Vapor pressure osmometers* are available with either digit LED or
band panel meter read out.
2. Thermocouple cleaning solution, blow clean pressure vessels or automatic head cleaning device.
3. Sample paper discs (6.4 mm diam., Whatman #1).

taut

4.

Stainless steel forceps.

5.

Standard 290

agent grade

NaCl

mOs NaCl

to 1000

solution: Prepare by adding 9.158 g of reml of boiled and cooled distilled H2O (0.1566 mo-

lal).

6.

Acetone.

7.

Lint-free paper tissues.

B. Standardization:
1.

Install

instrument

in

an area relatively free from large ambient temin the manufacturer's

perature changes and adjust the balance as specified


operating manual.

2. Pick up a single sample paper disc at the edge with clean, dry forceps
and immerse into the standard solution tl9.22(A.5)] long enough to saturate
the disc. Do not dip forceps. Allow approximately 2 sec for complete satura-

tion.

concave depression in sample holder. If


inadvertantly touched with sample, wipe
with acetone-impregnated tissue and resample.
4. Gently push sample slide into instrument and seal the chamber by
rotating the sealing knob clockwise.
5. When the beep tone sounds (1 10 sec) adjust the CALIBRATE knob
until the panel meter reads the standard solution milliosmolality (290 mOs).
6. Repeat steps 2-4 several times to assure that instrument repeatability
3.

Promptly transfer disc

outer surface of sample holder

is

290

to

is

mOs.

Repeat using distilled water. The "clean test" should read 20-40
mOs. Higher readings indicate thermocouple contamination. When the
"clean test" value reaches 70 mOs, the instrument thermocouple should be
cleaned using the manufacturer's instructions. Cleaning may be necessary
after every 100-200 samples.
7.

C. Procedure:
1.

Milk samples are introduced into the sample chamber

identical to that of the standard solution 119.22(B)] except

few seconds more to saturate the disc

*Wescor,

Inc.

459 South Main Street. Logan,

if

churned

UT

84321.

it

in

may

fat interferes.

manner

require a

235

Available Chlorine: Thiosulfate Titration

19.3

2. Paper tissues containing acetone are suggested for cleaning the


sample holder between samples. This increases the number of assays possible before thermocouple cleanup is required.

D, Calculation:
Calculate the per cent added water as follows:

% added

H2O = ^ ~

x 100

K.

R = Mean mOs/kg of reference authenticated milk samples which


known to be free of added water.
S = mOs/kg of sample.
Round data to nearest whole per cent.

are

E. Precautions:

While the vapor pressure osmometer has demonstrated several significant


advantages over traditional cryoscopic methods, large errors may result if
the thermocouple is not clean. The need for close observation of this requirement cannot be overemphasized.
19.3 Available Chlorine: Thiosulfate Titration
This method detects available chlorine

in

of sodium and calcium hypochlorite and

^^

commercially prepared solutions


chlorine rinse waters used to

in

check cleanliness of processing equipment."^

A. Apparatus and reagents:

5.

below are of reagent grade unless otherwise specified.


Volumetric pipets, delivery 25-, 50-, 100- and 200-ml.
Stoppered volumetric flask, capacity 100-ml or 1,000-ml.
Erlenmeyer flask, capacity 250-ml or 500-ml.
Graduated cylinder, 10-ml.
Buret, 50-ml capacity, graduated in 0.1-ml divisions.

6.

Analytical balance.

All chemicals listed


1.

2.
3.

4.

Mohr

pipet, 10-ml delivery, graduated in 0.1-ml divisions.


Beaker, 50-ml capacity.
9. Sodium thiosulfate (Na2S2035 H2O), O.OIN and O.IN solution.
10. Potassium iodide solution (KI) 10%: Weigh 10 g of potassium iodide
into a 50-ml beaker and dissolve with distilled water. Transfer to a 100-ml
stoppered volumetric flask and bring to volume with distilled water. Invert
stoppered flask numerous times to mix. Store in a dark place when not in use
7.

8.

and discard daily or when the solution develops a yellow color.


Starch, 0.5%: Make a thin paste from 2.5 g of starch and a little cold
1 1
distilled water. Stir in 500 ml of boiling distilled water and allow to stand

Use the clear supernatant liquid. To preserve the starch solution,


g of salicylic acid or 5 drops of chloroform. Use 0.5 ml per 100 ml of

overnight.

add

.25

solution.
12.

Distilled or deionized water.

CHEMICAL METHODS

236

B. Procedure:
1. Into an Erlenmeyer flask of appropriate size, pipet the correct
amount of sample, as indicated in Table 19:1, Sample Size.

Table

19:1.

Sample Size

Expected Chlorine

Sample Size

Concentration

(ml)

N of Sodium
Thiosulfate
Titrant

Less than 10

ppm

400

0.01

lOtoSOppm

200

0.01

ppm
100 to 200 ppm
200 to 300 ppm
300 to 400 ppm
400 to 500 ppm and

100

0.01

50 to 100

Add

2.

0.01
0.01

25

0.01

25

0.10

5 ml of cone acetic acid, and


yellow color after addition of the acid indicates presence of

ml of potassium iodide solution,

10

swirl to mix.

over

50
25

chlorine.

Titrate liberated iodine immediately

3.

upon addition of acid using the

thiosulfate solution of required normality (Table 19:1) until a pale yellow

color

is

4.

least

reached.

Add

ml of starch and

30 seconds.

If

color, immediately

titrate until the

blue color disappears for at

a solution, after addition of acid,

add the starch indicator and then

is

very pale yellow

in

titrate until the solution

remains colorless for 30 seconds.


5. Determine a blank titration using a volume of distilled water corresponding to that of the sample. If a blue color results, titrate with 0.01 ml of
thiosulfate to the same end point as the sample.
6. Before calculating the available chlorine, subtract the blank titration
from the sample titration result. If the blank requires more than 0.2 ml of
0.01 N solution per 50 ml of water, examine reagents and equipment for

source of contamination.

Calculation:

Available chlorine (ppm)

ml of thiosulfate x

19.4 Fat
19.41

Babcock

A. Milk:
1. Apparatus and reagents:

thiosulfate

ml of sample used

35.450

19.41

Fat:

237

Babcock

Milk pipet Std 17.6 ml

a.

TC

milk pipet meeting

AOAC

specifica-

tions.

Milk

b.

mm).

test

bottle NBS 8%, 18-g capacity, 6

0-1 IOC.
Acid-measure Device used

150-165

Thermometer

c.

d.

or pipet attached to

Swedish acid

17.5 ml.

Dividers or calipers

f.

Bottle holderfor test bottle.

g.

Trunnion

h.

Water bath for test bottles

for acid

60 C.

Reading

light

to

measure H2SO4, whether graduate


must be graduated to deliver

bottle,

for measuring

e.

i.

in. (total ht.

fat

column.

bottle.

able to maintain temperature

used as background when measuring

fat

at 55 to

columns.

Light should be diffused (soft green-colored preferred) and provide illumination from angles above and below level of fat column.
electrically heated, able to maintain air temj. Centrifuge or tester

perature of approximately 60 C.
k.
1.

Centrifuge speed indicator (tachometer).

Sponge.

m. Sulfuric acid (sp gr 1.83-1.83 at 20 C) tempered at 22 1 C.


Soft water maintained at constant temperature of 60 C or above.

n.
2.

Procedure:

Adjust both fresh and composite samples to approximately 38 C, mix until

homogeneous by pouring

into a clean receptacle

and back several times.

Immediately pipet 17.6 ml of sample into a milk test bottle. Do not release
the sample until the bulb of the pipet rests on the neck of the test bottle.
After active flow has ceased (about 30 sec) blow out the last drops from the
pipet tip into the bottle and remove the pipet. Hold the bottle at a slight angle
and add portion-wise about 17.6 ml of sulfuric acid, washing all traces of milk
into bulb. Immediately shake until all traces of curd disappear (reaction temperature should be 100-105 C)

Place bottle in heated (approx 55 C) centrifuge, counter-balance using a


second bottle, and turn on the centrifuge. After proper speed has been attained [19.41(A.3)] centrifuge for 5 minutes. Stop the centrifuge and add hot
soft water until bulb of bottle is filled. Centrifuge the sample for 2 minutes.
Stop the centrifuge and add hot soft water until the liquid column approaches the top graduation of the scale. Centrifuge the sample for 1 min longer.
Transfer the bottle to a water bath that is maintained at 55 to 60 C (prefer-

ably 57 C) and keep bottle there for a minimum of 5 minutes. The water level
should be slightly above the level of the fat column in the bottle. Remove the
bottle from the water bath, wipe it by drying the bottom of the bottle on a
sponge and with the aid of reading light, use dividers or calipers to measure
fat column in terms of per cent by weight to nearest 0.05% from lower surface to highest point of upper meniscus. Fat column at time of measurement

CHEMICAL METHODS

238

should be translucent, golden yellow or amber, and free from visible sus-

pended

particles.

cloudy (milky) fat column or showing the presence


of charred matter or curd, or where for any reason the reading is indistinct or
uncertain; repeat test, adjusting amount of H2SO4 added.
3. Note: Proper speed of the centrifuge may be determined from the
Reject

tests with a

all

"Diam

tabulation below.

of wheel"

is

the distance

between the inside bot-

tom of opposite cups, as measured through the center of rotation of the


centrifuge wheel while the cups are horizontally extended.

DiamofwheeKin.)
(cm)

No. ofrpm

10

12

25.4

30.48

1,074

980

18

20

35.56 40.64 45.72

50.8

800

759

14

909

16

848

22

24

55.88 60.96

724

693

B. Cream^:
1.

Apparatus and reagents:


a.

Cream

(15.24

meeting
b.

test bottles

cm) long neck; or

AOAC

Pipet

shortneck 6

g,

in.

(15.24 cm); or 9 g, 9 in.

18 g long neck, 9 in. (22.86 cm);

to

50%

specifications.

Mohr,

10 ml.

through the letter n same as 19.41(A.l).


with sensibility reciprocal of 30 mg.
o. Cream weighing scales
Weights
9
and
18
p.
g.
g
q. Medicine dropper (for dispensing glymol).
r. Glymol (red or blue reader)
sp gr not to exceed 0.85 at 20 C.
2. Preparation of sample:
Warm all samples to approximately 38 C in water bath. Just before weighc.

ing

sample for

test,

mix sample

until

homogeneous.

3. Procedure: Method 1
Accurately weigh either 9-g sample into a 9-g cream test bottle or 18-g

sample into an 18-g

bottle.

Avoid

spilling

cream on outside of

test bottle or

scale pan. Hold the bottle at a slight angle and add, in several portions, about
8-12 ml of sulfuric acid to 9-g bottle; or 14-17 ml to 18-g bottle, or add acid

mixture of cream and acid, after shaking, is chocolate brown. Rotate


the bottle slowly during the addition to rinse down any cream that may cling
in the neck. Immediately shake until all lumps completely disappear and add
5-10 ml of soft water and transfer to centrifuge. Proceed as in 19.41(A.2)
until

"Place bottle in heated centrifuge ..."


4. Procedure: Method 11
Accurately weigh 9 g of cream into tared test bottle. Pipet about 9 ml of
soft water and mix the water and cream thoroughly by swirling the bottle.
Hold the bottle at a slight angle and add about 17.5 ml of sulfuric acid,
rotating the bottle slowly during addition to rinse down any cream that may
cling to the neck. Immediately shake until all lumps completely disappear

starting with

19.42

Fat:

239

Gerber

and transfer bottle to centrifuge. Proceed as


."
"Place bottle in heated centrifuge
.

Whichever method
tained at 55 to 60

is

in

19.41(A.2) starting with

used, transfer the bottle to a water bath that is mainminimum of 5 minutes. The water

(preferably 57 C) for a

should be slightly above the level of the fat column in the bottle. Rethe bottle from the water bath and quickly dry the bottom of the bottle
on a sponge. Add 2 drops of glymol, allowing it to run down the inside neck
of the bottle to the top of the fat column. Immediately measure the fat column using dividers or calipers from the bottom of the lower meniscus to the
sharp line of demarcation between the glymol and fat. Read the percentage

level

move

of

fat directly.

fat column at time of reading should be translucent, golden yellow to


amber, and free from visible suspended particles.
Reject all tests with a cloudy (milky) fat column or that show the presence
of charred matter or curd, or where for any reason the reading is indistinct or
uncertain; repeat test, adjusting amount of H2SO4 added.

The

19.42 Gerber

19- 20

This method

is

applicable to raw, pasteurized, homogenized, and com-

posite (preserved) milks, exclusive of chocolate-flavored milks.

A. Milk:
1.

Apparatus and reagents:


Standard Gerber milk

a.

test bottle,

8% Clear,

transparent, color-

annealed and free from defects;


neck, body, flat tube, and bulb on a straight median axis, joined smoothly to permit free flow of liquid; wall thickness adequate to provide sufficient strength but not less than 0.9 mm at any point (see Fig. 19:1); and
less, resistant borosilicate glass, well

of the following dimensions:

Total length

190

Neck and body


Neck length

cylindrical

Neck,

Neck

15

ID

11.5

rim, optionally reinforced

2.5

1.0

mm

mm
mm

0.5

diam

total

not

thickness of external rim

more than

2.5

mm

GRADUATED AREA
FLAT TUBE
^^^^
Figure 19:1, Gerber milk

BODY
test bottle,

LOCK
STOPPER
lock stopper and key.

diam

CHEMICAL METHODS

240

Body,

Body

OD

not

capacity

21.5

more than 25.0


0.4 ml

mm

diam

Flat tube, uniform in cross-section

graduated length

not less than 70.0

mm

tube, uniform cross-section


beyond each terminal graduation
not less than 3.0
Flat tube, external width
not less than 10.0
Graduation lines, permanent, cleanly
Flat

etched, perpendicular to

flat

mm
mm

tube

pigment contrasting
sharply with color of fat; width of

axis, filled with

mm

0.10 to 0.20

lines

Bulb, tapered, and with matte surface

on side above graduations for


sample identification, O D
not more than
Graduated volume

in the flat

tube at 20

1.000 ml

is

mm on the flat tube and

extends not less than 70

is

15.0

13.546

mm
gHg).

It

divided into 80 equal

each equivalent in volume to 0.0125 ml. Error of total calibrated


is not to exceed V2 of the smallest graduation. Graduation
lines are uniformly centered on the flat tube face, with the 0. 1% lines not
less than 3 mm long, the 0.5% lines extending
mm on each side beyond
parts,

length

1%

the 0.
side

lines,

and the

beyond the 0.5%

.0%

lines,

lines

extending not less than

mm on each

or perhaps extending across the entire

flat

face.

Each whole-percent graduation is identified by etched and pigmented


numbers running serially from
to 8, at the right and just above the
whole-per cent lines, with the zero measurement nearest the body. Capacity of the bulb at 20

to the

are identified "Milk," with the

8%

line

name

is

distributor permanently inscribed on the


plied after recalibration

not less than 1.5 ml. Bottles

or symbol of the manufacturer or

where required

body and a State symbol apconformance with state

to attest

specifications.

Bottle support rack

b.

To hold bottles

in

a secure, vertical position.

Stainless steel (Series 300) racks consisting of

and a

solid

two perforated shelves

bottom, supported by solid sides, provide convenience and

durability. Optionally use racks with stainless steel "rapid

clamp-on"

covers to lock bottles securely when using shaking machines.


c.

Milk

test pipet

To

contain

11

ml. Clear, transparent, colorless

from defects and well annealed; bulb, suction and delivery tubes on a straight median axis; bulb cylindrical and tapered to
tube junctions to permit free flow of liquid; top of suction tube smooth
and perpendicular to the tube axis; and delivery tube wall straight above
the tapered tip, with dimensions as follows:

glass tubing, free

19.42

Fat:

241

Gerber

Wall thickness

not less than 1.0

Total length

340

Delivery tube length, including tapered

Tapered

tip,

length

Delivery tube,
Bulb,

tip

mm

mm
105
5 mm
.5 mm
7
6.5 0.5 mm
16.0 1.0 mm

20

OD

OD

Suction tube, to graduation

not less than 110

Suction tube, O D
Graduation to bulb

40
6.5

mm

mm
mm

0.5
15

Graduation line, 0.1-0.2 mm wide, is cleanly etched completely


around and perpendicular to the end of the suction tube, the line filled
with adherent dark pigment. The pipet may be filled to the mark with
water at 20 C to discharge within 5-8 sec, TC 11.07 0.03 ml at 20 C;

and is identified "TC 1 .07 ml at 20 C," with the name or symbol of the
manufacturer or distributor permanently inscribed on the bulb and a
State symbol applied after recalibration where required, to attest conformance with state specifications.
Concentrated commercial (technical) grade,
d. Sulfuric acid (H.2SO4)
1

clean, colorless, free from fat, sp gr 1.820-1.825 at 15.5

sponding to 90-919^ H2SO4

closed contain-

ers.

by weight, stored

in tightly

(60 F), corre-

Isoamyl alcohol for Gerber test Clean, clear, colorless or almost


colorless. CP primary isobutyl carbinol, free from water, acid, fat and
furfural: sp gr to be 0.814-0.816 at 15.5 C: more than 95% distills at an
atmospheric pressure of 760 mm Hg within a 2-C range between 128 and
e.

132

(it

evaporates, leaving no residue); store

in tightly

closed contain-

amber bottles, or in darkness.


Test each batch of isoamyl alcohol identified by the manufacturer as
suitable for Gerber milkfat tests by transferring 10 ml of H2SO4, 1 1 ml of

ers, in

water, and

ml of the isoamyl alcohol to a

test bottle.

Shake and

centri-

fuge bottles as required in the test for fat in milk [19.42(A.2)]. If oily

Use four numbered bottles, a


sample of milk for tests in the same bottles,

particles are recovered, discard the batch.


single pipet,

both with

and a single

new and

Determinations should

in-use isoamyl alcohol.

0.05% variation.
For H2SO4, use a measure to deliver 10 ml.
For isoamyl alcohol, use a measure to deliver ml. Optionally use a tilt

agree, with less than


f.

Reagent dispensers

measure, buret, or short cylinder for dispensing lO-ml quantities; opmeasure, semiautomatic syringe, or other suitable
apparatus to dispense 1-ml portions; use of pipets to measure H2SO4
and isoamyl alcohol is unsafe. Dispensers that operate by adjustable
positive displacement (syringes), when properly used, combine uniform
tionally use a

tilt

CHEMICAL METHODS

242

delivery speed with no loss of time for drainage. Burets and automatic

devices of various types are available.


The lock stopper
g. Lock stopper and key for bottle

is

of rubber or

synthetic resilient, reagent-resisting material, of standardized dimensions,

molded

to provide a seat for the ball or plug,

and a channel for the

key: rim and channel are reinforced by metal, plastic, or other firm binding or insert.

The key

is

of noncorrodible material suitable for inserting

into the stopper.


h.

Centrifuge for bottles

to rotate at 1.100

Standard

style,

with disc and trunnion head

100 rpm; or an angle head to rotate at 1,350

100

rpm. Trunnion-head models are supplied with heaters to maintain internal temperature at approximately 60 C. Use a centrifuge that attains full
rotating speed within 2 min of operation. If not equipped with permanent speed indicators, check the operating speed of all Gerber centrifuges under full load at monthly intervals, or more frequently if necessary. Bottles should receive an acceleration of 350 50 times the
acceleration of gravity.

Optionally use

Babcock centrifuges

for

9-in.

(22.8

cm)

bottles,

equipped with heaters and cup adaptors for Gerber bottles, and operate
at 1,100 100 rpm.
i.
Water bath Depth to permit vertical immersion of bottles to the
terminal bulb, equipped to provide intermittent or constant agitation
and to control temperature at 60-62.8 C.
To standardize test conditions and to
j. Shaking machine (optional)
save time. Available models may be readily modified to hold Gerber

racks.

k. Desk reader (optional)


Improvised to expedite reading of fat columns. Models with a shielded tubular electric bulb standardize background illumination for the bottles.
1.
Washing device (optional) Consisting of a rack and perforated cov-

er (Item b), expedites washing. After reading, return bottles to the rack,

remove the stoppers, lock

the rack cover; empty, wash, rinse and drain

bottles as a imit.
2.

Procedure:

Measure

10 ml of

H.SO4

0.2 ml at 15.5-21.1

properly prepared milk sample

at

into a test bottle.

more than 23.9 C, measure

not

From

the test

charge, using an 11-ml pipet. with the top of the meniscus resting on the

graduation line of the suction tube, and touch off any part-drop

Allow the milk


with the acid

to drain into the bottle

then

at first, to

permit the pipet to empty. Wait a

flow stops and blow out the last drop.


the lock stopper securely.

Add

it

(CAUTION HOT)

until the
at

curd

at

the

tip.

prevent reaction

full 3

sec after free

ml of isoamyl alcohol and insert

With stoppered end up, grasp the

graduated column and shake


the bottle

slowly,

is

test bottle at the

completely digested. Holding

stopper and neck, invert

it

at least four

19.42

Fat:

243

Gerber

times to mix the acid remaining

shaking of

filled,

in

the bulb with the contents. Machine-

scaled test bottles secured in racks equipped with quick-

locking covers insures safe, rapid, uniform treatment and optimal temperatures for centrifuging.

Prepare composite (preserved) and aged milks for measurement of the test
C and thoroughly disperse all fat before
transferring the test charge. To prevent charring of warm milk by acid, a.)
cool the acid to 10 C or below just before adding the warm milk charge; or
portion by heating the milk to 35-38

contents of the test bottle after adding the acid; or c.) transfer the
milk before adding the cooled acid. Use Method c.) when test bottles
are filled at one location and tests are completed at another. Chill the acid to
below 5 C and hold the test bottle at a 45 angle, with the plane of its flat
b.) cool

warm

column vertical, to displace milk from bulb and column with acid.
Before contents of the bottles have cooled below 60 C. place them in a
centrifuge and counterbalance the load as needed. Before operating the machine, close the cover and lock. After the required speed is attained, centrifuge for 4 minutes. Stop the machine and place the bottles in a water bath at

glass

60-62.8 C, leaving only the bulbs exposed. After

remove the

at least 5

bottles singly. Holding each bottle vertically,

fit

min

in

the bath,

the key into the

lock stopper, optionally using a desk reader [19.42(A.l)k]. Gently push the

bottom of the fat column upward so that it coincides with


whole-per cent graduation mark. Promptly read the scale at the
bottom of meniscus and the top of fat column to the nearest 0.10% graduation; subtract the lower from the upper reading and record the difference as
the fat content in each sample, before removing the next bottle from the
bath. Experienced operators can accurately estimate fat tests to the nearest
0.05%.
Repeat tests where fat columns are imperfect. As fat cools in the column,
the slope of the meniscus increases, tending to lower the per cent fat reading. Soft, glareless background lighting and a desk reader stand, which is
straight line at the

the nearest

readily adjusted to bring the

column

to tester

eye level, expedite rapid, accu-

rate readings.

Proper

columns contain no

fat

light or

dark particles or zones. They are

pale to strong yellow in color, depending on the season of the year and

feed given the animals.

When

test results are a

on

matter of public record, or to

determine adherence to standards or provide the basis of payment, test samples in duplicate, repeating the tests when duplicate determinations vary

more than 0.10%.


B.

Cream:
Apparatus and reagents;
5-g capacity, construca. Standard Gerber cream test bottle, 50%
the external width of
that
in
19.42(A.l)a
except
tion and dimension as
1.

the

flat

tube

is

not less than 13

mm.

CHEMICAL METHODS

244

Graduated volume

at

extends not less than 70


parts,

length

20

in the flat

tube

2.839 ml (38.457 g Hg). It


is divided into 100 equal

is

mm on the flat tube and

each equivalent in volume to 0.02839 ml. Error of total calibrated


is not to exceed V2 of the smallest graduation. Graduation

lines are

uniformly centered on the

flat

tube face, with the 0.59^ lines not

and the 5.0%

mm on each side beyond


mm equally
lines extending not less than

on each side beyond the 1%

lines, or perhaps extending across the en-

less than 3

the

0.5%

mm long, the

lines,

1.0%

lines

extending

tire flat face.

Each successive 5% graduation is identified by etched and pigmented


numbers running serially from to 50, at the right and just above the per
cent lines, with the zero measurement nearest the body. Capacity of the
body, at 20 C, from junction with the neck to the 0% line is 19.0 0.4
ml; capacity of the bulb, at 20 C. to the

Bottles are identified

"Cream,

50%

line is not less

g," with the

name

than 2.0 ml.

or symbol of the

manufacturer or distributor permanently inscribed on the bulb and a


State symbol applied after recalibration where required to attest con-

formance with State specifications.


b. Gerber cream test bottle, 25%
flat

tube

is

Graduated volume

at

20

in the

1.4195 ml (19.2285 g Hg). Graduation lines are uniformly

centered on the

flat

tube face, with the 0.2% lines not less than

mm

long, the 1.0% lines extending 1.5

mm

on each side beyond the 0.2%

line, and the 2.0% lines (and the 25% line) extending not less than 1 mm
on each side beyond the 1.0% line, or they may extend across the entire
flat face. Even-numbered per cent graduations should be numbered, in
addition to the 0% and 25% lines. Capacity of the body, at 20 C, from
junction with the neck to the 0% line is 20.5 0.4 ml.
Graduated volume at 20 C in the
c. Gerber cream test bottle, 15%
flat tube is 0.8517 ml (1 1.537 g Hg). Graduation lines are uniformly centered on the flat tube face, with the 0.2% lines not less than 3 mm long,
the .0% lines extending 1.5 mm on each side beyond the 0.2% line, and
the 2.0% lines (and the 15% line) extending not less than 1 mm on each
side beyond the 1.0% line, or they may extend across the entire flat
surface. Even-numbered per cent graduations should be numbered, in
addition to the 0% and 15% lines. Capacity of the body, at 20 C, from

junction with the neck to the

0%

line is 21.0

0.4 ml.

Balance for weighing cream Accurate, having a sensitivity reciprocal not greater than 0.03 g, equipped with a convenient taring or counterbalancing device, and with a pan bottle support on the balance to
provide firm vertical support for the bottles.
e. Weight, 5-g
One-piece, accuracy 5.000 0.005 g.
f.
Transfer device for sample A pipet of not less than 5-ml capacity,
or a syringe with a 10- to 14-gauge cannula, will be satisfactory.
As described in 19.42(A.l)b,d,l.
g. Other apparatus and reagents
d.

19.43

245

Fat: Roese-Gottlieb

Procedure: Measure

2.

cream

10

0.2 ml of

H2SO4

at

15.5-21

into a

Place the bottle in the support on the balance and tare.

test bottle.

Place the 5-g weight on the opposite pan and weigh 5.00 g of prepared cream
sample into the bottle. Remove the bottle from the balance and add 5 ml of

water

C and

15.5-21

at

ml of isoamyl alcohol. Insert the lock stopper se-

curely and proceed as in 19.42(A.2).

Read

the scale to the nearest

0.5%

graduation mark and record results.


C. Chocolate milk:

This method

is

applicable to sweetened, flavored milk and drinks.

Apparatus and reagents:

1.

Same

as for milk [19.42(A.l)b,e.l] and for

ing:
a.

cream

[19.42(B.l)d,f], also add-

Slowly add 94 parts by volume of cold sulby volume of cold water.


Weight Special one-piece 11.125 0.005-g weight, or an equivacombination of weights, kept clean and free from corrosion.
Sulfuric acid, diluted

furic acid to 6 parts


b.

lent

Procedure:

2.

Measure

10

0.2

ml of diluted H2SO4 [19.42(C.l)a]

at 15.5-21.1

into

a milk test bottle. Pipet the test charge, see 19.42(A.2). If viscous, tare and

place the 11.125-g weight on the balance pan, and weigh 11.125 g of properly
prepared sample into the bottle. Remove bottle from balance and add 2 ml of

isoamyl alcohol. Insert the lock stopper securely and proceed as in


19.42(8.2). Read the scale to the nearest 0.1% graduation mark and record
results.

19.43 Roese-Gottlieb

3'

32

A. Milk, cream, ice cream and frozen desserts, nonfat dry milk and dry

whole milk powder, and malted milk:


1. Apparatus and reagents:

Mojonnier G3** or #M-8865-S.^


#T-49, or Disposable pans^ can body, white

a.

Fat extraction flask

b.

Aluminum

dishes*

with clear coating, 309 x 307 x 207


c.

Cork stoppers,

to

fit

d.

Crucible tongs.

e.

Analytical balance.

f.

g.

h.
i.

j.

flask

mm.

#6.

Interval timer.

Mohr

pipet, or pipetting device, 10 ml.

Hot plate.
Water bath, maintained

at 100 C.

Beakers, 150 ml.

^Lurex Manufacturing Company, 1298 Northwest Boulevard. Vineland. NJ 08360


^Mojonnier Brothers. 4601 West Ohio Street, Chicago, IL 60644.
^Central States Can Company, 700-16th Avenue, SE, Massillon, OH 44646


CHEMICAL METHODS

246
k.
1.

Stirring rods.

Hood, or

volatile solvent

m. Automatic shaker

exhaust apparatus.

optional.

with Drierite (calcium sulfate).

n.

Desiccator,

o.

Suspension apparatus for holding

p.

Wash

filled

flasks.

bottle.

specially denatured, US formula #1 or #30.


hydroxide ACS grade, sp gr 0.90.
Distilled water.
Ethyl ether ACS grade, peroxide-free.
Petroleum ether ACS grade, boiling range 30-60 C.
Preparation of sample before ether extraction:
Milk Adjust both fresh and composite samples to approximately

q.
r.

Alcohol

Ammonium

s.
t.

u.
2.

a.

20 C, mix until homogeneous by pouring into clean receptacle and back


repeatedly and promptly weigh approximately 10-g sample directly into
If lumps of cream do not disperse, warm sample in
water bath to approximately 38 C and keep mixing until homogeneous,
using policeman, if necessary, to reincorporate any cream adhering to
container or stopper. Add 1.25 ml of ammonium hydroxide or 2 ml if
sample is sour, and mix thoroughly. Add 10 ml of alcohol and mix well.

extraction flask.

Follow directions under procedure 19.43(A.3).


b. Cream
Same as milk except weigh 5 g into a tared flask and
dilute with approximately 10.5 ml of H2O.
Accurately weigh 4-5 g of a thorc. Ice cream and frozen desserts
oughly mixed sample directly into fat extraction flask. Dilute with 10 ml
of water and rinse sample into lower chamber. Mix by shaking. Add 2
ml of ammonium hydroxide, mix thoroughly, and heat in water bath for
20 min at 60 C with occasional shaking. Cool and add 10 ml of alcohol to
sample and mix. Follow directions under section 19.43(A.3).
d. Nonfat dry milk, dry whole milk, powder, and malted milk
Quickly weigh approximately g of well mixed sample into small beaker.
Add ml of water and rub to smooth paste. Add 9 ml of additional water
and 1-1.25 ml of ammonium hydroxide and warm on steam bath. Transfer to fat extraction flask with 10 ml of alcohol and mix thoroughly. Cool
and follow directions under procedure 19.43(A.3).

3.

Add

Procedure:
25 ml of ethyl ether to sample, stopper with cork and shake very

vigorously one minute. Cool,

if

necessary, add 25 ml of petroleum ether and

repeat vigorous shaking. Caution


the flask during shaking, twist to

is

necessary, since pressure builds up

remove cork momentarily from

odically to release pressure. Centrifuge flask at approximately 600


it

stand until upper liquid

able flask or metal dish.

is

practically clear.

As stoppers

are

in

flask peri-

rpm or

Decant ether solution into

let

suit-

removed wash "inserted portion"

with equal parts of the two ethers and add washings to dish. Repeat extrac-


19.43

247

Fat: Roese-Gottlieb

ml of each solvent each time


necessary, but omitting rinsings with mixed solvents

tion of liquid remaining in flask twice, using 15

and adding water

if

necessary with nonfat dry milk

after final extraction. (Third extraction not

product.)

Evaporate solvents completely on hot plate or steam bath at temperature


does not cause spattering or bumping (boiling chips may be added).
Dry fat to constant weight in oven at 102 2 C or a vacuum oven at 7075 C under pressure below 50 mm Hg. Weigh cooled flask or dish, without
wiping immediately before weighing. Remove fat from container with 15-25
ml of warm petroleum ether, dry. and weigh as before. Loss in weight of
that

container

is equal to
Notes:

4.

a.

Correct

agents used.

fat
If

fat.

weight by doing a blank determination on ether

blank

is

re-

greater than 0.5 mg, reagents must be replaced

or purified.
b.

Difterence between duplicate determinations obtained by analyst

should not be more than 0.039^.


c.

Certain types of products such as listed below can be tested for

fat

using this ether extraction method

Animal feeds
Cheeses

Condensed milk products

Cream

(regular, sour

and imitation sour)

Cultured milk products

Dairy puddings

Dry and concentrated whey products


Dry chocolate milks
Dry milks
Ice cream mixes and toppings
Sherbets
Yogurt flavorings.
Roese-Gottlieb test equipment:''

5.

Equipment

is available to help conduct the Roese-Gottlieb fat extraction


and also moisture/solids tests. Laboratories not so equipped can use manual
procedures. It is no longer a requirement that the extraction flasks be centri'fuged
though the available equipment uniformly off'ers electric centrifuges. Hot plates, steam baths, desiccators, vacuum ovens and automatic
tilting pipets can be substituted for traditional tester components.
6. Procedures for using test equipment:
To prepare fat dishes, place clean dishes in a vacuum oven at 100 C for 10
minutes. Transfer the dishes to a desiccator for 10 minutes. Weigh on an
'

"Plans for testers and flask shakers are available from Kraft. Inc., Chicago, lihnois. Testers
Ohio Street, Chicago, Illinois 60644.
can be obtained from Mojonnier Brothers Co.. 4601

CHEMICAL METHODS

248

and keep in the desiccator until


pans should be weighed as near as possible to the time

analytical balance to obtain tare weight

ready to use. The

fat

of analysis.
a.

tester temperature controls at least

Turn on the

hr before begin-

ning fat tests. Accurately weigh 1 .0 to 1 .5 g of sample into a tared tester


boat on an analytical balance. Place tester boat, with sample, into a

clean dry fat extraction flask.

Add

b.

7 ml of distilled water at 60

to the flask, being careful to

wash down any sample still adhering to the sides of the flask. Cork the
flask. Keep it in an upright (vertical) position and shake vigorously for a
short time to uniformly suspend the sample. Shake with a swirling motion to

make

keep the contents

lower bulb. Care should be taken to


suspended before adding the ammonium

in the

sure the entire sample

is

hydroxide.

ammonium hydroxide and 10 ml of alcohol, mixing


between additions as follows: place the forefinger over
the cork, with the hand around the upper portion of the flask. Shake the
flask with horizontal and vertical rotary motion for about 90 seconds.
Remove the cork by twisting it off" carefully, and add the next reagent.
c.

Add

ml of

and shaking

d.

Add

in

25 ml of ethyl ether to the flask and cork

the mechanical shaker

if

one

is

it.

Place the flask in

available or shake manually for exactly

30 sec (each shaking device has an automatic timer operating with

While

flasks are shaking,

Remove

add solvents to another

unit).

set of four samples.

from shaker and remove cork from flask by twisting


good idea to form the habit of twisting the cork to the
right (clockwise) when placing the cork in the bottle and twisting it to
the left (counterclockwise) when removing it] to help groove the cork
for easy removal. Add 25 ml of petroleum ether to the flask and cork it.
Shake in the shaker for exactly 30 seconds. Remove flask from shaker
and remove cork carefully, thus releasing the pressure.
e.

it

carefully

flask

lit is

Remove

from shaker, remove cork from flask, and centrifuge


flask from centrifuge. Hold flask by small end
of the bulb. Raise to eye-level height and carefully decant the ether
layer into a tared fat dish. (In this step, avoid tilting the flask beyond a
straight horizontal position so that none of the aqueous layer will pour
off with ether layer.) Evaporate ether at 100 C with hood of the fat hot
f.

flask

for 30 seconds.

plate

Remove

down over

the sample.

For the second extraction, add 5 ml of alcohol to the flask. Keep it


in an upright (vertical) position and swirl in a rotary motion to mix the
sample. Repeat shaking with the cork in the flask, for about 30 seconds.
g.

h. Add 25 ml of ethyl ether and 25 ml of petroleum ether to the flask


of mechanical shaker after the addition of each reagent, corking the

19.44

Automated Turbidometry

249

and following steps d, e, and f. Centrifuge for 30 seconds. Remove


from centrifuge.
i.
The dividing line between the ether and aqueous layer must not he
above the top neck of the lower bulb of the flask. If the dividing line is
below the neck of the lower bulb of the flask, add distilled water dropwise from a pipet to raise it to within about Vk in. to 'A in. (0.32 cm to
0.64 cm) of the top neck of the lower bulb of the flask.
j. Hold flask by small end of the bulb, raise to eye-level height, and
carefully decant ether layer into the same dish used before. (Avoid tilting the flask beyond a straight horizontal position.) Evaporate ether at
100 C with hood of the fat hot plate down over the sample.
k. Place pan in vacuum for 10 min at 100 C. Vacuum should reach at
least 20-25 in. (50.8 to 65.5 cm). Release vacuum, transfer pans to desiccator for 10 min and weigh.
flask

flask

7.

Calculation

% Fat

gam

in

weight of tared pan

^^

weight or sample

B. Cheese (acid hydrolysis)

'.

Apparatus and reagents:

1.

Same

as 19.43(A.l) with the addition of

w. Litmus paper-red.
X.
y.
z.

2.

glasses, 50 mm.
Hydrochloric acid, ACS grade, sp gr 1.1.
Glass beads, anti-bumping discs, or boiling chips.

Watch

Procedure:

Accurately weigh approximately g of prepared sample into beaker. Add


9 ml of water and, if desired. 1 ml of ammonium hydroxide. Mix until
smooth; then warm mixture at low heat until casein is well softened. If am1

monium hydroxide was used, neutralize


Remove from hot plate.

with

HCI using

litmus paper as in-

dicator.

With samples in fume hood, cautiously add while stirring, 10 ml of HCI


and a few glass beads or other inert material previously digested with HCI to
prevent bumping, cover with watch glass and boil gently 5 min or place in a
beaker in a boiling water bath 20 minutes. Cool solution and transfer mixture
to fat extraction flask.

Rinse beaker successively with 10 ml of alcohol, 25 ml of ether and 25 ml


of petroleum ether and transfer rinsings to flask; mix thoroughly after adding

each reagent. Proceed as


pressure builds

in

19.43(A.3) beginning "caution

is

necessary since

.".

19.44 Automated Turbidometry

Recent electronic milkfat

test

instruments measure the turbidity or

scattering caused by the fat globules.

The sample

is first

light

homogenized

to

CHEMICAL METHODS

250

produce

fat

globules of uniform size. Next,

is

it

diluted quantitatively with

an alkaline solution of sodium tetraethylenediamine tetraacetate

(EDTA)

to

eliminate turbidity caused by the colloidal protein (casein) particles. Finally

passes through a photocell where the

light scattering is measured. The


which is proportional to the fat content of the milk, is registered on a read-out system calibrated to read the per cent of fat directly. All
operations are done sequentially in the instrument.
Milko-testers models Mark 11 and 111. and 111 Industrial," and the Anritsu
K373A and K373A1 Milk Checker are designed to test primarily raw unhomogenized milk and cream. Milko-tester Mark Ill-Industrial Model can be
used to test homogenized milk.
Although the method described herein refers to Milko-tester apparatus
and equipment, the Anritsu Milk Checker models K373A and K373A1 are
equally suitable for milkfat determinations and have received AOAC apit

light scattering,

proval.

A. Apparutiis and reagents:


1.
Milkfat turbidometer acceptable units include the Milko-tester

and

the Anritsu Milk Checker.^


2.

Manufacturer's instrument operation manual.


to maintain 37

3.

Water bath, thermostatically controlled

4.

Distilled water.

5.

EDTA

solution: Dissolve 45.0 g of

20 (Tween 20), and 7.6 g of

sodium

EDTA

EDTA.

0.5-1 ml of Triton

salt, 'Hiu

NaOH

EDTA,

in

EDTA,

Na^

10

C.

ml of polysorbate
(The di-

10 liters of distilled water.

can be used

in

place of the tetra sodium

X-100 and Antifoam Y-30 may be used with Mark

III unit.)

B. Calibration of instrument:

Each instrument
in triplicate

is

calibrated, using 20 representative milk samples tested

and ranging from

Wc

to

6%

in fat

content.

The

calibration milk

samples should be previously tested by either the ether extraction method


(19.43(A)] or a volumetric method [19.41(A)]. Fat samples shall be prepared
from raw milk less than 48 hr old." For fat standardization purposes do not
use cream which has gone through a separator as physical changes occur in
the fat.'-'* Directions for calibration and suggested limits of deviation have

been presented by Shipe.'"'


The averages for each sample obtained by each method (standard and
Milko-tester) shall be computed to the nearest 0.01%. Using the averages,
the standard deviation of the difference should be computed as follows:

S(D2)

Sd =

^Distributor. Foss American, Inc. Route 82, Fishkill,

American Metering Systems,

Inc. PC)

Box

129,

NY

Hopewell

12524.
Jet,

NY

12533.

19.44

Automated Turbidometry

where

D =

251

Average of standard method resuhs on a sample minus the average of Milko-tester results on the same sample, e.g.
((B,

+ B2 +

B3)/3)

- ((M, + M2 +

M,)/3)

= D

where

B = Babcock reading
M = Milko-tester reading
N = Number of samples tested.

If the specification of 20 samples is


exceeded, all samples tested must be included in the calculations except for those for which an error in one or more determinations can be proven,

and Sd= Standard deviation of difference.


be considered to be properly calibrated when the stanis equal to or smaller than the value shown in the
Table 19:11. Should the standard deviation of difference exceed values
shown in Table 19:11, the instrument must be adjusted in accordance with

The Milko-tester

will

dard deviation of difference

the manufacturer's instructions and the calibration procedure repeated by


retesting the

same samples.
and preparation of sample:

C. Collection
1.

Before testing, samples

2.

Samples

shall be stored at 0-4.4 C.


be prepared for testing by tempering at 37 1 C in a
thermostatically controlled water bath for a maximum of 15 min after temperature is reached. CAUTION: Samples should not be held in water bath
longer than 15-20 minutes.'^
3. Gently mix samples by inverting the sample about six times being
shall

sample instabili(Must have air space in container otherwise sample must be mixed by
passing from one container to another.)
4. Samples must be tested no later than 30 sec after mixing.
D. Procedure:
Obtain readings using manufacturer's instructions for instrument operation and maintenance.

careful to avoid over mixing or churning action resulting in


ty.

Table

19:11.

Maximum allowable standard deviation of difference between the


instrumental and the reference method

Reference method
^^^^

Instrument calibrated for use on


Individual

cow samples

Herd or composite samples

Mojonnier or ether
0.04

0.06

0.06

CHEMICAL METHODS

252

19.5 Moisture

19.51

and Solids

Vacuum Oven

A. Apparatus and reagents:


1. Thermometer, 0-110 C.
2. Desiccator filled with efficient desiccant such as Drierite, indicating,
4

mesh (calcium sulfate), calcium chloride, etc.


3. Aluminum moisture dishes: AOAC specifications, round,

tomed, at least 5 cm in diam, provided with close-fitting


#12715** or equivalent.
4.

Crucible tongs.

5.

Analytical balance.

slip-in

flat-bot-

cover,

Cenco

Vacuum oven equipped

with sulfuric acid Biichner flask gas washThese bottles should be arranged so that the moisture in the air
entering the oven from the outside is removed. Corning glassware #311760
6.

ing bottles.

or equivalent.
7.

Vacuum pump: Welch


minimum vacuum of

taining a

in.

8.

Steam bath.

9.

Osterizer or Waring blender.

10.

Sulfuric acid. Technical, or

B. Collection

type #1404 H, capable of maincm) in the oven.

distillation

26

(65

P.

and preparation of sample:

When natural cheese can be cut, take a narrow


wedge-shaped segment reaching from outer edge to center and place in container with a tight-fitting lid. When cheese cannot be cut, take sample with
cheese trier. Three plugs of cheese should be taken, one from center, one
from point near outer edge, and one from point halfway between the other
two. Cheese plugs should be placed immediately in container with tight fitting lid. Before taking sample for analysis, the wedge-shaped segment
should be cut very finely with the aid of a knife or spatula and mixed well. If
plugs are taken, either place all three side by side on a flat stainless steel or
glass plate and cross-cut into fine slices about V32 in. (0.77 mm) in thickness,
or mix plugs with the aid of an Osterizer blender for a total of about 15
seconds. Blender should be turned off" and sample and jar shaken manually
to help move larger particles toward bladeside of mixer. Prolonged mixing is
to be avoided as fat separation and mashing of product occurs.
2. Process cheese, cheese food and spreads: With the aid of a spatula,
cut cheese into small segments and place in a 4- or 8-oz (120 or 240 ml)
sample jar. (Avoid taking outer surface of cheese for sample use.)
1.

Natural cheese:

**Central Scientific, 2600 S Kostner, Chicago, IL 60623.

253

Vacuum Oven

Moisture and Solids:

19.51

3. Regular cheese spreads: With the aid of a spatula, thoroughly mix


approximately 5 oz (150 g) of sample of cheese in an 8-oz (240 ml) sample

jar.
4. Condiment cheese spreads: To get a more homogeneous sample, it is
necessary to mix at least a 5-oz (150-g) sample of cheese in an Osterizer
blender for four 15-sec periods, turning off and starting blender at the end of
each 15-sec period (making a total of one minute).

Cheese powders, graded and flavored: With the aid of a spatula,


thoroughly mix samples before sampling.
6. Milk, ice cream and malted milks: Same as item 5 above.
C. Preparation of moisture dishes:
The aluminum moisture dishes used shall be washed thoroughly, rinsed,
and dried in an oven for several hours at 100 C. Store in a clean desiccator
5.

until used.

D. Procedure:
1. Weigh about 2-3 g of sample into a tared dish on an analytical balance. Where possible, distribute sample evenly over the bottom of the dish.
Some samples require predrying, see 19.5 1(F. 2). Place dish with cover

on metal shelf in the vacuum oven for 5 hours. The oven


temperature specified for the particular sample, see
19.51(F.l), being tested with a minimum vacuum of 26 in. (15.24 cm). During
drying, admit into oven a slow current of air (about 2 bubbles per sec) dried
by passing through sulfuric acid washing bottles.
2. After 5 hr, shut off the vacuum pump and slowly readmit dry air into
the oven. Using tongs press cover lightly into dish and remove dish from the
oven. Cool in desiccator for at least 30 min, or until the dish has reached
room temperature. Weigh dish on analytical balance.
placed beneath

should be

at

it,

the

E. Calculations:

^
%

^,

Moisture =

Solids

Loss in weight (g) x 100


j7t
.^.
Weight, of^ sample (g)

100

Moisture

F. Notes:
1.

The 100

C vacuum oven

moisture method can be applied to

the following dairy-type products and ingredients:

Casein powders

Cheeses
Cheeses

natural, process, foods and spreads


powders, grated, flavored

Dairy animal feeds


Dried egg products
Dried milks whole and nonfat, and buttermilk

Gelatins
Ice

granules, crystals and powders

cream powder mixes

CHEMICAL METHODS

254

Malted and chocolate milks


Nuts and nut products.
The 70 2 C vacuum oven moisture method applies

to the following

products:

Food flavor and colors


Foods with a large amount of sugar
Fruit products

Sugars.

The following product types

2.

require samples to be either partially

dried on a steam bath before placing in oven; or samples can be pre-dried in

vacuum oven with door closed for 15 min at atmospheric pressure before
proceeding to turn on vacuum normally used in test procedure.

Liquid egg products


Soft natural type and high moisture type process foods and spread

cheeses.

19.52

Vacuum Oven: Sand Pan

A. Apparatus and reagents:


1 through 7 same as 19.5 1(A. 1-7).
8. Small glass rods with flattened tips,
9.

x 60

mm.

Atmospheric oven.

10.

Sulfuric acid, Technical, or

11.

Fine white sea sand.

P.

B. Preparation of sand dish (pan):


1. Prepare aluminum moisture dishes to be used by washing thorough-

and drying in an oven at 100 C for several hours.


Place about 25 g of clean dry sand and a small stirring rod into a
moisture dish. Place in atmospheric oven at 100 C for at least 5 hr or overly,

rinsing
2.

night to dry.
3.

Remove

dish from oven and store in a desiccator until used.

C. Procedure:
1

Weigh about

3 to 5

g of sample into a tared sand pan on an analytical

balance.

pan to keep the sample


from touching the sides of the pan. Weighing should be done as rapidly as
2.

Add sample

directly in the center of the sand

possible.

balance and mix sand and sample with the stirring rod
as thoroughly as possible. Leave stirring rod in pan throughout the determi3.

Remove from

nation.
4.

Place dish

in

the

vacuum oven for 5 hours. The oven should be at a


C and at a minimum vacuum of 26 inches (66 cm).

temperature of 100 2
During drying admit into the oven a slow current of air (about 2 bubbles per
sec) dried by passing through sulfuric acid.
5. After 5 hr shut off" the vacuum pump and slowly readmit air into the
oven. Using tongs, press cover lightly into dish and remove dish from the


19.53 Moisture and

oven. Cool

in

Solids:

255

Atmospheric Oven

desiccator for at least 30 min, or until the dish has reached

room temperature. Weigh

dish on analytical balance.

D. Calculations:

Loss

^
=
Moisture

Solids

...

100

x 100
p-

weight

in
.

Weight^

oi^

(g)

sample

(g)

Moisture

E. Note:

The 100

C vacuum oven

sand pan moisture method applies to the

following products:'

Butter and margarine

Condensed whey
Frozen desserts
Ice cream
Ice cream toppings and syrups
Sweetened condensed milk

Whey

concentrates

Yogurts.
19.53 Atmospheric

Oven

A. Apparatus and reagents:


1 through 5 same as 19.51(A.l-5).
6.

Atmospheric moisture oven.

7.

Steam

8.

Osterizer or Waring blender.

bath.

B. Collection

and preparation of sample:

When

narrow
and place in
container with a tight-fitting lid. When cheese cannot be cut, take sample
with cheese trier. Three plugs of cheese should be taken, one from center,
one from point near outer edge, and one from point halfway between the
other two. Cheese plugs should be placed immediately in container with
1.

Natural cheese:

natural cheese can be cut, take a

wedge-shaped segment reaching from outer edge

to center,

tight fitting lid.

Before taking sample for analysis, the wedge shaped segment should be
cut very finely with the aid of a knife or spatula

three side by side on a

taken, either place

all

and cross-cut into

fine slices

about V32

in.

flat

and mixed

well. If plugs are

stainless steel or glass plate

(0.77

mm)

in

thickness, or

mix

plugs with the aid of an Osterizer blender for a total of about 15 seconds.
(Blender should be turned off and sample and jar shaken manually to help

move

larger particles toward bladeside of mixer.) Prolonged mixing

avoided as

fat

is

to be

separation and mashing of product occurs.

Cottage cheese curd for manufacturing use and nuts: To get a more
it is necessary to mix at least a 5-oz (150-g) sample in
an Osterizer blender for four 15-sec periods, turning off and starting blender
at the end of each 15-sec period (making a total of 1 minute).
2.

homogeneous sample,

CHEMICAL METHODS

256
C. Preparation of moisture dishes:
The aluminum moisture dishes used shall be

and dried
until

in

an oven for several hours

at 100

washed thoroughly,

rinsed,

C. Store in a clean desiccator

used.

D. Procedure:
1. Weigh about 2-3-g sample into a tared dish on an analytical balance.
Where possible, distribute sample evenly over the bottom of the dish. Place
dish with cover under it on metal shelf in the atmospheric oven. Consult
I9.53(F.2) for oven temperature and length of time in oven for various products tested.
2. After specified time in oven, using tongs press cover tightly into dish
and remove dish from the oven. Cool in desiccator for at least 30 min or until
the dish has reached room temperature. Weigh dish on analytical balance.

E. Calculations:

Moisture =

Solids

Loss
,

100

^z

01

(g) x 100
7-sample (g)

weight

in

Weight

Moisture

F. Notes:
1.

The following products can be

spheric oven method:

liquid

tested for moisture using the atmo-

buttermilk, natural cheese, chocolate and

cocoa, cottage cheese, liquid creams, egg albumin liquid and powders,
milks liquid, condensed whole and skim milk, sweetened condensed skim,

nuts and toppings.


2.

Oven temperature and

Product item
Buttermilk, liquid

length of time in oven:

Dry on
steam bath

Oven temp

C 2

Hours in
oven

19.54

Solids

in Milk:

19.54 Solids

in Milk:

Inspectors in the

should use the

257

Lactometric Method

Lactometric Method at 39

field

AOAC

who have no

official

3- 21. 31

facilities for

procedure

if

temperature adjustment
C (51-70 F).

testing at 10-20

A. Apparatus ** and reagents:


1.

Lactometer, large type

'^'^

(see Fig. 19:2): Specifications in centime-

ters.
2. Lactometer, small type: Specifications in centimeters are approximate, since certain tolerances are allowable for glass hydrometers and since
this lactometer is undergoing slight changes: total length 15; stem length 4.5;
stem diam, 0.5; bulb diam, 3.5; scale length 2.3. The bulb displaces about 65

designed for whole milk over the range 24.5-35.0


degrees, the smallest division being 0.5 degree. [Before use and as often as

ml of

liquid.

The

scale

is

0.60

0.05
0.1

WALL

THICKNESS

PYREX, FLINTGLASS, OR EQUAL

3.40
0.05

0.2

WALL

THICKNESS

SCALE

BALLAST

FIRST QUALITY

LEDGER PAPER
Figure 19:2. Dimensions of 39

(102 F) glass lactometer.

**Suitable portable kits, including lactometer, bath, racks and cylinder, may be purchased
from the Talboys Instrument Corp, 313 Ackerman Avenue, Emerson, NJ 07630; or Wedco, Inc,
PO Box 223, Silver Spring, Md. 20907 Lactometers may also be purchased from leading manufacturers of thermometers, hydrometers, and similar equipment.
''"^Catalog No. 22261, Taylor Instrument Co. Rochester, NY 14603 or equivalent.

CHEMICAL METHODS

258

needed,

test

lactometers for accuracy by comparison with instruments of

known accuracy

at

15.5

two sp gr

(60 F) at

levels.

Discard those with

readings that exceed 0.0001 sp gr from that on the reference standard.]


3.

Water bath: To maintain bath at 39


C and equipped with oversome device to hold water at the proper level for heating milk
1

flow tube or

samples.
4.

Lactometer cylinder: ID

of lactometer to permit free

sufficiently greater than the largest

movement

diameter

of the lactometer; and having the

capacity necessary to float the lactometer.


5.

Butterfat test equipment: See

12th ed, 1975, pp. 259-260

'

for

AOAC

Babcock

test

Official

Methods of Analysis,

equipment.

B. Procedure:
1.

Lactometer

test:

Rapidly heat milk sample (about

oz (150 ml) for a

small lactometer or 10 oz (300 ml) for a large lactometer) to approximately

39

C by immersing

at intervals to

the sample in water at about 46 C. Gently shake sample


prevent overheating. The flask should be loosely stoppered to

reduce evaporation.
Transfer the sample with minimal agitation to a lactometer cylinder held in
a water bath with a temperature thermostatically maintained at 39 C. The
lactometer itself should be preheated for not less than 3 min before use by
immersion in a cylinder of water held in the same constant-temperature

Remove the lactometer and wipe dry immediately before use.


Slowly immerse lactometer in milk sample. The reading is taken at the top
of the meniscus after the lactometer comes to rest. It is important that the
stem of the lactometer be clean and dry above the milk surface. Repeat
readings can be made by withdrawing the lactometer just enough to enable
the operator to wipe the stem with a tissue or soft cloth before slowly immersing it again to the reading point.
Each lactometer degree on the scale is divided into 0.2 degree for the
larger type, and readings should be estimated readily to 0.1 degree. Successive readings should agree by 0.1 degree if the temperature remains constant. Temperature of the milk should be checked with an accurate

bath.

thermometer
2.

Fat

at

test:

the time of reading.

Conducted by the standard Babcock method or

Amounts should be

lent.

3.

its

equiva-

read to the nearest 0.05%.

General suggestions:

large

number of determinations can be done

rapidly by using several lactometer cylinders in a constant-temperature


bath.

The cylinders of milk can be

held at this temperature for about

with only negligible change in specific gravity, provided that the milk

is

hr

gent-

is immersed. This long holding method is not


which tends to become rancid or to oil off.
When milk samples are rather uniform in fat content, routinely it is unnecessary to wash and dry the lactometer between measurements; however, the

ly stirred

before the lactometer

suitable for use with old milk,

19.54

Solids in Milk: Lactometric

259

Method

in such instances, in which event


wiped
dry.
only the stem need be
Composite samples may be preserved for 8 days with only negligible
changes in specific gravity. Samples are refrigerated at about 4.5 C after the
addition of 0.5 ml of formalin (40% formaldehyde solution) per pint of milk.

instrument should be rapidly transferred

Calculations:

Milk,

total solids

where

F=

1.33

fat

F+

273

Milk,

Tables

19:111,

milk,

total solids

nonfat solids

'

^"^^

by Babcock method, and

L = lactometer reading in
Skim

-JT^^

0.33

1.33

F+
273

F+

19:IV and 19:V will aid

in

degrees.

273

L +

L
1000

0.40

use of the formula

when one

calculates the per cent of total solids. Table 19:111 gives values of 1.33

Ffor

determinations where F ranges from 2.50 to 6.95. Table 19:IV


273 L
- 0.4 for lactometer readings (L)
gives values (listed as M) of

Babcock

fat

from 25.5 to 34.9 covering the range for whole milk. Table 19:V gives values

Table

19:111.

Values for Babcock Fat Determinations


(Range, 2.50 to 6.95)

CHEMICAL METHODS

260

Table 19:IV.

M Values for Lactometer Readings, 25.5 to 34.9.


(Range for Whole Milk)

19.6

Organochlorine Pesticide Residues

in

19.6 Organochlorine Pesticide


for Multiple Residues)
Milk

may be contaminated

261

Milk

Residues

in

Milk (Examination

with one or more pesticide residues in the same

sample. Residues of the chlorinated hydrocarbon pesticides have the great-

appearing in milk and have received the greatest attention


development of analytical methods and in residue monitoring programs.
Pesticide residues frequently found in milk and milk products have been:
DDE, dieldrin, BHC, methoxychlor, heptachlor epoxide, DDT, hexachlorobenzene (HCB), and TDE in this approximate order of occurrence in
milk produced in the United States. With a decreasing use of several chlorinated hydrocarbon pesticides in general, and DDT and aldrin/dieldrin in particular, the overall pattern of residues in milk can be expected to change.
Residues of polychlorinated biphenyls (PCBs) have been found in milk
and continue to be a potential contaminant. This group of multi-component
and persistent industrial chemicals [trade named Aroclor(s)] must be considered as possible residues detectable in the examination of milk by the method given here.
The analytical method described here is essentially the same as that in the
est propensity for
in

12th edition. Association

of

Official Analytical

Chemists, Official Methods

of Analysis.^ In this method qualitative and quantitative determinations are


based on chromatographic principles that make possible simultaneous determination of multiple residues present in the same sample. Extraction and
cleanup procedures use solvent partitioning and column chromatography to
isolate the pesticide residues from sample material. These operations are
vital to quantitative recovery of the chemicals sought and to reliable performance of the subsequent gas or thin layer chromatographic determination. This method has been tested in interlaboratory collaborative studies
and has AOAC Official status for the following pesticide chemicals in milk
and dairy products: BHC, dieldrin, p,p'-DDE, p,p'-DDT, p,p'-TDE, heptachlor epoxide, lindane, methoxychlor, and perthane.^- '^' *^ Development
of this method, including reference to various studies and supporting data,
has been reviewed.^ Over 100 organochlorine pesticides and PCBs, certain
parent organophosphorus pesticides, and other non-polar pesticides are
completely or partially recovered by this method. A compilation of the behavior of these chemicals in the method is given in the Food and Drug Administration Pe^r/c/We A/za/j/Zcfl/ Mfl/7w/, Volume I.^The detection of PCBs
and organochlorine pesticide residues together in the same sample indicate
special additional procedures to separate them before determination. '

A. Extraction offat and pesticides:


+ 1 with H2O)
1. Milk: To 100 ml of fluid milk (dilute evaporated milk
in a 500-ml centrifuge bottle, add 100 ml of alcohol or MeOH and about 1 g
1

of

Na

or

oxalate, and mix.

Add

50 ml of ethyl ether and shake vigorously

CHEMICAL METHODS

262
for

min; then add 50 ml of petroleum ether and shake vigorously for 1


rpm for about 5 minutes. Blow off the

minute. Centrifuge at about 1,500

solvent layer with the wash bottle device into a 1-liter separator containing
500-600 ml of H2O and 30 ml of saturated NaCl solution. Reextract the aqueous residue twice, shaking vigorously with 50-ml portions of ethyl-petroleum ether (1 1), centrifuge, and blow off the solvent layer into the separator after each extraction. Mix the combined extractions with H2O
cautiously. Drain and discard the H2O. Rewash the solvent layer twice with
100-ml portions of H2O, discarding the H2O each time. (If emulsions form,
add about 5 ml of saturated NaCl solution to the solvent layer or include with
the H2O wash.) Pass the ether solution through a column of anhydrous
Na2S04 (22 X 50 mm) and collect the eluate in a 400-ml beaker. Wash the
column with small portions of petroleum ether and evaporate the solvent
from the combined extractions at steam bath temperature under air current
:

to obtain fat.

Butter:

2.

through a dry

Warm

at

about 50

until the fat

separates and decant the

fat

filter.

Cheese: Place 25-100 g of diced sample (to provide 3 g of fat), about


or K oxalate, and 100 ml of alcohol or MeOH in a high-speed
blender and blend for 2-3 minutes. (If experience with the product indicates
3.

2 g of

Na

that emulsions will not be

broken by centrifuging, add

ml of H2O for each 2

g of sample before blending.) Pour into a 500-ml centrifuge bottle, add 50 ml


of ethyl ether, and shake vigorously for 1 min; then add 50 ml of petroleum

min (or divide between two 250-ml bottles


and extract the fat from each by shaking vigorously for 1 min with 25-ml
each of eth^r) Proceed as in
above beginning at the sentence starting,
"Centrifuge at about 1,500 rpm for about 5 minutes."
ether and shake vigorously for

B. Acetonitrile partitioning:

Weigh <3 g of fat into a 125-ml separator and add petroleum ether so that
volume of fat and petroleum ether in the separator amounts to 15
ml. Add 30 ml of CH;jCN saturated with petroleum ether, shake vigorously

the total

for

min,

let

the layers separate, and drain the

containing 650 ml of H2O, 40 ml of saturated

CH3CN

NaCl

into a separator

solution, and 100 ml of

petroleum ether. Extract the petroleum ether solution in a 125-ml separator


with three additional 30-ml portions of CH3CN saturated with petroleum
ether, shaking vigorously for 1 min each time. Combine all extracts in the
-liter separator. Hold the separator in horizontal position and mix thoroughly for 30-45 seconds. Let the layers separate and drain the aqueous
layer into a second 1-liter separator. Add 100 ml of petroleum ether to the
1

second separator, shake vigorously for 15 sec and let the layers separate.
Discard the aqueous layer and combine the petroleum ether layer with the
petroleum ether in the original separator and wash with two 100-ml portions
of H2O. Discard the washings and draw off the petroleum ether layer through

19.6

Organochlorine Pesticide Residues

a 22 X 50

mm

in

263

Milk

column of anhydrous Na2S04

into a

500-ml Kuderna-Danish

concentrator. Rinse the separator and then the column with three portions of

petroleum ether approximately 10-ml each. Evaporate the combined extract


and rinses to about 10 ml in the Kuderna-Danish concentrator for transfer to
the Florisil column.

column cleanup:
Florisil column, ID 22 mm, containing, after settling,
4 in. (10.16 cm) (or weight based on adsorption of lauric acid) 19.6(H. 1 )] of
activated Florisil topped with about V: in. (1.27 cm) of anhydrous Na2S04.
Prewet the column with 40-50 ml of petroleum ether. Place the KudernaDanish concentrator with a calibrated collection tube under the column to
receive the eluate. Transfer the petroleum ether concentrate to the column,
letting it pass through at <5 ml per millimeter. Rinse containers and Na2S04
with two portions of petroleum ether of approximately 5 ml each, pour rinsC. Florisil

Prepare

column, rinse the walls of the tube with additional small porand elute at about 5 ml per min with 200 ml of 6%
eluting solvent. Change receivers and elute with 200 ml of 15% eluting
solvent at about 5 ml per minute.
Concentrate each eluate to a suitable definite volume in the Kuderna-Danish evaporator. NOTE: When a volume <5 ml is needed, use a 2-ball microSnyder or micro-Vigreaux column.
NOTE: The first eluate (6%) contains aldrin, BHC, DDE, DDD (TDE),
o,p'- and p,p'-DDT, heptachlor, heptachlor epoxide, lindane, and methoxychlor, and PCBs and is suitable directly for chromatography. If further
cleanup is necessary, repeat the Florisil chromatography, using a new column. The second eluate (15%) contains dieldrin and endrin. If further cleanup is necessary, proceed with MgO-Celite column cleanup [19.6(D)] and or
saponification [19.6(E)] which are applicable only to dieldrin and endrin in
the 15% eluate. Additional cleanup is usually required if thin-layer or paper
chromatography is used.
ings onto the

tions of petroleum ether,

D. MgO-Celite column cleanup:


This procedure is applicable only to dieldrin and endrin in 15% eluate
when additional cleanup is necessary.
Transfer about 10 g of MgO-Celite mixture to a chromatographic tube
without stopcock, using vacuum to pack. Prewash the column with about 40
ml of petroleum ether, discard the prewash, and place a Kuderna-Danish
concentrator under the column. Transfer the 15% Florisil eluate, concentrated to about 5 ml to the column, rinsing with small portions of petroleum
ether. Force the petroleum ether into the column with slight vacuum or pressure. Then elute with 100 ml of petroleum ether. Concentrate the eluate to
suitable volume. Proceed with determination, or saponification if required.

CHEMICAL METHODS

264
E. Saponification cleanup:

This procedure
eluate

umn

when

is

applicable only to dieldrin and endrin in

additional cleanup

[19.6(D)]

if

is

thin-layer or paper

Transfer the concentrated

15%

15%

Florisil

necessary. Follow with MgO-Celite col-

chromatography

is

used.

eluate to a 125-ml g-s flask, rinsing with

petroleum ether, and evaporate just to dryness. Add 20 ml of 2% alcoholic


NaOH or KOH and reflux for 30 min under an air condenser. Transfer to a
125-ml separator and rinse the flask with three 10-ml portions of petroleum
ether, transferring each to a separator. Add 20 ml of H2O and shake vigorously. Drain the aqueous layer into a second separator containing 20 ml of
petroleum ether, shake vigorously, allow the mixture to separate, discard
the aqueous layer, and add petroleum ether to the first separator. Wash the
combined petroleum ether extracts with three 20-ml portions of aqueous
alcohol (1 + 1). (If the initial aqueous alcohol wash causes heavy emulsions,
use H2O only for additional washes.) Discard the aqueous alcohol and dry
the petroleum ether layer through a column, 22 x 50 mm of anhydrous
Na2S04, rinsing with petroleum ether.
Concentrate the solution to suitable volume. Proceed with determination,
if necessary. NOTE: A microscale saponification is available and may be used instead of the procedure above to treat
the 15% eluate or an aliquot of it.^^
or MgO-Celite column cleanup

F. Determination:
1.

Electron capture gas chromatography.^-

^' ^

Microcoulometric gas chromatography.^- '"


^- '"
3. Thin-layer chromatography.^Use gas chromatography with electron capture detection as the primary
2.

means of determination. From

the range of detector sensitivities available,

select a detector sensitivity in conjunction with a weight of milkfat

injected for gas

chromatography

to provide a limit of quantitation

corder scale deflection) of 0.05-0.1

ppm

heptachlor epoxide

of quantitation for other chemicals will vary up or

its

in milkfat.

down from

heptachlor epoxide, depending on the detector response to them.)

Dependent on the purpose of the

may be

sample

(10%

re-

(Lim-

that for

NOTE:

analysis, a diff'erent limit of quantitation

desired.

suggested combination of detector sensitivity and gas chromatographic


sample weight is: detector sensitivity to obtain half-scale recorder deflection
for

one nanogram of heptachlor epoxide (10% scale deflection for 0.2 ng).
a cleaned up extract of 3 g of milkfat sample in 5 ml of solution, inject

From
5

fx\,

equivalent to 3

mg

of milkfat sample.

phase such as OV-101 (DC-200) or a mixture of OV210 (QF-1) plus OV-101 (DC-2()0) under conditions giving separation of a
mixture of lindane, heptachlor. aldrin, heptachlor epoxide, dieldrin, endrin,
and p,p'-DDT. Recommended operating conditions for a 1.8 m x 4 mm ID

Use a column

liquid

19.6

Organochlorine Pesticide Residues

in

265

Milk

column, of either 10% OV-101 or 1:1 mixture of 15% OV-210 plus 10% OV101 on 80/100 mesh Chromosorb W-HP: column temperature, 200 C; flow
rate 120 ml per min; injection temperature 225 C.
Tentatively identify peaks from residues by their retention times in comparison to standards. (A reference list of retention time data is given in the
FDA Pesticide Analytical Manual. Measure the peak height or area under
the pesticide residue peak and compare to same measurement made on a
known weight of appropriate reference standard. For most accurate determination, peaks for residue and standard should be of similar size. Chromatograph the standard immediately before or after the sample. Calculate ppm

residue in milkfat as follows:

nanogram standard x peak height (area) sample


peak height (area) standard x milligrams milkfat injected

Confirm the identity of residues identified by electron capture gas chromatography by appropriate combination of one or more additional tests such
as: thin layer chromatography, element-specific gas chromatographic detectors, preparation of a characteristic derivative; gas chromatography-mass
spectrometry provides the most certain means of identification of a residue.
G. Apparatus and reagents:

2.

Chromatographic tube: With Teflon stopcock and coarse fritted plate


mm ID x 300 mm.
Chromatographic tubes without stopcocks: 22 mm ID x 300 or 400

3.

Filter tubes:

1.

or glass wool plug; 22

mm.
Approximately 22

mm

ID x 100 mm, with short

deliv-

ery tube and coarse fritted plate or glass wool plug.


4. Kuderna-Danish concentrators: Capacity 500 ml with 5- or 10-ml volumetric or graduated receiving flasks (Kontes Glass Co. No. K-570000, K-

621400, or equivalent).
5.

Separators: 1,000- and 125-ml capacity, with Teflon stopcocks.

6.

Micro-Snyder column:

2-ball type

(Kontes Glass Co, No. K-569001

or equivalent).
7.

Micro-Vigreaux column: (Kontes Glass Co, No. K-569251 or equiv-

alent).
8. Wash bottle siphon: To siphon ofl" ether, use tube similar to delivery
tube of ordinary wash bottle but with intake end bent up into U shape in
higher than bottom
opposite direction to outlet end, with opening 6-12
of U, cut off" horizontally. (Avoid excessive constriction when bending.) Set

mm

can be raised or lowered. In


operating, adjust opening of U bend to about 3 mm above surface of aqueous
layer and blow ether layer off" by gently blowing through mouthpiece tube

delivery tube loosely

enough

in

stopper that

it

inserted in adjacent hole in stopper.


9.

See 19.6(H) for checking reagent suitability. Purify


as follows: To 4 liters of CH3CN add 1 ml of H3PO4, 30 g of

Acetonitrile:

technical

CH3CN

CHEMICAL METHODS

266
P2O5, and boiling chips;

distill in all-glass

Do not exceed 82 C.
Some lots of reagent-grade CH3CN

apparatus

at

81-82

(178-180 F).

are impure and require distillation.

Generally, vapors from such lots will turn moistened red litmus paper blue

when
odor

held over the


is

10.

mouth of

the storage container.

pronounced amine

detectable.

Acetonitrile saturated with petroleum ether: Saturate

CH3CN

(item

9 above) with redistilled petroleum ether (item 21 below).


11.

Alcohol: Ethanol,

USP,

reagent-grade; or

MeOH, ACS

specifica-

tions.
12.

Alcoholic alkali solution, 2%: Dissolve 2 g of

NaOH

or

KOH

in

alcohol and dilute to 100 ml.


13.

Celite 545: Manufacturer, Johns Manville Co.

Must be

free of elec-

tron-capturing substances.
14.

Eluting solvent,

6%: Dilute 60 ml of

ethyl ether (item 16) to

liter

with redistilled petroleum ether (item 21 below).


15.

Eluting solvent. 15%: Prepare as in item 14 above, using 150 ml of

ethyl ether.
16.

contain

Ethyl ether: Redistilled

2%

at

34-35

C and

stored under nitrogen; must

v/v ethyl alcohol and be peroxide-free. Also see 19.6(H)

below

for checking reagent suitability.


17.

Florisil:

PR

60/100

650 C-activated Florisil

is

grade, activated at 650

obtained

in

(1250 F).

When

bulk, transfer immediately after open-

ing to pint-size (500 ml) glass jars or bottles with g-s or foil-lined screwtops

and store
130

130

C
C

in

the dark. Heat

in g-s bottles

after 2 days.

18.

or

in a

See

>

5 hr at 130

desiccator

at

(266 F) before use. Store at

room temperature and

19.6(1) for evaluation

reheat at

and standardization.

Hexane: See 19.6(H) below for checking reagent

suitability; re-

agent-grade, redistilled in all-glass apparatus.


19.

Magnesium

oxide: (adsorptive magnesia, Fisher Scientific Co, No.

H2O, heat on a steam bath


approximately 30 min, and filter with suction. Dry overnight at 105-130 C
(221-266 F) and pulverize to pass through a No. 60 sieve. Store in a closed
S-120). Treat as follows: Slurry about 500 g with

jar.

20.

MgO-Celite mixture: Mix treated


1 by weight.

545 (item 13 above)


21.
ability;

22.

MgO

(see 19 above) with Celite

-I-

Petroleum ether: See 19.6(H) below for checking reagent


reagent-grade, redistilled in all-glass apparatus at 30-60 C.

Sodium

sulfate,

anhydrous, granular.

**Available from the Floridin Co, Berkeley Springs,

WV

25411.

suit-

19.6

Organochlorine Pesticide Residues

H.

in

267

Milk

of reagent for electron-capture detector:


must be purified, and final distillation conducted,

Suitability
^^

Solvents

in all-glass

apparatus.
Purity test:

1.

Electron-capture gas chromatography requires the absence of substances

causing detector response, as indicated by the following

test.

Place 300 ml of

reagent in a Kuderna-Danish concentrator fitted with a calibrated collection


vessel and evaporate to 5 ml. Inject 5
into the gas

/xl of concentrate from a 10-/u,l syringe


chromatograph under the same gas chromatographic conditions

used for analysis. The concentrated solvent should not cause recorder deflection > 1 mm from baseline for 2-60 min after injection.
/. Evaluation and standardization ofFlorisil chromatography:

may

Florisil

into the

6%

covered

in

On

eluate.
is

exhibit lot-to-lot variation in

and

159?^

ability to separate pesticides

its

eluates. Dieldrin and endrin should be completely re-

the 159? eluate; however, they

occasion, heptachlor epoxide

may sometimes be found


may be eluted in the 15%

in

the

6%

eluate.

It

also possible that dieldrin and endrin will be incompletely eluted by the

15%

eluate. Florisil should be

checked regularly to insure

pesticides has been effected and that recovery

is

that separation of

complete. Separation and

recovery of pesticides should be checked as follows:


Prepare a mixed pesticide standard solution in hexane to contain 1,1.1
and 2 ixg per milliliter, respectively, of aldrin, heptachlor epoxide, dieldrin,
and endrin.
Test the activated Florisil by placing
ml of mixed pesticide standard on
the prepared column and eluting as in 19.6(C).
Concentrate the eluates from the Florisil column to 10 ml.
Inject an aliquot of the appropriate eluate into the gas chromatograph and
determine quantitatively the recovery of each pesticide. Florisil that completely elutes aldrin and heptachlor epoxide in 69f eluate and dieldrin and
1

in \5% eluate is satisfactory.


The standardization procedure given below

endrin

[19.6(1.2)]

may be used

to ad-

just for variations in Florisil to achieve correct separation.

Apparatus and reagents:


Buret Capacity 25 ml. with graduations

1.

a.

A.
b.
c.

d.
e.

10-ml intervals, class

Pipets 10- and 20-ml transfer, class A.


Erlenmeyer flasks Capacity 125 ml, narrow mouth and 25 ml T.
Volumetric flasks Capacity 500 ml, class A.
Sodium hydroxide Dissolve 20 g of NaOH
reagent-grade)
at

(pellets,

water and dilute to 500 ml (IN). Dilute 25 ml of IN


with water (0.05N).
in

''^Suitable

products are available from Burdick

Mich. 49442, and from other manufacturers.

&

NaOH

Jackson Laboratories.

Inc.

to 500 ml

Muskegon,

CHEMICAL METHODS

268
f.

Laurie acid solution

CP quality,
500
g.

Transfer

10.000 g of lauric acid, purified to

to a 500-ml volumetric flask, dissolve in hexane.

ml = 20 mg).
Phenolphthalein indicator

and dilute to

milliliters (1

Dissolve

in

alcohol and dilute to 100

milliliters.
h.

Ethyl

alcohol

USP-grade

or absolute,

neutralized

to

phenol-

phthalein.

Hexane

i.

Reagent-grade,

redistilled in all-glass apparatus.

Standardization:

2.

a.

b.

Weigh 100-200 mg of

Add

lauric acid into a

125-ml Erlenmeyer flask.


3 drops of phenol-

50 ml of neutralized ethyl alcohol and

phthalein indicator; titrate to permanent end point.


c.

Calculate milligrams of lauric acid per milliliter of 0.05N

(about 10
3.

mg

per

NaOH

milliliter).

Procedure:

Transfer 2.000 g of Florisil to a 25-ml $ Erlenmeyer flask. Cover loosely


with aluminum foil and heat overnight at 130 C (266 F). Stopper, cool to

room temperature, add 20.0 ml of

lauric acid solution (400 mg), stopper, and


shake occasionally for 15 minutes. Let the adsorbent settle and pipet 10.0 ml
of supernatant liquid into a 125-ml Erlenmeyer flask. Avoid the inclusion of
any Florisil. Add 50 ml of neutral alcohol and 3 drops of indicator solution;
titrate with 0.05N NaOH to permanent end point.
4.

Calculation:

Calculate the amount of lauric acid adsorbed on Florisil as follows:

mg/g

Florisil

= 200 -

(ml required for titration x mg/ml).

Evaluation:

5.

lauric acid value of 110

is

the desired adsorptive capacity of 20 g of

which would fill a column 22 mm ID to a depth (after settling) of 4 in.


(10.16 cm) if its density (60-100 mesh) was about 30 lb per cubic foot (480.5
kg/m'^). Such a Florisil column would fulfill the requirements of 19.6(C).
To obtain an equivalent quantity of any batch of Florisil. divide 10 by the
lauric acid value for that batch and multiply by 20 g.
Experience with this procedure indicates that column weight adjustment
for Florisil with lauric acid adsorption values of about 80-120 generally results in proper elution of heptachlor epoxide, dieldrin. and endrin. Check
column of adjusted Florisil weight before use with elution test under 19.6(1).
Florisil.

J.

Evaporation techniques:

To concentrate extract to 5 ml or more:


Evaporate in a Kuderna-Danish concentrator fitted with a 3-ball Snyder
column and volumetric flask or a graduated collection tube; 20-mesh boiling
1.

chips are necessary.


2.

To concentrate

Evaporate to about
the concentrator and

extract to less than 5 ml:

ml as

fit

in

above.

Remove

the calibrated tube from

the tube with a 2-ball micro-Snyder or micro-Vi-

19.71

Protein:

Dye BindingAmido Black

269

greaux column. Evaporate to slightly less than the desired volume, permit
the condensate to drain into the tube, and remove the column. Minimum
attainable

volume

0.2

is

To evaporate

3.

0.4 ml.

solvents from fat extracted from dairy products:

beaker on an HgO bath at 35-40 C under a


heat and the air stream as soon as the
last of the solvent evaporates. Let residual H2O evaporate spontaneously.
Solvents may be evaporated from fats on a steam bath for short periods.

Evaporate the extract

stream of clean, dry

air.

in a

Remove from

19.7 Protein

Dye Binding

19.71

Since 1883, the Kjeldahl principle has been used as the standard method

measuring nitrogen and protein in foods and feeds. With greater emphabeing placed on the importance of protein values in foods today, newer
more accurate, specific, and rapid methods have been developed. Such

for
sis

methods as the manual and automatic dye binding

tests are being

dairy and other industries, and are included in this section.^*

A.

used

in the

^^' ^^' ^^' ^^

Amido
1.

black: See 19.71(A.6)d


Apparatus and reagents:
11.'
a. Pro-Milk
b. Operational Manual.

MK

c.

(2) 10-liter capacity. ^^


funnel powder type.

Plastic bottle

d.

Plastic

e.

Hot

plate, or gas plate.

f.

Refrigerator for milk samples.

g.

Syringe for dispensing dye.^^

h.

Plastic

squeeze bottles (3) 500 ml.^^

i.

Plastic bottles, 2-liter capacity,

j.

Plastic syringe 2

k.
1.

ml adjusted to 1.00 ml.''

Forceps."'

Kimwipes, or equivalent,
capacity.
beakers

Lint free tissues

m. Glass

fiber filters.

n.

Stainless steel

o.

Graduate, 1000-ml capacity,

p.

Distilled water.

(2)

3-liter

Chemicals for working liquid Foss America


tion can be made by the user.^)
r. Triton X-100, Foss America #PRM 507.''
ACS sp gr 0.90.
s. Ammonium hydroxide
q.

#PRM504

(This solu-

^Distributed by Foss America. Route 82, Fishkill, NY 12524


"Supplied with Pro-Milk unit. Replacements may be ordered from Foss America,
icals are in measured quantities to prepare 10 liters of dye.

Inc.

Chem-

CHEMICAL METHODS

270
2.

Solutions:
a.

Working O-liquid

Carefully measure 10

(buffered amino dye


liters

solution).

See 19.71(A.6)a.

room temperature into


container. Tap water is used

of tap water at

the

Mark the volume on the


for
calibrating only. Remove water and rinse jug with distilled water. Invert
to drain for a short period. The two components, dye and buflFer, are
plastic jug.

each dispensed into approximately 2 liters of distilled water. Bottles


should be thoroughly rinsed and rinsings added to respective solutions.
For complete dispersion of chemicals the solutions are heated to 70 C,
see 19.71(A.6)b.

When

dilution

is

ing the container in


ly a liter

complete, transfer solution to the 10-liter bottle rinswhich the chemicals were heated with approximate-

of water for each container, adding rinsings to combined solu-

mark with distilled water and add, by means of


ml of Triton X-100. Mix the solution thoroughly and

tions. Fill to the 10-liter

plastic syringe,

hold for 24 hr before using. Record date solution

and on
b.

book,

Should be made

in suf-

Standard liquid for checking "45'' reading

ficient quantity for

use with the entire dye

400 ml of dye, plus 1000 ml of


c.

made

in log

is

bottle.

Ammonia

Preparation by volume:

distilled water.

2% to 2

solution,

lot.

ml of ammonium hydroxide add 98 ml

of distilled water.
3.

Initial calibrations:
a.

Syringe calibration

continual use.

To

The capacity of the syringe

will

assure that delivery by the milk syringe

change with

is

constant,

it

checked before use, and daily for an initial period of 2 weeks. The
syringe must be adjusted to deliver 1.00 g of water at 20 C.
For testing use distilled water at 20 C. Insert the syringe tip a minimum of V2 in. (1.27 cm) into the water and pump the syringe five times to
expel air before filling syringe. (One must be certain no air is trapped in
the syringe.) If the cannula is in an U shape, less air volume variability
will result with some syringe systems. Invert the syringe and wipe the
cannula with tissue to remove any water adhering to the surface. Expel
the sample into a tared covered container of sufficient size to hold 10
samples from the syringe. Depress syringe plunger only once per delivery. When a total of 10 deliveries has been made, weigh the container
and obtain net weight. Calculate weight of a single delivery. Record this
is

weight
It is

for a

in log

book.

important that the sample be taken

maximum

in

the

same manner each time

uniformity of sample size.

Record of syringe delivery weight must be maintained.


vary more than 0.002

g.

When

records

show

It

should not

that this tolerance

is

maintained for a 2-week period, checking can be done on a weekly interval. If a new syringe is used or another employee is using the syringe.

19.71 Protein: Dye BindingAmido Black

271

the daily calibration shall again be inaugurated.

brated syringe shall be available


b.

Dye

delivery

It

is

at all

An

additional clean cali-

times.

important that the amount of dye delivered

(about 20 g at 20 C) be consistent. The average value is calculated from


the weight of 10 deliveries made into a tared container. Give a firm,
gentle uniform stroke on the dye dispenser handle with the dye opening

over the tared container. Repeat for a total of 10 discharges. A record is


maintained to assure consistency of delivery. Delivery should be
checked if dye delivery is altered to calibrate unit, and each time a new
dye solution is made.
Using a sample of homogec. Repeatability of operator's technique
nized milk, each operator is to make 25 replicate tests. The means of
each operator's sample should check within 0.02% protein of each
other with an individual standard deviation of 0.008% protein or less.
d. Calibration of instrument
Proteins shall be determined in duplicate by the Pro-Milk and by the Kjeldahl method of analysis on a minimum of 10 samples. Save a duplicate set of samples in freezer until
Kjeldahl results are received. The average differences in means shall
not be more than 0.02% protein if unit is in calibration.
If the Pro-Milk is giving protein values higher than the Kjeldahl, the
amount of dye should be increased. This is accomplished by activating
the dispenser adjustment screw on top of spring by turning it clockwise.
Turn only a quarter turn and check stock samples to evaluate the
change this adjustment has made.
If the Pro-Milk protein results are lower than those of the Kjeldahl,
the amount of dye should be decreased by turning the screw adjustment, counterclockwise. Continue the adjustment and repeating analysis until the 10 sample means are within the 0.02% protein tolerances.
Ten samples should be submitted monthly for duplicate tests by the
Kjeldahl method. The same tolerances should be maintained. Calibration should be repeated for each new batch of dye.
4. Measuring procedure:

a. Turn on instrument at least 1 hr before determining samples. Instrument can remain "on" if used daily.
b. Using a clean filter and tube, check reading on the scale using the
standard liquid [19.71(A.2)b]. The needle should rest on "45". Bring

needle to correct position using the 45 adjustment knob on the readout


unit.
c.

Replace

filter

and

clean

tube

and

check

[19.71(A.2)a] as described in the operational manual.

the

"0-liquid"

The needle should

on O, and adjustment to bring it into position is made with the O


knob.
If an adjustment is made on the "O" setting then the "45" setting
must be checked again and readjusted if necessary. Continue checking

rest

CHEMICAL METHODS

272

two solutions

the

The

alternately until both points are in correct adjustment.

O-liquid normally needs checking once a day, but the 45 standard

should be repeated every 50 samples.

If

major adjustment

is

required,

then the O-liquid test should be repeated.


d.

Determine

five replicate protein tests

sample. Carefully

fill

on an homogenized milk

the syringe with milk, discarding the

first

fill

after

The syringe should be handled in the same manner to fill


with milk by pumping five times and wiping tip.
The pumping action will assure that the sample is homogeneous when
distilled water.

changing milk samples. Milk samples are tested


are held in a bath in the refrigerator and

at

removed

20 to 37 C. Samples

in

small lots.

Temper

and mix samples thoroughly before testing (as is done for fat testing).
Samples in plastic bags in racks may be mixed by "rocking" the samples 10 times through an arc of 180.
Repeat determination of protein on the homogenized milk sample after each 25 milk samples throughout the day's operation. Record results. The difference should not exceed 0.02% protein.
If samples are not within the prescribed tolerances, a check on milk
syringe, dye syringe, age of dye and recheck of 45 and 0-liquids should
be made.
e. Place a clean filter and tube on platform.
f.
Dispense milk into dye with a single stroke of the milk syringe
plunger. Place stopper in position and fasten by turning the operation
knob.
g. Move the dye dispenser over to the tube, and with a gentle, firm
uniform stroke, dispense dye into the tube. Release the dispenser to the

upright position.
h. Mix the sample and dye by tilting the mixing assembly until it
touches the rim of the discharge tray and then return to upright position.

Repeat for a
is

total

of five

full arcs.

Apply pressure

firm in the netting. While sample

is filtering,

to rubber bulb until

clean and

fill

it

syringe with

next sample. Press the release button top of measuring unit, to bleed
pressure from mixing tube until

all air is

expelled.

Read

the per cent

when the reading has stabilized. Release the


operation knob and turn away the stopper. Turn the mixing assembly
until the lock catches, then press lever to release tube. Remove and

protein on the upper scale

replace

filter

paper, clean mixing tube by wiping with tissue placed on

the tube stand.


5.

Use same

tissue to

wipe mixing assembly.

Rinsing and cleaning:

a. The instrument is rinsed with ammonia solution [I9.71(A.2)c] after


each group of 50 samples has been tested, and when measurements are
terminated. Place a filter on the lower support for the mixing tube and
place tube on the filter. Fill V.i of the tube full with ammonia solution
and turn stopper in position over the mixing tube. Press the rubber bulb
and flush the cuvette with the entire amount of ammonia solution.


19.71

Protein:

273

Dye Binding Amido Black

Release pressure and turn stopper away from the mixing tube.
tube with distilled water and turn stopper
tube.

Apply pressure

in

Fill

position over the mixing

water comes out at


water remain in the in-

to the rubber bulb until clear

the discharge tray. Release pressure and

let

until the equipment is again used.


At termination of daily tests, rinse the milk syringe with ammonia

strument
b.

solution then distilled water.

Allow water to remain in syringe until the next series of measurements are made. The milk syringe should be dismantled at least monthly
and the individual components washed with a mild detergent solution,
followed by a thorough rinsing with tap and distilled water.
6.

Notes:

solution may become reduced in activity with results


lower values as the amount of dye in the bottle is reduced
and/or dye is over 45 days old.
The time for a dye change must be anticipated in advance, and a new
lot prepared and allowed to stabilize. Determine the protein, in duplicate, on the current homogenized sample of milk using the "old" dye
solution. Discard the remaining dye solution. Rinse bottle with distilled
water and allow to drain. Introduce new dye. Check zero and "45"
using freshly prepared solution. Make necessary adjustments. Deter-

The dye

a.

drifting to

mine

five replicate proteins using the same current homogenized milk


sample. Results should not vary from those obtained on the old dye by
more than 0.029^ protein.
If

"0" and "45" and retest the


made on delivery volume of milk and

greater variance does occur, recheck

milk sample. Recheck should be

dye syringes. If this does not rectify the difference the dye should be
discarded and a new lot made.
If variation occurs from one lot of dye solution to the next, it might be
advantageous to maintain a log on the dye weight, and the lot number.
b. Heating should be done in a well-ventilated area, preferably a

fume hood.
c. Pro-Milk dye binding method is not applicable for testing samples
preserved with potassium dichromate.-'^ This milk preservative can
cause a decolorization of dye solution.

d. Method is suitable for determining protein content of milk, cream


and other dairy products such as
Chocolate milk and chocolate drink

Creams
Cultured buttermilk

Half and half


Ice cream mixes
Nonfat dry milk (on an "as is" or reconstituted basis).
On these products the sample must be weighed and sample size must
be sufficient to provide about 60 to 100 mg of protein.

274

CHEMICAL METHODS

B. Protein: Acid orange 12:^-

Apparatus and reagents:

1.

Model
Color analyzer Model

Protein analyzer

a.

L, Part No. L-1000*** and the follow-

ing accessories:

101.

b.

f.

Cuvette for color analyzer.


Color analyser lamp, for Model 101.
Filter paper, 1.8-cm discs.
Drain tube, for cuvette.

g.

Polyethylene cover, for cuvette.

c.

d.
e.

Kimwipes or equivalent.

20 C.
Polyethylene bottle 8-oz (240 ml), with dropper cap.
m. Polyethylene bottle 2-oz (60 ml), with dropper cap.
Polyethylene bottle 16-oz (480 ml), to be used as a drain recepLint-free tissues

h.
i.

j.

k.

at

Dispensing bottle polyethylene, 5-gal (18.9 liters) capacity.


Automatic pipet, 40-ml.
Sampling syringe 0-5 ml, calibrated to deliver 2.240 ml of water

1.

n.

tacle.

p.

Closures gum rubber.


Tripod support for 5-gal (18.9

q.

Calibration test

o.

kit

liters)

dispensing bottle.

with instructions. Part No. SL-1511 (used for

preparation of standard curve).


r.

s.

tion
t.

u.

v.

Graph paper.

Graduate cylinder 1000-ml (used for preparing reagent dye solufrom a concentrate).

Hot plate.
Erlenmeyer

flask

2000-ml.
for wiping cuvette.

Lint-free cloth or paper

10-ml.

w. Beaker
X.
y.

Reagent dye solution Part No. SL-1209, 10, 11 or 12.


Reference dye solution Equivalent to 0.600 g of dye per

liter

(used to standardize the colorimeter to 42.0).


z.

aa.

Cleaner

for cuvette.
with instructions (used for preparing calibra-

Calibration test kit

tion curves)

SL

151

1.

bb. Distilled water.


cc.

Reagent dye solution

As supplied with the instrument,

to use without further preparation; contains 1.3

Additional orders for this solution

may be

PO Box

148, Boulder,

CO

of dye per

it

is

80302

ready

milliliter.

comwhich must be properly

placed, either for the

pletely prepared solution or for a concentrate

'Udy Analyzer Company,

mg

19.71

Protein:

Dye Binding

Acid Orange

275

diluted before use. Concentrates are available for preparing 20 liters

(#SL-1212) and 50

liters

(#SL-1211). Instmctions for dilution follow:

the dilution container (20-liter or 50-liter) about three-fourths

(1)

Fill

full

of distilled water.

Completely dissolve the buffering salts, enclosed with the reagent


dye concentrate, in about 1,500 ml of boiling distilled water and add
to the dilution container, mixing well by swirling.
(2)

Add

(3)

1,200 ml of glacial acetic acid to the 20-liter container, or

3,000 ml to the 50-liter container, and mix again.

Warm

(4)

the dye concentrate at 50

is no
Complete the

the solution
(5)
2.

(122 F) for about

hr. or until

longer turbid; then transfer to the dilution container.


dilution with distilled

water and mix thoroughly.

Preparation of colorimeter:
a.

The instrument should be allowed

to

use. In actual routine use. the instrument

warm up

is left

on

for 3-4 hr before

at all

times. 24 hr a

supply of color analyzer

day. except holidays, long weekends, etc.


lamps. Part No. SL-1107, should be kept on hand for replacement as
required.
b.

Before placing cuvette

in

color analyzer,

it

should be wiped clean

The index mark on

the funnel
and dry with
with
drain
is
in
place
cuvette
when
the
front
end should be facing to the
calibrating.
for
tube attached and ready
c. It is important that the discharge end of the drain tube be at an
elevation slightly above the mid-point of the main body or short-lightpath section of the cuvette proper. At this elevation the cuvette body
retains the correct amount of liquid by capillary action.
d. Fill the cuvette with water and remove all air bubbles by exerting a
momentary pressure by mouth on the funnel opening.

a soft cloth or lintless paper.

When

is not in use. the cuvette should be kept filled


with the liquid cleanser should be done as
cleaning
with water. Periodic
usage and experience.
equipment
on
based
often as required

e.

3.

the analyzer

Preparation of calibration curve:


a. Warm up the analyzer for 3-4 hr before use.

Fill the cuvette with reference dye solution by adding solution to


cuvette until a steady reading is registered on the meter.
funnel-top
the
meter to a scale reading of 42.0.
Adjust
the
c.

b.

d.

Fill

the cuvette, in turn, with each of the calibration test kit solu-

Record the reading obtained for each.


e. Plot on graph paper the concentration of the dye solutions versus
the meter reading obtained for that concentration. Connect the points
obtained with a smooth curve. The curve will not be a straight line. If
the meter reading is plotted on the logarithmic axis of semi-log paper
and the concentration on the arithmetic axis, a straight line should be

tions.

obtained.

CHEMICAL METHODS

276
4.

Preparation of reagent dye dispenser and syringe:


a.

Reagent dye dispenser

(1)

Place the dispensing vessel on the tripod stand.

(2)

Attach the automatic 40-ml pipet to the stand and dispensing vessel, using the tubing and clamps provided.

(3)

Fill

the dispensing vessel with reagent

dispensing

tip

with dye solution.

ready for use. The reservoir

is

dye solutions and

The automatic

pipet

automatically refilled as

it

each

fill

now

is
is

being

The pipet is calibrated at the factory to deliver 40.0 ml of


water at 20 C. which is equivalent to 40.44 g of reagent dye.
To check calibration of the dispenser, add a pipetful of dye from
each tip into separate tared 100-ml beakers, and weigh on an anadrained.

(4)

balance. Deliveries should be 40.44

lytical

.05 g of reagent

dye.
b.

Syringe

Using trial-and-error technique, adjust the syringe to deliver


2.234 .002 g of water at 20 C into a tared 10-ml beaker.
Weigh
beaker and water on an analytical balance. Adjust the sy(2)
ringe as often as necessary to arrive at this tolerance.
NOTE: Each person using the syringe must become familiar with the
equipment and develop a technique for effecting reproducible deliv(1)

eries.
5.

Procedure:
a.

Add. from a calibrated syringe, 2.24 ml of milk

at

26

into a 2-oz

(60 ml) polyethylene bottle.

Add 40 ml

b.

of reagent dye solution

at

25

to the bottle.

paper disc under the support disc in the tip cap of the
dropper and screw tightly on the bottle containing sample and dye.
c.

Place a

filter

d.

Shake the

e.

Standardize the color analyzer to read 42, using the reference dye

bottle vigorously for 30 seconds.

solution in the cuvette.


f.

Slowly squeeze a

sufficient

amount of

(60 ml) plastic sample bottle, through the

cuvette so that a steady reading

drops

will

be

is

the filtrate from the 2-oz

filter

paper

in the

cap, into the

obtained on the meter. Usually 15-20

sufficient.

g. Record the meter reading to the nearest 0.1 unit. If there is any
doubt regarding the reading, restandardize the instrument with the reference dye solution and pass another portion of the sample solution

through the cuvette.


6.

Calculation:

Convert the meter reading to milligrams of dye by using the prepared


bration curve:

vc Protein

52

mg of dye/ml)
7.0512

(42.24 X

cali-


19.8

Fat, Protein, Lactose: Infrared

277

Spectrophotometry

= total milligrams of dye available in 40.0 ml of dye solution;


= total volume of 40 ml of dye and 2.24 ml of milk; and
7.0512 = derived from multiplying the sample weight of milk,

Where 52

42.24

2.26 g, by 3.12, the conversion factor used to convert results to per cent

sample weight used shall be multiby 3.12 to obtain correct factor to be used in calculation of protein.)
7. Notes:

protein. (For samples other than milk, the


plied

a.

Method

is

suitable for determining protein content of other prod-

ucts such as

Chocolate milk and chocolate drink

Creams
Cultured buttermilk

Half and half


Ice cream mixes

Nonfat dry milk (on an "as is" or reconstituted basis).


the above products (products other than regular fluid and liquid skim
milk) the sample must be weighed and sample size must be suflficient to
provide about 60 to 100 mg of protein.
b. The dye binding method is preferably not applicable for testing
milk samples preserved with potassium dichromate.^ This milk preservative causes decolorization of dye solution.

On

19.8 Fat, Protein, Lactose: Infrared Spectrophotometry


A.

IRMA:^

(IRMA

^^'^)

is a double-beam spectrometer
wavelength and to produce signals which
are linear with component concentration. The instrument is used to determine the fat, protein and lactose content of milk. The principle for analysis
of milk by IR is based on absorption of infrared energy at specific wavelengths by carbonyl groups in ester linkages of fat molecules, by peptide
linkages between amino acids of protein molecules and by OH groups in
lactose molecules. Calibration procedures and factors influencing the analy-

The

infrared milk analyzer

equipped to make rapid changes

ses have

in

been discussed by Biggs.

'^

B. Milkoscan:^

Apparatus and reagents:


Milkoscan Model 300
and lactose).
1.

a.

b.

Ca propionate

nate
c.

and

H2O

stock

solution

Lactose stock solution


1

Dissolve

Dissolve

15

g of

(fat,

protein

Ca

propio-

liter.

50 g of lactose

H2O

in

water

liter.

^^''^Equipment for infrared milk analysis

Box

and protein) or Model 203

water and dilute to one

in

dilute to

(fat

441, Islington, Ont, Canada.

is

distributed by A. Reyrolle and

Company

(Ltd),

PO

CHEMICAL METHODS

278
2.

Principle of method:

Infrared analysis of milk

groups

in

is

based on absolution of IR energy by carbonyl

ester linkages of fat molecules, by peptide linkages

acids of protein molecules, and by

OH

groups

in lactose

between amino

molecules. With

Milkoscan instruments, the incident IR beam is split into two beams, one of
which is passed through an optical filter which transmits energy at the wavelength of maximum absorption for the component being measured. The other beam, the reference beam, is passed through a filter which transmits at a
wavelength where there is a minimum of absorption by the component. A
rotating chopper alternately recombines these beams. The resulting beam is
passed through the milk-filled sample cell and thence to a detector. Signals
from the detector are used to attenuate the reference beam to reduce its
energy level to that of the sample beam. The amount of attenuation required
is proportional to the difference in absorption at the two wavelengths and
hence to the concentration of the component. Interference effects result
from differences in scattering of energy at the sample and reference wavelengths and from differences in the absorptivity of water at these two wavelengths. Since the sample and reference wavelengths are close to each other,
these effects are not large but electronic corrections, nevertheless, are

made to compensate for them. Differences in the degree of homogenization


achieved by the instrument homogenizer, whether because a particular
sample

is

high

in fat

before analysis,

content or because the sample has been homogenized

may

or

may

not cause differences

tends to shift the wavelength of apparent


higher wavelength, a

phenomenon

refraction of milk fat

is

maximum

resulting

in results.

Scattering

absorption to a slightly

from the

fact that the index of

considerably higher on the high wavelength side of

the fat absorption band. Strategic location of the transmission of the fat

midway between

the absorption of well homogenized milk and


homogenized milk will provide considerable compensation
for differences in homogenizing efficiency. With peak transmissions for the
fat sample filter which are either at higher or lower wavelengths than that
required for the compensatory effect, different calibrations are required for
samples which have been previously homogenized as compared to those
which have not been so treated. Compensation for the lesser homogenizing
efficiencies attained with samples high in fat can be achieved by adjusting the

sample

filter

that of less well

linearity of the output signals.

Irrespective of the foregoing, the adequacy of analysis will depend upon


the following:
a.

Presentation of good quality samples to the instrument

at

temper-

atures between 35 and 45 C.


b.

99%

Maintaining adequate purging of previous samples

at a level

of

or better.

c. Proper adjustment of
ponent concentration.

linearity of output signals relative to

com-

19.8

Protein, Lactose: Infrared Spectrophotometry

279

Maintenance of uniform homogenization via the instrument ho-

(J.

mogenizer.

Adequate control of calibration with milk standards of known

e.

composition.
Maintaining a low water vapor content

f.

in

the console of the in-

strument.
3.

Preparation of samples:

Milk samples should be of reasonably good quality. They should not be


sour and the fat should not be oiled off. Either mercuric chloride or potas-

sium dichromate may be used as a preservative. Samples should be preheated to 40

before analysis to melt the

Incorporation of air

fat.

in the

sample by vigorous mixing or stirringjust before analysis should be avoided,


as air in the sample will cause erroneous results.
4.

Test for purging efficiency:


Fill

a.

teurized

10 sample vials with water and


homogenized milk.

Warm

b.

to 40

alternate pairs,

place

in

instrument sample rack

in

the order of

of milk and then of water.

Analyze the 20 samples, using automatic mode.

c.

Calculate the percentage purging efficiency as follows:

d.

W,,

C and

first

10 vials with aliquots of a pas-

W., represent the fat readings for

first

If M,, M-,.
and second results of alternate

pairs of milk and water, respectively,

purging efficiency, water to milk

milk to water

5.

= ^,

.'

ZM.y -

-hrr x 100

EW2

Z^/ - ^rr
ZM2
SWo

x 100

Linearity adjustments:

Linearity adjustments,

if

necessary, should be

made

before attempting to

calibrate the instrument.


a.

Mix measured portions of water with measured portions of cream,

stock solution of calcium propionate or stock solution of lactose, for


fat, protein and lactose, respectively. Use unhomogenized or homogenized cream, depending on which type of sample
is to be analyzed with the instrument. The dilutions should be made so
that the relative concentrations are known and so that the readings ob-

checking linearity of

tained adequately cover the anticipated concentration range of analysis

be done. Approximations of the dilution factors required can be estimated by obtaining readings on the stock solutions. About eight samples should suffice for the linearity check on each component.
b. Obtain readings on the diluted creams and diluted stock solutions.
to

Pump each solution into the instrument twice to avoid carryover effect
and use only the second reading. Plot these against the relative concen-

CHEMICAL METHODS

280
trations of the samples used. If the resulting plot

is

not linear, locate the

appropriate linearity adjustment potentiometer, as indicated

in

the in-

strument operating manual, and make a linearity adjustment.


c. Repeat (b) above until the readings obtained are linear with solution concentrations.

Homogenizing efficiency:
Homogenizing efficiency may decrease with time due to wear on the ball
and seat of the homogenizer valve or due to loss of tension in the spring
supporting the ball. The effect is to cause loss of linearity at the high end of
the fat calibration. Examine the ball and seat for wear. If they are in good
6.

condition,

it

may be

sufficient to replace only the spring.

Calibration procedures:

7.

Milkoscan 300 A reading is obtained by multiplication of the sigand protein by constant values, adding the results and then
adding a constant to adjust for the average interference effect of lactose
and minerals. For example, for fat
a.

nals for fat

Reading = a

-I-

bFj

-I-

cP;

where F, and Pj are the instrument signals and a,b,c are constants.
These constants are set by the manufacturer, but the intercept (a) may
need to be changed. The objective of calibration is to obtain a plot between instrument readings and reference standard values for calibration
milk samples which begins at the origin and has a slope of 1. The simplest way to do this is to determine the relationship of the prevailing
calibration line to the one required and then to make the necessary adjustments.

Obtain a series of eight or more milk samples of the type (homogenized or unhomogenized) to be analyzed by the instrument and with a
fat and protein content close to that of the population of milks
be analyzed. Herd milks are preferable if they can be obtained with
the required compositional range. Avoid abnormal samples with low

range of

to

lactose content.

Analyze these calibration samples, using accepted standard reference


methods.
Following directions in the operating manual, analyze these samples
in duplicate with the instrument. In subsequent calculations, use only
the second of duplicate readings, as this will avoid any carryover effects.

Either plot the instrument readings (vertical axis) against the standard
values, or do a least squares regression analysis, using the instrument

readings as the dependent variable. Determine the intercept and slope


of the calibration line either from the plot or the regression equation.

Reduce

first equation] by the amount of the


on the sensitivity control for the com-

the bias value |(a) in the

intercept. Adjust the reading

281

References

19.9

ponent to a value equal to

its

prevailing reading

h-

slope of the prevailing

calibration line.

Repeat the instrumental analysis of the calibration milks. If the origwas a long way from that required, a second adjustment
may be necessary.
Readings on the Milkoscan 203 are obtained in the
b. Milkoscan 203

inal calibration

same way

made

are

as on the Milkoscan 300, except that cross corrections also


for lactose content.

For example, for

fat

Reading = a + bFj + cPj + dLj


Calibration

may be achieved

in the

same way

as for the Milkoscan

300, either by plotting or by the use of simple linear regression analysis.


8.

Operation of the instrument:

Adequate operating and maintenance instructions are provided in an operating manual. However, particular attention should be given to maintenance
of a low moisture vapor content in the optical section of the instrument. The
silica gel desiccant bags used for this purpose should be changed frequently,
particularly if the relative humidity is high in the laboratory. Adjustments to
optical zero are

made necessary by changes in the moisture vapor content in


made at the end of a day's operation

the instrument. This change should be

Checking the magnitude of the


optical zero readings the next morning will indicate the total amount of
change in optical zero since the previous change in desiccant. Frequency of
change should be adjusted so that this value is not greater than 0.2.
to allow the instrument to stabilize overnight.

19.9 References
1.

American Public Health Association,


tion of Dairy Products llth ed.

Inc. 1960. Standard Methods for the ExaminaAmerican Public Health Association. New York.

2.

Anritsu. 1976. Milk checker equipment brochures on K373A and K373A1 models. American Metering Systems, Inc. P.O. Box 129, Hopewell Jet. NY 12533.

3.

Association of Official Analytical Chemists.

1975. Official

Methods of Analysis,

12th ed. Association of Official Analytical Chemists. Washington, D.C.


4.

Association of Official Analytical Chemists. 1975. First supplement to 12th ed (Oct.


14-17, 1974) Official Methods of Analysis. Association of Official Analytical Chemists.
Washington, D.C.
Dairy Sci. 50:799-803.

5.

Biggs, D.A. 1967. Milk analyses with the infrared milk analyzer.

6.

Biggs, D.A. 1977. Collaborator study of estimation of fat, protein, and lactose in milk with
Milko-Scan. Association of Official Analytical Chemists. Annual Meeting. Washington,

J.

D.C.
7.

Burke,

J.

A. 1971. Development of the Food and Drug Administration's method of analysis

for multiple residues of organochlorine pesticides in foods and feeds. Residue Rev. 34:5990.
8.

Carr, R.L.
dues

9.

1970. Collaborative study of a

in fatty foods. J.

England, C.W., and M. Neff.


Anal.

method

for multiple chlorinated pesticide resi-

Assoc. Off. Anal. Chem. 53:152-154.

Chem. 46:1043-1049.

1963.

The accuracy of cryoscopic methods.

J.

Assoc.

OflF.

CHEMICAL METHODS

282
10.

Food and Drug Administration.

11.

Revised July 1969, July 1970, April 1971, January 1972, September 1972, June 1973. March
1975, Food and Drug Administration, Washington, D.C.
II & III. Foss
Foss America, Inc. 1971-73. Milko-tester equipment brochure on

Pesticide Analytical Manual, Vol.

2nd ed. 1968.

I,

MK

12.

America, Inc. Route 82, Fishkill, NY 12524.


Foss Electric. Denmark. 1971. Technical report newsletter in cooperation with the agriculture institute, Moorepak, Fermoy, Ireland, on new application in protein analysis using

13.

GiNN, R.E.

Pro-Milk

II.

1974.

The Milko-tester and problems

related to

its

use.

J.

Milk Food Technol.

37:218-221.
14.

Heinemann,
1954.

B.J.,

CosiMiNi, E., Jack, L., Willingham,

Methods of determining the percent

total solids in

J. J.,

and B.M. Zakariasen.

milk by means of the lactometer.

J.

Dairy Sci. 37:869-876.


15.

Henningson, R.W. 1963. The variability of the freezing point of fresh raw milk. J. Assoc.
Anal. Chem. 46:1036-1042.
Johnson, L. 1%5. Collaborative study of a method for multiple chlorinated pesticide residues in fatty foods. J. Assoc. Off. Anal. Chem. 48:668-675.
Kaplan, E. 1963. U.S. Food and Drug Officials Quart. Bull. 27:101.
Krause, R.T. 1973. Determination of several chlorinated pesticides by the AOAC multiresOflF.

16.

17.
18.

method with additional quantitation of perthane after dehydrochlorination: collaboraAssoc. Off. Anal. Chem. 56:721-727.
Levowitz, D. 1960. An appraisal of the Gerber test for milk fat in milk and market milk
products. J. Milk Food Technol. 23:69-72.
Levowitz, D. and C.E. Hynds. 1958. Specifications for Gerber glassware for determining
the fat content of milk and cream. J Milk Food Technol. 21:259-261.
idue

tive study. J.

19.

20.

21.

Madden,

D.E. and J.R. Brunner. 1958. Comparative accuracy of small lactometers for
total solids of individual cow's milks. J. Dairy Sci. 41:783-788.
Milk Industry Foundation, 1949. Methods of analysis of milk and its products. Labora-

determining the
22.

tory Manual. Washington, D.C.


23.

Sherbon, J.W.

dye binding.

J.

for protein in milk.

J.

1974. Pro-milk for the determination of protein in milk by

Assoc. Off. Anal. Chem. 57:1338-1341.


24.

Sherbon, J.W.

1975.

collaborative study of the Pro-Milk

method

Assoc. Off. Anal. Chem. 58:770-772.


25.

Sherbon. J.W.

1977.

Does protein

testing give accurate results? Dairy Ice

Cream

Field.

Jan., 1977
26.

27.

Pensiripun, D., Campbell, E.C, and G.H. Richardson. 1975. A vapor pressure osmometer for determination of added water in milk. J Milk Food Technol. 38:204.
Richardson, G.H., Mortensen, M.S.. and R.G. Crockett. 1977. Quantitation of added
in milk using vapor pressure osmometry. 91st Association of
Chemists Meeting. Washington. D.C.

water

Official Analytical

28.

Shipe. W.F. 1958. Report on cryoscopy of milk.

29.

Shipe. W.F. 1956. The use of thermistors for freezing point determinations.

J.

Assoc. Off. Anal. Chem. 41:262-267.


J.

Dairy Sci.

39:916 (Abstr.).
30.

Shipe. W.F. 1969. Collaborative study of the Babcock and Foss Milk-tester methods for

measuring
31.

Watson.
J.

32.

fat in

raw

milk.

J.

Assoc.

Off. Anal.

Chem. 52:131-138.

P.D. 1957. Determination of the solids of milk by a lactometric method

at 102 F.

Dairy Sci. 40:392-402.

Weik, R.M.

Committee

C.J.

Young. S.J.. and Burke, J. A. 1972. Micro scale alkali treatment for use in pesticide
due confirmation and sample cleanup. Bull. Environ. Contam. Toxicol. 7:160-167.

resi-

1969. Report on dairy products. General Referee Report

Assoc. Off. Anal. Chem. 52:235-242.


33.

Chapter 20

RADIONUCLIDES

IN

MILK

G.K. Murthy, R.L. Morris, Rogert W. Cochran and


Kathryn Glynn

Introduction

20.1

The importance of radioactive substances in milk as a


from testing of nuclear weapons has been established. In
milk

is

result of fallout

the food chain,


considered the major source of radionuclides for children and a sig-

nificant contributor to radioisotope ingestion

by adults. Although several

radionuclides are present in the fallout, most of them either have a short half
life

or they are not metabolized by the cow; only a few of the radionuclides

They

are found in a measurable amount.

are strontium-89, strontium-90,

iodine-131, cesium-137, and barium-140. In the vicinity of a test


ever, radioiodine-133

found
served
limit

20.8 hr) and tritium-3

(ti/2

in

also

milk

site,

is

considerably lower and

of 200 pCi per

its

level

is

how-

may be
(ti,2 = 245

12.3 years)

Around nuclear processing plants, zinc-65


be present in the milk. The amount of tritium-3

in the milk.

may

days)

(ti/2

(^H) ob-

close to the detection

liter.

The concentration of strontium-89, strontium-90, iodine-131, cesiumand barium-140 reached levels of significance immediately following
testing but recently have fallen to lower levels. Occasionally,
testing has continued by the Chinese and the French. It is considered advisable to determine concentrations of these milkborne fission materials and to assess their dietary contribution to assist in evaluation of the
biological effects, if any, from man's ingestion of these elements. The appearance of nuclear power generating stations in milk production areas renders advisable the radioassay of milk for its environmental contaminants,
especially in view of public concern over low-level, long-term radiation effects, which are as yet inadequately defined.^- ^'
Procedures for milk ashing followed by chemical separation and sub137,

weapons
weapons

''*

sequent radioassay are as referenced.^"*^-^^-^^


of

'^'I

tested.

at

pCi per

liter

of milk level

is

method for determination


method has not been

available but the

^^

ISC Liaison: W.W. Ullmann


283

RADIONUCLIDES

284

IN

MILK

20.2 Sampling
Sampling of milk for analysis depends on whether samples are intended
by farm practices, b.) in the

for determination of radionuclides a.) as affected


vicinity of nuclear

To study

power generating

stations, or c.) at the

the effect of farm practices

on radionuclides

in

consumer

level.

milk, samples are

cows or a portion of the pooled milk is taken from


on the farm. Milk samples in the vicinity of nuclear power
generating stations are taken from within a discrete geographical area depending on meteorological conditions and the number of farms affected. The
sample may be from an individual farm or from groups of farms. To study
market milk, samples at each location are composites of subsamples from
each milk processing plant, the quantity being proportional to the plant's
collected from individual
the storage tank

average sales

in the

community served.

Frequency of sampling depends on the seriousness of the situation and the


radionuclide considered for analysis. With iodine-131 (half-life of 8.08 days)
it is necessary to collect milk samples every day or at least once a week.
With strontium-90 (half-life of 28 years) it is necessary to collect once a
week or at least once a month.
20.3 Milk Preservation
At

least

one gallon

preserved with

HCHO

(3.8 liters) of milk

110 ml of

40%

(raw or pasteurized) is collected,


per gallon (3.8 liters) of milk],

HCHO

Formaldehyde does not interfere


by gamma spectroscopy or ^'^Sr and *^*^Sr
by ion-exchange and/or by chemical methods. However, addition of HCHO
to raw milk causes transformation of ''''I to protein-bound '34. ^^ The enzyme, xanthine oxidase, in raw milk oxidizes HCHO to H2O2 in the presence
of oxygen and H2O2 oxidizes ^^^l to ^^%, which binds to protein. In pasteuand shipped

to the laboratory for analysis.

with analysis of

^'''I,

'*Ba,

and

rized milk or dried milk the

'^^Cs

enzyme

is

absent, consequently, addition of

HCHO to processed milk has no effect on '^'I.

Processed milk containing *^'I


from fallout or labeled in vitro can be preserved with HCHO and successfully used for determination of '^'I by ion exchange methods.^** However, if raw
milk is the sample of choice and
is the only radionuclide of concern, no
preservative should be added to the milk. Analysis can be simplified by passing milk through an anion exchange resin column at the sampling station and
''''I

then shipping the column to the laboratory for further processing.^" This

presupposes that

20.4

all

the '^4 in milk

Determination of

'Sr

and

is in

the inorganic form as the iodide.

-"*Sr In

Milk

By

Ion

Exchange

modified method for evaluation of stfontium 89 and strontium 90

in fluid

milk has been developed for public health surveillance.^^

A. Principle:

Fresh milk samples are preserved with formaldehyde


per gallow (3.8

liters)

of milk

[10 ml of 40%

and stored, preferably

at 4

HCHO

C, for up to

20.4 Ion Exchange: ^^Sr

285

and ^Sr

weeks to obtain yttrium ingrowth. After storage, yttrium, strontium and


barium carriers and citrate solution are added. The latter ions cause foiniation of an yttrium complex which is adsorbed on the anion-exchange resin
when the sample is passed through cation- and anion-exchange columns. ^^
The yttrium is desorbed from the anion exchanger and purified of radionuclides such as lanthanum by tributyl phosphate (TBP) extraction of yttrium. The yttrium is back-extracted in dilute HNO3 and precipitated as an
2

oxalate, which

is

weighed and counted for

^'Sr.

The radiostrontium is desorbed from the cation exchanger along with calcium and radiobarium, purified of calcium and rare earth radionuclides by
repetitive precipitation as nitrates in 16N (70%) HNO3, purified of radiobarium by barium chromate precipitation, precipitated as strontium carbonate, and counted. The total radiostrontium, less the ^"Sr by ^"Y measurement, yields a value for ^^Sr. Carrier recovery tests are made to account for
loss of radionuclides in the procedure.

B. Interferences:

Presence of radioactive barium and lanthanum will cause interference


without appropriate purification. Purification from calcium is important for
recovery tests (by gravimetric techniques) but need not be as thorough when
strontium recovery

counting

total

is

determined by

radiostrontium for

*^

^'^Sr

tracer or by flame photometry. If

determination,

^'^Sr

tracer should not be

used since it would interfere in beta counting of total radiostrontium. Spoilage of sample leading to curds which clog the exchanger columns should be
prevented by preservation and refrigeration or avoided by filtration before
sample flow through exchangers. Columns should be thoroughly desorbed
before reuse and periodically tested to assure complete desorption.

and equipment:
Ion-exchange apparatus

C. Apparatus
1.

as

shown

*: Assembled with stoppers and mounted


The apparatus consists of a 1 -liter graduated separa250-ml, 5-cm-diam separatory funnel having a fritted glass

in Fig. 20: 1.

tory funnel; a

disc as the cation-exchange column; and a 30-ml,

1.9-cm-diam separatory

funnel having a fritted glass disc as the anion-exchange column.

For mounting precipitates for counting the filassembly consists of a Teflon filter holder having specifications
given in Fig. 20:2 and plastic rings and discs * moulded of nylon, Zytel 101,
2.

Filtration assembly:

'

tration

according to specifications
3.

Filter paper:

in Fig. 20:3.

Whatman No.

541.

*The apparatus assembly may be obtained from various supply houses such as Kontes Glass
Company, Vineland, NJ 08360.
^Currently available from Fluorulon Laboratories, Box 305, Caldwell, NJ 07006.
*One supplier of the
Staten Island,

NY

rings

10303.

and discs

is

the Control Moulding Corporation, 84 Granite

Avenue,

RADIONUCLIDES

286

IN

MILK

r\
1 -liter graduated
separatoty funnel

250-ml separatory
.funnel with fritted
glass disc

cation
resin

30-ml separatory
.funnel with fritted
anion

glass disc

resin

Figure 20:1. Ion Exchange System

Mylar

Mylar film provides a convenient cover to protect preand storage. The film is about mil (0.025 mm) thick
and can be obtained in rolls IV2 in. (3.8 cm) wide.
5. Glass fiber filter paper: Filter paper No. 934-AH,
2.8 cm in diameter.
6. Glass centrifuge tubes: 40-ml capacity, heavy-duty, with short cone
bottom and pour-out lip.
7. Glass centrifuge bottles: 250-ml capacity.
4.

film

cipitates during counting

"

8.

Pipets: 1-ml volumetric, calibrated for accurate delivery.

9.

Separatory funnels: 1000-ml, 500-ml, 125-ml and 60-ml capacity.

10.

Analytical centrifuge.

11.

Analytical balance.

12.

Beta particle counter: Low-background.

D. Reagents:
Unless otherwise indicated, all reagents shall conform to specifications of
the Committee on Analytical Reagents of the American Chemical Society.
Other grades may be used, provided it is first ascertained that the reagent is

^Mfr's No. 50A, from E.

I.

du Pont de Nemours, Wilmington, Del 19801.

"Obtainable from H. Reeve Angel

chemical supply houses.

& Company, 9 Bridewell Place, Clifton, NJ 071 14, and other

20.4 Ion Exchange: ^^Sr

and '"Sr

287

Grooves approx.
1/32" wide by
1/32" deep

No. 30

drill

Inside surface

smooth polished

2"1/8'

1.125"

+0.005"
-0.000"

3/16"

WH09239
Figure 20:2. Teflon Filter Holder

of sufficiently high purity to permit

Water

the determination.

Ammonium

tate

(NH4CoH;jOo)

cial acetic
1

liter

2.
3.

acetate buffer,

in

its

use without lessening the accuracy of

refers to reagent (distilled) water.

pH

5.0:

Dissolve 153 g of ammonium acepH of solution to 5.0 with gla-

900 ml of water. Adjust

acid (sp gr 1.05) using a glass-electrode

pH

meter, then dilute to

with distilled water.

Ammonium
Ammonium

hydroxide cone, sp gr 0.90.


hydroxide, 6N: Dilute 400 ml of cone

NH,OH

to

liter

with water.
4.
5.

trate,

Anion-exchange resin: Dowex 1-X8 (CT form). 50-100 mesh size.


Barium carrier, 20 mg of Ba-+ per ml.: Dissolve 38.1 g of barium niBa (NO.i).2, in water, add ml of cone HNO.j, and dilute to liter with
1

water.
6.

size.

form by passing 1,500

Dowex 50W-X8 (Na^

form), 50-100 mesh


H+ form of resin is converted into the Na^
ml of 4N NaCI through 170 ml of resin placed in the

Cation-exchange

The commercially

resin:

available

RADIONUCLIDES

288

IN

MILK

0.950"

0.005"

0.040"

0.9325"

0.0025-

Figure 20:3. Rings and Discs

column and

rinsing

chloride-free

when

to

8.

Diethyl ether.

Ethyl alcohol, 95%.

10.

Formaldehyde

1 1

Hydrochloric acid, 6N:

pH

to

37%.

solution,

Add

500 ml of cone HCl to water and dilute

liter.

Hydrochloric acid, 2N: Add 167 ml of cone HCl to water and dilute

liter.

21N: sp gr 1.48

13.

Nitric acid, fuming,

14.

Nitric acid cone, 16N: sp gr 1.42,

15.

Nitric acid, 14N:

liter.

liter.

liter

Nitric acid,

16.

add 875 ml of cone HNO.-j

to

water and dilute to

HNOu

to

water and dilute to

Add

6.25 ml of cone

HNO.j

to

water and dilute to

with water.

Oxalic acid, 2N: Dissolve 126 g of oxalic acid (H2C2O4 R,0)

18.

warm

90% HNO,.
70% HNO,.

i.e.,

i.e..

6N: Add 375 mi of cone

Nitric acid, O.IN:

17.

water, cool, and dilute to

Sodium

20.

brown

in

liter.

Silver nitrate, 1%: Dissolve

19.

to 100 ml. Store in

g of silver nitrate

in

water and dilute

bottle.

chloride, 4N: Dissolve 234 g of

NaCl

in

water and dilute to

liter.

Sodium carbonate, 3N: Dissolve 159 g of Na2CO:i

21.
lute to

22.

to

is

NaOH.

9.

12.

with about 500 ml of water until the wash water

Citrate solution, 3N: Dissolve citric acid in water and adjust

7.

6.5 with dilute

to

it

tested with 1.0^ AgNOij.

in

water and

di-

liter.

Sodium chromate, IN: Dissolve

liter.

81

gof Na.,CrO.,

in

water and dilute

and

20.4 Ion Exchange: '^Sr

289

*<'Sr

Strontium carrier: 20 mg of Sr^"^ per milliliter. Dissolve 48.3 g of


1 ml of cone HNO3, and dilute to 1 liter. Standardize

23.

Sr (N03)g in water, add

carrier solution by pipetting 1-ml portions of Sr (N03)2 carrier solution into

40-ml centrifuge tubes containing 15 ml of water. Adjust pH of


6N NH4OH. Add, with stirring, 3-5 ml of 3N
Na2C03 and digest in hot water bath for 5 minutes. Cool to room temperature and process precipitate as in 20.4 (G. 15-17) or 20.4 (G.19).

each of

six

solution to 8.5-9.0 with

Sr,
24. Tributyl

mg/ml = (mg SrCOg) (0.5935)

phosphate (TBP) preequilibrated: Add 150 ml of water and

30 ml of 3N Na2C03 to 300 ml of TBP in a 1-liter separatory funnel. Shake


for 2-3 min and allow phases to separate. Discard aqueous lower phase. Add
150 ml of water to separatory funnel, shake for 2-3 min, and allow phases to

Add 150 ml of 14N HNO3 and shake


Allow phases to separate and discard lower aqueous phase. Re-

separate. Discard aqueous lower phase.


5 minutes.

HNO3 to assure equilibration.


Yttrium carrier: 10 mg Y^+/ml: Dissolve 12.7 g of yttrium oxide*
(Y2O3) in 50 ml of cone nitric acid by heating (avoid boiling). Dilute to 900 ml
with water, adjust pH to 2.0 with cone ammonium hydroxide, and dilute to
1 liter with water. Standardize carrier solution by pipetting 1-ml portions of

peat twice the treatment with 14N


25.

yttrium carrier solution into each of six 40-ml centrifuge tubes containing
15

ml of water. Add 5 ml of

NH4OH,

using a

pH

2N

oxalic acid and adjust

meter. Digest

in

pH

to 1.5 with

6N

a hot water bath for 10 min and cool to

below room temperature. Centrifuge and discard supernatant

fluid.

Process

precipitate as in 20.4 (F. 12-16) or 20.4 (F.19).

E. Procedure for removal ofradioelements in milk by ion/exchange resin:


1.

Freshly drawn sample

is

preserved with about

solution for each liter of milk and refrigerated for

ml of formaldehyde

two weeks

to allow yttrium

ingrowth.

Thoroughly mix the preserved sample of milk which has been stored
nonhomogeneity. If homogeneous, transfer 1 liter of subsample to the separatory funnel used in the ionexchange apparatus, [20.4(C.l)]. If the milk is nonhomogeneous, it should
first be filtered through a loose bed of borosilicate glass wool to prevent
clogging of the resin column.
3. Combine 1 ml each of yttrium, strontium and barium carriers with
10 ml of citrate solution in a small beaker or vial. Quantitatively transfer
carriers to milk sample in funnel and mix well.
2.

for ingrowth of yttrium. Inspect milk for signs of

^Yttrium oxide Code

1 1

18,

American Potash and Chemical Corp, West Chicago, 111 60185; or


Code 1118 may require purification because of

equivalent. Yttrium oxide of purity less than


radioactive contamination.

RADIONUCLIDES

290

IN

MILK

0~

3.5

liter level

254mm

47mm

89mm

11

1mm

Figure 20:4. Cross section of reentrant sample beaker

4.

Add

Prepare exchanger columns

170 ml of

ion-exchange apparatus [20.4(C.l)].


column by pouring the
with water and add 15 ml of Dowex 1-X8

Dowex 50W-X8

in

[20.4(D.6)] to upper

column filled
column in a similar manner.
5. Connect funnel to ion-exchange system (Fig. 20:1). Place beaker to
collect effluent. Open stopcocks of funnel, anion column and cation column
in that order. Control the effluent flow at 10 ml per min with the anion column stopcock. Check effluent and adjust flow periodically.
resin into the

[20.4 (D.4)] to lower

Stop flow when milk level reaches top of resin in each bed. Discard
RECORD
TIME as the average period of effluent flow, as in
20.4(E.5,6). This time is taken as the beginning of yttrium 90 decay (there
should be no unnecessary delay during steps 20.4(E.5,6).
7. Connect separatory funnel containing 300 ml of warm distilled water
and continue elution as in 20.4(E.5,6).
6.

effluent.

F.

''"F

MEAN

separation, purification

and determination:

Connect a separatory funnel containing 100 ml of 2N HCl to top of


anion column. Open funnel stopcock, then column stopcock, and control
1.

20.4 Ion Exchange: ^^Sr

and 5Sr

291

yttrium chloride effluent to 2 ml per minute. Collect first 15 ml of eluate.


Close both stopcocks and remove separatory funnel. Stir resin thoroughly
with a glass stirring rod. Wash stirring rod with a small amount of 2N HCl,
which is added directly to the resin column. Connect separatory funnel and

2N HCl. Collect a total of 70 ml of yttrium eluate.


Continue treatment of eluate as in 20.4(F.3).
2. Adjust elution rate to 10 ml per min for remaining 30 ml of acid to
recharge the column. Discard effluent. Wash resin with 100 ml or more of
distilled water until a negative AgNOg test for chloride is obtained. The column is now ready for another determination.
3. Add 5 ml of 2N oxalic acid to eluate from 20.4 (F.l) above and adjust
pH to 1.5 with 6N NH4OH, using a pH meter. Stir, heat to near boiling in
water bath, cool in an ice bath, centrifuge, decant, and discard the supercontinue elution with

natant

fluid.

4.
in

If

fresh fission products are

known

ml of 6N HCl, add 15 ml of water,

filter

to

be absent, dissolve precipitate


Whatman No. 541

solution through

paper into a 40-ml centrifuge tube, wash

filter

ings in a centrifuge lube, discard

filter

filter

paper, collect wash-

paper, and continue as given

in

20.4(F.11,19).
If

5.

fresh fission products are present, add 10 ml of cone

HNO3

to the

centrifuge tube to dissolve yttrium oxalate precipitate. Transfer solution to a

60-ml separatory funnel. Wash centrifuge tube with 10 ml of cone HNO3,


add washings to the separatory funnel, and then add 10 ml of pre-equilibrated

TBP.

6.

Shake

for 2-3 min, then allow phases to separate;

draw

off

and

dis-

card the lower acid phase.

Add 15 ml of 14N HNO3 to separatory funnel and shake for 2-3 minAllow phases to separate and again discard the acid phase.
8. Repeat the preceding step, [20.4(F.7)]. These treatments purify yt7.

utes.

trium of light lanthanide elements,

Add

in particular '^"La.

ml of water to the separatory funnel and shake for 2-3 minutes. Allow phases to separate and drain the aqueous phase containing most
9.

15

of the yttrium into a 40-ml centrifuge tube.

Repeat the preceding step, [20.4(F.9)], using 15 ml of O.IN HNO3.


the aqueous phase of the preceding step and of this step,
20.4(F.9 and 10).
11. Add 5 ml of 2N oxalic acid to the purified yttrium solution from
20.4{F.4 or 10) and adjust pH to 1.5 with cone NH4OH. using a pH meter.
Digest the solution in a hot water bath for 10 min, mixing occasionally. Cool
in an ice bath, centrifuge, and discard supernatant fluid. Proceed as in
20. 4(F. 12-18) or alternatively proceed as in 20.4(F.19). Standardize carrier
by whichever alternative technique is chosen for unknowns.
12. Place a 2.8-cm glass fiber filter on a stainless steel planchet and
weigh together. Transfer tared filter paper into filter holder (Fig. 20:2) and
assemble filtration apparatus.
10.

Combine

RADIONUCLIDES

292

IN

MILK

With water spray, transfer yttrium oxalate precipitate quanminimum of vacuum so that the precipitate is distributed uniformly over the filter area. Increase vacuum as necessary uniformly over the filter area. Increase vacuum as necessary after most
13.

titatively to the filter funnel, using a

of the precipitate

Wash

14.
ter,

is

on the

filter.

the precipitate three times with 10-ml portions of

warm wa-

three times with 959c ethyl alcohol, and three times with diethyl ether.

Continue vacuum for about 2-3 minutes.


15. Remove filter paper carefully and place it on the original stainless
steel planchet. Allow to stand at room temperature for 10-15 minutes.
16. Weigh sample and calculate yield of yttrium oxalate [likely
Y2(C2 04)39H2 0] by dividing observed weight by weight of yttrium oxalate
obtained on standardization of carrier [20.4(D.25)].
17. Remove filter paper from planchet, place on top of nylon disc, cover it with a piece of Mylar film, place nylon ring over Mylar film, and press
ring onto nylon disc. Cut off excess Mylar film.
18. Count yttrium 90 activity in a low-background anticoincidence beta
counter without undue delay. Repeat counting after 3 days to confirm purity
of yttrium 90 by its rate of decay. RECORD DATES AND TIMES OF
COUNTING. Calculate strontium 90 activity from yttrium 90 count as given
in20.4(H.l).
19.

An

alternate procedure to prepare yttrium oxalate for weighing and

counting consists of dispersing the precipitate from Section 20.4(F.ll) twice


with 20-ml portions of

warm

water, cooling to below

room temperature,
Then

centrifuging and discarding the supernatant fluid after each wash.

transfer the yttrium oxalate precipitate quantitatively to a tared stainless


steel dish.

Disperse the precipitate uniformly over the dish bottom. Dry un-

der infrared

light to

constant weight and count

in

an internal proportional

counter or a thin end-window counter. Standardize carrier by the same

method chosen

for the

unknown.

G. ^^Sr separation purification and determination:


1. Connect a 1-liter separatory funnel containing one
,

liter

of

4N NaCl

cation-exchange column. Open the funnel and column stopcocks in


and adjust eluate flow to 10 ml per minute. Collect about 1 liter of
eluate in a 2-liter beaker but leave resin covered with several milliliters of
to the

that order

Continue treatment of eluate as in 20.4(G.3).


cation-exchange column with 500 ml of water from a separatory funnel, using a flow of 10 ml per minute. Discard wash water. If resin
becomes clogged with milk solids, backwash column or transfer resin to a
beaker and clean by water agitation and decantation.
3. Dilute the eluate from 20.4(G.l) to 1,500 ml with water, heat to 8590 C on a hot plate, and add 100 ml of 3N NagCOg with gentle stirring. Resolution.
2.

Wash

293

20.4 Ion Exchange: ^^Sr and ^Sr

Table 20:1. Recovery of Strontium and Calcium from a Single Precipitation

,_,^

HNO3

Sr Recovery

Ca Recovery

^^

14N
16N
18N

81

98
100

move from

hot plate and cool to

1.4

11

1.7

51

4.0

2.6

room temperature. Decant bulk of

0.9
2

clear

Transfer precipitate quantitatively to a 250-ml centrifuge


bottle, using water. Centrifuge and discard supernatant fluid.
4. Add 50 ml of water and disperse the precipitate. Centrifuge and dissupernatant

fluid.

card the supernatant

6.
7.

of

6N

fluid.

Repeat the preceding step, 20.4(G.4).


Dry precipitate in oven at 110 C for 4 hr and cool.

5.

Dissolve precipitate slowly with vigorous stirring, using about 4 ml


HNO3 (a magnetic stirrer is helpful). Filter solution through Whatman

No. 541

filter

with 4 ml of

paper into a 40-ml graduated centrifuge tube. Rinse the tube


the washing through the filter. Discard filter

6N HNO3 and pour

paper.

Add

21N HNO:j

and cool in an ice bath,


then centrifuge, and discard the supernatant fluid. Strontium nitrate precipitations are critical to good recovery of strontium and adequate separation of
^^^'^
calcium. Recovery of strontium and calcium from a single precipitation
is shown in Table 20:1.
9. Dissolve precipitate in 5 ml of water. Adjust the pH to 5.0 with cone
NH4OH, using a pH meter. Add 5 ml of ammonium acetate buffer.
10. Heat the solution in a water bath, add 1 ml of IN Na2Cr04, and mix
well. Digest in water bath for 5 minutes. Centrifuge and decant the supernatant fluid into a small beaker. Evaporate to about 2 ml, add 2 ml of 6N
HNO3, and transfer to a 40-ml centrifuge tube, using 3 ml of rinse water.
11. Add 20 ml of 21N HNO3, stir, cool in an ice bath, centrifuge, and
8.

20 ml of

to the filtrate. Stir

discard the supernatant fluid.


12.

Add

ml of water and

ml of 6N HNO3 to dissolve the precipitate,


cool in an ice bath, centrifuge, and discard
THE TIME. This is the beginning of yttrium

then add 20 ml of 21N

HNO3,

the supernatant fluid.

RECORD

stir,

90 ingrowth.
13.

Dissolve precipitate

8.5-9.0 with

a few milliliters of water and adjust pH to


3-5 ml of 3N Na2C03 to precipitate strontium

in

6N NH4OH. Add

carbonate, centrifuge, and discard the supernatant fluid.


14. Disperse precipitate in 10 ml of water, centrifuge, and discard the
supernatant fluid. Proceed as in 20. 4(G. 15-18), or alternately proceed as in
Section 20.4(G.

19).

RADIONUCLIDES

294

IN

MILK

15. Place a 2.8-cm glass fiber filter on a stainless steel planchet and
weigh together. Transfer filter paper to a filter holder (Fig. 20:2) and assemble filtration apparatus.
16. Disperse precipitate in water and transfer it quantitatively to filter
funnel, using a minimum of vacuum, so that the precipitate is distributed
uniformly over the filter. Increase vacuum as necessary after most of the
precipitate is on the filter.
17. Wash the precipitate three times with 5- to 10-ml portions of water,
transfer filter sample to the planchet previously used, dry in oven at 110 C
for 30 min, cool in desiccator, and weigh.
18. Transfer filter sample to a nylon disc, cover with Mylar film, press
on a nylon ring, trim oflf excess Mylar, and count radiostrontium in a thin

end-window beta counter.

RECORD TIME OF COUNTING.

Calculate Sr

according to 20.4(H).
19. An alternate procedure consists of washing the precipitate twice in
about 10 ml of water, with centrifugation and decantation after dispersing
the precipitate, then transferring the precipitate quantitatively to a tared
stainless steel dish. Dry in an oven, cool in the desiccator, weigh, and count

an internal proportional counter or a thin end-window counter.


TIME OF COUNTING. Calculate ""^Sv according to 20.4(H).
in

RECORD

H. Calculation:

I.

cpm

Srac.ivky.pCi/li.er

r^e^d,I,,V

where cpm = net beta count per minute of

cpms
Ls

+ bkgr

= Standard

^P^^tJkgr

+ bkgr

'^'^Y,

deviation of net count.

l-bkgr

recovery of yttrium carrier, decimal fraction,


counter efficiency for ^''Y as Yg (C204)3, cpm/pCi,

R,
E,
D,

decay correction factor e"-^" for '"'Y, where t is the time of


separating the ^"Y from Sr [20.4(E.6)] to the time of counting
120.4(F. 18,19)].

ly

= ingrowth

um

correction factor

e"'''

for the degree of equilibri-

ingrowth period, where t is the


time from the start of the ingrowth period [20.4(E.l)] to the
time of separating ^"^Y from Sr [20.4(E.6)] and

V =

attained during the

sample volume,

in liters.
1

2.

^''Y

^^Sr activity, pCi/liter

E.D.

cpmg

rTv

cr

Cs(AsEs

Eyly)

20.5

295

TCA: ^Sr and ^Sr

where Es = counter
Ds

efficiency for

correction factor

e"*^'

*^^Sr

as SrCOg, cpnVpCi,

for ^^Sr decay,

sample collection [20.4(E.l)]


[20.4(G.18 or

a =

cpm^hkgr
ts

where

is

the time from

to the time of

counting

19)].

= Standard

'^P"^hkgr

+ bkgr

deviation of net count.

IbRgr

Rs = recovery of strontium carrier, decimal fraction,


cpm = net counts per minute of observed radiostrontium,
V = sample volume, in liters.
Cs = ^Sr activity, pCi/liter,
As = absorption factor for ^"Sr as SrCOa obtained from

a self-ab-

sorption calibration curve (self-absorption curves for ^^Sr and


for ^"Sr are derived by precipitating a series of carrier

SrCOa

concentrations over the expected "recovery range"

in the

presence of a constant amount of

and yttrium-free ^'^'Sr.


respectively. The ordinate to the curve is the ratio of the
count rate for each thickness to the count rate at zero sample
thickness, and the abscissa is the sample weight for the given
type of sample mount).
Es = counter efficiency for ^''Sr as SrCOa. cpm/pCi.
Ey = counter efficiency for '^"Y as Y2(C204)3. cpm/pCi. and
ly = correction factor 1 - e"''' for degree of equilibrium attained
during ^"Y ingrowth period, where t is the time in which ^*^Y

was separated from SrCOg

**^Sr

[20.4(G.13)] to the time of count-

ing [(20.4(G.18 or 19)].


/.

Precision

and accuracy:

samples containing known amounts of


added radionuclides, eleven collaborators determined **^Sr and ^"Sr.^
The average recoveries of added ^^Sr (29.0 and 197 pCi/1) from the two
milk samples were 31.0 pCi/1 (range 26 to 36 pCi/1) and 199.6 pCi/1 (range
164 to 290 pCi/1); standard deviation of 2.9 and 7.2 and per cent error of 10.0
and 3.4, respectively. For milk containing 32.4 and 151.2 pCi/1 of ^^'Sr. results were: average 32.5 (range 27.2 to 38.0 pCi/1) and 149.9 pCi/1 (range
125.3 to 170.3 pCi/1); standard deviation of 0.32 and 4.17 and per cent error
of 0.9 and 2.8, respectively.
There was greater dispersion in the higher levels for *^^Sr but this was
expected; even though the per cent error for the level of **^Sr was greater
than the higher level, the standard deviation was acceptable. Both ^^Sr and
In a collaborative study of milk

""Sr determinations

showed

little

20.5 Determination of ^^Sx

bias.

and

^^Sr in Milk

A method for determining

by

TCA

strontium 89 and strontium 90 in preserved fluid


"^' i"-"-is given below.
milk for routine public health surveillance
"^^

RADIONUCLIDES

296

IN

MILK

A. Principle:

Strontium and barium carriers are added to milk preserved with formaldehyde. Milk is coagulated with trichloroacetic acid (TCA) and filtered
through a Biichner funnel. The curd is washed by being slurried with dilute
TCA and refiltered. Alkaline earth elements in combined filtrates are precipitated with carbonate at pH 8.5-9.0, separated by centrifugation, and dissolved in a minimum of HNO3. Strontium is separated from most of the
calcium and phosphorus by strong nitric acid precipitation and centrifuga-

Radiobarium, radium and lead contaminants, when present, are recoprecipitation, with barium carrier as the chromate. Ferric chloride is added and strontium is further purified of polyvalent radionuclides by
ferric hydroxide scavenging. Strontium is precipitated as carbonate and dissolved in hydrochloric acid. Yttrium carrier is added, and the solution is
stored for 2 weeks for ''"Y ingrowth. Yttrium is then precipitated as the hydroxide and is converted to the oxalate for mounting and counting.
The supernatant fluid from the Y(0H)3 precipitation contains radiostrontium. It is precipitated and counted for total radioactive strontium. The
diff'erence between total radioactive strontium and the ^*'Sr equivalent of ^"Y
is taken as a measure of ^^'^Sr, with due allowance for diff'erences in selftion.

moved by

absorption of radionuclides present.

B. Interferences:

Calcium, phosphorus, radioactive barium and lead are removed and hence
interfere with the determination, although repetitious purification
steps are time-consuming and unless done with care may lead to excessive

do not

loss of carriers

and the radioactivity sought.

C. Apparatus

and equipment:

Glass centrifuge containers: Tubes, 40-ml capacity, heavy-duty,

1.

with short cone bottom and spout. Also bottles, 250-ml capacity.
Pipets: 1-ml, volumetric,

2.

which assure accurate delivery of solutions

standardized for chemical yield calculations.


3.

Filtration assembly.*^

4.

Teflon

filter

holder.^

Rings and discs**: Plastic rings and discs are molded of nylon,
Zytel 101, according to dimensions given in Fig. 20:3. Discs are molded as
cups. This form allows discs to be used for mounting solid samples, using the
5.

ring, or for

evaporating liquid samples

in the

cup.

^As specified by one supplier, Fluoiiilon Laboratories, Box 305, Caldwell, NJ 07006.
**Present sources of supply include Control Molding Corporation, 84 Granite Street, Staten
Island,

NY

10303.

TCA:

20.5

^^Sr

Mylar

6.

and ^Sr

297

Mylar

film^^:

film

is

used to cover precipitates to protect them


in. (0.025 mm) thick and P/^-in. (3.81 cm)

during counting and storage. Film 0.001

wide

in rolls.

Diameter 2.8 cm, filter paper No. 934-AH


or equivalent. Check background count of paper.
8. Centrifuge, with heads for 40-ml tubes and 250-ml bottles.
7.

Glass-fiber

9.

Analytical balance.

10.

filter

paper^*:

Low-background

/3-counter, thin

end-window ^-counter, or

inter-

nal proportional counter.

Whatman No.

11.

Filter paper,

12.

Biichner funnel.

42.

D. Reagents:
Analytical reagent-grade chemicals which meet ACS specifications or
equivalent shall be used. Other grades may be used, provided it is ascertained that the chemicals are of sufficient purity to permit their use without
decreasing accuracy of the determination.

Water means

distilled or

Ammonium

deionized reagent water.

pH 5.0: Dissolve 153 g of ammonium ace(NH4C2H,(02) in 900 ml of water. Using a glass electrode, adjust the pH
to 5.0 with cone acetic acid (sp gr 1.05) and dilute to 1 liter with water.
2. Ammonium hydroxide, cone (sp gr 0.90).
3. Ammonium hydroxide, 6N: Dilute 400 ml of cone NH4OH to 1 liter
1.

acetate buff'er,

tate,

with water.

Barium

0.2N: Dissolve 26.135 g of barium nitrate IBa (N03)2]


liter with water.
ml of cone HNOu and dilute to
5. Ferric chloride, O.IN: Dissolve 9.01 g of ferric chloride (FeCla^GHgO)
water, add 1 ml of cone hydrochloric acid, and dilute to 1 liter with water.
4.

in

in

water, add

7.
8.
1

to

water and dilute

liter.

Nitric acid, fuming: 21

9.

10.
11.
1

Formaldehyde, 37%.
Hydrochloric acid, cone (sp gr 1.19)
Hydrochloric acid, 6N: Add 500 ml of cone HCl

6.

to

nitrate,

N HNO3

(sp gr 1.48)

cone (sp gr 1.42), 16N


Nitric acid, 8N: Add 500 ml of cone
Nitric acid,

HNO3

to

water and dilute to

liter.

12.

Methyl orange indicator solution: Dissolve

0.

g of methyl orange in

100 ml of water.
13.

Mixed

indicator: Dissolve 0.1 g of

phthalein in 100 ml of

50%

^'^Available as Manufacturer's

**H.

Reeve Angel and Co.

thymol blue and

0.1 g of phenol-

(v/v) ethyl alcohol.

No. 50A, E.

I.

duPont de Nemours, Wilmington. Del. 19899.

Inc. 9 Bridewell Place, Clifton,

NJ

07105.

RADIONUCLIDES

298

MILK

Oxalic acid, 2N: Dissolve 126 g of oxalic acid (H2C2042H20) in

14.

warm

IN

water, cool, and dilute to

liter.

Phenolphthalein indicator: Dissolve 1.0 g of phenolphthalein

15.

100 ml of

50%

in

(v/v) ethyl alcohol.

159 g of sodium carbonate


water and dilute to 1 liter.
Sodium hydroxide, 6N: Dissolve 240 g of sodium hydroxide pellets

Sodium carbonate, 3N: Dissolve

16.

(Na2C03)
17.

(NaOH)

in

in water, cool,

and

dilute to

liter.

81 g of sodium chromate (NagCrOJ


water and dilute to 1 liter.
19. Strontium carrier, 40 mg Sr++ per milliliter: Dissolve 96.6 g of strontium nitrate [Sr(N03)2] in water, add 1 ml of cone nitric acid, and dilute to
1 liter with water. Standardize the carrier solution by pipetting a 1-ml sample
of Sr(N03)2 carrier into each of six 40-ml centrifuge tubes containing 15 ml
of water. Add 1 drop of mixed indicator and adjust the solution to pH 8.5-9.0
with 6N NH4OH. Add, with stirring, 3-5 ml of 3N Na2C03 and digest in a hot
water bath for 5 minutes. Cool to room temperature, centrifuge, and discard
the supernatant fluid. Wash the precipitate by dispersing it in 25-30 ml of
18.

Sodium chromate, IN: Dissolve

in

wash water. Transfer each precipitate quana separate tared stainless steel planchet. Dry under an infrared
lamp to constant weight when the procedure in 20.5(G.5) is used; or in an

water, centrifuge, and discard


titatively into

oven

if

the procedure in 20.5(G.6)


Sr,

is

used.

mg/ml = (mg SrCO3)(0.5935)

TCA, 50%

solution: Dissolve 500 g of trichloroacetic acid


water and dilute to 1 liter.
21. TCA, 5% solution: Dilute 100 ml of reagent [20.5(D.20)] to 1 liter.
22. Yttrium carrier, 10 mg per milliliter: Dissolve 12.70 g of high-purity
yttrium oxide (Y2O3) in 50 ml of cone HNO3 by heating gently (avoid boiling); dilute to 900 ml with water, adjust pH to 2.0 with cone ammonium
hydroxide, and dilute to 1 liter with water (see ^''Sr by ion exchange,
20.

(CCI3COOH)

in

[20.4(D.25)].

Standardize the carrier by pipetting a 1-ml portion of the yttrium carrier


each of six 40-ml centrifuge tubes containing 15 ml of water. Neutralize
the acid with 6N NH4OH, using a drop of phenolphthalein as indicator. Digest in a hot water bath until flocculant yttrium hydroxide precipitate forms.
into

an ice bath, centrifuge, and discard the supernatant fluid. Dissolve


ml of 6N HCl, then add 5 ml of 2N oxalic acid and adjust pH
to 1.5 with 6N NH4OH, using a pH meter. Centrifuge and discard the supernatant fluid. Wash precipitate twice by dispersing in 25-30 ml of water. Cen-

Cool

in

precipitate in

and discard wash water. Transfer precipitate quantitatively into a


steel planchet. Dry under an infrared lamp to constant
weight. A period of 30 min should suffice for drying. Calculate average yttrifuge

tared

stainless

trium residue per milliliter of carrier.

20.5

TCA:

^'Sr

and ^Sr

299

Alternatively, standardize carrier according to 20.5(G.7).

E. Procedure to
1.

One

liter

remove protein by coagulation and filtration:

of fresh milk or milk that has been refrigerated with or

without 3 ml of formaldehyde preservative

is heated to room temperature in


ml of Sr(N03)2 and Ba(N03)2 carriers, mixing well.
Add, while stirring, 300 ml of 50% TCA and promote coagulation by

a 3-liter beaker.
2.

Add

occasional stirring for 30 minutes.


3. Filter, using a No. 42 Whatman filter paper, in a Biichner funnel.
Press the moist curd with the bottom of an Erlenmeyer flask to remove
strontium-bearing water.

4. Transfer curd to original beaker, add 300 ml of 5% TCA, mix thoroughly with a mechanical stirrer, and filter slurry as in preceding step

20.5(E.3).
5. Combine filtrates from steps 20.5(E.3,4) preceding in a 3-liter beakadd 4-5 drops of mixed indicator, adjust pH to 8.5-9.0 with 6N NaOH
(the end point is reached when the color of the indicator changes to purple),
and then add 50 ml of 3N NasCOg. Mix solution well and allow precipitate of

er,

alkaline earths to settle for several hours, or preferably overnight.


6.

Siphon off" the clear supernatant fluid with vacuum and discard. Cenremaining slurry in a 250-ml bottle and discard the clear super-

trifuge the

natant fluid. Disperse residue in 200 ml of water, centrifuge, and discard the
supernatant fluid.
7.

Dissolve residue in 10 ml of

8N HNO3 and

quantitatively transfer

solution to a 250-ml beaker.

F. Procedure to purify strontium nuclides:


1. Evaporate solution on a hot plate to near dryness. Dissolve residue
ml of warm water and, if necessary, 1 ml of cone HNO3.
2. Transfer solution quantitatively into a 40-ml glass centrifuge tube.
Add 20 ml of 21N HNO3 (use a portion of this acid to rinse the beaker). Cool
in an ice bath for 15 min, centrifuge, and discard the supernatant fluid.
3. Add 3 ml of water and 10 ml of cone HNO3 to the residue (largely
strontium and barium nitrates). Dissolve by heating in a water bath. Add
10 ml of 21N HNO3 and continue heating for 10 min, with occasional swirling of
the tube. Cool in an ice bath for 15 min, centrifuge, and discard the super-

in 5

natant fluid (effective precipitation of cold strontium nitrate with a

of calcium interference occurs in 15N to 16N


4.

Add 3 ml of water and warm to dis6N NH4OH, using 1-2 drops of mixed in-

Invert tube to drain ofl"HN03.

solve. Adjust
dicator. If

pH

to 8.5-9.0 with

no precipitate forms, continue with 20.5(F.7) below. If there is


is is likely to be due to presence of phos-

appreciable precipitate at this point


phate.

minimum

HNO3).

RADIONUCLIDES

300

5.

IN

MILK

Centrifuge and decant the supernatant fluid into a 150-ml beaker,

retaining

for 20.5(F.7).

it

add a few milliliters of water and 15 ml of 3N


a hot water bath for 30-45 min, stirring occasionally.
Cool, centrifuge, and discard the supernatant fluid.
7. Dissolve precipitate in 1-2 ml of 6N HCl and retest for phosphate
6.

To

the residue,

NagCOg. Digest

in

with 6N NH4OH as in 20.5(F.4) above. If appreciable precipitate forms, repeat 20.5(F.5) above through this step 20.5(F.7). If a small precipitate persists, it may be silica, which should be discarded. Centrifuge and combine
the strontium-bearing supernatant fluids in 20.5(F.5 and

7).

Slowly add, with stirring, 5 ml of 3N NagCOa to the combined supernatant fluids. Allow mixture to stand for 5 min, with occasional stirring.
Centrifuge, test the supernatant fluid with Na2C03 for completeness of pre8.

cipitation,
9.

and discard the supernatant

fluid.

Dissolve carbonate precipitate in about 2 ml of 6N

HCl and

heat in a

water bath to evolve CO2. Add 6N NH4OH dropwise to neutralize the


sample (methyl orange end point). Dilute to 20-25 ml with water, add 10 ml
of ammonium acetate buffer, 1 ml of IN Na2Cr04, and mix well. Digest in a
water bath for 5 min, add 5 drops of 0.2N Ba(N03)2, and digest for another
1-2 min, with occasional stirring.
10. Filter through No. 42 Whatman filter paper and collect filtrate in a
150-ml beaker. Wash centrifuge tube twice with 5-ml portions of water and
use these washings to wash precipitate on the filter. Discard precipitate.
Adjust pH of filtrate to 8.5-9.0 with 6N NH4OH, using a pH meter, and
precipitate strontium with 3-5 ml of 3N NaaCOg. Centrifuge and discard the
supernatant fluid after testing it for completeness of precipitation with
Na2C03. Add 10 ml of wash water, centrifuge, and discard washings.
11. Dissolve precipitate in 2-3 ml of 6N HCl, warm and dilute to 20-25 ml

with water.

NH4OH

Add

1-2 drops of O.IN FeCla, heat in a water bath, add

6N

and digest for a few minutes. Filter through No. 42


Whatman filter paper and collect filtrate in a 150-ml beaker. Wash the centrifuge tube and the precipitate with 5 ml of water and discard the precipitate.
until basic,

NOTE THE TIME WHICH

IS

THE BEGINNING OF

INGROWTH.

ml of 3N Na2C03 to filtrate to precipitate strontium carbonate. Centrifuge, and discard supernatant fluid after testing it for completeness of precipitation with Na2C03. Wash the residue with 25 ml of water
containing a drop of 3N Na2C03. Centrifuge and discard the washing (total
strontium radioactivity may be determined at this time or as in 20.4(G.6).
Dissolve the residue with a few drops of 6N HCl, add 1.0 ml of yttrium
carrier and mix well. Set the solution aside for 2 weeks for ^Y ingrowth.
12.

(The

^"^Y

Add

ingrowth reaches

97%

of equilibrium in 2 weeks.)

G. Separation of ^^Y:
1. After 2 weeks, dilute the solution, obtained in 20.5(F.12), to 20 ml.
Add 1-2 drops of phenolphthalein indicator and 6N NH4OH dropwise to the

20.5

TCA: ^Sr and

*Sr

301

indicator end point. Digest in a hot water bath for 5 min, cool in an ice bath
to

below room temperature, centrifuge, and decant the supernatant

fluid into

a 150-ml beaker for total radiostrontium determination [20.5(G.6)].

CORD DATE AND TIME AS THE BEGINNING OF '^"Y DECAY.

RE-

Contin-

ue treatment of yttrium residue.


2. Dissolve yttrium residue

in 1 ml of 6N HCl and dilute to 20 ml with


drop of phenolphthalein indicator and 6N NH4OH dropwise to
the indicator endpoint. Digest in a hot water bath for 5 min, cool in an ice
bath to below room temperature, centrifuge, and combine the supernatant
fluid with that saved from Section 20.5(G.l) preceding.
3. Repeat 20.5(G.2) preceding.
4. Dissolve yttrium residue with 1 ml of 6N HCl and dilute to 20 ml with
water. Add 5 ml of 2N H2C2O4 and adjust pH to 1.5 with 6N NH4OH, using a
pH meter. Digest in a hot water bath for 10 min, cool in an ice bath to below
room temperature, centrifuge, and discard the supernatant fluid. Wash the
residue twice by dispersing it in 20-ml portions of hot water. Cool to below
room temperature, centrifuge, and discard the supernatant fluids. Transfer
the yttrium oxalate residue quantitatively to a tared stainless steel planchet
and disperse it uniformly. Dry under an infrared lamp to constant weight
(about 30 min of drying should suffice). Count ^Y activity in a thin endwindow or low-background anticoincidence beta counter. Repeat count
3 days and 5 days after precipitation to confirm purity of ^"^Y by its rate of

water.

decay.
5.

Add

RECORD TIME AND DATE OF EACH COUNT.


An

alternate procedure for mounting yttrium oxalate for counting

is

given in Section 20.5(G.7) below.


6. Determine total radiostrontium by evaporating supernatant fluids
from 20.5(G. 1-3) to 20 to 25 ml. Add 6N NH4OH dropwise to adjust the pH to
8.5-9.0 and then add 5 ml of 3N NagCOg. Allow sample to stand for 5 min,
with occasional stirring. Centrifuge and discard the supernatant fluid. Wash
the residue twice, each time by adding 20 ml of water, centrifuging, and

discarding the supernatant fluids. Transfer the residue to a tared, stainless


steel planchet,
tivity

dry under an infrared lamp, cool, weigh, and count beta ac-

of SrCOg.

7.

An

RECORD DATE AND TIME OF COUNTING.

alternate procedure for mounting precipitates of yttrium oxalate

or strontium carbonate for counting follows: Place a 2.8-cm glass fiber filter
paper on a stainless steel planchet and weigh. Transfer filter paper to filter
holder of filtration assembly (Fig 20:2). Assemble filtration apparatus. Transfer yttrium oxalate or strontium carbonate precipitate quantitatively to filter
funnel, using water. Filter the clear liquid, then transfer the slurry of residue. Allow gravity settling before using a minimum of vacuum. Increase
vacuum as filtration rate decreases. Wash residue three times with 10-ml
portions of warm water and three 10-ml portions of 95% ethyl alcohol. Continue vacuum for an additional 5 minutes. Remove filter paper carefully and
place it on the original stainless steel planchet. Dry to constant weight in an
oven at 110 C. Weigh to determine yield of yttrium oxalate as compared to

RADIONUCLIDES

302

Standardization of carrier yttrium, or of strontium carbonate as


carrier strontium standardization.
Remove filter paper from planchet and place

covering

it

IN

compared

on top of a nylon

it

MILK
to

disc,

with a piece of Mylar film (Fig. 20:3). Place nylon ring over Mylar

and snugly fit it over the nylon disc. Cut off excess Mylar film. Count
sample in a low-background beta counter.
8. An alternate procedure to recover strontium so it can be determined
by flame spectrometry^^ consists of dissolving the residue from Step
20.5(0.6) in 6N HCl and diluting to 500 ml with water in a volumetric flask.
film

H. Calculations:

The

1.

efficiency, E, of a beta counter

try, G; radiation backscatter,

sample

a function of counter

is

E = GBT. The observed

solids, T; i.e.,

geome-

B; and transmission of radiation through

verted to pCi by dividing by 2.22 ER, where

recovery of carrier and by definition

counting rate (cpm)

is

is

con-

the fractional chemical

pCi has 2.22 disintegrations per min-

ute.
2.

^"Y has a half

served activity

life

at time,

t,

of 64.2 hr and

A =
Ao =
t

=
=

^''Sr

from

^Sr

by the relationship:

Aoe-^'

observed activity,
activity at time of separation,

between time of separation of ***' and counting, and


the decay constant and is equal to 0.693 divided by the half-life
Qf 90Y xhe counting rate Aq is converted to absolute units by
correcting for the loss of both Sr and Y carriers and by use of
interval

formulae
3.

necessary to correct the ob-

is

to time of separation

A =
where

it

in

20.5(H.l) above.

activity, pCi/liter

^^^ ~ ^

RsR,E,DvI,V
where cpm = net beta count

(J-

Cpm^

+ hkgr

rate of

'^^'Y,

see 20.5(G.4 or

5),

Cpm^kgr

t.s

Rs =
Ry =
Ey =
Dy =

bkgr

thkgr

recovery of Sr carrier, decimal fraction,


recovery of Y carrier, decimal fraction,
counter efficiency for ""Y as yttrium oxalate, cpm/pCi,
decay correction factor e"^' for ""Y, where t is the time of
separating '"'Y from Sr, [20.5(G.l)] above to the time of
counting I20.5(G.4,5)J,

ly

= ingrowth

- e"''' for degree of equilibrium


ingrowth period, where t is time from

correction factor

attained during the

'"'Y

20.5

TCA:

^Ssr

and ^"Sr

303

the start of the ingrowth period [20.5(F.ll)] to time of sepa-

from
sample volume

rating "Y

V =
4.

^''Sr

Sr, [20.5(G.l)],

^P"^^" - Q(AsE. +

activity, pCi/liter

R.V

E.D.

where Es = counter
Ds

efficiency for ^^Sr as

= correction
from

sample collection

cpms

+ hkgr

Is

SrCOa, cpm/pCi,
where

factor e"^' for strontium-89 decay,

120.5(0.6 or

o-

and

in liters.

[20.5(E.l)]

to

time

E,I,)

t is

time

of counting,

7)],

^ Standard

'^P"^h'<gr

+ hkgr

deviation of net count.

t(5i4gr

= recovery of Sr carrier, decimal fraction,


cpm = net counts per minute of observed radiostrontium,
Rs

120.5(6.6 or

V =

7)],

sample volume

in liters

Cs = ''''Sr activity, pCi/liter,


As = absorption factor for ''^Sr

as strontium carbonate obtained

from a self-absorption calibration curve, (self-absorption


curves for ^^Sr and for ^"Sr are derived by precipitating a series of carrier strontium carbonate concentrations over the
expected "recovery range" in the presence of a constant
amount of *^^Sr and yttrium-free ^^Sr, respectively. The ordinate to the curve is the ratio of the count rate for each thickness to the count rate at zero sample thickness, and the abcissa is sample weight for the given type of sample mount),
Es = counter efficiency for ^"^Sr as SrCOa, cpm/pCi,
Ey = counter efficiency for ^''Y as yttrium oxalate, cpm/pCi, and
ly

correction factor

e~^' for

degree of equilibrium attained

during the ^^Y ingrowth period, where t


separated from SrCOs, 120.5(0.1)] to
120.5(0.6 or

/.

Precision

time the ^''Y was


of counting,

time

7)].

and accuracy:

In a collaborative study'^ of

known

is

two

sets of paired milk samples, containing

additons of radionuclides, five laboratories determined

*^^Sr

and

^"Sr.

The results of **^Sr from one laboratory were rejected as an outlier.


The average recoveries of added "^Sr (68 to 482 pCi/liter) from the four
samples were 87, 96, 89, and 87%. For the two sets of paired samples, accuracy of the method was 12 and 14%, and the precision (random error) was
4 and 23%. The method was biased on the low side but it was not considered
serious.

RADIONUCLIDES

304

IN

MILK

The average recoveries of added *"^Sr (50.2 to 1 16.2 pCi/liter) from the four
samples were 82, 83, 88, and 88%. For the two sets of paired samples, accuracy of the method was 14 and 22%, and the precision (random error) was
2 to 22%. The method was biased on the low side, but not seriously.
20.6 Determination of ^^ii, i40Ba and i37Cs

in IVIilk

by

Gamma

Ray

Spectrometry
The

from presence of nuclear fission


animal forage and food are radiostrontium (^^Sr, ^"Sr), raradiobarium and daughter (^^''Ba + '^La) and radiocesium

principal radionuclides found in milk

products

in dairy

dioiodine C^'I),

('"Cs). Naturally occurring radioactive potassium

(^*'K) is

also present. Oth-

er fallout radionuclides of moderately long half-life are rarely found in milk.

Except for radiostrontiums, these radionuclides emit gamma rays with photopeak energies sufficiently separated from each other to make gamma ray
spectrometry an attractive means of quantitative measurement.^' ^' ^' ^"
A. Principle of met hod:
A known volume of milk contained
tioned over and around a

in a vessel (Marinelli

beaker)

is

posi-

right cylindrical scintillation crystal detector (Nal)

gamma

Gamma

radiation is counted for a


photon energy ranges of gamma ray-emitting radionuclides '^4, '^Ba + '^"La, '^^Cs and ^K are separated from each other and from background radiation by simultaneous equa-

of a multichannel

spectrometer.

given time and accumulated pulses

tions (matrix system).**-

in selected

*^' ^"

The two requirements of

are (1) knowing which radiospectrum and (2) having available a radionuclide photopeak which is in a different energy region of the
total spectrum of radionuclides present. Analysis of milk samples for radionuclides is particularly suited to solution by simultaneous equations because

nuclides are or

may be

the matrix

present

biological discrimination of the

forage and feed has limited the

in

the

method

gamma

many fission radionuclides


number of gamma-emitting

present

in

dairy

radionuclides in

milk to radioiodine 131, radiobarium 140 and daughter radiolanthanum 140,

and radiocesium

137, with

potassium 40 appearing as a natural element.

B. Interference:

Any gamma

ray-emitting radionuclide, other than those under examinaor as a contaminant of the sample or sample vessel has

tion, present in milk

interference potential by contributing counts in one or more of the photopeak ranges used. Similarly, any gamma ray-emitting contaminants in reference solutions used to calibrate the spectrometer, or any mechanical, electrical

or electronic instability of

component

parts of the spectrometer, will

contribute errors to measurements.

few special instances, newly formed fission products may be present.


The most important of these are the short-lived radioiodines ''^^I and *^^I.
In a

20.6

Gamma

These

Ray Spectroscopy:

fission

products

'^M, ^"^Ba,

may cause

and "^Cs

interference either through direct over-

lapping of photopeaks or by contributing

such instances the interference

305

Compton-continuum counts.

may be minimized by

In

waiting for decay of

the short-lived radionuclides or

by additional counting following decay.

Chemical separation may also be

helpful.

The estimate of parent-daughter pairs such as '^''Ba and '^"La is often


based on an assumed equilibrium of parent and daughter. Such equilibrium
frequently does not exist; hence its assumption may cause errors. Only '''"Ba
is reported in this method, but '^"Ba and '^'^La are in equilibrium during calibrations.

Apparatus:
Counting instrument: Low-level gamma spectrometer consisting of a
shielded thallium-activated sodium iodide scintillation detector coupled to a
multichannel pulse-height analyzer and an appropriate readout system.^"
For routine analysis, a detector 3 in. high by 3 in. (7.62 cm x 7.6 cm) in diam
is reasonably satisfactory for a 2-liter sample, but greater precision may be
achieved with 4 x 4-in. (10.16 x 10.16-cm) detectors for larger samples.
Subsequent directions are made on the basis of a 4 x 4 in. (10.16 x 10.16
1

cm) detector system and a sample

size of 3.5 liters.


Vessel (Marinelli beakei^^): For the 4 x 4-in. (10.16 x 10.16-cm)
detectors, a beaker with a capacity of 3.5 liters and dimensions such as given
2.

in Fig.

20:4

is

recommended.

Alignment sources: Gamma ray energies, at least one near either end
of the spectrum of major interest, shall be used for alignment. The source or
sources should have well-known energies and an abundance of gamma rays
in the photopeaks for alignment. Solid sources, about 0. 1 fxC'i in amount, are
preferred over liquid sources. Bismuth 207 is a satisfactory single source
with two photopeaks; cesium 137 and cobalt 60 have proved to be a good
3.

pair of alignment sources.

D. Reagents:
1.

Carrier solutions of iodine (Nal), barium (BaCla), and cesium (CsCl):

water to provide 10 mg of carrier element per


in polyethylene or glass bottles.
2. Stock standard solutions of '^'I, '^Ba + '^"La, '^^Cs and natural potassium for spectrometer calibration. A suitable sample for calibration is
about 10,000 pCi.
3. Calibrating solutions: Add 3 to 5 ml of carrier solution, [20.6(D.l)],
to 3 liters of distilled water, mix, add a stock radionuclide standard, mix, adjust pH, and dilute to 3.5 liters. For the radioiodine solution, adjust pH to
Dissolve carrier
milliliter

salt in distilled

of solution. Store carriers

^^Marinelli beakers are available from suppliers of laboratory appliances

laboratory ware suppliers such as Bel-Art Products, Pequannock,

NJ

and various

07400.

plastic

RADIONUCLIDES

306

IN

MILK

Table 20:11. The Photopeak and Photon Energy Range of Radionuclides*


Photopeak,
^r
MeV

Photon Energy
r.
a^
Range, MeV

'=^4

0.364

0.32-0.40

'^Ba

0.49

0.46-0.54

''^^C

0.66

0.60-0.72

^K

1.46

1.40-1.52

..
...
Radionuchde

*Source: Hagee. Karches

&

Goldin.*

For cesium and barium solutions, adjust pH to 3.5-4.5. No pH adis necessary for the potassium 40 solution, which is a solution of
reagent-grade potassium chloride.

8.0-9.0.

justment

E. Calibration of gamma spectrometer

and examination of milk:


Using alignment sources centered on the detector, adjust the spectrometer to cover the range of at least between
and 2 MeV, in intervals
(channels) of 10 or 20 KeV. Adjust voltage or gain control so that the two
gamma photopeaks of the standard fall into their appropriate channels.
Check and adjust alignment daily.
1.

Transfer 3.5

2.

liters

of a calibrating solution, 120.6(0.3)] to a Marinelli

beaker, place beaker over detector, and count the standard for a time

(10-100 min) sufficient to reduce the counting error to about 1%.

tilled

Repeat
Repeat

procedure for each calibrating solution.


procedure for a beaker containing only 3.5
water, to obtain instrument background.

3.

4.

5.

this

Calibrate

quently
6.

this

if

gamma

gamma-ray

of dis-

spectrometer, [20.6(E.2-5)], yearly, or more

fre-

resolution changes.

Test milk samples by transferring 3.5

has been adjusted to

liters

room temperature

liters

of well-mixed milk which

into a Marinelli beaker, place the

beaker over detector, and count for 100 min, or a time

sufficient to give the

desired counting statistics. Read the counting data.


F. Calculations:
1. Counter efficiency: Total individual counts observed in channels of
photopeak range for a calibrating solution, such as shown, for example,
in Table 20:11, and subtract the total background count for the same photopeak range. Divide residual or net count by time of counting in minutes and
amount of radionuclide in picocuries and record the cpm per pCi of radio-

the

'^

nuclide.
2. Interference coefficients: On reviewing individual spectra of radionuclides'"- ^" in relation to photon energy ranges selected for counting as

listed in

Table 20:11,

it

becomes

clear that potassium 40 in a sample will

20.6

Gamma

Ray Spectroscopy:

^^ii,

iMga, and ^"Cs

contribute a counting rate to each of the other photon energy ranges.

307

It is

cesium 137 and iodine 131 will contribute a counting rate to all
photon energy ranges except potassium 40, and that the barium-lanthanum
140 combination will, like potassium 40, contribute a counting rate to all
photon energy ranges listed. When counting a standard solution of only potassium 40, the ratio of net counting rate in each of the photon energy ranges
for the other radionuclides to the net counting rate in the potassium photon
energy range gives the fractional interference coefficients for the respective
energy ranges due to potassium 40. Similarly, count data of each standard
solution of iodine 131, cesium 137, and barium-lanthanum 140 are obtained
and are used to determine the numerical value of fractional interference coefficients for each radionuclide in each of the four photon energy ranges. The
value of this coefficient for a radionuclide in its selected photon energy range
is unity. The 0.49 MeV photopeak of '^"Ba has proved to be satisfactory,
principally due to the higher counting efficiency obtained. (See Table 20:11)
Designate counting rates for iodine 131, barium 40, cesium 137 and potassium 40 by the symbols I, B, C and K, respectively, and the net counting
rates (observed less background counting rate) in their respective photon
energy ranges by the symbols Ni, N,, and N^. and Nk, respectively. Then, if
also clear that

the value of

f,

the fractional coefficients or contributions of a nuclide in a

is designated by a pair of lower case subscripts, the first of


which designates the nuclide photon energy range (column) and the second

particular range,

the nuclide (row), the following four equations

=
Nh =
N, =
Nk =

Ni

a.

b.
c.

d.

-f

fu,

fi,

f,k

fi

+
+
+

C + fkiK
C + fkK
B +
C + fkc-K
B + f,k C +
K

B +

fei

B +
f,

fhk

f,h

can be simultaneously solved by matrix algebra, using inversion--' to provide numerical values for constants W, X, Y and Z in equations e, f, g, and
h, below.

These new values are used

to

determine individual concentrations of four

an unknown milk sample.


The net counting rate for each radionuclide is

or fewer of these nuclides

e.
f.

g.

h.

in

= I = WjNi + W.N,, + W3N, + W4Nk


= B = XjNi + X;n, + X^N, + X^Nk
137CS = c = YjNi + Y2N,, + Y3N, + Y,Nk
^"K = K = Z,Ni + Z.Nh + ZgNe + Z4Nk
'3'I

'^oBa

Calibration of an instrument to derive numerical values for constants in


e, f, g, and h is applicable as long as instrument alignment and
of operation remain constant and gamma-emitting nuclides are limited

equations

mode

to the four

elements

in

the matrix.

The long-hand inversion of a 4 x 4 matrix


It is therefore recommended

tends to be tedious and easily subject to errors.

RADIONUCLIDES

308

that a

computer be used

through

h.

ming counts

in

MILK

to provide numerical constants for equations e

Thereafter, desk calculations to determine

unknown samples can be

IN

done

readily

in

''"I.

the absence of a

'^"Ba and '''^Cs in


computer by sum-

each of the photopeaks. subtracting background, and apply-

ing equations e through h.

Milk sample activity: From spectral gamma counts of a milk sample


[20.6(E.6)], substitute net values in equations e through h and convert the
net counts per minute for each radionuclide to picocuries per liter of milk at
3.

the time of counting, using the formula:


pCi/liter

net

cpm/EV

where E = counting efficiency per picocurie from standard solutions, and

V = volume

of sample,

in liters.

''"1
and '^"Ba have a half life of 8.04
observed
values
and
must be corrected for decay to
some original time (sampling or production) by the relationship:

Correction for decay: Because

4.

12.8 days, respectively,

A =
where

A -

the activity remaining after time,

A,)= activity at
t

=
=

Ae-^'

some

interval of time
(the

radionuclide.

t,

original time,

between

decay constant)

The time and

A,,

and A, and

=-^

the half

where T,
life

must be

.,

in

the half

is

the

same

life

of the

units, ie,

seconds, days, years, etc.


G. Precision and accuracy:
In a collaborative study of

two milk samples, containing known amounts


''"I, ''"Cs, and '^"Ba in

of added radionuclides, 25 laboratories determined


triplicate.''*

The average recoveries of added '"! (98 and 633 pCi/liter) for the two
samples were 101 and 100.3%. The precision (random error) of the method
was 5.4 and 9.0% for the two samples. The method was considered satisfactory for the analysis of

'^'I.

The average recoveries of added ''^^Cs (52 and 305 pCi/liter) for the two
samples were 101.9 and 97.3%. The precision (random error) of the method
was 5.9 and 10.2%. For the sample containing 305 pCi/liter, the method had
a slight bias on the low side, but it was considered not significant.
The average recoveries of added '^"Ba (72 and 515 pCi/liter) for the two
samples were 105.6 and 101.6%. The precision (random error) of the method
was 17.5 and 7.5%. The method was biased on the low side but it was considered not serious.
In all instances, there

was

a greater dispersion of results in the sample

containing low level radioactivity but this was considered as normal.

20.7 References

References

20.7.
1.

American Public Health Association. 1971. Standard Methods for the Examination of
Water and Wastewater, 13th ed. Part 300 (Radioactivity). American Public Health Association,

2.

309

New

Ayers,

York.

Theory and Problems of Matrices. Schaum's Outline

F., Jr. 1962.

Publishing Co., 257 Park Av.,


3.

1,

USDHEW,

Baratta,

PHS, BRH, Analytical Quality Control

Service, 109 Holton Street, Winches-

E. 1974. Ion exchange determination of strontium 89 and strontium 90 in milk:

Crouthmel,

New
6.

Schaum

Mass. 01890.

Collaborative study.
5.

Series,

York.

Baratta, E., and F.E. Knowles. 1968. Collaborative study on the ion exchange and TCA
methods for the analysis of strontium 89 and strontium 90 in milk. Tech. Exper. 68-MKAPter,

4.

New

J.

Assoc. Off. Anal. Chem. 57:37.

Gamma-Ray

C.E., Ed. 1960. Applied

Spectrometry, Vol

2.

Pergamon

Press,

York.

FAO/WHO

Expert Committee. 1959. Methods of radiochemical analyses. Tech. Report


No. 173. World Health Organization, Geneva.
Federal Radiation Council. 1960. Background material for the development of radia-

Series
7.

tion protection standards. Staff

ton,
8.

DC

Federal Radlation Council.


tion protection standards. Staff

ton,
9.

DC

Heath, R.L.
2.

11.

13.

for the

development of

radia-

G.J. and A.S.

gamma

Goldin.

1960. Determination of

'^'I,

'"Cs, and

spectrometry. Talanta 5:36.

1964. Scintillation spectrometry

gamma

ray spectrum. IDO-16880, Vols

&

Tech. Information Div., Atomic Energy Commission, Washington, D.C.

Common

Interlaboratory Technical Advisory Committee.

1968.

struments for measurement of radioactivity. Report No.

Bureau of Radiological Health,

USDHEW, PHS
12.

Background material

1961.

Report #2. Supt. of Documents, Govt. Ptg. Off., Washing-

20402.

Hagee, G.R., Karches,


''"Ba in fluid milk by

10.

Report #1. Supt. of Documents, Govt. Ptg. Off., Washing-

20402.

2,

laboratory

in-

Pub. No. 999-RH-32.

Kahn, B. 1965. Determination of picocurie concentration


Food Chem. 13:21-24.
Knowles, F.K., Underwood, R.J., and E.J. Baratta.
analysis of radionuclides by

gamma

of iodine-131

in

milk.

J.

Agr.

1969. Collaborative study for


spectroscopy using simultaneous equations. Tech Ex-

USDHEW,

PHS, BRH, Analytical Quality Control Service, 109 Holton


Mass 01890.
NBS Handbook 69. 1963. Maximum Permissible Body Burdens and Maximum Permissible
Concentrations of Radionuclides in Air and in Water for Occupational Exposure. U.S.
Department of Commerce, Washington, D.C.
MuRTHY, G.K. and J.E. Campbell. 1959. A method for the rapid ashing of milk for radioper

69-MKAP-l,

Street, Winchester,

14.

15.

nuclide analysis.
16.

ashing
17.

J.

Dairy Sci. 42:1288-1293.

MuRTHY, G.K., CoAKLEY,


in

J.E., and J.E.

strontium 90 determination

MuRTHY, G.K., Jarnagin,


of radionuclides in milk ash.

18.

19.

Murthy, G.K. and

J.E.

protein-bound iodine

in

MuRTHY, G.K.
formaldehyde.

20.

J.

Campbell.

L.P., and A.S.


J.

1960.

A method

for the elimination of

Dairy Sci. 43:151-154.

in milk. J.

Goldin.

1959.

A method

for the determination

Dairy Sci. 42:1276-1287.

Campbell.

1966. In vitro transformation of inorganic iodine to

milk by formaldehyde.

J.

Dairy Sci. 49:1190-1196.

1969. Status of iodine in milk preserved with

sodium metabisulphite and

Dairy Sci. 52:124-125.

National Center for Radiological Health.


environmental samples.

USPHS Pub

Jan. 1967. Radioassay procedures for


No. 999-RH-27. Supt. of Documents, Govt. Ptg. OflF.,

Washington, D.C. 20402.


21.

Parker, W.V., and J.C. Eaves.

1960. Matrices.

The Ronald Press Co.,

New

York.

20.7

310
22.

REFERENCES

Porter, C.R., Augustine, R.J., Matusek, J.M., Jr.. and M.W. Carter. 1965. Procedures for the determination of stable elements and radionuclides in environmental samples.
USPHS Pub. No. 99-RH-lO. Supt. of Documents, Govt. Ptg. Off., Washington, D.C.
20402.

23.

24.

25.

26.

Porter, C.R., and B. Kahn. 1964. Improved determination of strontium 90 in milk by ion
exchange method. Anal. Chem. 36:676.
Sunderman, D.N., and C.W. Townley. 1960. The radiochemistry of barium, calcium and
strontium. NAS-NS 3010, pp 5 & 8. National Academy of Sciences, Printing and Publishing
Office. 2101 Constitution Avenue, Washington, D.C. 20418.

United States Atomic Energy Commission Health & Safety Laboratory. 1957.
Manual of Standard Procedures. Report No. NYO-4700. AEC, Washington, D.C.
WiLLARD, H.H. and E.W. Goodspeed. 1936. Separation of strontium, barium, and lead
from calcium and other metals by precipitation as nitrates. Ind. Eng. Chem., Anal ed,
8:414-418.

CHAPTER

21

ALTERNATE VIABLE COUNT METHODS*


A.R. Brazis, H. Behney, W.S. LaGrange

andD.Y.C. Fung

Introduction

21.1

Standard Plate Counts to determine viable bacteria in dairy products,


while relatively accurate, are expensive and time-consuming. Direct microscopic counts and reduction tests of raw milk are not sufficiently accurate.
The following methods have merit as alternative tests for determining viable
bacterial counts.

The oval tube count (OTC) and

plate loop count

(PLC) procedures

are

applicable specifically for examination of raw milk. Although both are single

loop measurements (0.001 ml), the limited growth area of


oval tube limits precision

when more than

medium

in

each

100 colonies develop while plate

loop counts are satisfactorily precise within the range of 30-300 colonies.
Limitations of the plate loop method,

been

when counts exceed 200,000/ml have

reported.*^

The

spiral plate

counting procedure (SPLPC)

is

applicable to raw milk,

pasteurized milk and milk products, and has been applied to other foods.

The SPLPC

is

a mechanical

method which requires

manpower,
Count (SPC) pro-

less skilled

time, equipment, and space than does the Standard Plate

cedure.

21

.2

Collection and Storage of

Collect and store samples


3.23, 3.24

21

.3

and

in

Samples Before Testing

accordance with the procedures specified

in

5.5.

Statistical Protocol for

an Analyst

Before any alternate viable count method is used, the individual analyst
should test his ability to perform that method against the SPC. The alternate

ISC Liaison: G.H. Richardson


*The Chapter Committee wishes to acknowledge, with thani<s, the assistance of J.E.
Campbell, Jr. and J.E. Gilchrist. Bureau of Foods. Food and Drug Administration, Cincinnati,
Ohio, in the preparation and comprehensive review of the chapter manuscript.
311

ALTERNATE VIABLE COUNT METHODS

312

method and the SPC should be done on the same samples of


The following protocol is recommended for the single analyst to eval-

viable count
milk.

uate his performance:


in duplicate by both the alternate method and the SPC
samples give plate counts of from 30 to 300 colonies by the SPC and
the PLC methods, or 30-100 colonies by the OTC method. Dependent on the
bacterial count range per ml, a count of 20-75 colonies in a segment or
wedge, or greater than 20 colonies on the total plate surface, [21.6(1)], can be
used for the SPLPC method. All samples do not have to be plated on the
same day: however, all preparations (both alternate and SPC methods) on
one sample should be done at the same time. Duplicate counts from the

Samples are plated

until 25

alternate technique are converted to

log,,,

counts: the difference between

squared and the squared values are added


for all samples. In addition, the logi,, counts are added together and divided
by 50 to obtain the mean logio count for each alternate count method. The
SPC results are tabulated and analyzed in the same manner.
For each alternate method to be satisfactory, the mean logio count for the
login

counts for duplicate plates

is

50 observations for the alternate method must not differ from the same value
for the SPC by more than 0.036. For satisfactory duplicate data, the sum of
the squared differences (log variances)

SPC methods,

alternate and

between duplicate samples

in Table 21:1 showing


determine compliance with the two criteria.

Examples of data are included


to

21.4 Oval

for both

divided by 50 should not be greater than 0.005.

how

to evaluate results

Tube Method ^

A. Equipment and supplies:


1. Oval tube: Round neck, plugged with cotton or preferably capped
with stainless

steel closures (Bellco B-16);

modified form, with 19.1

-mm

bulb dimensions, 100 x 17 x 27

neck (Fisher

Scientific

mm,

Co. No. 14-928).

Water bath: See 5.2(L).


Loop, 0.001 ml: Preferably welded, calibrated to contain 0.001 ml,
made of B & S gauge No. 26 platinum-rhodium (3.5%) or platinum-iridium
(15%) wire, true-circle loop, ID 1.45 mm 0.06 mm, attached to a 7. 62 -cm
length of wire (Scientific Products N-2075-2) and mounted in a suitable
holder. (Loops may be reshaped by being fitted over the top of an awl, then
checked over a No. 54 twist drill. Loops of proper size should not fit over a
No. 53 twist drill.)
2.

3.

Racks: Noncorrosive wire, for holding oval tubes during sterilizainoculation and incubation. Size for oval tubes, 40 or 50
spaces, 2.43 x 3.175 cm mesh; and two shelves, the top shelf to have a
4.

tion, cooling,

1.25-cm supports on one side (long dimension)


placed on

its

to

tilt

the rack slightly

side during incubation; or other suitable racks.

when

21.3 Statistical

e
3
o

U
>

S
u

S
3
O

o
o

Comparison Table

313

ALTERNATE VIABLE COUNT METHODS

314

5.

Containers: Sample, wide mouth, otherwise same as 3.11(C).

6.

Others: Colony counter, incubator, incubator room,

age space, refrigerator, thermometer,

tally,

work

area, stor-

autoclave, hot-air sterilizing ov-

en; See 5.2.

B. Materials:

See 4.9(F), 5.3(A,B).

C. Procedure:
1.

Shake each raw milk sample thoroughly 25 times through a 30.5-cm

arc in 7 seconds. Flame-sterilize the 0.001-ml standard loop and allow

it

to

cool. Transfer a loopful of milk to an oval tube containing 4 ml of sterile

melted agar, tempered to 44-46 C, being careful to dip the standard loop
below the surface of the milk, in an area as free from foam as
only 2-3

mm

Hold the plane of the loop in a vertical position when withdrawing


from milk and move the loop back and forth several times through the agar
to insure removal of all milk from the loop.
2. Replace the closure, mix agar and milk thoroughly by swinging the
tube back and forth rapidly through a small arc for 5 sec (about 25 complete
excursions), and lay the oval tube flat on the table or, preferably, slant it
possible.
it

slightly. After the

agar has solidified, incubate oval tubes horizontally

wire rack (with agar adhering to upper side of the container) for 48
32

in

3 hr at

C.

D. Examination and reporting results:

Count colonies by placing the tube on a colony counter. Record colony


counts from tubes having 30-100 colonies [unless excluded by 5. 1 1(1)], compute the number per milliliter, and record the results as "Oval Tube Count
per milliliter" (OTC/ml). If an oval tube yields fewer than 30 colonies, record the actual number of colonies and record the count as "Estimated Oval

Tube Count per

milliliter"

(EOTC/ml).

If

a tube prepared from any sample

has no colonies and inhibitory substances have not been detected, record the

count as "less than 1,000 EOTC/ml". If a tube has a colony count in excess
of 100 colonies, record the count as "> 100,000 EOTC/ml".
If spreaders are detected, proceed as described in 5.11(1.1,2). Where
tubes prepared from samples 1.) have excessive spreader growth, see
5.1 1(1.2); or 2.) are known to be contaminated, mislabeled or are otherwise
unsatisfactory, record as "Spreaders" (Spr) or "Laboratory Accident"
(L.A.). If inhibitory substances have been detected (Chapter 9), record the
OTC as "Growth inhibitors" (G.I.). Do not record the count/milliliter on
such samples.

21.5 Plate

Loop Method

21.5 Plate

315

Loop Method ^

A Equipment and supplies:


.

Total equipment assembly cannot be purchased as a unit, but can be

made

up from the following components:


1.
Loop, 0.001 ml: Same as 21.4(A.3). Make approximately a 30 bend
about 3-4 mm from the loop, with the loop opening toward the hub. Kink the
opposite end of the wire in several places.
2. Luer-Lok hypodermic needle: 13-gauge (sawed oflF 24-36 mm from
the point where the barrel enters the hub). Insert the kinked end of the
wire shank into the sawed-off needle to a point where the bend is about
12-14 mm from the end of the barrel.
3. Cornwall continuous pipetting outfit: Becton, Dickinson & Co. No.
1251 (consisting of a metal pipetting holder, a Cornwall Luer-Lok syringe,
and a filling outfit), 2-ml capacity. Adjust to deliver 1.0 ml. Rubber tubing,
0.42 X 0.21 cm, attached to the syringe and extending into a capped dilution
blank should be of suflficient length to facilitate the sampling process,
(Fig. 21:1). Repeated autoclaving will cause the rubber tubing to deteriorate;
therefore, additional tubing should be kept on hand. Optionally, attach rubber tubing to glass tubing, which extends through the plastic cap, above dilution bottle approximately 5.08 cm and into the dilution blank about 1.27 cm
from the bottom of the bottle. A rubber grommet should encircle the glass
tubing at the junction with the plastic cap to prevent contamination.
Attach the Luer-Lok hub to the Luer-Lok fitting. The apparatus and other parts should be assembled and sterilized by autoclaving at 121 C for
15 min or boil in water for 10 minutes.
4. Container, sample, wide mouth, otherwise same as 3.11 C.
5. Cover, protective glass test tube for 0.001-ml loop during sterilization and handling.
6. Other equipment and supplies, see 5.2.
B. Materials:

See

5.3.

Procedure:
After sterilization of the assembled pipetting outfit, allow equipment

1.

Depress syringe plunger rapidly several times to pump dilution wa(which has previously been adjusted to deliver 1 ml
with each depression of the plunger).
2. Before initial transfer is made in examining a series of samples, briefto cool.

ter into glass syringe

in a clean, high-temperature gas flame) and


more. Discharge several 1-ml portions of water to
waste; then discharge 1-ml portion of water into first petri dish. Label this
dish "sterile instrument control". Shake samples, see 5.6(B); carefully dip

ly

flame the loop (preferably

allow

it

to cool 15 sec or

ALTERNATE VIABLE COUNT METHODS

316

Figure 21:1. Plate loop

outfit,

showing

vertical

the loop into the sample (avoiding foam) as far as the

removal of loop

bend in the shank (the


bend serves as a graduation mark and also permits vertical removal of the
loop). To measure the sample, insert the loop vertically into the sample three
times, moving the loop with a uniform up-down movement over a distance of
about 2.54 cm. Avoid dilution water droplets that may rinse oflF the loop.
Each downward and upward cycle should be at a rate of about 55-60 beats
per minute. A metronome or watch may be used to establish uniform timing.

21.6 Spiral Plate Count

Method

317

The speed of removal of the loop from the surface of the milk sample affects
accuracy of the measurement. Removing the loop slowly causes less than
0.001 ml to adhere; jerking the loop out rapidly causes more than 0.001 ml to
adhere. It is necessary to use wide mouth sample bottles and good illuminaremoval of the loop during inoculation of plates.
Raise the cover of a sterile petri dish, insert loop and depress the
plunger causing 1 ml of sterile dilution water to flow across the charged loop
tion to facilitate
3.

and thus washing a measured 0.001 ml of sample into the dish.


CAUTION: Do not depress the plunger so rapidly that water fails to follow
the shank and flow across the loop.
4. Normally, the residue remaining on the loop after discharging the
sample is not significant. However, small imperfections in the welding of the
loop or in smoothness of the metal surface may lead to incomplete rinsing.
This possibility of significant residue retention should be determined for
each loop by doing a series of control plates, a minimum of at least one for
every 20 samples plated. If the loop is determined to be free-rinsing, no
flaming between samples is necessary.

Pour plates with

5.

32

ml of agar and incubate 48

12-15

hr at

C.

D. Examination and reporting counts:


Count plates, unless excluded by 5.11(1), with colony counter, having 30
to 500 colonies on each plate and record Plate Loop Count per milliliter
(PLC/ml) results as 1,000 times the number of colonies per plate. If plates
have fewer than 30 colonies each, record actual number of colonies, multiply
by 1,000 and record count as "Estimated Plate Loop Count per milliliter"
(EPLC/ml). If plates have no colonies, and inhibitory substances have not
been detected, record count as < 1,000 EPLC/ml. If plates have greater than
300 colonies, proceed as described in 5.11(H), except record results as

EPLC/ml.
21.6 Spiral Plate Count

Method ^^^

A. Equipment and supplies:

\ adjusted to deliver a total sample volume of 0.035 ml.


Colony Counter with special grid for relating deposited
sample volumes to specific portions of petri dishes.
1

Spiral Plater

2.

Spiral

3.

Vacuum

act as a

vacuum

'

vacuum bottle of 2-4 liters


vacuum source of 50-60 cm of Hg).

trap for disposal of liquids (a

reservoir and a

4.

Disposable micro beakers,

5.

Petri dishes, plastic or glass, 150

^Available
land 20760.

to

5 ml.

x 15

mm.

Spiral Systems Marketing, 1200 Quince Orchard Boulevard, Gaithersburg, Mary-

ALTERNATE VIABLE COUNT METHODS

318
6.

Standard Methods Agar, see 4.9(F).

7.

Calculator, optional, inexpensive electronic hand calculator recom-

mended.
Polyethylene bags, about 30 cm x 20 cm x 40 cm.
solution,
hypochlorite
sodium
Commercial

8.

9.

5% NaOCl
10.

Sterile dilution water.

11.

Syringe, with Luer tip, capacity not critical.


Other equipment and supplies, see 5.2.

12.

approximately

(bleach).

B. Preparation of agar plates:

An

automatic dispenser with a

sterile

delivery system

is

recommended

prepare agar plates. The agar volume dispensed into plates

is

to

reproducible

and the contamination rate is low compared to hand pouring of agar in an


open laboratory. When possible, a laminar air flow hood should be used in
conjunction with an automated dispenser.
The same quantity of agar should be poured into all plates so that the same
height of agar will be presented to the Spiral Plater stylus tip to maintain

contact angle. The agar plates should be level during cooling.


is

a suggested
1.

The following

plates:

Use an automatic dispenser or pour a constant amount (between

40-45 ml) of
2.

method of prepouring agar

sterile agar, at

Allow agar

60-70 C, into each

to solidify

on a

petri dish.

level surface with

poured plates stacked

no higher than ten dishes.


3.

Place the solidified agar plates in polyethylene bags, close with ties

or heat-sealer, and store inverted at 0-4.4 C.


4.

Bring prepoured plates to

C. Preparation of samples:
Shake samples as described

room temperature before

in 5.6

inoculation.

and 21.4(C).

D. Description of spiral plater:

Count (SPLPC) plater (Fig. 21:2) inoculates the surface of


measurement of numbers of bacteria in solucontaining between 500 and 500,000 bacteria per ml. An operator with

The

Spiral Plate

a prepared agar plate to permit


tions

minimum

of training can inoculate 50 plates per hour. Within the range

stated, dilution bottles, or pipets

required. Required bench space

is

and other auxiliary equipment are not


minimal, and time to check instrument

is less than two minutes.


The plater deposits a decreasing amount of sample in the form of an Archimedean spiral on the surface of a prepoured agar plate. The volume of
sample on any portion of the plate is known. After incubation, colonies ap-

alignment

pear along the

line

If colonies on a portion of the


spaced from each other, they are counted on a special

of the spiral (Fig. 21:3).

plate are sufficiently

21.6 Spiral Plate Count

Method

319

ul.L!l|liP!|ll|p

Figure 21:2. Spiral plater dispensing crystal violet dye onto surface of

15-cm agar

plate.

which associates a calibrated volume with each area. The


is estimated by dividing the number of
a defined area by the volume contained in the same area. Studies

grid (Fig. 21:4)

number of
colonies

in

bacteria in the sample

have shown the method proficient not only with milk analysis but also with
other foods.'E. Plating procedure:

Check

the stylus tip angle daily and adjust,

hold a microscope cover


slip

plane

is

parallel at

slip against the

about

necessary. (Use

vacuum

tip; if

tip is

moved through

to

the cover

mm from the surface of the platform, the tip

properly oriented.) Liquids are


Cleanliness of the stylus

if

face of the stylus

is

vacuum.
sec using sodium

the system by

assured by a rinse for

hypochlorite solution followed by sterile dilution water for

sec before

ALTERNATE VIABLE COUNT METHODS

320

Figure 21:3. Spiral plates representing 500 to 500,000 Escherichia coli/ml

and estimate counts of 5 and 50

million.

sample introduction. This rinse procedure between processing of each


sample minimizes cross contamination.
After rinsing, the sample is drawn into the tip of the Teflon tubing by the
vacuum applied to the two-way valve. When tubing and syringe are filled
with sample, the valve attached to the syringe

is

closed.

An

agar plate

is

placed on the platform, the stylus

tip placed on the agar surface, and the


During inoculation the petri plate lid may be labeled. After
the agar has been inoculated, the stylus lifts from the agar surface, and the
spiral plater automatically stops. The inoculated plate is removed from the
platform and covered. The stylus is moved back to the starting position. The
system is vacuum-rinsed with hypochlorite, water, then a new sample introduced. Invert plates and promptly place in an incubator for 48 3 hr at

motor

32

started.

C.

F. Sterility controls:

Check

sterility

of the spiral plater, for each series of samples, by plating

sterile dilution water.

21.6 Spiral Plate Count

Method

Figure 21:4. Spiral plate inoculated with Bacillus

321

subtilis

77,000/ml.

Two

segments of opposite wedges are outlined.

G. Cautions:

Prepoured plates should not:


1. Be contaminated by a surface colony.
2. Be below room temperature; condensed water welling up from agar
can occur.
3. Be excessively dry as indicated by large wrinkles or excessive glazed
appearance.
4. Have water droplets on surface of agar.
5. Have differences greater than 2 mm in depth of agar.
6. Be stored at 0-4.4 C for longer than one month.
Reduced flow rate through tubing indicates obstructions or material in system. Obstructions may be cleared by removing valve from syringe, inserting

322

ALTERNATE VIABLE COUNT METHODS


Table 21:11. Determination of Volume Constants for

150-mm Plates

Method

21.6 Spiral Plate Count

323

spirally plated bacterial concentrations, will indicate

how much volume was

deposited on that particular grid area. The following formula should be used:

,,

Volume
,

r,

(ml) tor grid area

Spiral colonies

counted

in

area

Bacterial count/ml (SPC)

If

the

SPC is not used, a count between 50 and 200 colonies on the whole
may be used to determine the concentration of the plated bacte-

spiral plate

suspension; this can be done by dividing the colonies counted by 0.035 (ml).

rial

The concentration of

the other 10 dilutions of plated suspension

may be

calculated by using the appropriate dilution factor.

One example
given

in

Table

of the calculation required to determine the area volume

is

21:11.

Examination and reporting spiral plate counts:


Counting rule of 20: After incubation, the spiral plate is centered
over the grid by adjusting the holding arms on the viewer. Choose any wedge
and begin counting colonies from the outer edge of the first segment toward
the center until 20 colonies have been counted. The count is completed by
counting the remainder of the colonies observed in the segment in which the
20th colony occurs. This number is recorded along with the number of the
segment marked on Fig. 21:4 as 1,2, 3, and 4, that included the 20th colony:
/.

1.

in this
all

counting procedure, the preceding numbers refer to the total area

the segments

from the outer edge of a wedge

illustrated in Fig. 21:4.

Any count

controlled by counting the


ing results. If there

wedge, then

number of

all

If

the

in the

sample composition are

opposite wedge and record-

not a total of 20 colonies in the four segments of the

of the colonies on the whole plate should be counted. The

bacteria

ume contained

is

irregularities in

same segments

in

to the designated arc line as

is

estimated by dividing the count obtained by the vol-

in all the

segments counted.

number of colonies exceed

75

in

the 2nd, 3rd or 4th segment which

also contains the 20th colony, the estimate of

number of bacteria

will

gener-

be low because of coincidence error associated with crowding of colonies. It is recommended that the count then be made by counting each of the

ally

all eight wedges, counting at least


two segments of a wedge contain 19 colonies and the
3rd segment contains the 20th and 76th (or more) colony, count the colonies
in all circumferentially adjacent 1st and 2nd segments in all eight wedges.
Calculate the contained volume in the counted segments of wedges and di-

circumferentially adjacent segments in

50 colonies,

e.g., if

the

first

number of colonies as shown below:


shows an example of a spirally inoculated plate which demonstrates the method for determining a bacterial count. Two segments were
counted on opposite sides of the plate with 21 and 33 colonies, respectively.
vide into the
Fig. 21:4

ALTERNATE VIABLE COUNT METHODS

324

From Table

21:11, the grid area

21

33

volume suggested

77,000

is

0.0007 ml.

SPLPC/ml

0.0007

When

counted on the total plate, record


per ml". If the colony count
exceeds 75 in the first segment of a wedge, record results as "greater than
500,000 Estimated SPLPC per ml".
3. Do not count spiral plates with irregular distribution of colonies
caused by dispensing errors. Report results of such plates as laboratory acci2.

less than 20 colonies are

results as "less than 500 Estimated

SPLPC

dent (LA).
4.

a spreader covers the entire plate, the plate should be discarded.

If

When

a spreader covers one-half of the plate area, count colonies only

when

well distributed in spreader-free areas, see 5:11(1).


5.

Compute SPLPC,

stances

in

5.11(1.3).

highest

unless restricted by detection of inhibitory subsample, excessive spreader growth or laboratory accidents, see

Record only the

first

number only when

two left-hand

digits; raise the digit to the

the third digit from the

left is five

next

or greater and

digit toward the right from the second digit.


Report counts as the Spiral Plate Count or the Estimated Spiral Plate
Count per ml or as applicable.

use zeroes for each successive

21.7 References
1.

Campbell,

J.E.,

and J.E. Gilchrist. 1973. Spiral plate method for counting bacteria

in

milk and other foods. Dev. Ind. Microbiol. 14:95-102.


2.

Donnelly,

C.B., Gilchrist, J.E., Peeler, J.T., and J.E.

Campbell.

1976. Spiral plate

count method for the examination of raw and pasteurized milk. Appl. Environ. Microbiol.
32:21-27.
3.

Gilchrist, J.E., Campbell, J.E., Donnelly, C.B., Peeler, J.T., and J.M. Delaney.
1973. Spiral plate

4.

5.

6.

7.

8.

method

for bacterial determination. Appl. Microbiol. 25:244-252.

Gilchrist, J.E., Donnelly, D.B., Peeler, J.T., and J.E. Campbell. 1977. A collaborative study comparing the spiral plate method with the aerobic plate count. J. Offic. Anal.

Chem.
Myers, R. P., and J. A. Pence. 1941. A simplified procedure for the laboratory examination
of raw milk supplies. J. Milk Technol 4:18-25.
Peeler, J.T., Gilchrist, J.E., Donnelly, C.B., and J.E. Campbell. 1977. A collaborative study of the spiral plate method for examining milk samples. J. Food Prot. 40:462-464.
Thompson, D.I., Donnelly, C.B., and L.A. Black. 1960. A plate loop method for determining viable counts of raw milk. J. Milk Food Technol 26:156-171.
Wright, E.O., and G.W. Reinbold. 1970. Prediction of standard plate count of manufacturing-grade raw milk from the plate loop count. J. Milk Food Technol. 33:168-170.

Appendixes

Supplemental microbiological control methods appear in Appendix A,


whereas supplemental chemical methods are described in Appendix B.
Some of the methods listed in the appendixes have only recently been developed and are presented in this section of the book because they have not yet
been evaluated adequately. It is hoped that inclusion of methods in this section of the book will stimulate studies to further evaluate these procedures.
Other methods are presented because members of the several committees
and of the Intersociety Council felt they would be useful to Dairy or other
laboratories. Methods in Appendixes A and B should not be considered
"Standard" as are procedures in the various chapters in this book.

325

APPENDIX A

SUPPLEMENTAL MICROBIOLOGICAL CONTROL


METHODS
J.J.

Redys, J.W. Messer, S.E.

Gilliland, R.T. Marshall,

G.W. Ronald, and C.H. White.

Appendix A contains "non-standard" but useful microbiological methods


examine dairy products, materials used in their manufacture and the environment associated with their production. Several methods which appeared
in the previous edition of this book are being studied collaboratively for
possible insertion as "standard" in the next edition. It is hoped that others
will be studied with the same purpose in mind.
to

A1

Aureomycin-Rose Bengal Agar Method


and Yeasts

.1

An

acidified

medium

to

making

Molds

recover molds and yeasts, often

bacteria, has been used for years.

medium

to Detect

in the presence of
There are several disadvantages to the

spreading mold colonies, bacterial growth, precipitation of casein,


it

difficult to distinguish

between colonies and curd

particles,

and

some yeasts and molds to grow at the low pH of the medium. As


early as 1944, rose bengal was used to reduce the rate of growth of fastgrowing molds. A medium which employed an antibiotic in combination
with acid and rose bengal was recommended in 1950 for estimating numbers
inability of

-^

of

soil fungi. ^

Rose Bengal Agar, or Potato Dextrose Agar,

fortified

with chlor-

tetracycline (Aureomycin) has been found suitable to recover molds and

yeasts from dairy products.'--^

A Equipment and supplies:


.

1.

Count

Glassware and other equipment: The same as the Standard Plate


15.2].

2.

Medium: Cooke Rose Bengal Agar.

3.

Antibiotic: Chlortetracycline hydrochloride (Aureomycin).

ISC Liaison: W.W. Ullmann


327

SUPPLEMENTAL MICROBIOLOGICAL CONTROL METHODS

328

B. Procedure:
1.

Preparation of medium: Dissolve

distilled water,

which

g of chlortetracycline in 99 ml of

in the refrigerator.

Before each use,

with a

racycline per milliliter.


2.

then stored

membrane filter. Add sufficient


melted and cooled medium to provide 20-35

sterilize the solution

tion to sterile

is

It is

not necessary to adjust the

pH

antibiotic solu-

of chlortet-

/xg

of this medium.

Plating and incubation: Prepare and plate samples as for the Stan-

dard Plate Count

seven days

^-^

Incubate petri dishes at 21 or 25


for
count
as
the Standard Plate Count [5.1
and
[5.6, 5.8].

for five or

1].

A1.2 References
1.

Cooke, W.B., and A.R. Brazis.

2.

Mycopathol. Mycol. Appl. 35:281-289.


J. A. KoBURGER. 1970. Fungi in foods. I. Effect of inhibitor and incubation temperature on
enumeration. J. Milk Food Technol. 33:433-434.

3.

J. P.

Martin.

estimating
4.

5.

1950.

The use of

soil fungi. Soil Sci.

1968.

Occurrence of molds and yeasts

acid, rose bengal

and streptomycin

in

in

dairy products.

the plate

method

for

59:215-232.

Overcast, W.W., and D.J. Weakley. 1969. An aureomycin-rose bengal agar for enumeration of yeast and mold in cottage cheese. J. Milk Food Technol. 32:442-445.
Smith, N.R. and V.T. Dawson. 1944. The bacteriostatic action of rose bengal in media
used for plate counts of fungi. Soil Sci. 58:467-471.

Citrate-Azide Agar Method for Enterococci

A2.1

The coliform count has proved

to be

in

Butter

an effective tool when used as a lineis churned

run test to indicate improper plant sanitation up to the time butter

and salted. It is not a reliable index for sanitary quality of churned butter.
Data reported by Saraswat et al.^ and by Blankenagel et al.' emphasize the
value of the enterococcus count for that purpose.

A Equipment and supplies:


.

1.

Count
2.

Glassware and other apparatus: As used for the Standard Plate


[5.2].

Plating

medium:

Citrate Azide

Agar of Reinbold

et al.^-

B. Procedure:

Prepare samples of butter for plating as described in Chapter 1. Prepare


samples of cream, milk, water, etc, as outlined in the appropriate chapters of
this book.
After petri dishes are inoculated, add 10 to 12 ml of melted and cooled
(45 C) Citrate Azide Agar per plate. Plates must be on a level surface and the
medium allowed to solidify. Then add 3 to 4 ml of the plating medium as an
overlay which completely covers the surface of the previously solidified
agar. After solidification of the overlay, incubate plates at 37 C for no more
than 72 hours. If necessary to facilitate counting of colonies, place a thin
sheet of white tissue paper under the petri dish on the illuminated colony
1

Cylinder Count Method

A3.1

329

counter. This enhances contrast between colonies and background. Count

only blue colonies.


C. Results

and significance:

Widespread occurrence of enterococci in dairy processing plants and their


resistance to the unfavorable microenvironment of butter have been demonstrated.'- ^ This method provides a good measure for evaluating sanitation in
a butter manufacturing plant.
milliliter

of butter

is

An enterococcus

attainable and

is

count of less than 10 per

not believed to be unduly restrictive for

a well-managed butter manufacturing plant.

A2.2 References
1.

Blankenagel,

G., Gibson, D.L., and C.N. Shih. 1967. The enterococcus count of butter
creamery sanitation. Canad. Dairy Ice Cream J. 46:17-19.
Reinbold, G.W., SwERN, M., and R.V. Hussong. 1953. A plating medium for the isolation and enumeration of enterococci. J. Dairy Sci. 36:1-6.
Saraswat. D.S.. Clark. Jr. W.S., and G.W. Reinbold. 1963. Selection of a medium for
the isolation and enumeration of enterococci in dairy products. J. Milk Food Technol.
for evaluating

2.

3.

26:114-118.
4.

Saraswat,

D.S.,

Reinbold, G.W., and W.S. Clark,

enterococcus, coliform and yeast and mold counts

Jr. 1965.

in butter. J.

The

relationship between

Milk Food Technol. 28:245-

249.

Cylinder Count Method to Determine Plate Count of


Pasteurized Milk Products

A3.1

The Standard Plate Count (SPC) is reliable but relatively expensive and
time-consuming. The Plate Cylinder Count (PCC) has been proposed as a
simplified method to determine plate counts of pasteurized milk products.'
Methodology is basically similar to that of the Plate Loop Method (PLM)^
which uses a continuous pipettor except that 0.01 ml of the sample is transferred to a petri dish with a specially designed cylinder machined from a
13-guage cannula rather than with a 0.01 -ml loop.

A Equipment and supplies:


.

1.

Sampling cylinder:

A 2.8-mm

cylinder

is

fashioned

at the distal

of a 13-gauge cannula, using an electric handgrinder; a vertical

mm

slit

in

end
the

wide permits free rinsing.'


2. Luer-Lok 2.0-ml continuous pipettor (Cornwall syringe)^: Equipment is adjusted to deliver 1.0 ml of dilution water. Sampling cylinder is
attached to pipettor and is autoclaved in kraft paper wrapping at 121 C for
cylinder about 0.8

15

minutes.
3.

Dilution blanks.

4.

Petri dishes.

5.

Standard Methods Agar.

SUPPLEMENTAL MICROBIOLOGICAL CONTROL METHODS

330

B. Procedure:
in original container and pour 80-90 ml into a
100-ml disposable plastic beaker. Dip cylinder three times into
sample at approximately one dip per second. Expel portion retained on the
third dip from cylinder into a petri dish with 1.0 ml of phosphate buffered
dilution water from a dilution blank which is attached to a continuous pipettor. Pour plates, incubate, and examine in accordance with Standard Methods (Chapter 5). Test for precision by doing SPC and FCC on the same

Mix pasteurized milk sample

sterile

samples of milk before using the simplified method exclusively.

A3. 2 References
1.

Donnelly, C.B., Leslie,


Read,

products.
2.

J.E.,

1970. Cylinder count

Jr.

J.

Thompson,

Messer, J.W., Green, W.T., Peeler, J.T., and R.B.


method for determining plate count of pasteurized milk

Dairy Sci. 53:1187-1193.


D.I.,

Donnelly, C.B., and

mining viable counts of raw milk.

J.

L. A.

Black.

1960.

Detection of Sulfa Drugs and Antibiotics

A4.1

This method
cillus

'

is

megateriitm

plate loop

method

for deter-

Milk Food Technol. 26:167-171.

in

Milk

a disc assay procedure which uses the spore-forming 5-

ATCC

9855. This organism

drugs, to bacitracin, and eight other antibiotics

sensitive to eight sulfa

is

commonly used

in mastitis

therapy.

A Equipment and supplies:


.

ATCC

1.

Test organism: B. megaterium

2.

Prescription bottles: Capacity 6 oz (180 ml).

3.

AK

4.

Incubator: Providing 37-C temperature.

Sporulation

9855.

Medium #2.

Mueller-Hinton agar: See 4.1.


Filter paper discs: Diameter V2
Carl Schleicher and Schuell Co.
7. Spectrophotometer.
8. Plastic petri dishes: ID 90 mm.
5.

6.

9.

in.

(1.27 cm), obtainable

from the

Centrifuge: Refrigerated.

10.

Refrigerator.

11.

Water

bath:

Capable of 50-C temperature.

B. Preparation of spore suspension:


1.

Transfer cells of B. megaterium

surface of sterile

AK

sporulation

ATCC

medium #2

9855 by streaking the entire

in a

6-oz (180 ml) prescription

bottle previously prepared.


2.

Incubate inoculated prescription bottles of agar for 48 hr

3.

After incubation,

agar with buffered

MS

wash spores and vegetative

water and centrifuge

at

an

RCF

at

35 C.

from surface of
of 5,000 for 15 min

cells

Assay

331

A5.1

Penicillin Detection: Disc

at 3

C. Repeat the washing and centrifugation process three times.


4. Store spore suspension in buffered MS water at 4 C until used.

C. Testing procedure:

Prepare Mueller-Hinton Agar and keep

1.

it

liquid in a

water bath

50 C. Fresh agar should be prepared each day of testing.


2. Adjust the inoculum to an optical density calculated to give a

concentration of about 5 x

Add

3.

10^

spores per

milliliter

at

final

of agar.

the spore suspension (to give the approximate concentration

called for in paragraph 2 preceding) to the liquid agar at 50 C.

Mix agar and spore suspension by

4.

swirling gently to avoid air bubble

formation.
Pipet 4 ml of inoculated

5.

medium

into petri dishes

and distribute over

dishes by rotation. Allow agar to harden.


6. Proceed to test milk samples, using V2-in. (1.27 cm) discs dipped in
milk samples and placed on the agar surface. Space discs so that overlapping
of zones will not occur. Incubate plates at 37 C for 4 to 5 hours. This in-

cubation temperature
ity to sulfa

is

recommended because

it

affords

maximum

sensitiv-

drugs.

D. Results:
Any clear zone around the periphery of the V2-in. 1 .27 cm) disc is considered a positive test provided proper controls are used. NOTE: A V2-in. (1.27
cm) disc impregnated with 50 /xg of para-aminobenzoic acid (PABA) may be
used to identify sulfa drug inhibitors. Discs with that concentration of PABA
(

the inhibitory properties of sulfa drugs in concentrations of at

overcome
least 5

/Ltg

per disc. Identification discs are prepared by adding

aqueous solution

to

them, followed by drying the discs

at

PABA

in

40-44 C.

A4.2 Reference
Read,

1.

Jr. R.B.,

Bradshaw,

tection of sulfa drugs

Swartzentruber, A. A., and A.R. Brazis.


in

This procedure
the

0.0025 unit/ml

skimmed

is

qualitative
in

1971. De-

milk. Appl. Microbiol. 21:806-808.

Detection of Penicillin in Milk by a Disc Assay


international Standard Fil-IDF 57:1 970^

A5.1

for

J.G.,

and antibiotics

Technique-

based on the method of Galesloot and Massing^ and is


detection of penicillin when present in excess of

raw milk, processed

liquid milk, partially

skimmed

milk,

milk, and reconstituted dried milk.

A Equipment and supplies:


.

Agar slant: 2 g of yeast extract, 5 g of peptone, g of meat


of sodium chloride, 15 g of agar, and 1000 ml of distilled water.
120 C for 15 minutes. The final pH should be 7.4 0.1.
1.

extract, 5 g
Sterilize at


SUPPLEMENTAL MICROBIOLOGICAL CONTROL METHODS

332

2. Culture medium: 1 g of yeast extract, 2 g of tryptone, 0.05 g of glucose, and 1000 ml of distilled water. Sterilize at 120 C for 15 minutes. The

final

pH
3.

should be 8.0 0.1.

Agar medium:

15 g of agar,

The

final

final

pH

pH

2.5 g of yeast extract, 5 g of tryptone,

and 1000 ml of distilled water.


should be 8.0

0.

Sterilize at 120

g of glucose,

for 15 minutes.

Count Agar with a

Alternatively, use Plate

of 8.0 0.1.

Prepared using a glass still.


quality, with flat bottoms of uniform thickness
minimum internal diameter about 110 mm.
6. Forceps; Fine pointed.
7. Antibiotic assay paper discs: Diam 12-13 mm Whatman AA discs are
4.

Distilled water:

5.

Petri dishes:

Good

from pure cellulose (over 98% alpha-cellulose) with ash


1
and 2 ppm,
respectively. The base weight of the paper is 440 g per square meter and the
thickness 0.88 mm. Do a blank test on nine discs from each batch of paper
discs, using sterile distilled water to verify that the discs do not give a clear
zone when incubated at 55 1 C for 5 hr on the assay plates, [A5.1(C)].
8. Penicillin: Crystalline sodium or potassium benzyl penicillin. Follow
suitable. Prepare

content below 0.006% and copper and iron contents below

label directions for storage of the standard. Prepare a standard solution of


penicillin of 100 units/ml
in sterile distilled

water

by dissolving sodium or potassium benzyl


in a suitably

only on the day of preparation; store


9.

stoppered

label directions for storage.

penicillin

Use solution

C when

in a refrigerator at 5

Penicillinase concentrate: Available

Follow the

sterile bottle.

not

in use.

from biological supply houses.

Prepare a penicillinase solution of

1,000 units/ml by dissolving penicillinase in sterile distilled water. Store in a

C for a maximum of two weeks.


Milk free from inhibitory substances: Prepare a 10% solution of inhibitor-free milk by reconstitution in sterile distilled water of non-fat dry

refrigerator at 5
10.

milk previously tested and found to be free from inhibitory substances. Al-

and found to be
from inhibitory substances may be dispensed in bottles, heated for one
hr at 100 C and stored in a refrigerator at 5 C for a maximum of one week.

ternatively, a sufficient quantity of fresh bulk milk tested


free

1 C.
var. calidolactis
organism:
stearothermophilus
Test
Use Bacillus
maintain
the culstrain C953 (Netherlands Institute for Dairy Research). To
11.

Incubator: Thermostatically controlled at 55

12.

ture, streak

an agar slant, [A5.1(A.l)],

in

a tube and incubate for 48 hr at


it a little way into
The stock culture

55 C. After incubation, flame the cotton stopper and force


the tube. Close the tube with a sterile rubber stopper.

obtained can be stored several months


13.

Water

at 5

C.

bath: Thermostatically controlled at 55

C.

B. Preparation of ^. sitdLVoXhQvmophWw^ suspension:


1. Aseptically transfer 10 ml of culture medium, [A5.1(A.2)] to a sterile

150-ml Erlenmeyer

flask.

A5.1

Penicillin Detection: Disc

2.

Assay

Inoculate the culture

333

medium

in the flask

with a loop of stock cul-

ml of a preceding liquid culture may be


used as the inoculum, provided the liquid culture is no more than 36 hr old
and has been kept in a refrigerator at 5 C.
ture, [A5.1(A.12)]. Alternatively, 0.1

NOTE. When

using a stock culture or a liquid culture which

is

more than

36 hr old as the inoculum, do the above subculturing procedure at least twice


with no more than 36 hr between subcultures.
3.

Incubate for 16-18 hr

at 55

C and

use

in

A5.1(C). After incubation

the liquid culture should have a viable colony count of 50-100 x 10^/ml

when incubated

at 55

uniformly turbid and

for 48 hr

if it

on Plate Count Agar. The culture must be

contains flocks or sediment

it

should be discarded

and a new culture prepared.

C. Preparation of assay plates:


1.

Prepare and sterilize appropriate amounts of agar medium,

tA5.1(A.3)].
sterile medium to 55 C.
Add part of freshly prepared liquid culture to five parts of agar
medium at 55 C in a sterile test tube or bottle and mix thoroughly.
2.

Cool the

3.

4.

test

of

Transfer a quantity calculated to give a layer 0.8-1

thickness of 0.8

they

.0

mm thick of the

medium to a sterile petri dish previously heated to 55 C. For a petri dish


10-mm internal diam about 8 ml of agar medium is necessary to obtain a

mm.

5.

Allow the agar

6.

Preferably use assay plates on the day they are prepared; however,

may be

to solidify

on a

level surface.

kept several days provided they are cooled immediately after


in a sealed polythene bag in a refrigerator at 5 C.

preparation and kept

D. Preparation of penicillin controls:


1. Prepare a working solution of penicillin by diluting 1.25 ml of the
standard penicillin solution to 1,000 ml with sterile distilled water. This
working solution contains 0.125 unit/ml.
2. Prepare 50 ml of a solution containing 0.0025 unit penicillin/ml by
adding inhibitor-free milk, [A5.1(A.10)] to one ml of the working solution
[A5.1(D.l)] and mixing.
3. Prepare 50 ml of a solution containing 0.005 unit penicillin/ml by adding inhibitor-free milk to 2 ml of the working solution and mixing.
4. Prepare solutions A5.1(D.l-3) on the day the test is done.

E. Preparation of penicillinase control:


1. Thoroughly mix the sample of milk to be

examined and transfer apwidemouth bottle.


ml of penicillinase solution [A5.1(A.9)] and mix.

proximately 10 ml to a suitable
2.

Add about

0.4

sterile

SUPPLEMENTAL MICROBIOLOGICAL CONTROL METHODS

334

F. Test procedure:

Mark

1.

off

mm

and number squares of 25

assay plate, [A5. 1(C)] using a ruler and

wax

on the outside bottom of the

pencil or other suitable means.

Thoroughly mix the sample of milk to be examined. Liquid samples

2.

should be tested as soon as possible and preferably within 10 hr of sampling,


maintaining the samples at as low a temperature as possible and

than 5

in

the meantime. If

it

is

they should be held frozen (-30 to


icillin

at

not

more

not possible to test the samples within 10 hr,

-15 C)

to

minimize inactivation of pen-

and should be tested within 24 hours.

Dip a paper disc [A5.1(A.7)] into the milk by means of a clean dry
pair of forceps. Remove any excess milk by touching the disc against the
side of the sample bottle.
4. Place the disc flat on the agar surface at the center of the marked
square and press down gently with the forceps.
5. Repeat steps 3 and 4 with another disc.
6. Repeat steps 3 and 4 in triplicate, using the penicillin control contain3.

ing 0.0025 unit of penicillin/ml instead of the milk sample.


7.

Repeat steps

and 4

in

duplicate, using the penicillin control contain-

ing 0.005 unit penicillin/ml instead of the milk sample.


8.

Repeat steps

and 4

in duplicate,

using the penicillinase control

in-

stead of the milk sample.


9.

When

random
10.

all

nine discs have been placed on the assay plate, [A5. 1(C)] in

fashion, invert the plate and incubate

After incubation, examine the plate(s)

it

at

55

in front

for 2.5-5 hours.

of a suitable light

source for clear zones of inhibition around the discs.


1 1

Determine the average diameters of the clear zones of inhibition for


and the penicillinase control.

the milk sample, the penicillin controls

G. Interpretation of results:
1. Clear zones around discs containing the penicillin control corre-

sponding to 0.0025 unit/ml should be just perceptible. There should be somewhat larger zones around discs containing the penicillin control corresponding to 0.005 unit/ml.
2.

The presence of

clear zones around discs containing the sample in-

dicates the presence of substances inhibitory to the test organism.


3.

If

there are no clear zones around discs containing the penicillinase

control but there are clear zones around discs containing the sample, the
inhibitory substance in the sample
4.

If

is

penicillin in

penicillinase control

is

equal to the average diameter of clear zones around

discs containing the sample, penicillin


5.

If

excess of 0.0025 unit/ml.

the average diameter of clear zones around discs containing the

is

not present.

the clear zones around discs containing the penicillinase control

are smaller in average diameter than clear zones around discs containing the

335

IDF Disc Test

Penicillin Detection: Modified

A6.1

sample, the sample contains penicillin and other inhibitory substances. The
around the discs containing the penicillinase control are attribut-

clear zones

able to inhibitory substances other than penicillin, while those around the
discs containing the sample are attributable to penicillin and other inhibitory

substances.

NOTE Growth

of B. stearothermophilus var. calidolactis

is

and to a lesser and varying extent by


other antibiotics and inhibitors, including those which occur naturally in
inhibited particularly

by

penicillin,

milk.

A5.2 References
1.

2.

International Dairy Federation. Commission

E. 1970. Detection of penicillin in milk

by a disk assay technique. Internatonal Dairy Federation, Brussels, Belgium. 7 pp.


Galesloot, Th.E. and F. Massing. 1%2. A rapid and sensitive paper disc method for the
detection of penicillin in milk. Neth. Milk Dairy J. 16:89-95.

Modified IDF Disc Test for Penicillin

A6.1

This disc assay method

Milk^^

which specifies Bacillus stearothermophilus

ation Procedure
lactis as the test

in

a modification of the International Dairy Feder-

is

organism. This assay

is

sensitive to as

little

var. calido-

as 0.002 unit of

penicillin/ml of milk.

A Equipment and supplies:


.

1.

2.

Test organism: B. stearothermophilus var. calidolactis.


Storage medium: 2 g of yeast extract, 5 g of peptone,

sodium chloride, 15 g of agar, and 1000 ml of


should be 7.4 0.1.

extract, 5 g of

The

final

pH

g of meat

distilled water.

Seed broth: 10 g of yeast extract, 20 g of tryptone, 0.5 g of glucose,


and 1000 ml of distilled water. The final pH should be 8.0 0.1.
4. Standard Methods Agar: (Chapter 4) adjusted to pH 8.0 0.1.
3.

5.

Plastic petri dishes: 85

mm

ID.

paper discs, nonsterile blanks: Diameter V2 in. (1.27 cm), nontoxic to B. stearothermophilus var. calidolactis, high absorbance, for
sample application.
7. Filter paper discs, penicillinase-impregnated: Diameter V2 in. (1.27
6.

Filter

cm), high absorbance, to identify penicillin.


8.

Penicillinase concentrate: Available

from biological supply houses.

Store at 0-4.4 C.
9.

10.

not use
11.

Forceps: Dissecting, with fine points.


Penicillin: Crystalline potassium penicillin G. Store at -10 C.

beyond expiration date.


Water bath: Thermostatically

12.

Incubator: 55

13.

Dilution bottles: (Chapter

C.
5).

controlled.

Do

SUPPLEMENTAL MICROBIOLOGICAL CONTROL METHODS

336

14. 1% phosphate buffer, pH 6.0 0.1: Dissolve 8.0 g of monobasic


potassium phosphate and 2.0 g of dibasic potassium phosphate in distilled
water and dilute to 1,000 ml with distilled water.

B. Preparation of B. stQarothtrmophWus suspension.


1.

Transfer a loopful of culture from a stock slant culture to 50 ml of

seed broth, [A6.1(A.3)]


2.
is

in

Incubate for 18 hr

a dilution bottle, [A6.1(A.13)].


at

55

C and

refrigerate at 0-4.4

for 5 days. This

the stock seed broth.

3. Inoculate 50 ml of seed broth in a dilution bottle with 0.1 ml of the


5-day-old broth culture.
4. Incubate for 18 hr at 55 C. Transfer 1.0 ml of this culture to 50 ml of
seed broth in a dilution bottle. Discard the remaining 49.0 ml of culture.
5. Incubate for 6 hr at 55 C. Transfer 1.0 ml of this culture to 50 ml of
seed broth in a dilution bottle. Store the remaining 49.0 ml of the culture at
0-4.4 C for 5 days. This culture can be used as the stock seed broth.
6. Incubate for 18 hr at 55 C and use in A6.1(C).

C. Preparation of assay plates:


1.

Prepare and

justed to a final

sterilize, in

pH

of 8.0

0.

60-ml amounts, Standard Methods Agar ad1

Cool the sterile medium to 55 C.


Add 2 ml of stock seed broth to 60 ml of Standard Methods Agar.
4. To petri dishes add 7.0 ml of the seeded agar. These plates can be
used immediately or stored in a plastic sleeve at 0-4.4 C up to 8 days.
2.

D. Preparation of control s:
1. Negative control: Autoclave 99-ml amounts of pasteurized, antibiotic-free homogenized whole milk in dilution bottles for 10 min at 121 C. Cool
rapidly and store at 0-4.4 C.
2. Positive control: Dissolve an accurately weighed portion of potassium penicillin G [A6.1(A.10)], in buffer [A6.1(A. 14)] to give a concentration

Use this stock solution immedimore than 24 hours. On the day of use, dilute
one ml of the stock solution to 100 ml with buffer; further dilute one ml of
of 10,000 units/ml. This
ately or store at 0-4.4

this solution to 100

tions of 0.004

is

the stock solution.

for not

ml with buffer.

Make

and 0.002 unit/ml with

additional dilutions to concentra-

sterilized, pasteurized antibiotic-free

whole milk [A6.1(D.1)J.


E. Test procedure

Divide the outside bottom of the assay plate, with a marking pencil
between discs will be at least 20 mm, and label
each with an identifying mark.
1.

into sections so the distance

A6.1

Penicillin Detection: Modified IDF Disc Test

2.

337

With a clean, dry forceps remove a paper disc from a vial or other
and touch the edge to a well mixed sample of milk.
Allow the milk to wet the disc by capillary action.
Touch the edge of the wetted disc three times on the underside of the

suitable container
3.

4.

petri dish lid.


5.

the

Place the disc,

marked
6.

side

flat

down, on the agar surface near the center of

section.

Place separate discs [A6.1(A.6)] wetted with the negative control

[A6.1(D.l)] and positive controls, [A6.1(D.2)] on each plate.


7.

Invert plates and incubate at 55

8.

After incubation examine plates

in front

for 3.5-5 hours.

of a suitable light source for

around the discs.


9. Presence of clear zones around discs wetted with milk samples indicates the presence of substances inhibitory to the test organism.
10. To determine if the clear zone resulted from penicillin or another
inhibitor proceed as in A6.1(G).
clear zones of inhibition

F. Penicillinase control:
1.

Add

0.05 ml of penicillinase concentrate [A6.1(A.8)] to 5.0 ml of

milk samples(s) found positive

in

A6.1(E).

2.

Mix

3.

Alternatively, use penicillinase discs [A6.1(A.7)].

well.

G. Identification of penicillin:
1.
Wet a blank disc [A6.1{A.7)] with the milk sample found positive
A6.1(E).
2.
3.

in

Allow the milk to wet the disc by capillary action.


the edge of the wetted disc three times on the underside of the

Touch

petri dish cover.


4.

the

Place the disc

flat

side

down on

the agar surface near the center of

marked section and press down gently with the forceps.


5.

6.

Repeat steps 1-4.


Repeat steps 1-4 in

triplicate, using the 0.002-unit penicillin/ml

trol

[A6.1(D.2)] instead of the milk sample.

trol

[A6.1(D.2)] instead of the milk sample.

7.

8.

Repeat steps 1-4


Repeat steps 1-4

in duplicate,

in

con-

using the 0.004-unit penicillin/ml con-

duplicate using the penicillinase-treated portion

of the sample or penicillinase discs wetted with the same sample.


9.

When

all

nine discs have been placed on the assay plate, A6.1(C),

in

and incubate at 55 C for 3.5-5 hours.


10. After incubation, examine the plate(s) in front of a suitable light
source for clear zones of inhibition around the discs.
1
Determine the average diameters of the clear zones of inhibition for
the milk sample, penicillin controls and the penicillinase control.

random fashion,

invert the plate

SUPPLEMENTAL MICROBIOLOGICAL CONTROL METHODS

338

H. Interpretation of results:
1. Clear zones around discs containing the

penicillin control corre-

sponding to 0.002 unit/ml should be just perceptible on fresh assay plates


NOTE On older assay plates this concentration may not produce a perceptible zone. There should be somewhat larger zones around discs containing the penicillin control corresponding to 0.004 unit/ml.
2. If there are no clear zones around discs containing the penicillinase
control, but there are clear zones around the discs containing the milk

sample, the inhibitory substance is penicillin.


3. If the average diameter of clear zones around discs containing the
penicillinase control is equal to the average diameter of the clear zone

around discs containing the sample, penicillin is not present.


4. If clear zones around discs containing the penicillinase control are
smaller in average diameter than clear zones around discs containing the
milk sample, the milk contains penicillin and other inhibitory substances.
Reporting:

/.

1.

Report the presence of penicillin only.

2.

To

estimate the concentration of penicillin, use the positive controls

[A6.1(D.2)] as a guide.
3.

If

the test indicates the presence of an inhibitor other than penicillin,

assay by other methods before reporting as other inhibitor.


4.

Report the absence of

penicillin as negative for penicillin.

A6.2 References
1.

2.

International Dairy Federation. Commission

E. 1970. Detection of penicillin in milk

by a disk assay technique. International Dairy Federation, Brussels, Belgium. 7 pp.


Kaufmann, O.W., 1977. A practical sensitive test to detect penicillin in milk. J. Food Prot.
20:250-251.

A7.1

A
in

DelvotestP

sensitive qualitative agar diffusion test (Delvotest P) to detect penicillin

raw milk has been described

and has been successfully used

in

some

laboratories.^'^

A. Equipment and supplies:


1. Seeded agar ampules: Ampules of plain agar seeded with Bacillus
stearothermophilus var. calidolactis. Store at 0-4.4 C.
2.

Nutrient tablets: Each containing 0.5

cose, 2.0

mg of non-fat

dry milk and 0.025

mg

mg

of tryptone, 5.0

mg

of glu-

of bromcresol purple. Store at

room temperature.
3.

Forceps: For transferring nutrient tablets.

4.

Dosing syringe with single service disposable

dispensing 0.1-ml samples of milk.

tips:

For sampling and

Delvotest P

A7.1

5.

339

Penicillinase cone: Available

from biological supply houses. Store

at

0-4.4 C.

Water

bath: Thermostatically controlled at 63-66 C.


Metal or wire rack: For holding ampules.
8. Non-fat dry milk: Antibiotic-free.
9. Penicillin: Crystalline sodium or potassium penicillin G. Store
at -10 C. Do not use beyond expiration date.
10. Dilution bottles: (Chapter 5).
11. \9c phosphate buffer, pH 6.0 0.1: Dissolve 8.0 g of monobasic
potassium phosphate and 2.0 g of dibasic potassium phosphate in distilled
water and dilute to 1,000 ml with distilled water.
12. Test tubes: 15 x 100 mm.
6.
7.

B. Controls:

As

a check on proper functioning of reagents, both penicillin-positive and

-negative control samples must be included with each series of samples test-

ed by the screening and confirmatory procedures. A single positive and negative control will suffice for each series of samples tested.
in

1. Negative control: Autoclave 100-ml amount of reconstituted (100 g


1000 ml of distilled water) antibiotic-free non-fat dry milk [A7.1(A.8)] in

screw-capped pyrex dilution bottles for


store at 0-4.4 C.

When

the entire solid test


2.

line

tested, this milk

10 min at 121 C. Cool rapidly and


must produce yellow coloration of

medium.

Positive control: Dissolve an accurately weighed portion of crystal-

sodium or potassium

penicillin

buffer [A7.1(A.ll)] to give a

[A7.1(A.9)] in sufficient

known concentration

1% phosphate

of 100-200 units per ml.

This solution

may be

this solution,

prepare a reference concentration of 0.006 unit per ml with

stored refrigerated for no

more than two days. From

antibiotic-free reconstituted non-fat dry milk. Dispense 5

tubes [A7.1(A.12)].

be stored

When

at

-10 C

Cap and

freeze at

for six months.^

tested, this milk

-IOC.

ml into

15

x l(X)-mm

This positive control

For use thaw

at

may

room temperature.

must produce purple coloration of the

entire solid test

medium.
C. Screening test procedure:
1
Place the required number of ampules [A7. 1(A.
be tested in a suitable rack [A7.1(A.7)].

ampule

and

2.

Identify each

3.

Remove

4.

With the clean forceps add one nutrient

legibly

1)]

for the samples to

indelibly.

the tip of each ampule.


tablet [A7.1(A.2)] to

each

ampule.
5.

With the syringe [A7.1(A.4)] add

0.1

ml of a well-mixed milk sample

or positive [A7.1(B.2)] or negative [A7.1(B.l)] control sample to each identified

ampule. Use a clean disposable

tip for

each sample and control trans-

SUPPLEMENTAL MICROBIOLOGICAL CONTROL METHODS

340

ferred.

Extreme care must be taken not

when sampHng and


6.

to

contaminate the multiuse syringe

dispensing.

Place the rack containing the ampules

two and a

in

a water bath at 63-66

for

half hours.

7.

After incubation observe the color of the solid medium.

8.

purple coloration of the entire or part of the solid test

medium

in

any of the milk sample ampules indicates the presence of substances inhibitory to the test organism.
9. Samples of raw milk which give a purple coloration of part or all of
the solid medium must be heated to 82 C for 2-3 min^ cooled, and retested
as in A7.1(D).

D. Confirmatory test procedure:


1. Add 0.05 ml of penicillinase concentrate [A7.1(A.5)] to 5.0 ml of
heated, cooled milk sample(s) found positive in A7.1(C).
2.

Mix

3.

Inoculate separate identified ampules [A7.1(A.l)] with 0.1 ml of the

well.

peniciilinase-treated, and untreated heated sample.


4.

Incubate as

in

[A7.1(C. 6)].

E. Interpretation of results:
1.

medium

yellow coloration of the entire solid

of the test ampule

containing the peniciilinase-treated portion of the sample and purple coloration of part of or the entire solid

portion of the

same sample

is

medium containing

the untreated heated

a positive test for penicillin.

sample contains heat-stable inhibitory substances and


negative penicillin confirmation by this test may occur.
2.

purple coloration of part of or the entire solid

NOTE If

the

penicillin, a false-

medium

of the am-

pule containing the peniciilinase-treated portion of the sample and a purple


coloration of part of or the entire solid

medium of the ampule containing the


may indicate the presence of an

untreated heated portion of the same sample


inhibitory substance.

Assay by other methods before

interpretation.

F. Reporting:

Report the presence of penicillin only.


If tests of heated milk samples indicate the presence of an inhibitor
other than penicillin, assay by other methods before reporting.
3. Report the absence of penicillin as negative for penicillin.
1.

2.

A7.2 References
1.

DicKES, G.J. 1973. The disappearance of residual

penicillin in milk.

J.

Assoc. Public Anal.

11:36-38.
2.

Baughman, R.W., Nelson, F.E., and P. A. Hartman. 1%1. Rapid antimethods u%\ng Bacillus stearothermophilus J. Milk Food Technol. 24:143-146.

Igarashi, R.T.,
biotic assay

BR Foss

A8.1

3.

Test

341

Packard, V.S., Tatini, S.. and R.E. Ginn. 1975. An evaluation of methods for detecting
and comparative incidence of penicillin residues in diflFerent types of raw milk. J. Milk Food
Technol. 38:601-603.

4.

Pater, B. 1977.
concentrations

5.

Van

collaborative study of the Delvotest-P

Food

Os, J.L.. Lameris. S.A..

test for the

A8.1

in milk. J.

Doodewaard,

determination of antibiotic residues

BR Foss

Test*

3-

method

to detect

low

penicillin

Prot. 40:23-24.
T.,

and J.G. Oostendorp. 1975. DiflFusion


Neth. Milk Dairy J. 29:16-34.

in milk.

4 5

This qualitative agar diflfusion procedure for inhibitory substances in milk


based on brilliant black reduction by Bacillus stearothermophilus var. calidolactis C953.
is

A. Equipment and supplies:


1. Agar powder: Each bottle containing 4.5 g of Antibiotic
Store at room temperature.
Glycerin: 2 ml per

3.

4.

Equipped with a 40-mm magnetic


Glass flask: Conical, 200 ml with wide neck.

vial.

Store at

1.

room temperature.

2.

Hot

Medium No.

plate:

stirrer.

5.

Glass beaker:

6.

Thermometer: 0-100 C.

7.
8.

Cornwall syringe: 2 ml with largest tip.


Rubber roller: For applying plate sealing tape.

9.

Plate sealing tape: Tesafilm.

liter.

10.

Water baths: Thermostatically controlled with and without

11.

Bacterial culture:

agita-

tion.

lidolactis

C953 containing

dried culture oiB. stearothermophilus var. ca-

10

mg

of brilliant black. Store at

room temper-

ature.
12.

Test plates: With small cells each having a volume of 0.2 ml.

13.

Aluminum

14.

Microliter syringe:

foil:

For wrapping test plates and flask closure.


0.1-ml Marburg 100 (Eppendorf Instrument

Manufacturing) or equivalent.
15. Penicillin: Crystalline sodium or potassium

Do

penicillin

G. Store at -10 C.

not use beyond expiration date.


16.

Store at

Penicillinase

0^.4 C.
1% phosphate

cone:

Available

pH

from

biological

0.1: Dissolve

supply

houses.

8.0 g of

monobasic

potassium phosphate and 2.0 g of dibasic potassium phosphate


water and dilute to 1,000 ml with distilled water.
18. Dilution bottles: (Chapter 5).
19. Test tubes: 15 x 100 mm.

in distilled

17.

buffer,

6.0

*System Enterotox
^NR. 5531 Baierdorf AG., Hamburg or equivalent.

SUPPLEMENTAL MICROBIOLOGICAL CONTROL METHODS

342

B. Preparation of test plates:

Dissolve the agar powder [A8.1(A.l)] and glycerine [A8.1(A.2)]

1.

in

110 ml of distilled water in a flask [A8.1(A.4)] during slow stirring.

Cover opening of flask with

2.

C. Leave stirring bar

at 121

Cool the

3.

sterile

medium

55 C. During cooling lightly

foil

[AS. 1(A. 13)] and autoclave for 15 min

in flask.

to 55

stir

in

a glass beaker containing water at

the agar.

Dissolve by heavy shaking the bacterial culture [A8.1(A.l

4.

1)] in

10 ml

of sterile water.
5. Add the suspension [A8. 1(B.4)] to the agar at 55 C and stir slowly for
two minutes. Avoid foam.
6. Using a sterile Cornwall syringe add 0.1 ml of the agar suspension
mix [A8.1(B.5)] to each cell in the test plate(s). All plates should be filled
within 10-15 minutes. An extended dispensing time might cause preincubation. During filling of cells, keep the flask in a water bath at 60 C.

Close the

7.

test plates [A8.1(B.6)]

the rubber roller [A8.1(A.8)] and

Store plates with sealing tape

8.

with sealing tape [A8.1(A.9)] using

wrap the sealed

plates in

downwards

at

aluminum

foil.

0-4.4 C. Test plates

should be used within one month.


C. Preparation of controls:

As

a check on proper functioning of reagents, both penicillin-positive and

-negative control samples must be included with each series of samples tested

on each test plate.


1. Negative control: Autoclave 99-ml amounts of pasteurized, antibiotic-free homogenized whole milk in dilution bottles [A8. 1(A. 18)] for 10 min at
121 C. Cool rapidly and store at 0-4.4 C. When tested, this milk must produce yellow coloration of the entire solid test medium.
2. Positive control: Dissolve an accurately weighed portion of crystalline sodium or potassium penicillin G [A8.1(A.15)] in sufficient 1% phosphate

buff'er

[A8.1(A.17)] to give a

known concentration of 100-200 units/ml.


more than two days. From

This solution

may be

this solution,

prepare a reference concentration of 0.006 unit per ml with


whole milk [A8.1(C.l)]. Dispense 5 ml each into

stored refrigerated for no

sterilized antibiotic-free
15

X 100

control

mm

tubes [A8.1(A.19)].

may be

temperature.

stored at

When

-10 C

-10 C. This

For use, thaw

positive
at

room

must produce a black-violet coloration

medium.

D. Screening

test

procedure:

1.

Remove

the

aluminum

2.

Identify each well.

3.

Peel

off"

freeze at

for six months.'

tested, this milk

of the entire solid test

later use.

Cap and

foil

wrapping of the

the sealing tape from the test plate.

test plate.

Save the sealing tape for

A8.1

BR Foss
4.

343

Test

With the syringe A8.


[

1(

A.

14)]

add

0.

ml of a well mixed milk sample

or positive [A8.1(C.2)] or negative [A8.1(C.l)] control sample to each identified well.


5.

After

filling,

the test tray

is

placed unsealed for

hr in a refrigerator

for predififusion of inhibitory substances.

Drain the milk from the tray by shaking the test plate.
test plate thoroughly and carefully with clean running tap
water. Shake off the excess water.
6.

7.

Rinse the

8.

Dry

9.

Re-fix the sealing tape

10.

the test plate using tissue.

Incubate for 2 hr

sealing tape upwards,

in

removed

in

A8.1(D.3).

a waterbath at 60

2 C. Float the test plate,

on the surface. The waterbath should have no

agita-

tion.
1

1.

12.

medium

After incubation observe the color of the solid


Little or

no change

in

of each well.

the original black-violet color in any of the

milk sample wells indicates the presence of substances inhibitory to the test

organism.
13.

Samples of milk which give a black-violet coloration of part of or all


medium must be heated to 82 C for 2-3 min,' cooled and retested

of the solid

as in A8.1(E).
E. Confirmatory test procedure:
1.

Add

0.05 ml of penicillinase concentrate [A8.1(A.16)] to 5.0 ml of

heated, cooled milk sample(s) found positive in A8.1(D).


2.

Mix

3.

Inoculate separate identified wells with 0.1 ml of the penicillinase-

treated,
4.

well.

and untreated heated sample.


Proceed as in A8.1(D. 5-11).

F. Interpretation of results:
1.

yellow coloration of the entire solid

taining the penicillinase-treated portion of the

ation of part of or the entire solid

portion of the

same sample

is

medium

medium containing

the untreated heated

a positive test for penicillin.

sample contains heat -stable inhibitory substances and


ative penicillin confirmation by this test may occur.
2.

of the test well con-

sample and black-violet color-

NOTE:

If

the

penicillin, a false-neg-

black-violet coloration of part of or the entire solid

medium

of the

well containing the penicillinase-treated portion of the sample and a blackviolet coloration of part of or the entire solid

medium

of the well containing

same sample may indicate the presence


substance. Assay by other methods before interpretation.

the untreated heated portion of the

of an inhibitory

G. Reporting:
1. Report the presence of penicillin when demonstrated.

SUPPLEMENTAL MICROBIOLOGICAL CONTROL METHODS

344

If tests

2.

of heated milk samples indicate the presence of an inhibitor

other than penicillin, assay by other methods before reporting.

Report the absence of inhibitor as negative.

3.

A8.2 References
DiCKES. G.J. 1973. The disappearance of residual penicillin

1.

in

milk.

Assoc. Public Anal.

J.

11:36-38.

Baughman, R.W., Nelson, F.E., and P. A. Hartman. 1961. Rapid antimethods using Bacillus stearothennophilus J. Milk Food Techno). 24:143-146.
Kraack. J., and A. Tolle. 1967. Brilliant black reduction test with Buc. stearothenno-

Igarashi, R.T.,

2.

biotic assay
3.

philus var. calidolactis for evidence of inhibitors

MuLLER,

in

milk. Milchwissenschaft 22:669-673.

The brilliant black reduction test with Bacillus stearothermophilus


variant calidolactis C953 for evidence of active antibiotic substances. Das Lebensmittellaund Kaesezeitung.
bor, Nr. 4, Beilage Der Zeitschrift deutsche Milchwirtschaft, Molkerei

4.

F.J.

1973:

6 pp.

Shraudy.

5.

E..

and W. Rauscher. 1972.

milk with the brilliant black reduction

in

A9.1

The

contribution to the determination of antibiotics

test.

Archiv fur Lebensmittelhygiene 6:117-121.

Disintegration Method to Determine the IVIicrobial Content


of Paper Container Materials
disintegration

method

is

applicable to paper, paperboard or molded

pulp items in intermediate or complete stages of conversion.


not applicable to items fabricated from plastic, dry-waxed or
per, metal and/or combinations of these materials
is

where the

The method is
wet-waxed paplastic or metal

the food-contacting surface. For these latter items the rinse or

od

swab meth-

be used.
Procedures for obtaining and testing finished single-service containers and
closures are contained in the Manual of Recommended Methods for Sampling and Microbiological Testing of Single Service Food Packages and
shall

Their Components.^

A Equ ipment and s upp lies


.

1.

Pipets, sterile: Capacity 10-ml, with tip opening cut to about 3

for passage of pulp.

20-ml large-bore pipet

is

mm

preferable for plating paper

pulp.
2.

Petri dishes, sterile.

3.

Plating

4.

Phosphate-butfered dilution water,

5.

Scalpels or scissors, sterile.

6.

Forceps,

7.

Balance.

8.

Kraft paper or envelopes, sterile: For protection of samples

medium: Standard Methods Agar.


sterile.

sterile.

in transit

to laboratory.
9.

Disintegrator:

Sterile

interior,

high-speed,

electrically

operated,

with corrosion-resistant metal (stainless steel preferred) cup, capacity about

500 ml. Optionally, use 500 ml

in

a 1,000-ml cup.

Microbial Content of Paper Container Materials

A9.1

345

B. Sampling procedure for paper stock:


Paper stock shall be sampled before it enters into any converting operation. Partially converted items may also be sampled in intermediate stages of
conversion. Sampling methods for these items shall, in general, comply with
sampling methods for paper stock or finished products. Items fabricated
from plastic or plastic-laminated paper or paperboard, dry-waxed paper,
wet-waxed paper, metal and/or combinations of these materials are usually
not sampled before conversion; therefore, sampling methods for finished
products only would apply.
1.

Roll stock sampling: Either of the following

two methods

is

accept-

able.
a.

Roll stock shall be sampled as a butt

roll,

with a

minimum

radial

cm) of paper on the core and a minimum roll


width of 12 in. (30.48 cm), except where mill roll width is less than 12 in.
(30.48 cm) and where the full width of the mill roll constitutes the sample. The cut web shall be firmly taped before removal of the butt roll
from the roll stand. If the width of the roll is over 12 in. (30.48 cm), for
thickness of

in.

(2.54

roll may be cut to a minimum of 12 in. (30.48 cm),


by using either a handsaw or a power band saw, with care to avoid any
unusual contamination. The roll stock sample shall be wrapped carefully in wrapping paper and sealed with tape.
b. Samples which are to be cut from the roll of paper shall be cut
with a clean sharp knife as follows: Cut into roll two laps deep and
discard these first two layers. Then cut into roll six laps deep to a size
of approximately 8 in. x 10 in. (20.22 x 25.40 cm), grasp sample by
top and bottom sheets near corners, remove and tape in three or four
places to keep sheets in alignment, thus protecting inner sheets which
will be tested. When cutting samples from the roll of paper, three sides
of the sample may be cut first, thus forming a flap. This flap may then
be stapled or taped to prevent undue exposure and riffling and the hinge
side of the sample finally cut as assembled sample is removed. Place
in a clean paper envelope, seal, and insert into a stout manila or brown
kraft envelope. For sample protection, envelopes should be of a type
which can be sealed, preferably with a pressure-sensitive adhesive
rather than one which must be moistened.
2. Sheeted stock sampling: Final samples shall consist of a minimum of
six sheets, each approximately 8 in. x 10 in., (20.22 x 25.40 cm). Sheeted
paper selected for sampling shall be taken from any portion of the stock
except the bottom or top 2 in. (5.80 cm). Large sheets shall be reduced to a
final sample size, using a guillotine-type cutter or equivalent means not involving direct handling of any sample sheets. Stacks of cut sample sheets
shall be taped and packaged as in paragraph b above. Top and bottom sheets
shall be considered as protective covers during sampling and shipping, and

convenience the butt

shall

be discarded before testing.

SUPPLEMENTAL MICROBIOLOGICAL CONTROL METHODS

346
C.

Sample preparation:

With

sterile scalpel or

other sterile cutting device, cut representative por-

tions to make 100 g from rolls of paper or paperboard, sheeted stock, nested
containers, blanks or closure discs. With sterile forceps, transfer cut por-

wrappers or envelopes. Using sterile forceps, place


and
other similar closures directly into sterile envelopes.
bottle caps, hoods
from
moisture
or other contamination during transit to the
Protect samples
laboratory. Because some papers contain few bacteria, take great precautions to avoid dust, sputum or other contamination of samples. With sterile scalpel or scissors, cut the middle portion of the sample sheet into pieces
[maximal size 3 ^/s in. (8.6 cm)] and collect the pieces in a sterile petri dish.
With sterile forceps to handle stock, weigh 3 g of sample directly into a
sterile petri dish. Put 300 ml of sterile buffered dilution water into a largesize sterile mixer cup, add 3 g of paper, and re-cover the mixer. In larger
cups, use 500-mI portions of sterile buffered dilution water and 5 g of paper.
Place the cup on the driving mechanism, operate at low speed for 15 sec and
then at high speed for a total time of operation usually not to exceed 2
minutes. Time of operation will depend on type of paper and may require 5
minutes. After 30 sec of high-speed operation, stop the machine. By revolving the cup at a slight angle, rinse all pieces of paper or paperboard from cup
tions to sterile kraft

walls into water.

Resume

disintegration at high speed, but at intervals re-

examine the interior to be sure


remain on sides of the cup.

that

no pieces of undisintegrated material

D. Plating:
Because few bacteria per gram are found in high-quality paperboard used
in making bottle caps, closures and milk containers, and because these bacteria are largely spore-forming types that usually produce spreading colonies on agar plates, accurate counts are often difficult to obtain. Protect plating operations from all dust or other forms of contamination. For high-grade
virgin-stock paperboard, no further dilution is necessary.
With a sterile 10-ml pipet having a large bore at the tip, divide 10 ml of
disintegrated paper or paperboard approximately equally

among

three sterile

C
Pour with Standard Methods Agar, incubate plates at 32
for 48 3 hr, and count colonies. When introducing medium, rotate the
plate so as to mix medium thoroughly with the disintegrated paper or
paperboard. When analyzing board that contains secondary stock, higher
petri dishes.

dilutions are usually required.


5 or

more colonies per

Keep

the pulp so concentrated as to yield

plate. Optionally, use five instead of three plates per

sample.
E. Results, reporting

sum

and standards:

of colonies developing on three or five plates from 0.1 g


of paper or paperboard stock by 10 and report the result as the number of

Multiply the

Preliminary Incubation (PI) for

A11.1

Raw

347

Milk

gram of stock. Recently set industry standards allow not more


than 250 colonies per gram of disintegrated paper or paperboard stock in
three of the last four analyses of these products taken at the mill from difcolonies per

A standard of not more than 250 colonies per gram has been
used for some time and, being calculated as the logarithmic average of plate
counts of the last four analyses taken during a grading period, is somewhat
ferent batches.

more

lenient than the percentage-compliance standard.

F. Test for conforms:

The procedure

to be

used

is

outlined in the manual previously cited.'

A9.2 Reference
1.

Russell. R.T.

Manual of recommended laboratory methods for sampling and microand their components. Syracuse UniversiResearch Corporation, Syracuse. N.Y. 18 pp.
1967.

biological testing of single service food packages


ty

A10.1
Agar

Moseley Keeping-Quality Test

on freshly processed milks provide little information


life of the product. Even if few psychrotrophic bacteria are present, microbial growth may occur during refrigerated storage,
resulting in increased bacterial counts and frequently in detectable flavor
changes. The combination of a preliminary incubation period and an agar
plate count can provide valuable information about the extent of post-pasplate counts

about the potential shelf

teurization contamination of milk products.

A. Procedure:
After making a Standard Plate Count, store containers of freshly proc-

C for 5 or 7 days, then replate.


count between the first and second plating suggest that keeping-quality problems can be expected during refrigerated storage of the product. For specific illustrations of this test under various condiessed milk or cream

Large increases

in

a refrigerator at 7

in bacterial

by Elliker et al.' Many plants


have eliminated the initial Standard Plate Count and only plate samples after
5 or more days of storage.
tions of plant sanitation, consult the paper

A10.2 Reference
1.

Elliker, P.R., Sing, E.L., Christensen.

L.J.,

and W.E. Sandine. 1%4. Psychrophilic


J. Milk Food Technol. 27:69-75.

bacteria and keeping quality of pasteurized dairy products.

Al 1 .1 Preliminary Incubation

(PI) for

Raw

Milk

Count of raw milk may not provide an accurate evalu^- ^


ation of conditions under which milk was produced.'Johns ^ has suggested that the efficient cooling systems used on today's dairy farms can
mask careless practices which may have been used in collecting and han-

The Standard

Plate

'^-

SUPPLEMENTAL MICROBIOLOGICAL CONTROL METHODS

348
dling the milk.

preliminary incubation treatment for samples of raw milk

has been proposed ^ in which milk


mining the Standard Plate Count.

is

incubated 18 hr

at 12.8

before deter-

A Equipment and supplies:


.

1. Glassware and other apparatus: In addition to those needed for the


Standard Plate Count, 10-ml pipets and an incubator set at 12.8 C.
2. Media: Standard Methods Agar.

B. Procedure:

be analyzed should represent more than two milkings from


If only one or two milkings are represented it is suggested
that the sample be held at 4 C for an additional 24 hr before starting the
preliminary incubation at 12.8 C to obtain full benefit of the test."*

The sample

to

a given producer.

After thorough mixing, pipet 10 ml into a sterile plastic vial (capacity

about 30 ml). Temper samples to 12.8 C and incubate at that temperature for
18 hr; then determine Standard Plate Count (or Plate Loop Count) in the
usual manner.

If

a large

number of samples

are to be plated, cool

them to4 C

or lower before plating.

and significance:
The value of preliminary incubation is based on the theory that microorganisms making up the normal flora of the udder do not grow well at
12.8 C, whereas many saprophytic contaminants can grow actively at this
temperature.^ The total count for milk in a bulk tank might not be affected
C. Results

too greatly by use of a milking machine (or other piece of equipment) that

is

The contaminants would, however, probably grow


C than the normal flora of the milk as it comes from the

grossly contaminated.

much

better at 12.8

cow. Thus a high count after preliminary incubation suggests use of careless
has
handling practices which allowed contamination of the milk. Johns
proposed a maximum allowable count of 200,000/ml following preliminary
incubation. Others have narrowed the maximum allowable count to
100,000/ml ^ and at least one company has successfully used 5000/ml as the
basis for bonus payments to its producers. ^^
"^

A11.2 References:
1.

Druce, R.G. and S.B. Thomas.


bacteriological examination.

2.

1968. Preliminary incubation of milk

and cream prior to

review. Dairy Sci. Abstr. 30:291-307.

Johns, C.K. 1960. Preliminary incubation for raw milk samples.

J.

Milk Food Technol.

23:137-141.
2a.

Johns, C.K. 1975. Use of counts after preliminary incubation to improve raw milk quality
Denver plant. J. Milk Food Technol. 38:481-482.
Johns, C.K., Clegg, L.F.L., Leggatt, A.G., and J.M. Nf.sbitt. 1964. Relation between
milk production conditions and results of bacteriological tests with and without preliminary
for a

3.

incubation of samples.

J.

Milk Food Technol. 27:326-332.

A13.1

349

Microbiological Quality of Air

Reinbold, G.W., Johns, C.K., and W.S. Clark, Jr. 1969. Modification of the preliminary
incubation treatment for raw milk samples. J. Milk Food Technol. 32:42-43.
WiLDASiN, H.L. and A.F. Eraser. 1973. Preliminary incubation (PI) as a supplementary
bacteriological test to improve the quality of raw milk. Presented at the Vermont Dairy

4.

5.

Industry Association Conference Sept.

A12.1

12, 1973.

Screening Method (Nutrient Broth Modification) For


Retail Milk Containers

poured from three containers, tested by the Standard Method (Secemploying either buffered rinse solution or Nutrient Broth, sometimes yield no colonies. To determine the frequency with which such containers are encountered, a large number of samples must be examined.
Inoculate 20-ml portions of Nutrient Broth into 50 containers and shake them
Plates,

tion 16.31)

to rinse their surfaces thoroughly. If

cubate containers

at

32

(89.6 F);

space

if

is

available in the incubator, in-

not, incubate

them

at

room temper-

ature. After 48 hr, note the percentage of containers with turbid broth

and

thus demonstrate bacterial growth. Calculate the percentage of containers


exhibiting growth.

A13.1 Screening Tests for Microbiological Quality of Air


A. Selection of method: ^
Viable bacteria, yeasts and molds

in air

may be determined by sedimenta-

and by several types of air samplers.

tion

by particle size and by the velocMicroorganisms that do not settle onto agar from air are not included in the count
obtained by the sedimentation method.
Air samplers to determine the number of microorganisms are chiefly impactors, impingers, centrifuges, ^ filters,*^ and electrostatic precipitators.^
With impaction methods, organisms from a specific volume of air are deposited on agar or on coated or dry surfaces. Under satisfactory conditions,
the agar method is relatively efficient, but it has limitations because too high
a count causes excessive colony growth and hence counting difficulties. Air
temperature must be above freezing. Too long an exposure to airflow during
sampling causes dehydration of the agar surface, which reduces adhesion of
particles and hence fewer colonies will develop. Coated and dry surfaces
In sedimentation, results are influenced

ity

and direction of air currents in relation to the agar plates.

may

not retain all the organisms.


Liquid impingement consists of depositing organisms from air into a liquid, such as buffered distilled water or Nutrient Broth. The method is not

numbers of airborne organisms. Not all organisms are reand some may be destroyed with high impingement velocity. With
extended sampling, air movement tends to reduce the volume of liquid in the
sampler. This problem is aggravated when the liquid contains materials
which tend to produce foam.
suited to small
tained,

SUPPLEMENTAL MICROBIOLOGICAL CONTROL METHODS

350

The

viability

and number of microorganisms

in air

may be

influenced by

temperature, humidity and light rays. Air currents, the number of micro-

organisms on various surfaces, ambient air conditions surrounding the area,


the number and activity of people, other activities, and sanitary conditions
also affect viable counts. These factors should be recorded when air is sampled.

B.

Agar plate preparation:

Using equipment, supplies and procedures described for the Standard


Plate Count, for psychrotrophic counts, or for yeast and mold counts,
aseptically pour 10-ml portions of sterilized tempered agar into sterile petri
plates. Replace covers and allow agar to solidify on a level surface. Hold
plates at 2 to 7 C in a manner which will prevent dehydration of the agar.
These plates with agar are used for sedimentation or impaction methods of
estimating numbers of microorganisms in air.
C. Sedimentation:

Place a new paper towel or parchment paper at the location where air is to
be sampled and set the petri plate horizontally on a flat surface. Expose
Standard Methods Agar or selective medium by removing the plate cover
and, without inversion, place it on the paper beside the bottom of the petri
plate. After exposure of the agar medium for 15 min, replace the cover and
incubate the plate according to appropriate Standard Methods. Colonies are

then counted and results expressed as the

number per square

foot per minapproximately 1/15 sq ft. (61 .8


cm'-^). Standard Methods Agar or Acidified Potato Glucose Agar are generally
used. Other selective media are occasionally used, but not all species for
which a selective agar was designed will grow on it after their recovery from
air. For example, some aerosolized Escherichia coli cells do not grow on
Desoxycholate Agar, but will produce colonies on Standard Methods Agar.
ute, since a standard petri plate

bottom area

is

D. Impaction onto agars: '^-^-^


Several types of commercial samplers are available. Three common designs are the single-stage, the six-sieve, and the cascade impactor. To attain
optimum results, attention must be given to correct airflow rate, distance of
agar from slit, volume of air sampled, and concentration of viable microorganisms.
E. Liquid impingement:

Connect impinger

to

vacuum pump by means of adequate

tubing. Adjust

airflow rate through impinger as specified for the unit. Place sterile buffered

MS

water with antifoam into the impinger and sterilize. Place the sampling
probe in the proper location for procurement of the air sample. Connect
impinger to the suction tube and conduct sampling for Vi hr or for a specific

A14.1

Swabbing Methods

351

time in accordance with the expected number of microorganisms


pare dilutions, plate and incubate

in air. Pre-

accordance with appropriate Standard

in

Methods. Count colonies and record as the number per cubic foot of

air.

A13.2 References
1.

Anderson,

A. A. 1958.

air-borne particles.
2.

J.

New

sampler for the collection, sizing and enumeration of viable

Bacteriol 76:471^84.

Anderson, A. A. and M.R. Anderson. 1%2. A monitor

for air-borne bacteria. Appl. Mi-

crobiol. 10:181-184.
3.

CowN, W.B., Kethley, T.W., and

E.L. Fincher. 1957, The critical-orifice liquid impinger

as a sampler for bacterial aerosols. Appl. Microbiol. 5:119.


4.

Decker. H.M., Juehne, R.W., Buchanan, L.M., and

R. Porter. 1958. Design

and evalua-

tion of a slit-incubator sampler. Appl. Microbiol. 6:398.


5.

DiMMiCK. R.L. and A.B. Akers.


Wiley

6.

&

Sons, Inc.,

Ehrlich.
brane

R. 1955.

filters. J.

New

1969.

An

introduction to experimental aerobiology. John

York.

Technique

for microscopic count of

microorganisms directly on mem-

Bacteriol. 70:265.

8.

Kraemer, H.F. and H.F. Johnstone. 1955. Collection of aerosol particles in presence of
Chem. 47:2426.
May, K.R. 1945. The cascade impactor: an instrument for sampling coarse aerosols. J. Sci.

9.

Olson, H.C. and B.W. Hammer.

7.

electrostatic fields. Indust. Eng.

Instr. 22:187.

plants.
10.

J.

1934.

Number

of microorganisms falling from air of dairy

Dairy Sci. 17:613-623.

Sawyer, K.F. and W.H. Walton.


for air-borne particles.

J. Sci. Inst.

1950.

The conifuge

size separating

sampling device

27:272.

A14.1 Swabbing Methods: Calcium Alginate, Dacron


(Polyester) and Rayon Swabs

The swab-contact method using cotton may be employed to determine


conformance with efficient sanitation practices. The fibers of cotton swabs
mechanically trap some organisms, so that not all of those displaced from
the swabbed surface are liberated into the rinse solution. Swabs made from
calcium alginate, polyester and rayon minimize this problem.
A. Procedure:

Swabs made of calcium alginate fibers ' ^' ^are soluble in aqueous
solutions (rinse, culture media, etc.) containing 1% of sodium hexametaphosphate (or sodium glycerophosphate, or sodium citrate, or 1% of
any mixture of these). All the organisms dislodged from swabbed surfaces
are thus liberated. When using calcium alginate swabs,* prepare rinse solu"*

tion vials to contain 4.5

10%

*^

ml (rather than 5.0 ml) after

sterilization.

Prepare a

sodium hexametaphosphate or other solubilizing compound,


package in appropriate vials, and autoclave. Follow the standard procedure but after swabbing the final (fifth) 8-sq in. (51.6 cm) area and depositing the swab head in the rinse vial
add 0.5 ml of sterile solubilizing solusolution of

tion.

*Obtainable from Consolidated Laboratories, Inc., Glenwood.

III.

60425.

SUPPLEMENTAL MICROBIOLOGICAL CONTROL METHODS

352

Swabs made of Dacron (polyester) fibers have been found to rinse out
more completely than those made with cotton.'^
Rayon swabs are also available for comparable laboratory use."

A14.2 References
Angelotti.

1.

R..

Foter, M.J., and K.H. Lewis.

1958.

comparative evaluation of meth-

2.

ods for determining the bacterial contamination of surfaces. Food Res. 23:175-185.
Barnes, J.M. 1952. The removal of bacteria from glass surfaces with calcium alginate,

3.

guaze. and absorbent cotton-wood swabs. Proc. Soc. Appl. Bacteriol. 15:34.
Cain, R.M. and H. Steele. 1953. The use of calcium alginate soluble wool for the examiJ. Pub. Health 44:464^67.
HiGGiNS. M. 1950. A comparison of the recovery of organisms from cotton-wool and calcium alginate wool swabs. Ministry of Health and Public Health Laboratory Service (Gt

nation of cleansed eating utensils. Canad.

4.

Monthly Bull. 9:50-51.


HoLLiNGER, N.F. and L.H. Lindberg. 1958. Delayed recovery of streptococci from throat
swabs. Amer. J. Pub. Health 48:1162-1169.
Tredinnick, J.E. and J. Tucker. 1951. The use of calcium alginate wool for swabbing
Brit)

5.

6.

dairy equipment. Proc. Soc. Appl. Bacteriol. 14: 85.

A15.1 Testing of Sterilized or Microbiologically Stable Milk

Products
been an appreciable increase

In recent years there has

in the

number and

types of milk products offered for sale that are labeled "sterilized" or "longlife" or the equivalent.

uct

is

The designation "long-life"

microbiologically stable in that, although there

present, they are in a state

where they

indicates that the prod-

may

be viable bacteria

either will not multiply or

multiplication will be so slow as to be insignificant over the shelf

product. Refrigeration
ilized;

however,

it

is

may

2.

may

not be required for products

where
of the

marked

ster-

required for those that are marked long-life.

A Procedure for sterile


.

or

life

or sterilized products:

Incubate the product

in the original

container for one week at 32 C.

Plate 0.2 ml of undiluted product in each of

two

petri dishes

and

prepare pour plates with Standard Methods Agar.


3.

at

Incubate one plate aerobically and the other anaerobically for 48 hr

32 C.

4. Count both plates and record the results.


Caution should be observed in interpreting the significance of low numbers of colonies in either the aerobic or the anaerobic plate. If the product
was not sterile, the numbers of viable bacteria should have increased to
several million per milliliter during the incubation period. This would give an
almost solid mass of colonies where 0.2 ml of undiluted product was plated.

^Obtainable from Scientific Products, McGaw Park, 111; Curtin Scientific Company, Houston,
Tex; or Scientific Glass Apparatus Company, Bioomfield, N.J. 07003.
"Obtainable from Fuller Laboratories, Inc., 7900 Fuller Road, Minneapolis, Minn. 55401.

353

Lactic Culture Bacteriophage

A17.1

few colonies on either or both plates may be the

result of inadvertent

contamination by the analyst.


B. Procedure for long-life or equivalent products:

For those products marked long-life or the equivalent, microbiological


prevent the consumer from receiving either a microbiologically decomposed or a hazardous product. The following procedure is
stability is required to

believed useful

in

detecting undesirable microbial growth:

in the original container at 10 C for one week.


Prepare
1:100
dilution
of the product and plate in 1-ml amounts in
2.
a
each of two plates. Add Standard Methods Agar as in routine testing of
pasteurized milk (see Chapter 5).

Incubate the product

3.

Incubate one plate aerobically and the other anaerobically

at

32

C for

48 hours.
in the usual manner and record results.
requirement that the product be microbiologically stable,
fewer than 5,000 bacteria should be present per milliliter of incubated product (< 50 colonies on either the aerobic or the anaerobic plate). To be meaningful, the sampling program for these products must include samples found

4.

To

Count colonies

satisfy the

in stores.

A16.1 Automated Plate Loop Count Procedure

A machine # is available which automates the plate loop count procedure


(PLC) (Chapter 21). It adds a constant amount of agar, inoculates 0.001-ml
sample, mixes, cools, identifies and stacks plates at up to 300 per hour. The
standard error of difference between duplicates has been reported to be
lower using the machine than that produced between PLC duplicate differences or between PLC and the instrument. The unit also plates 0.01-dilutions, however, a large loop has not been found satisfactory.^ Either a cylinder should be considered or additional comparative and collaborative
studies are needed before approval can be given to the larger loop part of the
machine.

A16.2 Reference
Donnelly, C.B., Leslie,

1.

Read,

Jr.

products.

J.E.,

1970. Cylinder count


J.

Messer, J.W., Green, M.T., Peeler. J.T., and R.B.


method for determining plate count in pasteurized milk

Dairy Sci. 53:1187.

A17.1 Detection and Enumeration of Lactic Culture

Bacteriophage
Cheese plants which use known

strains of lactic culture need to occasionmonitor for build-up of bacteriophage (phage) particles during cheese
manufacture. It is best to use whey collected near the end of the production

ally

*The

unit

is

distributed by Foss

America

Inc., P.O.

Box

504, Fishkiil,

New York

12524.

SUPPLEMENTAL MICROBIOLOGICAL CONTROL METHODS

354

example at milling of Cheddar cheese. The whey can be filtered, to


remove host bacterial cells, and then phage particles can be enumerated

cycle, for

using standard dilution procedures and a plaque forming technique. Alternatively the effect of diluted

phage

filtrate

upon acid production

in sterile re-

constituted nonfat dry milk substrate containing an acid-base indicator and


the host bacterial strain can be used to estimate

rich

medium

phage

particle

numbers.

incorporating /3-glycerol phosphate allows superior lactic

organism growth and development of phage plaques of all strains tested:^


The dilution method of enumerating phage is conducted by filling capped
test tubes with 9 ml of 9.5% nonfat dry milk and 0.015% brom cresol purple
(BCP), heating at 121 C for 15 min, cooling to 30 C and inoculating with
0.1 or 0.2 ml of the host bacterial strain. Litmus milk may be used in place of
the

BCP

milk.'

Suspect whey

is passed through a suitable filter device which retains bacand allows phage passage. A disposable plastic syringe fitted with a
sterile 0.45-^tm filter cartridge ^ is adequate (sterile Millex disposable filter
unit, 0.45 /xm, 25-mm diameter). One milliliter of filtrate is added to contents
of the first tube and mixed. One milliliter from the first tube is then transferred to the 2nd tube, etc. Inoculated tubes should be covered and in-

teria

cubated

at 32

C. Inhibition of acid production

is

evident following 3.5 to 8 hr

of incubation. Control tubes without phage inoculation can be used to assure

normal acid production and proper operation of the test.


Tubes which indicate acid production compared with those which indicate
inhibition of acid production can be used to estimate phage titer in the original filtrate.

For qualitative screening, when phage enumeration is not required, a drop


filtrate from the filter unit can be added directly to tubes of BCP or litmus
milk that contain the host culture. Replacement of lactic strains which become ineffective through phage activity can be simplified through recently
developed strain selection procedures.
of

A17.2 References
Frazier,

W.C, Marth,

E.H., and R.H. Deibel. 1968. Laboratory manual for food micro-

biology. Burgess Publication Co. Minneapolis, Minn. Page 79.


2.

Heap. H.A. and R.C. Lawrence.

New
.1.

Zealand

J.

1976.

The

selection of starter strains for cheese making.

Dairy Sci. Technol. 11:16-20.

Terzaghi. B.E. and W.E. Sandine.

1975.

Improved medium

their bacteriophages. Appl. Microbiol. 29:807.

'Millipore Corporation. Bedford Mass. 01730, or equivalent.

for lactic streptococci

and

APPENDIX B

SUPPLEMENTAL CHEMICAL CONTROL METHODS


L.J.

J.A. Burke, D.

Acidity: Titratable

B1.1

B1

Bianco, G.H. Richardson, J.W. Sherbon,


R. Ginn, and D.T. Liden

Lehman, W. Roth,

.11

Dry Whey, Buttermilk, and

Whey Concentrate

Products

A. Apparatus and reagents:


1. Pipets: Standard milk, 17.6 ml TC, and 25-ml volumetric
2. Buret: 25-ml capacity, graduated in 0.1-ml divisions, preferably a
precision-bore type. Equipment with bottle assembly top openings should
be protected with soda lime tubes. Optional: Nafis
3. Sodium hydroxide, 0.1 N.
4.

titration.

Phenolphthalein. 1.0%.

carbon dioxide-free: Prepare by boiling water for


by cooling without agitation. As an alternate method, bubble
a rapid stream of air (which has been passed through a soda lime tube)
through the water at room temperature until a 200-ml portion of the water
yields a pink color on addition of 1 drop of O.IN sodium hydroxide using
1% phenolphthalein indicator. Store carbon dioxide-free water in a stop5.

Distilled water,

5 min. followed

pered container.
6. Torsion balance or equivalent, 0.1-g sensitivity
7. Volumetric stoppered flask, 100 ml
8. Beakers, 100 ml, 250 ml
9. Glass stirring rod with rubber policeman
10. Erlenmeyer flask, narrow mouth, 125 ml; or casserole, porcelain
70 x30 mm. 50 ml; or 100-ml beaker
11.

Wash

12.

Graduated cylinder, 100-ml capacity

bottle

ISC Liaison: G.H. Richardson


355

SUPPLEMENTAL CHEMICAL CONTROL METHODS

356
B. Procedure:

Weigh accurately 6.5 g of dry whey or 7.5 g of dry buttermilk, or 2 g of


whey concentrate into a tared 100-ml beaker. Add about 25 ml of carbon
dioxide-free distilled water and

pend sample
umetric

Make up

flask.

stir

slowly with glass rod to dissolve or sus-

Transfer with the aid of a wash bottle to a vol-

in solution.

volume and mix

to

well.

whey or dry buttermilk, or


whey concentrate into an Erlenmeyer flask. Rinse pipet with
an equal amount of distilled water and add rinse to sample in flask.
Add 0.5 ml of phenolphthalein indicator and titrate with O.IN sodium hyPipet 17.6 ml of the prepared sample of dry

pipet 25 ml of

droxide to the

first

permanent (30

change from clear

sec) color

to pink.

C. Calculations:

Acidity

is

expressed as

lactic acid (1

ml of

0.

N NaOH =

0.009 g of lactic

acid).

Reconstituted basis: (dry

whey and

^
%

ml of O.IN

.,.

Acidity

buttermilk)

NaOH

x 0.009 x 100

18

Dry

basis: (dry

~
%

whey and
.

,.

Acidity, dry

buttermilk)

whey =
,

Acidity, dry buttermilk

Acidity,

acidity, reconstituted basis

whey concentrate =

-,

= ml
D. Note: The tabulation below

is

results to reconstituted basis (based

(weight of aliquot sample)

of O.IN

NaOH

x 0.30

useful in converting dry basis dry

on 6.5%

liquid

whey

solids):

Reconstituted

Dry

basis

basis

0.11

1.7

0.12

1.9

0.13

2.0

0.14

2.2

0.15

2.3

0.16

2.5

0.17

2.6

0.18

2.8

whey

357

B1.13 Fluid Whey, Milks, Ice Creams, Sherbets, Ices, Yogurts

Whole and Nonfat Dry


A. Apparatus and reagents:

B1

2 Dry

Same

as

F/t

in.

B 1.1 1(A)

Electric

13.

and Chocolate Malted Milk Products

with the addition

mixer,*

diam x V4

Milk

in.

(replace

(4.34 x 0.63

of:

steel

cm)

with round Teflon

disc

stirrer,

thick).

B. Procedure:
10 g of nonfat dry milk or chocolate or malted milk; or 13 g of dry

Weigh

250-ml beaker. Add about 100 ml of carbon dioxidesample and mix for no more than 1 min to avoid
formation of excessive foam.
hour. With stirring rod, gently but thoroughly mix
Allow to stand for
sample. Pipet a 17.6-ml sample into an Erlenmeyer flask or casserole. Rinse
out the same pipet with 17.6 ml of distilled water and add to sample in flask

whole milk

into a tared

free distilled water to the

or casserole.

Add

0.5

ml of phenolphthalein indicator and titrate with O.IN sodium hyfirst permanent (30 sec) color change from clear to pink.

droxide to the

C. Calculations:

Acidity

is

expressed as

lactic acid (1

ml of 0.

N NaOH =

0.009 g of lactic

acid)

_
%

...

acidity

ml of O.IN
rr-

NaOH

ml of O.IN

NaOH

X 0.009 X 100
f

% acidity

or

20

B1.13 Fluid Whey, Milks, Ice Creams, Sherbets,


A. Apparatus and reagents:

Same

r^;

18 (weight of sample)

Ices,

and Yogurts

as Bl. 11(A).

B. Preparation of sample:

Mix milk thoroughly by shaking or stirring gently. If milk is lumpy or


C in a thermostatically controlled water bath.
2. Ice cream, ice cream mixes and sherbet samples should be first
warmed to room temperature to eliminate the high viscosity that may be
1

frozen, heat to 37

encountered.
C. Procedure:

Weight 9- or 18-g sample into a tared 100-ml beaker. If an 18-g sample is


used, add carbon dioxide-free distilled water in accordance with directions
in B1.13(E.l) to the sample and mix gently but thoroughly.

Hamilton Beach #936. Racine, WI

43401.

SUPPLEMENTAL CHEMICAL CONTROL METHODS

358

Add

0.5 ml of phenolphthalein indicator

droxide to the

first

permanent

D. Calculations:
Acidity expressed as

and titrate with O.IN sodium hychange from white to pink.

(30 sec) color

lactic acid (1

ml of O.IN

NaOH

= 0.009 g of

lactic

acid)

^
%

% acidity
%

.^.

acidity

ml of O.IN

NaOH

x 0.009 x 100
or

2^

weight or sample

ml of O.IN
(using 18 g sample)

acidity (using 9 g sample)

NaOH

20

ml of O.IN

NaOH

10

E. Notes: The amount of distilled water used to dissolve or suspend


sample will vary with the product type as indicated below:
1. Fluid whey: When 18-g sample is used, rinse pipet with 18 ml of distilled water; when 9-g sample is used, rinse pipet with 9 ml of distilled water.
2. Whole, skim and chocolate milks: Rinse pipet with two volumes of
distilled water.
3.

Condensed and evaporated

ml of

18

4.

milk:

When

pipet with 36 ml of distilled water.

When

18-g sample

9-g sample

is

is

used, rinse

used, rinse pipet with

distilled water.

^Ice

Cream and

ice

cream mixes

Same as

in

condensed and evapo-

rated milk.

Same as condensed and evaporated milk.


Same as condensed and evaporated milk.

5.

^Sherbets and ices

6.

^Ycgu?ts

in

in

B1.2 References
1.

American Dry Milk Institute,


methods of

2.

Doan,

Inc. 1971. Standards for grades of dry milk including

analysis.

F.J. et al. 1957

Procedures for making acidity tests of

fluid

milk products.

J.

Dairy

Sci. 40:1643-1644.

B2.1 Acidity: Potentiometric,


B2.11

pH

Cheese

A. Apparatus and reagents:


1.

2.

pH

meter.
Beaker, 50 ml.

^On samples of chocolate-colored and

different flavored ice

cream, sherbets and yogurts,

water instead of 36 ml. Also, if sample is dark (as strawberry,


may be necessary after addition of distilled water, to add 2 ft (60.9 cm) of

rinse pipet with 45 ml of distilled

raspberry, cherry)

it

The
room temperature and proceed with

white yarn and boil for 10 min or until yarn has absorbed the color.

slight residual color will

fade during the titration. Cool to

titration.

B2.1 Acidity: Potentiometric

pH

359

3.

Thermometer, 0-100 C, graduated

4.

Wash

5.

Kleenex or Kimwipes or equivalent.

6.

Electrodes: Glass electrode used in conjunction with calomel elec-

in

1-C divisions.

bottle.

trode (alternative, combination electrode).

ACS

7.

Benzene,

8.

Buffer solutions,

grade.

pH

4.00 and

pH

7.00 (do not use buffer concen-

trates).

Potassium chloride, saturated: Used to refill electrodes.


10. Distilled water, carbon dioxide-free: Prepare by boiling water for
5 min and then cooling without agitation. Alternatively, rapidly bubble a
stream of air that has been passed through a soda lime tube through the
water at room temperature until a 200-ml portion of water yields a pink color
after addition of 1 drop of 0. IN sodium hydroxide, using 1% phenolphthalein
9.

indicator.
1

Potassium chloride solution: Dissolve 35 g

in

100 ml of carbon diox-

ide-free, preferably deionized, distilled water.

Mild soap or detergent solution: For washing electrodes.


B. Instruction for care and use of electrodes:
1. Handle carefully. The electrode is the most important and sensitive
part in a pH measurement, and affects the accuracy of measurement.
12.

2. Before using a new glass electrode, soak it for at least 2 hr in distilled


water to assure proper functioning.
3. The electrode must be kept exceptionally clean. Rinse electrode between buffer and samples with distilled water and blot with tissue.
4. When working with cheese samples containing fatty substances,
which may reduce sensitivity of the electrode, it may be necessary to remove the film by swabbing bulb with benzene solvent or mild soap solution.

Rinse thoroughly with water and blot with tissue.


5. When used in measuring or standardizing, the electrode should be
sufficiently immersed in the sample to cover the pH-sensitive bulb.
6. Between measurements the electrodes should be stored in distilled
water. This insures that electrodes are always ready for measurement. If
electrodes are not to be used for several months, they may be stored dry. In
this instance, the glass electrode must be kept in water for a few days before
reuse, to regenerate the gel layer.

fill

7. The protective sleeve on a glass electrode must be moved below the


portion of the electrode to insure proper operation.
8. The combination electrode calls for the following additional proce-

dures:

Vent the reference side of the electrode before use, by removing the rubfilling tube. This permits slow leakage of solution from the
electrolyte bridge, insuring good electrical contact at the liquid junction and
ber cap at the

preventing reference contamination.

SUPPLEMENTAL CHEMICAL CONTROL METHODS

360

Since the potassium chloride solution will diflFuse outward, it is necessary


potassium chloride through the refill aperture. Use a

to replace saturated

saturated potassium chloride solution purchased for this purpose.


Store electrode between periods of use by immersing the pH-sensitive tip
in

potassium chloride solution. This maintains the necessary hydrated sur-

face of the glass electrode and insures against leakage. Storage in distilled

water can result


in

in

a high-resistance junction, which

may cause

inefficiency

the operation of the electrode.

C. Standardization of
1.

pH meter:

The pH meter should be turned on V^ hr before use so

the in-

strument stabilizes.
2. The pH meter must be standardized with buffer solutions before

making determinations.
3. Pour approximately 25 ml of buffer solution into a 50-mI beaker. The
pH of buffer solution should be as close as possible to the pH of the sample
being tested.

both

pH

If

pH determination range varies, standardize meter, using


pH 7.0 buffers [B2.11(E.l)] and follow instructions in pH

the

4.0 and

meter manual.
D. Procedure:
1.

Immerse combination electrode

the pH-sensitive bulb

is

directly into cheese

sample so that

covered.

2. Take temperature of sample and set temperature compensator to


correspond to temperature of sample. For best results, the temperature of
the sample should be 25 3 C. Make sure electrode is in contact with
sample for at least 45 seconds.
3. Activate the pH meter and read the pH directly [B2.11(E.2)].
E. Notes:
1. For accurate work, use freshly prepared buffer solution. Buffer solution bottles should be capped when not in use to prevent evaporation and
contamination. Never pour buffer solutions back into bottle. Discard at the

end of day.
2.

The pH meter should be restandardized

operation.

When

used for at
procedure.

least

periodically during the day's


changing types of samples, or when meter has not been

'/-^

hr,

it

is

advisable to repeat the regular standardization

B2.12 Butter and Margarine


A. Apparatus and reaj^ents:

Same

as B2. 11(A) with addition of:

13.

Beakers, 30, 40 ml

14.

Centrifuge, see 19.41(A.l)j.

15.

Refrigerator

B. Instruction for care

Same

as B2. 11(B)

and use of electrodes:

B3.1 Alkalinity of

Ash

361

C. Standardization of
Same as B2. 11(C)

pH

meter:

D. Preparation of sample:
1. Preparation of serum: Heat about one quarter pound (113.5 g) of butter or margarine in a 400-ml beaker to 46-52 C in a water bath, so that the fat
melts and rises to the top while the serum goes to the bottom.
(possibly 45 min),

little

time

may be

required for this separation to occur. The sample


should not be agitated during this period. When separation is complete, re-

move

the serum by plunging a pipet through the fat layer and drawing up the
serum, which should then be placed in a 30-ml beaker and centrifuged for
3

min

in

Babcock centrifuge.

Place the beaker containing the serum in a refrigerator for approxi-

2.

mately

2 hr,

which

will

permit any

fat

remaining

in the

serum

to rise to the

top and solidify.

After fat has formed a hard layer, plunge a pipet through it and draw off all
serum possible, transferring it to a clean 30-ml beaker. Warm the serum to
25 C and make the pH determination.
E. Procedure:
Same as B2. 11(D) except immerse directly into the serum from butter or

margarine.

B3.1

Alkalinity of
Milk

Ash

in

Dry Buttermilk, Nonfat and Whole Dried

A. Apparatus and reagents:


1.

Graduated cylinder, 50 ml.

2.

Pipet, volumetric, 50 ml.

3.

Burette,

Mohr

type, 50 ml, and bottle assembly, openings protected

with soda lime tubes.


4.

Beakers, 400 ml.

5.

Wash

6.

Stirring rods with rubber policemen.

7.

Watch

8.

Desiccator,

bottle.

glasses, 90
filled

mm.
with efficient desiccant such as Drierite (calcium

mesh, calcium chloride, etc.


Evaporating dish, porcelain, Coors #4.
10. Wire gauze with an asbestos mat center.

sulfate), 4
9.

1.

Bunsen burner.

12.

Iron tripod with support for dish.

13.

Tongs, muffle and crucible.

14.

Analytical balance.

15.

Muffle furnace, thermostatically controlled.

16.

Steam bath.
Hot plate, thermostatically controlled.

17.

SUPPLEMENTAL CHEMICAL CONTROL METHODS

362

Hydrochloric acid,
Sodium hydroxide,

18.

19.

0.

IN.

0.

IN.

Calcium chloride solution 40%. Dissolve 885 g of calcium chloride


(CaCl2-2H20) in 783 ml of distilled water. Add a few drops of phenolphthalein indicator and adjust to neutrality with 0. IN hydrochloric acid. Filter and
20.

store in a rubber-stoppered bottle.


21. Phenolphthalein indicator,

1.0%

B. Procedure:

Weigh accurately

2 g of dry milk into a porcelain evaporating dish. (Later

if exactly 2 g are weighed.) Place sample on an


asbestos gauze over a Bunsen burner and slowly carbonize. (The flame
should be adjusted to a height whereby carbonization is complete in about
30 minutes. The sample should not be carbonized to such a point where any

calculations are simplified

part of

it

becomes

Place dish

in

white.)

muffle furnace regulated at 540 to 550

for

hour.

(It is

very

important that the specified temperature range be maintained. If temperature


exceeds 550 C or is allowed to drop below 540 C, chances are that some of
the samples will be incompletely ashed as indicated

by carbon specks pres-

when the ash is dissolved. This condition causes erroneous results.)


Remove dish from furnace and cool. Moisten ash with a small amount

ent

of

water to form a paste and gently crush the ash particles with a rubber policeman. To remove any adhering ash particles, rinse policeman with a
few milliliters of distilled water and allow rinsings to drain into dish. Evaporate sample solution on steam bath. Remove dish and dry contents on a hot
plate regulated at 100-120 C. (If hot plate is too hot, there is danger of ash

distilled

spattering due to residual moisture present.

sample, temperature of the hot plate

may be

After partial drying of the


increased to assure thorough

drying of the ash.)


Place dried sample dish back in muffle furnace, observing the same precautions as noted above and re-ash for 1 hour. Remove dish and place in
desiccator to cool. Re-weigh dish. This dish weight may be used to calculate
is not used in the alkalinity calculations.
Moisten the ash with a small amount of distilled water and transfer, with
the aid of a rubber policeman, toa400-ml beaker. Pipet 50 ml ofO.lN hydrochloric acid and add to sample in beaker, cover with a watch glass and heat
to boiling. Boil gently for 5 min and avoid spattering! Allow beaker to cool
and rinse the watch glass with recently hoilcd distilled water. Allow rinsings
to drain back into the beaker. Add 30 ml of 40% calcium chloride solution,

the per cent ash in the sample, but

cover with a watch glass and allow to stand for 10 minutes.


of phenolphthalein indicator solution and titrate with
IN sodium hydroxide. The end point is marked by a faint pink color which

gently

Add

0.

stir,

10 drops

persists for 30 seconds.

363

B4.1 Chlorides

C. Calculations:
All results are expressed as

"ml of O.IN hydrochloric acid per 100 g of dry

milk".
If exactly 2 g of sample are weighed out and the hydrochloric acid and
sodium hydroxide solutions are exactly O.IN, the calculation is simplified
and may be expressed as:

ml O.IN HCl/lOO g = 50 X (50-ml of sodium hydroxide used)


weight of the sample

If the

is

not exactly 2 g, and the solutions are not

exactly O.IN, the following formula

is

used:

ml O.IN HCl/lOOg =
1000

Weight of sample

X (50 X normality of hydrochloric acid


,.
,.
^
normality of sodmm hydroxide)
,

or
50

- ml of O.IN sodium hydroxide x

100

Weight of Sample

B3.2 Reference
1.

Association of Official Analytical Chemists.

1975. Official

12th ed. Association of Official Analytical Chemists. Washington.

B4.1
B4.11

methods of analysis,

DC.

Chlorides
Milk

and Cream

A. Apparatus and reagents:


1. Buret, glass stoppered, 50-ml bottle assembly, or 50-ml buret, 0.1-

ml divisions.
cream, volumetric, 9 ml.

2.

Pipet, standard

3.

Glass

4.

Porcelain dish, Coors,

5. Pipet,

stirring rod.

USA

#1.

Mohr, 10-ml graduated

in 0.1 ml.

6.

Distilled water.

7.

Silver nitrate, O.IN.

8.

Potassium dichromate indicator, 10% solution.

B. Procedure:

cream into a porcelain dish.


volume of water into the same

Pipet 9 ml of milk or
pipet with an equal

Add

If

sample

is

cream, rinse

dish.

ml of potassium dichromate indicator. Titrate with O.IN silver


red-brown color, lasting 30 seconds.

trate to the first perceptible pale

ni-

SUPPLEMENTAL CHEMICAL CONTROL METHODS

364

C. Calculations:

^
%

^,

Chlorides

ml of

silver nitrate

of silver nitrate x 3.55

Weight of sample
35.46 X 100
rzr^

, ^^
Factor 3.55 =

-,c

.^

where 35.46

is

the molecular weight

D. Note:
The weight of sample is assumed to be 9 grams. This is not exact but the
salt content of milk or cream is so low that any diflference due to error in
weight of sample is beyond the accuracy of the test.

B4.12 Cottage Cheese


A. Apparatus and reagents:
1.

Torsion balance, 15-mg sensitivity, or analytical balance.

2.

Beaker, 250 ml.

3.

Graduated cylinder, 25 ml.


Thermometer,
to 100 C.

4.
5.

Electric mixer.

6.

Wash

7.
8.

Volumetric flask, 100 ml.


Glass funnel, 75 mm.

9.

Filter paper,

bottle.

Whatman #2

folded, 12.5 cm.


narrow mouth, or casserole 70 x 30 mm.

10.

Erlenmeyer

11.

12.

Volumetric pipet, 25 ml.


Buret, 50-ml graduated in 0.1-ml divisions.

13.

Spatula, stainless steel.

flask, 125 ml,

14.

Distilled water.

15.

Silver nitrate, O.IN.

16.

Potassium chromate, 10%.

B. Procedure:

With the aid of a spatula weigh exactly 10 g of sample on a torsion balance


250-ml beaker. Add about 15 ml of warm distilled water (50-55 C) and
mix to a thin paste with an electric mixer. Add about 25 ml of distilled water
and mix until sample is dispersed.
Transfer solution to a 100-ml volumetric flask. Rinse beaker with distilled
water several times and add rinsings to volumetric flask. Make to volume
and mix thoroughly. Filter through folded filter paper, and collect approximately 50 ml of filtrate.
Pipet 25 ml of filtrate into a clean flask or casserole. Add
ml of potassium
dichromate indicator and titrate with O.IN silver nitrate to the first perceptible pale red-brown color, lasting 30 seconds.
into a

C. Calculations:

^
%

,.

,,

..

Sodium chloride =

ml of

silver nitrate

of silver nitrate x 5.85

weight of aliquot (2.5 g)

B4.1 3 Cheeses: Volhard

365

D. Notes:
c

T^

oc

58.45 X 100

1.

Factor 5.85

2.

Aliquot sample weight

dilution

CO .c

where 58.45

^^^

the molecular weight.

is

is determined by dividing sample weight by


and multiplying by volume pipetted.

grams of sample x volume


ml of dilution
B4.13 Cheeses: Volhard
A. Apparatus and reagents:
Balance, analytical.

1.

2.

Beaker, 50 ml.

3.

Boiling discs, porcelain, Coors (42401, size #1).

4.

Bottle, wash.

5.

Buret, 50 ml, 0.1-ml divisions.

6.

Buret, for dispensing nitric acid, 100 ml.

7.

Filter paper,

8.

Flask, Erlenmeyer, 250 ml.

9.

Flask, volumetric, 1000 ml.

Whatman #2

folded, 12.5 cm.

mm.

10.

Funnel, 75

11.

Graduate, 25 ml.

12.

Hood

13.

Hot

14.

Pipet, automatic with

15.

Pipet,

16.

Stirring rod with flat end, stainless steel.

or adequate air exhaust area.

plate.

T joint,* 5-ml

Mohr, 10 ml, 0.1-ml

17.

Thermometer, 0-100 C.

18.

Tongs.

19.

Distilled or deionized water.

20. Ferric

ammonium
ACS,

sulfate,

capacity.

divisions.

FeNH4(S02)212H20, ACS.

sp gr 1.42.

21.

Nitric acid,

22.
23.

Potassium permanganate, ACS.


Potassium thiocyanate, ACS.

24.

Silver nitrate,

25.

Sucrose.

ACS.

B. Solutions:
1.

2.

1-liter
3.

4.

Ferric

ammonium

sulfate, saturated, standard indicator.

Potassium permanganate, 5%: Dissolve 50 g in


volumetric flask. Make up to volume and mix.
Potassium thiocyanate, O.IN.
Silver nitrate, O.IN.

^Catalog #JP-6875B, Scientific Glass Apparatus, Bloomfield,

NJ

distilled

07003.

water

in

SUPPLEMENTAL CHEMICAL CONTROL METHODS

366

C. Procedure:

Accurately weigh about 2 g of sample into a tared 50-ml beaker. Add about
20 ml of warm (50-55 C) distilled water and break up particles with the flat
end of a stainless steel stirring rod until a uniform slurry is formed. Quan-

250-ml Erlenmeyer flask. Rinse beaker with


C and add to flask.
O.IN
silver
nitrate
from a buret to the sample
25
ml
of
Add exactly
[B4.13(E.l)]. Immediately add 10 ml of halogen-free nitric acid and about 50
ml of water. Add one clean boiling disc. Place on hot plate and allow to boil
[B4.13(E.2)]. Add in 5-ml portions, as the solution boils, a sufficient amount
(usually 15 ml) of potassium permanganate until the solution turns brown
and remains brown for a minimum of 5 min of gentle boiling [B4.13(E2,3)].
Continue heating until brown color disappears with a resulting clear, strawcolored solution. If the solution is not clear, add more 5-ml portions of permanganate until the solution becomes clear (indicating complete oxidation of
titatively transfer slurry to a

about 10 ml of distilled water at 50-55

organic material).
filter paper into a clean 250-ml Erpaper thoroughly with hot distilled water. Cool
the solution to room temperature. Add 2 ml of ferric ammonium sulfate indicator and titrate excess silver nitrate with 0. IN potassium thiocyanate to a
red-brown color change lasting 30 seconds. Subtract blank value obtained
using 2 ml of HgO in place of 2 g of cheese from final titration obtained.

Filter the hot solution

lenmeyer

flask.

Wash

through folded

filter

D. Calculations:

Sodium chloride =
58.5

(ml X

silver nitrate)

(ml x

potassium thiocyanate) x 1000 x 100

grams of sample

Chloride ion

=
35

(ml x

silver nitrate)

(ml x

potassium thiocyanate) x 1000 x 100

grams of sample
E. Notes:

Method can be used

1.

added

nitrate

enough

to

sample

will

for

most dairy foods. The amount of

vary for

diff"erent

products.

It is

silver

important to add

have an excess.
2. Addition of potassium permanganate to the boiling solution is hazardous. Add the permanganate down the side of the flask in small portions.
Direct addition of permanganate into the center of the boiling solution could
cause the solution to "pop" and spray the operator with hot acid.
3. If too much permanganate is added to the solution and the brown
color does not clear in 15-20 min, add a very small amount of sucrose.
4. Boil the solution in an area where the air exhaust system is adequate.
silver nitrate solution to

367

Extraneous Matter

B5.1

B4.2 Reference
Association of Official Analytical Chemists.

1.

methods of analysis, 2nd

1975. Official

supplement to 12th ed. Oct 13-16. 1975.

Extraneous Matter

B5.1

Cheeses
A. Apparatus and reagents:

B5.11

1.

ical

Natural

Balance, torsion or equivalent, 0.1-g sensitivity.

2.

Beaker, 400 ml.

3.

Beaker, 2

liter

or quart, stainless steel with slotted cover for mechan-

liter

or quart, Bain Marie^ stainless steel #7872, with cover

mixing.
4.

Beaker, 2

#7904.
5.

Blender, Osterizer or equivalent with

6.

Bottle, glass-stoppered.

7.

Bottle, wash.
Cellophane envelopes,"

8.

Ys

in.

quart (946 ml) jar.

3 in. (4

x 7.6 cm) B5.12(D.l).

Extraneous matter record cards.


Filter flask, with side arm, 1000 ml or 2000 ml.
11. Graduate, 1000 ml, 250 ml.
12. Infrared lamp or atmospheric oven.

9.

10.

13.

Knife or spatula, stainless

steel.

14.

Lintine disc,*

cm) diam.

15.

Microscope,

16.

Pipet,

17.

Specified standard test chart.'

18.

Stirring

19.

Hot

Mohr,

!4 in. (3.1

minimum x

12 magnification.

0.5 ml.

motor apparatus, such as Thermolyne

multi-stirrer.

plate, thermostatically controlled.

apparatus to hold 1 Va in. (3.1 cm) Lintine discs.


0-150
21. Thermometer,
C.
22. Distilled water (filtered).
23. Sodium citrate, 10%: Dissolve 100 g of sodium citrate in distilled
water in a 1-liter volumetric flask and dilute to volume. Filter through lintine
20.

Suction

filtering

disc before using.

B. Preparation of sample:

With a knife cut processor

soft natural

cheese into

Hard natural cheese should be blended (Osterizer)


the motor switched off and on during the mixing.

cubes (1.27 cm).


few seconds with

Vi-in.

for a

C. Procedure:

With a torsion balance weigh an 8-oz (240 ml) sample into a tared 400-ml
beaker. Transfer sample to a 2000 ml stainless steel beaker containing
^Minimum order

6:

Vollrath Co, 1236

18th St.

Sheboygan, Wisconsin 53081

llSediment Testing Supply Co., 20 E. Jackson Blvd., Chicago,

'USDA

Sediment Standard Chart for Milk and Milk Products,


^Filter Fabrics, 814 JefiFerson, Goshen, Indiana 46526.

111.

60604.

No

CFR

58.2728.

SUPPLEMENTAL CHEMICAL CONTROL METHODS

368

1000 ml of
agitate for

10% sodium
hr at 55
1

citrate solution. Place


3

C.

The

ideal

on hot plate and mechanically


is 55 C. (Should sample

temperature

be heated to more than 60 C, the protein material will coagulate producing a


slime which can clog the filter pad.) The beaker should be covered during
agitation to prevent outside contamination.
After 1 hr remove the agitator and rinse with filtered distilled water allowing rinsings to drain

using

medium

(Should

filter

back into the

rest of

sample

in the

beaker. Filter solution

suction through an extraneous matter disc

waffle

side up.

within 5 minutes.) Rinse beaker with about 150 ml of filtered

water and pour rinsings through the same disc.


disc from the filtering apparatus and dry with an infrared lamp or
an atmospheric oven at 65 C for 20 minutes. The disc should be protected
while drying to prevent contamination. Enclose the disc in a cellophane envelope and mount on an extraneous matter record card bearing its identity.
Examine the disc under a microscope of at least x 12. Identify and record
distilled

Remove

on the card the kinds of extraneous matter present, using the appropriate
standard.^

B5.12 Processed Cheeses


A. Apparatus and reagents:
Same as in B5. 1 1(A) but without item 23 10% sodium citrate and adding:
23. Pepsin,** 1-10,000 granular. Refrigerate pepsin. Remove from
cooler before use and equilibrate with room temperature before opening

container.

Pepsin-phosphoric acid: To approximately 200 ml of distilled water


85% phosphoric acid and 6.5 g of
pepsin. Swirl to dissolve pepsin. Make up to volume with distilled water.
Filter solution through a lintine disc.
B. Preparation of sample:
Manually dice cheese by cutting with knife or spatula, into Vi in. (1.27 cm)
24.

a 1000-ml volumetric flask, add 30 ml of

in

or smaller cubes.
C. Procedure:

With a torsion balance weigh an 8-oz (240 ml) sample into a tared 400-ml
beaker. Transfer the sample to a 200-ml stainless steel beaker containing
1000 ml of pepsin-phosphoric acid solution. Place on a hot plate and mehr at 52 4 C. The ideal temperature is 52 C.
Proceed as directed in B5. 1(C) beginning with ''Should sample be heated
to a temperature in excess of 60 C, etc."
chanically agitate for

B5.2 Reference
1.

Spicer, D.W. and W.V. Price. 1938. Test for extraneous matter

in

21:1-6.

American

Laboratories, 4110 S 102nd St.

Omaha, Nebraska 68127.

cheese.

J.

Dairy Sci.

B6.1

Fat: Modified

Fat: Modified

B6.1

369

Babcock

Methods

Babcock
Over the years, much effort has been expended to refine the original Babcock method to increase its accuracy and appHcabihty when used to determine the fat content of various types of milk, cheese, whey, and other related dairy food products. These refinements consist basically of: a.) using a
better grade of H2SO4 acid (cone) instead of a commercial grade (1.84 sp gr
B6.11

instead of 1.82-1.83 sp gr); b.) using distilled water maintained at specified

temperatures;

c.) controlling

and standardizing the technique used

in

adding

acid to samples to obtain the heat evolution necessary to disintegrate completely non-fatty material while at the same time releasing liquefied milkfat

measurement. [The technique involves rapid addition of acid accompanied by a smooth rotary swirling of the bottle, first in a clockwise direction
immediately followed by a counter-clockwise movement. Time allotted for
addition of acid to the sample should not exceed 20 sec as heat loss due to
time lag causes incomplete digestion of sample resulting in cloudy fat column (undissolved curd).]; d.) employing a semi-automatic shaker to aid in
digestion of non-fatty materials and in quick release of fat globules; e.) con-

for

trolling variability in

temperature of product-acid mixture.

With refinements incorporated in the following Babcock methods, improved accuracy resulted as was shown by collaborative studies conducted

among

different laboratories.*

A. Homogenized milk

':

Apparatus and reagents:


a. Milk pipet, TC 17.6 ml.
b. Milk test bottle NBS 8%, 18-g capacity, 6-in. (15.24 cm) long.
c. Thermometer 0-110 C.
d. Acid-dispensing bottle with trunnion, factory calibrated to deliver
17.5 ml; laboratory calibrated and marked on side of delivery tube in

1.

subdivisions suitable for determining acid additions used. Suitable alternative acid measuring devices
e.

Column meter,

used only
f.

g.

if

meter

is

may be

substituted.

preferred for reading fat column; dividers to be


not available.

Bottle holder for test bottle.

Water bath, capable of maintaining a temperature 55

to

60

(pref-

erably 57 C).
h.
i.

Bottle shaker^' for test bottle.


Centrifuge, electrically heated, capable of maintaining air temper-

ature of 5=55.
j.

k.

Speed indicator (tachometer),


Sponge.

^^Garver Manufacturing Co, Union City, Indiana 47390.

SUPPLEMENTAL CHEMICAL CONTROL METHODS

370

Sulfuric acid (cone) sp gr 1.84,

1.

ACS grade (used at ambient

temper-

ature).
Distilled water, maintained at constant temperature of 60

m.

or

above.
2. Procedure:
Mix sample thoroughly. Pipet 17.6 ml of sample into a milk test bottle. Do
not release the sample until the bulb of the pipet rests on the neck of the test
bottle. After active flow has ceased (about 30 sec) blow out the last drops

from the
in

tip into the bottle.

Hold the

bottle at a slight angle

and add steadily

small portions (approx 5, 3, and 2 ml) about 10 ml of sulfuric acid, rotate

the bottle using the technique described in paragraph

item

Babcock,

B6.1i,

3.

Place the bottle

in the

mechanical shaker for about

min or

until all traces

of curd have disappeared. Transfer the bottle to the centrifuge and proceed
as in 19.41(A.2).
3.

Note:

Due to the nature of milk solids in different localities, the amount of acid
may be varied by about 0.5 ml. Once the correct amount of acid has been
established for the geographic location, this amount should be used as the
guideline.

B. Chocolate milk

and

drink:^

Because of the nature of the nonfat milk solids in chocolate milk and
drinks, it is extremely difficult to obtain complete fat separation using the
regular Babcock procedures, thus a Roccal solution is used which acts as a
wetting agent. This helps in dispersion of the protein layer enclosing the fat
globule, thus allowing a

more complete separation of protein from

fat

which

reduces charring of the sample.


1.

Apparatus and reagents:

Same

as B6. 11(A), with the following changes and additions:

a.

Milk

test bottles,

capacity 18

NBS

8%, 6V2-in.

(16.5

cm) long graduated

to

g.
**

50% concentrate of Benzalkonium chloride USP.


add ml of 50% Roccal to 200 ml of concentra-

b.

Roccal,

c.

Roccal solution

II

Mix well and cool before using. Roccalkeep about 2 weeks. At that time a dark amappear, indicating that the Roccal has dissipated and will

ted sulfuric acid (sp gr 1.84).


sulfuric acid solution will

ber color will

not be effective in testing. Discard the solution


to

brownish

2.

Procedure:

Proceed as

in

3, 2,

**Hilton Davis Chemical Co, Division of Sterling

Ohio 45202.

it

becomes yellow

B6.11 item 2 except add 12 ml of the Roccal-sulfuric acid

solution in small portions (approx. 5,

nati,

when

in color.

and 2

ml).

Drug Co, 2235 Langdon Farm Road, Cincin-

Fat: Modified

B6.1

Cream

C.

1.

371

Babcock

cheese, processed cheese, processed cheese foods

and spreads:^

Apparatus and reagents:


Mohr pipet, 10 ml (measuring)

a.

b.

Cream

test bottles, capacity 9 g,

NBS

(0-50%

in

0.5%

divisions),

6 V2 in. (16.5 cm) long.


to 110 C.
c. Thermometer,

Beaker, capacity 50 to 100 ml borosilicate glass.


Acid-dispensing
bottle with trunnion, factory calibrated to deliver
e.
17.5 ml; laboratory calibrated and marked on side of delivery tube in
subdivisions suitable for determining acid additions used. Suitable alternative acid measuring device may be substituted.
d.

f.

g.

h.
i.

Stirring rod, glass.

Medicine dropper for glymol.


Beaker tongs.
Bottle holder, for test bottle.

Column meter,

preferred for reading fat column; dividers to be


j.
used only if meter is not available or not permitted in State.
k. Torsion balance.
1.
Water bath, capable of maintaining a temperature at 55 to 60 C
(preferably 58 C).

m. Bottle shaker,^^ for


n.

test bottle.

Centrifuge, electrically heated, capable of maintaining air temper-

ature at 55-60 C.
o.

Speed indicator (tachometer).

p.

Weight, 9

q.

Sponge.

r.

g.

Sulfuric acid, (cone)

ACS

grade, sp gr 1.84 (used at ambient tem-

perature).
(red or blue reader), sp gr not to exceed 0.85 at 20 C.

s.

Glymol

t.

Distilled water, maintained at constant

temperature of 60

or

above.
2.

Procedure:

Using a torsion balance, accurately weigh 9 g of well-mixed sample into a


tared beaker. Pipet about 10 ml of hot distilled water into the beaker. Mix
well using glass stirring rod, to break up lumps, wet and emulsify cheese.
Hold the beaker at a slight angle with tongs and add about 15.0 ml, see
B6. 1 item 1 of sulfuric acid in successive portions of approximately 4, 4, 4,
and 3 ml. Quickly mix sample and acid with the glass stirring rod after each
,

addition.

Carefully transfer contents of the beaker to a fat test bottle and rotate
the bottle

using the technique described in Modified Babcock Section

B6.1, item

3.

Rinse beaker with about 8 ml of hot

Garver Mft. Co, Union City, Ind. 47390.

distilled

water and add

SUPPLEMENTAL CHEMICAL CONTROL METHODS

372

rinse to the test bottle. Place bottle in mechanical shaker for about 5
until all traces

min or

of curd have disappeared.

Transfer bottle to centrifuge, counterbalance bottle, and turn on centri-

Proceed as in 19.41(A.2).
Note:
Cream cheese can be tested also using the acid hydrolysis technique

fuge.

3.

[19.43(B)].

D. Natural cheese:
1. Apparatus and reagents:

Same

as section B6.1(C.l) excepting items d and h are not applicable.

Preparation of sample:

2.

When cheese can be cut, take a narrow, wedge-shaped segment reaching


from outer edge to center. Cut this segment into strips about 2 in. long
and V4 in. (5.08 x 0.64 cm) wide and use representative strips from each of
the three plugs for the sample.

Procedure:

3.

On

a torsion balance, accurately weigh 9 g of cheese strips into a tared fat

test bottle,

being careful to drop the cheese through the neck of the bottle so

as not to touch the sides of the glass. Pipet about 10 ml of hot distilled water

Wet the cheese with distilled water and mix to thoroughly


suspend cheese before addition of acid.
Holding the bottle at a slight angle, disperse the water and cheese by manually shaking for several seconds in a rotary motion first clockwise and then
counter-clockwise. Repeat this shaking sequence. Proceed as in section
into the bottle.

B6.11(C.2).

2% fat) and whey:^


encountered in obtaining fat columns free of
some charred matter or curd when using H2SO4 alone for testing low fat
products, a Roccal solution is used in combination with the H2SO4. The
Roccal is used because of its wetting properties which help disperse the
protein layer that encapsulates fat globules thus freeing the fat so it can
E. Fluid skim milk

Because of the

(<

0.5 -

difficulty

combine more readily with other


umns.
1. Apparatus and reagents:

Same

as B6.11(A.l) with the following changes and additions:

a.

tle

free fat globules to give clearer fat col-

for

skim milk 0.5 to 2% or more fat, use test botFor fluid whey and skim milk less
use double-necked 18-g NBS bottles
to 0.5% in 0.01%

Test bottle

fluid

specified in section B6.11(A.l).

than 0.5%

fat

divisions.

50% concentrate of Benzalkonium chloride USP.


add ml of 50% Roccal 100 ml of concentrated

b.

Roccal,

c.

Roccal solution

sulfuric acid.
tion will

II

Mix

to

well and cool before using. Roccal-sulfuric acid solu-

keep about

weeks. At that time a dark amber color

will ap-

pear, indicating that the Roccal has dissipated and will not be eflfective

B6.1

Fat: Modified

in testing.

Babcock

373

Discard the solution

when

it

becomes yellow

brownish

to

in

color.

Procedure:

2.

Proceed as in B6.11(A.2), with the following changes and additions:


For fluid skim milk with less than 0.5% fat or for whey pipet 17.6 ml of
sample into the skim milk test bottle through the large neck slanting the
bottle in a position to facilitate the escape of air, thus preventing any tendency for the large neck to clog or overflow. Blow the last drop of milk out of the

pipet.

F. Chocolate milk

This procedure

is

and drink (Pennsylvania

The reagents used


clear fat column in the

are N-butyl alcohol and sulfuric acid

Apparatus and reagents:


as for cream 19.41(3.1) items b through
test bottle,

s.

Ammonium

t.

Normal

u.

which provide a

testing of ice cream, chocolate milk and drink.

Same

and additions:
a. Milk

a test for fat in dairy products containing sugar or choco-

late.

1.

modification):^'

NBS

with the following changes

8%, 18-g capacity,

hydroxide

(NH4OH-28

to

29% NH3).

butyl alcohol, bp 117C.

Sulfuric acid, diluted (sp gr 1.72-1.74)

mix

3.5 parts of

H2SO4

one part of water.


Measuring burettes or pipets for measuring ammonium hydroxide
and normal butyl alcohol.
(sp gr 1.82-1.83) to
V.

2.

Preparation of sample:

Thoroughly mix the sample, warming if necessary, to disperse the fat,


taking care not to churn the product and to bring all the cocoa fiber into
suspension.
3. Procedure:
Accurately weigh 18 g of well mixed sample into a tared milk test bottle
taking precautions to prevent chocolate from settling in the well-mixed
sample. Hold the bottle at a slight angle, add 2 ml of NH4OH and rotate
the bottle slowly to thoroughly mix reagent with sample. Add 3 ml of normal

butyl alcohol and mix sample and alcohol thoroughly by swirling the bottle.

Then add

17.6 ml of diluted sulfuric acid following the

[19.5 1(B. 3)], beginning with

"Hold

tionwise about 17.6 ml of diluted sulfuric acid, washing


the bulb. Immediately shake

cream procedure

the bottle at a slight angle and add porall

traces of milk into

."

Note:

4.

Temper

test bottles in

water bath for not

less than 3

min rather than

min

as specified in 19.41(B.2).

G. Ice cream and ice cream mixes (Pennsylvania modification):

Apparatus and reagents:


as for cream, 19.41(B.l), items b through r, and B6. 12(A) chocolate
milk and drink, items s through v with the following exceptions:
1.

Same

SUPPLEMENTAL CHEMICAL CONTROL METHODS

374
a.
in.

Ice

cream

test bottle

20% NBS

9-g graduated in

V5%

divisions, 6V2

(16.5 cm),

w. Waring or Osterizer blender.


Preparation of sample:
a. Melt ice cream at room temperature and, if necessary, heat to
eliminate foam. Warm to about 20 C.
b. Reduce any large particles in fruit and nut ice cream to a finely
2.

divided state with a blender.


3.

Procedure:

Accurately weigh 9 g of sample into a tared ice cream test bottle. Precautions should be taken to keep the fruit or nut ice cream thoroughly mixed
to get a representative sample.
Follow the chocolate milk and drink procedure B6.11(F.3) beginning with

"Hold the

bottle at a slight angle

and add 2 ml of

NH4OH

rotating

."

H. Skim milk and buttermilk or whey {Pennsylvania modification):


1. Apparatus and reagents:
Same as Milk apparatus and reagents [19.41(A.l)] except: substitute in
b-Skim milk test bottle for milk test bottle listed. Double necked 18-g graduated test bottle with 0.01% divisions. Add the following:
o. Normal butyl alcohol.
2.

Procedure:

Measure

normal butyl alcohol into the 18-g skim milk test bottle.
whey sample to the bottle
through the large neck slanting the bottle in a position to facilitate escape of
air, thus preventing any tendency for the large neck to clog or overflow.
Add 7 to 9 ml of sulfuric acid (it may be necessary to vary the amount of
acid to obtain a fat column of golden yellow to light amber color) to the test
bottle. Mix contents of the bottle thoroughly and centrifuge for 6 min at the
proper speed, see 19.41(A.3), and temperature, see 19.41(A.2).
Add sufficient hot soft water (60 C or above) until bulb of bottle is filled.
Centrifuge the sample for 2 minutes. Stop the centrifuge and add hot soft
water until the liquid column approaches the top graduation of the scale.
Centrifuge the sample for 2 min longer.
Transfer the bottle to a water bath that is maintained at 55 to 60 C (preferably 57 C) and hold bottle in bath for a minimum of 5 minutes The water level
should he slightly above the level of the fat column in the bottle.
Remove bottle from water bath, dry bottom of bottle on a sponge and with
the aid of a reading light, use dividers or calipers to measure fat column as
per cent by weight to the nearest 0.05%; measure from lower surface to the
highest point of upper meniscus. Fat in the column at time of measurement
should be translucent, golden yellow or amber, and free from visible suspended particles. Double the reading to obtain the percentage of milkfat
since a 9-ml sample was used in an 18-g bottle.

Add

2 ml of

exactly 9 ml of skim milk, buttermilk, or

375

B6.2 Fat: Gerber

/.

Milk and milk mixes: In-plant production control (Roccal)^


Apparatus and reagents:

1.

Same
2.

Mix

as B6. 11(A).
Procedure:
the sample thoroughly by shaking and swirling the bottle. Pipet

17.6 ml of sample into a milk test bottle.

Do not

release the sample until the

bulb of the pipet rests on the neck of the test bottle. After active flow has
ceased, blow out last drops from

Hold the
proximately
solution.

tip

of pipet into the bottle.

and add steadily in small portions (apand 4 ml), 17.6 ml of the prepared Roccal-sulfuric acid

bottle at a slight angle


5, 5, 4,

Rotating the bottle using the technique described in section

Acid used should be at room temperature. Place the bottle in


and shake for minute.
Transfer bottle to centrifuge [19.41(A.3)] heated to approximately 60 C.
Add distilled water (60 C or above) until liquid column approaches top
graduation of scale. Centrifuge sample for 1 min, stop centrifuge, remove
bottle, wipe bottom of bottle with sponge and place on the column meter.
Immediately read results by measuring fat column from extreme bottom of
the column to the extreme top of the upper meniscus. Read percentage of fat
directly to the nearest 0.05%.
Repeat all tests with a cloudy fat column or one showing the presence of
charred matter or curd, or where for any reason the reading is indistinct or
B6.11, item

3.

the mechanical shaker

uncertain.

B6.2 Fat: Gerber


Frozen desserts: In-plant production control
High concentrations of nonfat milk solids, sugars, stabilizers, emulsifiers,
and homogenizing treatment make vigorous shaking necessary to liberate fat
after addition of reagents. Prolonged action of H2SO4 and isoamyl alcohol

B6.21

appears to increase its solubility in fat, thus often resulting in higher fat
determinations than those by the Roese-Gottlieb method. For this reason,
use of the Gerber test for frozen desserts and mix should be restricted to
processing control tests. Where critical data are required, use of the RoeseGottlieb or an equivalent solvent extraction method is necessary.

A. Apparatus and reagents:


Same as Sections 19.42(A.l)b,e,l; 19.42(B.l)d; 19.42(C.l)a; plus the

fol-

lowing:

Gerber ice cream test bottle, 20%: Capacity 5 grams. Construction and
dimensions should be as described in 19.42(A.l)a, except that external
width of the flat tube is not less than 11 mm.
Graduated volume at 20 C in the flat tube is 1.1356 ml (15.3828 g Hg). It
extends not less than 70 mm on the flat tube and is divided into equal parts,
each equivalent to 0.01136 ml, error of the total calibrated length not to
exceed half of the smallest graduation, plus or minus. Graduation lines are

'

SUPPLEMENTAL CHEMICAL CONTROL METHODS

376

mm

uniformly centered on the flat tube face with 0.2% lines not less than 3
on each side beyond the 0.2% line; and
long; 1.0% lines extending 1.5

mm

2.0% lines extending not less than mm on each side beyond the 1.0% line.
They may extend across the entire flat surface.
Each successive 2% graduation is identified by etched and pigmented serito 20, at the right and just above the per cent lines, with
al numbers from
nearest the body. Body capacity at 20 C, from junction with the neck to
the
the 0% lines, is 20.5 0.4 ml. Bulb capacity at 20 C, to the 20% line, is not
less than 1.75 ml. Bottles are identified ''Ice Cream, 5 g" with the name or
1

symbol of the manufacturer or distributor permanently inscribed on the


body and a State symbol, where required to attest conformance with specifications, applied after recalibration.

B. Procedure:

Measure 10 0.2 ml of diluted H2SO4 [19.42(A.l)d] at 15-21 C into an


ice cream test bottle. Place bottle in support on balance and tare. Place 5-g
weight on opposite pan and weigh 5.00 g from a properly prepared sample into the bottle. Remove bottle from balance and add 5 ml of water and
1
ml of isoamyl alcohol. Insert lock stopper securely and proceed as in
19.42(A.2) except that the bottle should be centrifuged for 5 min at operating
speed for the machine. Read the scale to the nearest 0.2% graduation and
column. If the
second reading diflFers by more than 0.2% from the first, the initial shaking
was inadequate and the test must be repeated. When the second reading is
less than 0.2% above the first, report the result of the first test.
record the result. Recentrifuge, retemper, and reread the

fat

B6.2 References
1.

2.

Bianco L.J. Kraft Inc. Chicago, 111.


Milk INDUSTRY FOUNDATION, 1949. Methods

of analysis of milk and

its

products. Labora-

tory Manual. Washington, D.C.


3.

Newlander, J.A. and H.V. Atherton, 1977. The Chemistry and Testing of Dairy Products.

AVI

B7.1

Publishing Co.. Westport, Ct.

Fat,

Moisture and Salt

in

Butter and Margarine (Modified

Kohman Method)
A. Apparatus and reagents:
1.

Analytical balance.

2.

4.

Beakers, aluminum, with pouring spout, 200 ml.


Buret, 25- or 50-ml, calibrated in 0.1-ml divisions.
Volumetric pipet, 25 ml.

3.

5.

Crucible tongs.

6.

Hot

7.

Volumetric

8.

Stirring rods, glass, with rubber policeman.

9.

Graduate, 100 ml.

10.

plate, thermostatically controlled at


flask,

Erlenmeyer

450 F (232 C).

250 ml.

flask, 125 ml.

B7.1 Fat, Moisture, Salt: Butter

11.
12.

and Margarine (Kohman Method)

377

Beaker, 250 ml.


Medicine dropper.

13.

Distilled water.

14.

Petroleum ether, boiling range 30-60 C, Mallinckrodt #6128 or

equivalent.

16.

Silver nitrate, O.IN.


Potassium chromate indicator, 10%.

17.

Water bath, cold

15.

(7 to 13

C)

B. Procedure for moisture:


1. Using the analytical balance, weigh a clean, dry aluminum beaker.
Record as weight A.
2. Accurately weigh about 10 g of sample into the beaker. Record total
weight of beaker and sample as weight B.
3. Using crucible tongs, place the aluminum beaker on the hot plate at

approximately 232 C. Swirl constantly during heating to avoid spattering


and burning of milk solids.
4. Continue evaporation until all foaming has ceased and the milk solids

brown color. Care should be taken to obtain the same


each test.
5. Cool beaker to room temperature by dipping in cold water using crucible tongs. Dry the outer surface thoroughly with a towel.
6. Weigh the beaker and moisture-free residue on the analytical balance. Record as weight D.
and

salt

take on a light

degree of color

in

^
%
where

A =
B =
C =
D =
E =

^M
Moisture =

B minus D

Beaker weight,
Weight of beaker and sample,
Sample weight (B minus A),
Weight of beaker and moisture-free residue, and
Weight of beaker and fat-free residue, see B7.1(C. 8)

Procedure for fat:


Add about 125 ml of petroleum ether

1.

the

,^^

x 100

aluminum beaker.
2. Use a glass rod

to

break up the residue

plete fat extraction. Rinse the rod with a

before removing

to the moisture-free residue in

few

in

the beaker to insure

milliliters

com-

of petroleum ether

it from the beaker.


beaker at a 45 angle by placing it on top of a 250-ml beaker
with the lip at the low point and allow it to remain undisturbed until solids
have collected at the bottom.
4. Decant the petroleum ether solution of the fat, taking care not to
disturb the solids (milk solids and salt) which have settled at the bottom of
the beaker. To be sure that no solids are decanted, use a rubber policeman to
push back floating solids.

3.

Tilt the

SUPPLEMENTAL CHEMICAL CONTROL METHODS

378

Repeat extraction two more times, using about 75 ml of petroleum


ether each time.
6. Evaporate residual petroleum ether from the beaker by warming
very slowly over a hot plate. Do not allow the beaker to touch the hot plate,
as rapid heating will cause the solids to pop out of the beaker, thereby destroying the accuracy of the test. Do not use an open flame when using
petroleum ether. During heating, hold the beaker at a 45 angle and tap lightly on the hot plate to break the cake of solids and salt. Continue heating until
the residue is dry, free from solvent odor, and assumes a uniform powdery
5.

appearance.
7. After the solvent has evaporated, cool and dry the beaker as before.
8. Weigh beaker and residue. Record as weight E.

Fat

minus E

,^^

X 100

D. Procedure for salt:


1. Transfer residue in the beaker to a 250-ml volumetric flask with hot
water (about 65-70 C), using about three washings.
2.

Cool to room temperature and make up to volume with water. Mix

thoroughly.
3.

The

salt will

dissolve and milk solids will settle to the bottom of the

flask.
4.

Pipet 25 ml of the solution into a 125-ml Erlenmeyer flask.

5.

Add

ml of potassium dichromate indicator and

silver nitrate solution to the first

deep red or orange-red, etc, but discontinue


manent tinge of red has appeared.

NaCI =

with standard

titrate

permanent color change.

Do

not titrate to

titration after the first per-

nlofAgNO,xNAgN03x58.5

,^

E. Notes:
1

if

a cloudy petroleum ether layer develops and solids are not precipi-

1
drop of ethyl ether, using an eye dropper, directly into the
middle of the petroleum ether layer in the beaker. Wait 15 sec and tap the
bottom of the beaker with the stirring rod fitted with a rubber policeman.
2. Repeat the above step 10 or 15 times, waiting about 30 sec in be-

tating, release

tween each

repetition.

By

this

time the solution should be entirely clear.

B7.2 Reference
1.

American Butter Institute.

1937. Laboratory

methods of

analysis. Chicago, Illinois.

379

Hydrolytic Rancidity

B8.1

Hydrolytic Rancidity

B8.1

An

increase in appearance of rancid flavor in raw milk supplies has been

associated with pipeline milkers. Hydrolytic rancidity develops

when

milk-

has undergone lipolysis, one measure of which is the acid degree value
(ADV). Normal raw milk has been reported to have an ADV of 0.25 to 0.40.

fat

When

the

ADV

following test

is

reaches about

1.2, the

The

rancid flavor can be detected.

applicable to raw milk and cream.'

A. Apparatus and reagents:


1. Hypodermic syringe, 50 ml: Graduated, fitted with No. 15 needle or
a standard 17.6-ml milk pipet can be used as an alternative.
2. Babcock milk test bottle: Capacity 18 g, 8% milk; or 9 g, 20% ice

cream

cream test bottle 50%, 9 g.


ml or automatic pipetting device adjusted to deliver 10 ml

test bottle;

Pipet, 10

3.

per cycle.
4. Reagent: 30 g of Triton X-100 (a nonionic surface-active agent) and
70 g of sodium tetraphosphate (or sodium hexametaphosphate if uncaked)
made up to 1 liter with distilled water.
5.

Boiling water bath: With rack or tray to hold test bottles.

6.

Centrifuge:

test bottle at the

Babcock or any other centrifuge

that will

proper centrifugal force, see 19.41(A.3).

accommodate a

An unheated

cen-

trifuge is satisfactory.

Aqueous methyl

7.

and

alcohol: Equal

volumes of absolute methanol, CP,

distilled water.

bath: Controlled to a range of 55-60

8.

Water

9.

Syringe, tuberculin type:

10.

Erlenmeyer

flask:

(preferably 57 C).

ml, with a 4-in. (15.2

cm) No.

19 needle.

50 ml.

11. Fat solvent: 4 parts of petroleum ether (Skellysolve B) to 1 part of


n-propanol (reagent grade) by volume.
12. Indicator solution: 1 g of phenolphthalein dissolved in 100 ml of

absolute methanol.

KOH:

KOH

in absolute methanol. StanPrepare 0.02N


dardize solution frequently against standard potassium acid phthalate or oth13.

Alcoholic

er suitable standard.
14.

Microburet, 5-ml capacity, calibrated

in 0.1

-ml divisions.

B. Recovery offat:
Using either a 50-ml syringe or a 17.6-ml pipet, place 35 ml of milk or

type of test bottle [B8.1(A.2)] add 10 ml of reagent and mix


thoroughly. Place bottle in a bath of gently boiling water (water should cover
the base of test bottles), agitate contents thoroughly after 5 min, and again

cream

in either

after 10
at least

min in the bath. (CAUTION: Bottles should remain


5 min more to effect clear fat separation.)

in boiling

water

SUPPLEMENTAL CHEMICAL CONTROL METHODS

380

After the test bottle has been in boiling water for 15-20 min, remove bottle

and centrifuge

it

for

minute.

Add

sufficient

aqueous methyl alcohol

to bring

the fat column well within the graduated portion of the test bottle neck.

Centrifuge for an additional

minute.

Place the bottle in water bath at 55-60

above

the water level

(preferably 57 C) for 5 min, with

the top of the fat column.

C. Titration procedure:

ml of fat at 57 C from the test bottle to a 50-ml Erlenmeyer


flask, using the 1-ml syringe, and weigh the exact volume transferred. Weigh
1.0 ml of fat into a tared Erlenmeyer flask. (If exactly 1.0 ml can be removed,
a 1.0-ml volumetric pipet may be used, in this event rinse with two 1.0-ml
portions of fat solvent and reduce the amount of solvent in the next step by
the amount used to rinse a pipet, namely 2.0 ml). Dissolve the fat in 5 ml of
fat solvent and add 5 drops of indicator solution.
2. Titrate to the first faint definite color change with standardized alcoholic KOH solution, using a 5-ml microburet. (NOTE: If turbidity develops
in the solvent-fat mixture during titration, 2 or 3 ml of additional solvent
should clear the mixture.) Make blank titrations on the fat solvent (in the
absence of fat), using 5 drops of indicator on each new batch of solvent.
Check blank values periodically.
1.

Transfer

D. Calculation of Acid Degree Values (ADV):


Calculate the grams of milkfat in the sample titrated by multiplying the
milliliters of milkfat by the density of milkfat at 50-60 C (preferably 57 C).

Then,
g of milkfat

= ml of

milkfat x 0.90

Calculate the acid degree value as follows:

ADV

(ml

KOH

for sample

- ml

of

KOH

Weight of

N
Titration blank

=normality of alcoholic

= titration

KOH

for blank)

x 100

fat

solution

the absence of fat and using


drops of phenolphthalein as indicator. (Blank determinations should be done on each new batch of solvent.)

value of fat solvent

in

Example: Assuming that 0.75 ml of fat required 0.70 ml of alcoholic KOH


having a normality of 0.0203, and that the blank titration value was 0.03.
Then:
0.67 x 0.0203 x 100
(0.70 - 0.03) X 0.0203 x 100

ADV

0.75 X 0.90
^36

0.675

2.015

0.675

(ADV

should be expressed to the second decimal place only

results, e.g., 2.02 in the

example

just given.)

in

reporting

B9.1

Lactose

in

Cheese

381

E. Interpretation:

Express results
100 g of

fat).

terms of ADV (the amount of IN base required to

in

The following values

= Normal
= Borderline (indefinite)
1.2 = Slight rancidity
and above - Unsatisfactory (extreme

<

titrate

are suggested:

0.4

0.7 to 1.1

1.5

hydrolytic rancidity)

B.8.2 Reference
1.

Thomas,
simplified

Nielson D.J. and J.C. Olson, Jr. 1955. Hydrolytic rancidity in milk
method for estimating the extent of its development. Amer. Milk Rev. 17:50-58,

E.L.,

85.

B.9.1

Lactose

Cheese

in

A. Apparatus and reagents:


1. Volumetric flask, 250 ml, 500 ml.
2. Volumetric pipet, 25 ml, 50 ml.
3. Graduated cylinder, 10 ml.
4. Erlenmeyer flask, 250 ml.
5. Beaker, 50 ml, 250 ml, 400 ml.
Funnel, 125

7.

Watch

8.

Glass stirring rod, with rubber policeman.

9.

Wash

10.

mm.

glasses, 95

bottle.

Desiccator,

dicating, 4

4.25

mm.

6.

with efficient desiccant such as Drierite,

filled

mesh (calcium

11.

Suction flask,

12.

Gooch

13.

Filter paper,

calcium chloride,
1000 ml, or 2000 ml.
sulfate),

crucibles, porcelain,

Coors #3.

Whatman

cm

#1, 24.0 cm; or glass fiber


#226820-20.
Reeve Angel #X-934-AH,

14.

Rubber crucible holder.

CM

apparatus.

15.

Suction

16.

Wire gauze with asbestos center.

17.

Iron tripod.

filtering

18.

Bunsen or

19.

Interval timer. General Electric Co, or equivalent.

Terrill

burner or equivalent.

20. Stainless steel stirring rod with flattened end.


21.

22.
23.

Beaker tongs.
Hot plate, thermostatically controlled.
Atmospheric oven, gravity convection type.

24.

Analytical balance.

25.

Thermometer,

26.

Ethyl alcohol,

27.

Ethyl ether,

approximate range.
formula #30 or 3A.

to 100 C,

USSD,

ACS

grade.

in-

etc.

filter

paper,

SUPPLEMENTAL CHEMICAL CONTROL METHODS

382

29.

Asbestos, long-fiber, acid-washed and ignited.


Filter-aid, Celite analytical, Johns-Manville.

30.

Copper

28.

sulfate,

ACS

grade.

32.

sodium potassium tartrate, ACS grade.


Sodium hydroxide, pellet, low in carbonate, CP Baker.

33.

Nitric acid,

34.

Distilled water.

31. Rochelle salts,

ACS

grade, sp gr 1.42.

B. Solutions:

Copper

1.

distilled

water

sulfate, Fehlings' A: dissolve 34.639 g of


in

a 500-ml volurrietric flask.

thoroughly. Filter through

Make up

to

copper sulfate in
volume and mix

Whatman #1 filter paper into a bottle suitable


may be prepared and stored.

for

storage. Large quantities of solution

2. Alkaline tartrate, Fehlings' B: dissolve 173 g of Rochelle salts and


50 g of sodium hydroxide in distilled water in a 500-ml volumetric flask.
Make up to volume and mix thoroughly. Allow to stand for two days and
filter

through asbestos. Store

store

more than one month

Nitric acid, 1:3: dilute

3.

tilled

in

rubber-stoppered reagent bottle.

Do

not

as solution deteriorates on standing.

one part of

nitric acid

with three parts of dis-

water.

ofGooch crucibles (Asbestos Gooches):


suspension of asbestos and distilled water and keep ready to
use. Place Gooch crucible in rubber crucible holder in filter flask. Shake the
asbestos mixture and immediately fill crucible to within Vs in. (3.17 mm) of
the top. Apply vacuum. An asbestos pad should be formed about Vs in. (3.17
mm) thick, which covers all the holes in the crucible.
Wash thoroughly with hot distilled water to remove fine particles of
C. Preparation

Prepare a

asbestos.

1:4

Pour hot solution out offilter flask. Follow with 10-ml portions of

alcohol and ether. Dry the prepared crucible in an atmospheric oven at


100

for 30 minutes.

Cool

in

desiccator and weigh on an analytical balance

to obtain tare weight.

To

reuse the asbestos mat, dissolve the cuprous oxide precipitate in the

crucible with 1:3 nitric acid.

Wash

thoroughly with

lytical

balance to obtain tare weight.

D. Preparation of Gooch crucible (Glassfritted Gooch):


A medium porosity, glass filtering crucible may be used

Gooch

Dry the
on an ana-

distilled water.

crucible in an atmospheric oven, cool in desiccator and weigh

crucible. Certain precautions

must be taken with

this

in

place of the

type of crucible

since the hot alkaline tartrate solution attacks the sintered glass disc.

clean glass crucible

is

not weighed before being used. The precipitate

The
col-

washed, dried and weighed as directed for the Gooch


Then the glass crucible is cleaned, dried and weighed. The

lected in the crucible,

crucible, B9. 1(C).

is

difference in weight represents the weight of the precipitate.

Lactose

B9.1

in

After each
there

is

Cheese

383

filtration, the

bottom of the glass disc should be examined.

If

a red color visible, the crucible should be replaced since the crucible

pores have become too large to retain the red cuprous oxide precipitate.
E. Procedure:

Weigh accurately on an analytical balance, a 25-g sample of processed


American cheese [B9.1(G.l)] into a tared 50-ml beaker. Add a few milliliters of warm distilled water (about 50 C) to the sample and break up the
cheese with the flattened end of the stainless steel stirring rod until cheese
takes on a smooth paste-like texture.
Transfer the suspension to a 500-ml volumetric flask with the aid of water

from a

bottle.

Rinse beaker thoroughly and add rinsings to flask. Dilute soluml with distilled water and mix thoroughly.

tion to about 400

Add

copper sulfate solution to precipitate protein from sample. If


more copper
sulfate solution,
ml at a time, until protein does precipitate from the solution. Usually about 10 to 15 ml is sufficient. If necessary, place the flask with
the sample in a bath of hot water to aid in coagulating the protein material.
Make up to volume with distilled water and mix thoroughly.
Filter through Whatman #1 filter paper into an Erlenmeyer flask, discarding the first few milliliters of filtrate. The filtrate should have a slight
blue color as evidence of copper in solution. Collect about 150 ml of filtrate.
Pipet 25 ml of copper sulfate (Fehlings' A) and alkaline tartrate (Fehlings' B) solutions into a 400-ml beaker. Mix well by swirling. Pipet 50 ml of
sample filtrate into the same beaker. Immediately heat the solution on an
asbestos gauze over a Bunsen burner for 6 minutes. It is necessary that the
flame be regulated so that boiling begins in exactly 4 min; boiling is continued for an additional 2 minutes [B9.1(G.2)].
Filter hot solution at once through a prepared tared crucible using medium
vacuum. Rinse the beaker thoroughly with hot distilled water, using a rubbertipped glass stirring rod to remove the last traces of cuprous oxide adhering
to the beaker. Filter rinsings through the crucible. Wash the cuprous oxide
precipitate thoroughly with hot (about 60 C) distilled water followed by 10 ml
10 ml of

protein in the solution does not coagulate or precipitate, add


1

of alcohol. The

filtrate

tion using a smaller

should be blue;

sample so

if

it

that the 25

is

colorless repeat the determina-

ml of copper sulfate gives an ex-

cess of copper ion.

atmospheric oven at 100 C for 1 hour. Remove from oven. Cool in


at least 30 min or until crucible has reached room temperature. Weigh and record weight as cuprous oxide residue.
Place

in

desiccator for

F. Calculations:
1.

Use

the

Munson Walker

table

to convert the weight of cuprous

oxide determined into milligrams of lactose. For example, if the residue value is 0.2788, the procedure to use is as follows:
a. Multiply the residue weight of cuprous oxide by 1000 to convert

grams

to milligrams for use in the table.

SUPPLEMENTAL CHEMICAL CONTROL METHODS

384

0.2788 g X 1000
b.

The fourth

figure

is

278.8 milligrams

not given in the table. Obtain milligrams of

lactose by interpolation as follows:

Cuprous Oxide

Lactose

2780
2788 (weight of residue)
2790

189.5

value to determine
190.2

Use the ratio between the values given to determine the lactose value.
The difference between 2780 and 2790 is 10, the difference between 2780
and 2788 is 8. The difference between 189.5 and 190.2 is 0.7 and x is the
value to determine.
8/10

Solving for x =
c.

tose

Add
=

u)~~

volume of cheese

x/0.7

^-^^

the figure 0.56 to 189.5 to obtain milligrams of lac-

190.06 which

The per cent

2.

is

the lactose value for cuprous oxide

lactose

No. 2788.

multiplied by a constant to correct for the

is

in solution.

Lactose (process cheese) =


milligrams of lactose x 0.97 (factor)

X 100

aliquot sample weight in milligrams

Lactose (natural cheese) =


milligrams of lactose x 0.94 (factor)

X 100

aliquot sample weight in milligrams

Explanation of factors:

3.

a.

(in

Aliquot sample weight

x 50 x 1000
= grams of sample
z^-r

milligrams)

b.

Factors 0.97 and 0.94

Because

insoluble material in cheese and

some space in the flask, it is necessary to correct


for this volume change. From the average composition of processed
cheese, the volume of precipitate was calculated to be 14 ml or 97% of
the 500-ml volumetric flask. The volume of precipitate from natural
cheese was calculated at 94% of the volumetric flask.
precipitate occupies

385

Moisture and Solids

B10.1

G. Notes:

The percent of lactose in cheese varies with the type of cheese. The
can be used as a guideline for weight of samples to be used in the
below
chart
1.

test

procedure:

Cheese

Grams

Natural

50

Processed American,

25

Swiss, etc.

Cheese Food Spreads


Grated American
Modified Cheese Powders
2.

served.

It is

To

15

10
5

imperative that directions for boiling the solution be strictly obregulate the burner for this purpose, make preliminary tests using

50 ml of reagent and 50 ml of distilled water.

9.2

Reference
Association of Official Analytical Chemists.

1.

ed. 43.011

Bianco,

2.

B10.1

pp 720-725. Association of

1960. Official

Official Analytical

methods of analysis, 9th

Chemists, Washington, DC.

L.J. Kraft, Inc., Chicago, Illinois.

Moisture and Solids

Microwave Oven: Cheese'*


is based on use of microwave energy to remove moisture.
The microwave oven uses an internal balance, recirculating water system,
and a variable power control for adjusting microwave energy.
A. Apparatus and reagents:
B10.11

This method

1.

Osterizer or equivalent blender.

2.

Analytical balance.

3.

Pyrex

4.

Spatula.

5.

Apollo microwave oven: 10-g Mark IV (mechanical) or 10-g Mark

(digital

petri dish, 4 in. (10.16

cm)

in

diameter.

read out) models.'"

B. Preparation of sample:
Natural cheese: to obtain a
1.

more homogeneous sample,

it

is

neces-

sary to mix cheese in blender for about 15 seconds. (Blender should be


turned off and sample and jar shaken manually to help move larger particles

toward bladeside of mixer. Prolonged mixing

shall

be avoided as

fat separa-

tion occurs.)

"Apollo

Microwave Products, 6204

Official

Road, Crystal Lake,

Illinois

60014.

SUPPLEMENTAL CHEMICAL CONTROL METHODS

386
2.

Process cheese:

blender as product

is

It is

not necessary to mix this type of cheese in a

relatively uniform. Strips or large pieces of cheese

may

be used to make up 10-g sample.


C. Procedure:
1
Place into a pyrex petri dish cover, a glass filter paper and transfer to
sample platform inside the microwave cavity. Using the tare knob on the
control panel (location of tare

knob depends upon model),

to zero. Place 10 g of the prepared

sample on

paper

tare the readout

dish and
cover with a second filter paper to avoid spattering. (Make certain that the
sample is spread out evenly before covering.)
2. Subject the sample to microwaves for 2V2 min at a power setting of
filter

in petri

54, see BlO.ll(E).

D. Calculation:
At the end of the

can be read directly in per cent


determined by the type of unit used.
taken, a new sample may be introduced into the

test cycle, the result

moisture.

The method of readout

After the

final

reading

is

is

oven.
E. Note:
It is

important that this method be periodically checked against the vacu-

um oven method

to

make

certain that calibration of instrument

is

being

maintained.

B10.12 Moisture Balance: Dry Milk Products


A. Apparatus and reagents:
1.

Cenco moisture balance.**

2.

Polyethylene cup, 2 oz (60 ml).

3.

Spatula, scoop style. ^^

2- 3

B. Procedure:

Light the scale lamp by means of the toggle switch at the right of the
Cenco balance. Rotate scale until 100% mark is at the index by turning the
scale adjusting knob on the right side of the unit. (This establishes a reference point.) Bring pointer to index by turning the pointer adjusting knob on

"zeros" the balance.) Rotate scale


amount of unbalance or amount
of sample needed.) The pointer will now be above the index.
Raise lamp housing and carefully distribute the sample to be tested on
sample pan until pointer returns to the index. (This amount of material then
corresponds to 100 divisions of the scale). Lower lamp housing and light
heat lamp by means of toggle switch at left. Set timer for 8 minutes, see
10.12(D). Adjust the powerstat control to 80. (This may vary for specific
materials being tested; however, a setting of 80 works well for dry milk prodleft

side of the unit. (This calibrates or

until

0% mark

is at

index. (This presents the

**Central Scientific, 4401


^'Sargent

W 26th

St,

Chicago, IL. 60623

Welch Cat #S-75290, 7300 N. Linder, Skokie,

IL. 60076

BIO. 13

Refractometer:

ucts.

As

Turn

off heat

Whey Concentrate

387

the sample loses moisture, the pointer will rise above the index.

lamp

after 8 minutes.

To determine per

cent reduction in

weight due to loss of moisture, rotate scale by turning scale adjusting knob

on the

right until pointer returns to the index.

C. Calculation:

Read

directly

Solids

from scale as per cent moisture.

100 minus

moisture.

D. Note:

The length of time in the infra-red oven may need to be varied from the
min depending on amount of moisture in different types of samples tested. Once correct drying time in the oven has been established for a
product type, it is desirable to maintain this same drying time for all like
stipulated 8

samples tested.

B10.13 Refractometer: Whey Concentrate^


A. Apparatus and reagents:
1. Hand refractometer, Bausch and Lomb scale range 40
Zeiss Model #0-85 with double scale complete with stand.
2. Soft paper tissue for wiping refractometer prisms.
3.
rial

Solvent for cleaning prisms, alcohol or xylol are suitable.

being examined

is

water-soluble, use distilled water.

Do

to

If

85%

or

the mate-

not use acetone

or similar solvent. (Such solvents attack prism cement.)


4.

Distilled

water for checking refractometer. (Must be free of impu-

rity.)

spoons for transferring sample on prism.

5.

Plastic

6.

Light source (illuminator) for refractometer.

B. Procedure:

Place about

to 2 drops of

warm sample

concentrate, taken from plant

operation pan, on prism of refractometer [B10.13(D.3)]. Close the cover

and immediately raise the refractometer


source of

to

eye level and point toward a good

light.

Observe the intersection of

field-dividing line

and

scale.

Make

the neces-

sary temperature correction indicated on thermometer on the side of refrac-

tometer.
C. Calculation:

Solids

[B10.13(D.6)

= Reading on

refractometer scale minus tem-

perature correction factor

D. Notes:
1. Immediately after taking refractometer reading above, wash prism
thoroughly with distilled water to remove whey product. Allowing product
to dry on prism is harmful to surface of prism.
2. Care should be taken to rinse refractometer prisms with distilled water and wipe dry with soft paper tissue. Do not touch prism with glass stirring rod, metal spoon or other abrasive equipment.

SUPPLEMENTAL CHEMICAL CONTROL METHODS

388

Test must be read quickly, as crystals form upon cooling and

3.

in-

accurate results are obtained.


4. Any air incorporated in sample will cause erroneous results. Samples
must be thoroughly but carefully mixed.
5. Accuracy of test is about 0.5 to 0.6% if all the steps in the proce-

dure are followed explicitly.

This method

6.

(40 to 60%).

is

only applicable to regular whey concentrate

correction factor must be applied to

whey concentrate

with a

higher solids content.

B1 0.2 References
2.

Bianco. L.J.
MiCKLE, J.B.

3.

Tech. Bull. T-67


Newlander, J. a. and H. V. Atherton, 1977. The chemistry and testing of dairy prod-

1.

Kraft, Inc., Chicago, Illinois.


et al. 1957.

Methods of determining

the total solids in fluid milk. Okla. Exp.

Sta.

ucts.

AVI

Pub. Co., Westport, Conn.

PiEPER, H.,

4.

Stuart

Jr., S.A.,

and W.R. Renwick. 1976. Rapid determination of moisture

cheese by microwave energy, collaborative study. Presented meeting. Association of


Official Analytical Chemists, Washington, D.C.
in

B1 1 .1 Somatic Cell Count


B1

Membrane Filter-DNA

1 .1 1

The

3.

io

MF-DNA test for abnormal

milk is a chemical assay system for determining the concentration of somatic cells in milk. The test consists of passing a milk sample through a special membrane filter thus trapping the somatic cells on the surface. The filter is then placed in an acidic indole solution
and heated. Upon incubation, cells lyse and the indole reacts with cellular
DNA to form a colored complex with a maximum absorption at a wavelength of 490 nm. Since the amount of DNA in each cell is uniform, the DNA
complexed with the indole can be related to the number of somatic cells
present in the original volume. In the MF-DNA test, the intensity of the
absorption at a wavelength of 490 nm is directly related to the concentration
of somatic cells in the milk sample.
A. Apparatus and reagents:
1. Multiple filter-holding manifold: every sample well capable of hold-

ing 25 ml.***
2.

Two

-liter

vacuum

flasks for moisture traps.

4.

Rubber hose for vacuum


Vacuum pump.

5.

Mastitis

3.

catalog

test

flasks.

membrane

#EMWP 02500).

^Millipore Corp., Bedford,

MA

01730.

filter:

25-mm

diam

(Millipore

Somatic

B11.1

Cell

Count

389

6.

Forceps: smooth tipped, stainless

7.

Spectrophotometer: Digichrome/^^
Automatic diluter dispenser: capable of handling 22.5
Disposable test tubes: 13 x 100 mm.

8.

9.

10.
11.

steel.

ml.^^^

Polypropylene test tube support.


Timer.

12.

Wash

13.

Two

bottle,

liter.

water baths: one

65

at

C, the other

at

90

with gable

cover.
14.

Heat-tempered storage

15.

5-ml dispensing pipettor.

graduated cylinder.

16.

17.

Amber

18.

Concentrated HCl.

-liter

bottles.

storage bottles.

19.

Indole (Millipore Corp.).

20.

Trisodium

ethylenediaminetetraacetic

acid

(EDTA)

(Millipore

Corp.).
21. Distilled or deionized water.
22. Triton

X-100 (Millipore Corp.).

Standard filters: A freeze-dried filter with a known number of


somatic cells trapped on the surface used to check the operation of the system (Millipore Corp.)
24. Color reference solution: A colored cobalt solution with an equivalent somatic cell concentration used for the daily calibration of the spectrophotometer (Millipore Corp.)
23.

B. Solutions:
1.

Stock HCl

(1 liter):

Pour 584 ml of

HCl

gently stirring to avoid excessive heat.


2.

Diluting agent

with enough water to

60

water into a pyrex bottle


add 416 ml of concentrated

distilled

suitable for hot, highly reactive solutions. Slowly

(1 liter):

make

ml of Triton X-100 and 4.5 g of EDTA


It is best to heat the water to
will be evenly dispersed. The solution will

Mix

liter

before adding the Triton so

it

of solution.

will clear upon cooling.


Mix 0.60 g of indole with enough water

be cloudy at this temperature, but


3.
1

liter

4.

Stock indole

(1 liter):

to

make

of solution.

Color developing solution: Mix the above solutions

proportions: stock HCl,


solution should be

made

part; stock indole,

fresh each day and stored in

The above reagents may

in the

amber

also be purchased, premeasured,

pore Corporation.

^^^Gilson Medical Electronics, Middleton,

WI

53562

following

part; water, 2 parts. This


bottles.

from the

Milli-

SUPPLEMENTAL CHEMICAL CONTROL METHODS

390

C. Procedure:

The

MF-DNA

test

procedure consists of the following basic steps:

Preparing equipment and reagents

Mixing milk with diluting solution


and diluting solution
Developing color
Analyzing color
1. Preparing equipment and reagents: Mix reagents as previously described. If pre-measured chemicals are purchased follow the manufacturer's
directions. Pour the diluting agent into heat-tempered storage bottles and
place in a water bath at 65 C. The diluting agent remains here for the entire
test. Set the other water bath at 90 1 C for use in color development.
Assemble the sampling manifold based on the manufacturer's directions.
Use the tubing to connect the manifold to the vacuum source through the
1-liter trap flask. Number samples and incubation test tubes to correspond
with sample wells on the sampling manifold. Unscrew handwheel and remove top plate of the sampling manifold to expose the filter support screens
in each sample well.
Carefully open the box of Millipore EMWPO2500 membrane filters. Notice that each of the membrane filters is sandwiched between two paper separators. One is blue and the other is light colored. Always make sure that the
side facing the blue separator paper is facing up in the manifold. The membrane pore structure is more effective when the proper side is up. Place one
filter on each filter support screen. If the filter support screens are first wet
with water, filters will stay in place better. The color development reaction is
affected by the filter and reagents, as well as by somatic cells. Therefore,
include at least three filter blanks with each set of samples that undergo color
development. Treat filter blanks exactly like sample filters, omitting only the
Filtering milk

milk sample.
Periodically (at least once a week) include one high and one low Somatic
Cell

already trapped.

#MBOOO

000 MS) to verify the reaction


known quantity of somatic cells
Milk samples should be allowed to warm to room temper-

Count Standard (Millipore Cat

chemistry. These standards are

filters

with a

ature.

Place the tubing from the large reservoir of the Digidil diluter in the hot

and connect the tubing from the small reservoir to the handchamber of the Digidil to 20 ml (5 ml x 4),
and the smaller chamber to 2.5 ml. Check to see that these volumes are
actually dispensed by discharging a test sample into a graduated cylinder. If
the diluter is not preheated it will cool the hot diluent and thus render it
ineffective. Dispense hot diluent back into the diluent supply bottle until the
large chamber is too hot to touch. The diluent will be cloudy if it is at the

diluting agent,

held dispensing probe. Set the large

proper temperature.
2.

Mixing milk with diluting agent: Swirl the milk sample to ensure unitip of the dispensing probe from the dilutor into the

formity and immerse the

B11.1 Somatic Cell Count

391

sample. Push the thumb button. The diluter will then draw up 20 ml of dilutand 2.5 ml of the milk sample into the respective reservoirs. Place

ing agent

the tip of the dispensing probe into the

first sample well of the sampling


manifold and press the button a second time. This will both mix and deliver
the milk-diluting agent into the sampling well. Repeat the last two steps for

each milk sample.


3. Filter milk and diluting solution: Turn on the vacuum pump, set the
vacuum to read 20 in. of Hg on the vacuum gauge, and open the valve on
each Sampling Manifold. The vacuum is to be left turned on until after top
is removed, as described later in method.
Because of sample sediment, a few samples may clog a filter before the
entire sample has been filtered. If this happens, puncture the filter to let the
remaining sample through. Set that sample aside and redo it after all other
samples have been filtered. Discard the punctured filter. If a milk sample
repeatedly clogs test filters, adjust the diluter to take up .25 ml of milk, redo
the sample, and then multiply the final results by two. Rinse the walls of
each sample well with water to wash down any clinging cells and residual
diluent. Unscrew the hand wheel and remove the top plate of the Sampling
Manifold. Leave the vacuum on to hold the filters in place until after the top
plate is removed. Then shut off the vacuum.
4. Developing color: Use a smooth-tipped forceps to individually transfer each filter into a 13 x 100-glass test tube. This should be done while the
filters are still wet. Keep the side of the filter with the cells on it facing in, so
cells are not wiped off on the test tube opening. Place the tubes in a test tube
rack. Dispense 5 ml of color development reagent into each tube, using the
fluid stream to wash the filter to the bottom of the tube. Filters must be fully
immersed in the reagent.
Incubate test tubes in a water bath at 90 1 C for 25 minutes. It is best to
cover or loosely cap tubes to keep foreign matter or condensate from falling

plate

into them. After incubation, cool tubes quickly to

room temperature

in tap

water.
5. Analyzing color: Although any dual-beam spectrophotometer could
be used, the following procedure is based on use of a Gilson Digichrome
Spectrophotometer. It is a vacuum-aspirating, flow-through design and is
well suited for rapid testing of consecutive samples. It measures the difference in optical density between the sample solution and a reference solution (pooled filter-blank solutions), and may be calibrated to read directly in
millions of somatic cells per milliliter of milk.
Allow the spectrophotometer to warm up for 15 minutes. Make sure there
is a moisture trap between the vacuum pump and the spectrophotometer to
prevent moisture from being drawn into the pump. Set the vacuum pump at
5 in. of Hg. To draw liquid into the sample (active) cell, immerse the tip of
the hand sampling probe and press the button on the handle. The reference
cell has a separate probe which is activated by the green button on the instrument panel.

SUPPLEMENTAL CHEMICAL CONTROL METHODS

392

Immerse both probes in 5% detergent solution (5 ml of liquid detergent


mixed with 95 ml of water). Flush the cells by activating both probes several
times. Flush each cell 5-10 times with water. Purge both cells with air by
activating the probes in the air. Make sure the active cell is drawing up 4 ml
of liquid. Immerse the tip of the hand probe in a graduated cylinder that
contains exactly 10 ml of water and note how much is left after the probe is
activated and withdrawn. Sampling volume can be adjusted by turning the
"sample time" screw on the back of the instrument. Test and adjust in this
way until the probe is consistently drawing up 4 ml. The reference cell will
then automatically take up the correct amount of liquid. Purge with air between each sample.
To "zero" the instrument, place both probes in a beaker of water and
activate both probes. Repeat several times. Adjust the balance control knob
until the digital readout is 0.00. The last digit may flicker a little. With water
in the reference cell, draw color reference solution (Millipore Cat #MBOOO
000 MC) into the active cell. The digital readout should correspond to the
cell count on the bottle. If not, adjust the "scale factor" screw on the back
of the instrument until it does. Next, recheck the "zero" as above, adjusting
the balance control knob if necessary. Then check once more with color
reference solution.

If the reading has changed, adjust the scale factor again.


Repeat the above procedure to zero instrument until both readings are correct. The unit is now ready for operation. Flush both cells with several rinses
of water and purge with air. Pool contents of all test tubes having filter-blank
solution, mix well, and draw this solution into the reference cell.
D. Calculation:
To measure a sample, insert the tip of the hand sampling probe into the
desired test tube and press the button on the handle. Wait for the digital
reading to stabilize, and then record the number as millions of cells per milliliter

of milk.
for example, the reading

If,

is

1.04, record

it

as 1,040,000 cells per ml of

milk.

E. Notes:
1

Purge the active

cell

with air before proceeding to the next sample to

prevent cross contamination.


2.

At the end of each day's testing, flush both cells with 5% detergent
and rinse with water to prevent the build-up of fat residue.

solution

B11.12 Optical: OSCC-llTechnicon '^-^-e-s


A prefixed milk sample is automatically measured into, then sequentially
and automatically reacted with, a reagent matrix which dissolves fat, carbohydrates, etc, in milk, leaving somatic cells in suspension. The mixture is
then pumped into a cell counter which counts cells as they pass through the
stage of a darkfield microscope. Samples are compared with a reference suspension of turkey erythrocytes and results are recorded on a strip chart.

393

B11.1 Somatic Cell Count

A. Apparatus and reagents, see Bl 1 .12 (I):


1. Automatic analyzer "*: AutoAnalyzer with following modules: Sampler IV, proportioning pump III. constant temperature bath cartridge with a
3.5-min hold time at 65 C, optical somatic cell counter, and recorder with
strip chart

Pipets: Disposable, 4

2.

ml for sampling and

HCHO

4.

of absolute methanol.
5. Diluent/wash solution:
liter

ml graduated to 0.1 ml.

H.O.
Mix 40 ml of 37%
in 500 ml
Triton
X-100
Mix
ml
of
50%:
500
Methanolic Triton X-100,
Fixative:

3.

Add

solution with 60 ml of

2 ml of methanolic Triton

X-100, 50%, to

of water and mix.

Methanolic potassium hydroxide: Dissolve 350 g of KOH in absolute


liter with absolute methanol.
methanol and dilute to
hydrochloride: Dissolve 120 g of
hydroxylamine
7. Methanolic

liter with
of
absolute
methanol, and dilute to
in
900
ml
NH2OH HCl
6.

methanol.
8.

to

EDTA,

liter

and

tetrasodium

filter

through

salt:

Dissolve 200 g of Na4EDTA


I paper.

in

H2O,

dilute

Whatman No.

Cool 600 ml of methanolic KOH, (item 4)


portions
to 1.5 liter of methanolic NH2OH,
in
100-ml
add
to 2 C. Slowly
after
each addition. (Caution: Do not let
vigorously
(item 5), cap, and shake
to
10
container.)
Cool
C and shake every 15 min during
in
pressure build up
9.

Clarification reagent stock:

cooling. Refrigerate overnight. Filter with

Add

vacuum through coarse-poros-

liter of methanolic
mix.
If a precipitate
and
Triton X-100. add
at 10 C.
for
30
days
is
stable
Solution
refilter.
standing,
appears upon
volumes
of clarisix
Dilute
solution:
working
reagent,
10. Clarification
ity fritted-glass funnel.
1

liter

of

fication stock (9) with four

2 liters of filtrate to

EDTA

solution, cap,

volumes of water.
Fixed turkey erythrocytes"*. Lots

may vary:
Reference cells:
use.
number
before
lot
known
against
a
lot
number
check
each
therefore,
five
with
compare
and
counts,
and
cell
curves
threshold
comparative
Check
milk samples having various somatic cell counts, against direct microscopic
11.

somatic

cell

counts.

B. Analytical system:

Use standard Solvaflex pump tubing throughout system, unless otherwise


specified.

Pump sample
with air

pumped

at 0.32
at 0.32

ml/min.

Pump

diluent at 1.20 ml/min

ml/min: add to sample

in

HO

fitting.

and segment
Pass diluted

sample through double Kel-F mixing coil to resample fitting where clarification reagent is introduced through Solvaflex tubing at 2.50 ml/minute.
Pump stream through single Kel-F mixing coil into heating bath at 65 C.
Pass clarified, diluted sample through debubbler tube, and then through
flowcell in optical somatic cell counter.

***Technicon Instruments Corp, Tarrytown.

NY

10591.

SUPPLEMENTAL CHEMICAL CONTROL METHODS

394

C. Start-up procedure:

reagent lines in appropriate solutions and sampling line in wash


H2O. Turn on power switch for each module. Latch platen into position and

Place

all

pump

reagents through system until oscilloscope on cell counter indicates


particle-free flow stream (ca 10 minutes). Fill PC-1 valve V3 full by inverting
it

for a short time.

Check

flowcell pull-through line to insure that

no bubbles

are being pulled through. Adjust resample fitting so that a small portion of
every air bubble is pulled through. Examine for leaks. Check bath temperature (65 1 C). Set threshold control to 21. Turn on recorder chart drive.

O and record baseline 5 minutes.


procedure:
Shut-down
D.
Turn off" sampler manually, or with stop pin, after last sample has been
aspirated. Turn off" recorder chart drive when pen returns to baseline after
last sample peak. Place clarification reagent line in 30% methanol wash solution. Place diluent and sampling lines into wash water. Pump cleaning solutions through system 20 minutes. Remove all lines from wash solutions. Release platen, relax pump tubes, and turn module switches off. Heating bath
may be left on to eliminate warm-up time the following day.
E. Checks and calibration:
Proceed as in Bll. 12(C) Start-up Procedure, obtain reagent baseline,
and clamp off sample waste. Thoroughly mix one tube of reference cells by
inverting 30 times, letting siliconized glass beads fall completely through
liquid until cells are evenly distributed. Place sampling probe into reference
tube, keeping end of probe > 5 mm from bottom of tube, and continually
aspirate reference material into system. When cell pulses appear on the oscilloscope, adjust recorder pen to 50% full scale, using cell counter sensitivity control. Record for 2 min, turn off chart drive, set threshold control to 8,
wait 2 min, turn on chart drive, record for 30 sec and turn off chart drive.
Manually advance paper 0.5 in. (1.27 cm). Set threshold control to 24, wait
2 min, turn on chart drive, record for 30 sec, and turn off chart drive. Manually
advance paper 0.5 in. (1.27 cm). Set threshold to 21, wait 2 min, turn on
chart drive, and record for 2-5 minutes. Return probe to sampler arm, and
turn off chart drive. Difference between readings at threshold settings of 8
and 24 should be <5% full scale deflection. Excessive threshold difference
may be due to blockage in flowcell or tube system, optics out of focus, or
Set reagent baseline to

inadequate mixing of reference

cells.

F. Preparation of sample:
Pipet 4 ml of fresh milk (<36 hr from time of collection) into sampling

tubes to which 0.1 ml of fixative reagent has previously been added, stopper,

and invert 10 times for mixing. Let tubes stand 18 hr

at

20

or

hr at

55 C.

G. Procedure:
Place

H2O

in first

cup and reference

cells in

next three cups. Place un-

treated milk sample, for reagent control (response should be similar to

response), in

fifth

cup. Continue with samples, inserting

H2O cup

H2O

every

B11.1 Somatic Cell Count

395

10-15 samples and reference cell cup every 30 samples. Set sampling rate to
80/hr, (or 120/hr^) with 4:1

sample-to-wash

ratio.

Activate sampler.

As

first

reference peak appears on chart, adjust peak to correspond to assigned val-

ue of reference tube, using sensitivity control. Each scale division on strip


chart should be equivalent to 20,000 somatic cells, e.g., reference assigned
value of 1.4 X 106 = 70% full scale.

H. Calculation:
Calculate results for each sample by multiplying peak height in scale divisions X 20,000. Report as optical somatic cell count (OSCC)/ml. If peak

height exceeds

full scale,

report as

OSCC =

not clear, e.g., from instrument lag time

peak height

is

high cell count

is

>2,000,000/ml.

when sample with

If

following by sample with low cell count at a high sampling rate, reanalyze

doubtful sample to obtain well defined peak height.

Notes:
Consult 1975 AOAC laboratory safety sections 51.037, 51.040, 51.066
when working with sodium and potassium hydroxide, toxic solvents and
methanol.
/.

1.

the

2. If damping of the signal is required when operating instrument, move


damping control from the rapid position to 80, 60 or 40 (depending on the

rate of analysis), as
at

soon as the pen has reached

its

maximum and

is

plotting

steady state. The amount of damping time varies with the rate of analysis

from 20 sec at 80/hr to about 30 sec at 40/hour. Be sure the damping is not
used on either the wash in or wash out. Ideally, damping should end at about
2-3 sec before washout begins. Be sure the damping switch is not activated
unless the timer switch is in the set position. When no damping is desired, be
sure that damping switch is in the rapid position and the timer switch is at
set. No damping is possible if sampling is being done at 120 or 200 samples
per hour.
3.

Prevent samples from freezing before analysis. Freezing irreversibly

coagulates protein, resulting in incorrect high counts.


4.

Milk samples should be no older than 36 hr before "fixing" and

should be analyzed within 48 hr after "fixing".


5. Once a vial of OSCC reference has been opened, no more than eight

samplings should be taken from


6.

Do

it

and

it

should not be held more than 2 days.

not freeze or refrigerate standards. Refrigeration causes cells to

clump and freezing causes lysis.


7. If the sample comes from a "Twirlpack" or other small
tial

shaking

is

bottles, ini-

important. Twenty-five times in 7 sec through an arc of

necessary to obtain satisfactory counts.


8. Threshold checks should be done daily

at

ft is

the beginning of the day's

if there is a sudden
system should be examined to locate malfunction.
9. Samples with a count of over 2 million may be estimated by reducing
the sensitivity setting and recalibrating accordingly.
10. When preparing working clarification reagent, protect hands with

operation.

change

If

the threshold

in sensitivity, the

is

not within specifications or

SUPPLEMENTAL CHEMICAL CONTROL METHODS

396

Wash any

gloves and eyes with safety glasses.

spills

on bare skin with

copious amounts of water.


11. Extreme care should be taken not to contaminate samples with extraneous particulate matter.
12. For "Screening" purposes, any satisfactory sampling rate
used,^ but for confirmation of sample counts, the 80/hr sampling

may be
cam must

be used.

B11.13 Fluorescent Dye: Automated, '^^ Foss


A. Principle:
Milk samples are preheated and fed to the instrument. The sample is taken
automatically, mixed with preheated buflFer and dye and is stirred extensively.

Part of the mixture

is

then transferred to a rotating disc which serves as

The mixture on the wheel is illuminated


by a xenon arc lamp which excites the somatic cell complex to emit fluorescent light. Each cell produces an electrical pulse which is counted and the
total cell content per milliliter is printed after each sample.
B. Apparatus and reagents:
1. Fossomatic ^^^ consists of heating coils, rotating table, stirrer, syringes for delivering buffer and dye, rotating disc and a fluorescent microthe object plane for the microscope.

scope.
2.

Standard solution:
a.

Dye

solution,

idium bromide

is

0.1% ethidium bromide (C2iH2oBrN3):

diluted in

liter

1.00 g of eth-

of distilled water. Heating to 40-50

helps dissolve the powder. Store in a light-proof and air-tight bottle no

longer than 60 days.


b.

(1%) 10 ml of Triton X-100 is dissolved in liter of


C may be necessary. This solution can be
an air-tight container for a maximum of 25 days.

Triton X-100

distilled water.

stored in

Heating to 60

c. Buffer Potassium acidphthalate (C8H5KO4) (0.025M): 51.0 g of


potassium acidphthalate plus 13.75 g of potassium hydroxide (KOH) are
dissolved in 10 liters of distilled water. Heating to 50 C helps dissolve
the powder. Add 10 ml of 1% Triton X-100 solution. Store no longer
than seven days.
3.

Working
a.

Dye

solution:

solution

20 ml of 1% ethidium bromide solution per

liter

of

buff'er.

pure

b.

Diluting solution

c.

Rinsing liquid add 10 ml of

ammonia

buffer.

1%

Triton X-100 and 25 ml of

25%

solution to 10 liters of distilled water.

C. Analytical system:

The Fossomatic

is

started

by pressing the

"POWER ON"

button and the

5Manufactured by Foss Electric, HillerOd, Denmark, and available from Foss America,
Inc.,

P.O.

Box

504, Fishkill,

New York

12524.

B11.1 Somatic Cell Count

"LINE FAILURE"

397

button and after 30 sec, press the

button on the front panel. Check that the xenon lamp

"LAMP IGNITION"

(the green lamp in


and press the button once more. The
lamp should stay on. Make sure "Liquid Heaters" switch has been turned
on. Cycle the instrument six times.
Pretreatment of the samples is limited to heating to 40 C (approximately 5
min for a 10-15-ml sample) in a water bath and inversion to mix the contents
of the sample bottles. Caps are then removed and bottles placed on the automatic feeding rack or the samples are transferred to containers of the type
for which the instrument is adjusted. To ensure that somatic cells are distributed evenly throughout the milk, samples are first stirred by an external
stirrer while they are still on the rack.
Press the "START" button and the instrument will start its automatic
procedure which will continue until the last sample has been measured and
the result printed. The pipet then dips into the bottle and draws out 200 /xl of
the sample after which it rises and swings to a position over one of the six
glasses in the rotary table. One syringe measures the buffer while the other
syringe measures the dye solution. The sample is transferred from the bottle
to the glass in the rotary table by means of another syringe.
The 200-/Ltl sample plus 1.8 ml of buffer is discharged into the glass together with 2 ml of dye solution. Immediately before being discharged into the
glass, both buffer and dye solutions are heated by passing them through

the bottom

is lit

on). If not, wait 10 sec

is

electrically-heated coils, the resulting temperature of the mixture in the glass

being 63 C. This temperature, which

tween the dye and

DNA,

is

is

important for complete reaction be-

controlled by

means of a sensor

in the heating

coil circuit.

The rotary

table

now

turns to bring the glass one position further in the

sequence. Here the mixture

is

stirred vigorously

to

keep somatic

evenly distributed and to break up any bacterial clusters which

formed

in the milk.

In the next position, the glass

through a tube

in the sealing

tube, through a

charge

cells

may have

filter

cap.

is

sealed and compressed air

The mixture

is

is

introduced

forced out through a second

and valve and out via the measuring syringe and

dis-

jet.

After 1.5 to 2.0 ml of the mixture have flushed the tubing, the measuring
syringe, controlled by a motor, dispenses 20

/xl

of the mixture through the

discharge jet to the periphery of the disc. This takes place at a constant

speed for 12 seconds.


After this period, the valve opens and cleaning liquid

is

forced up through

same
back through the filter. Flushing the system through in this way eliminates any possibility of transfer error.
The next rotary movement brings the glass to where it is rinsed and the
rinsing liquid is then almost completely drawn away. At the last position, it
is drawn out completely, ready to receive the next sample mixture.

the measuring syringe and out through the discharge jet and at the
time,

SUPPLEMENTAL CHEMICAL CONTROL METHODS

398

When

passing under the microscope, somatic cells which have reacted to

the dye will be excited

The red fluorescent

by the blue

signal

is

light

and

emit red

will

light.

picked up by the photo multiplier and ampli-

A discriminator is set

fied

before being fed to the readout display and printer.

at a

prescribed setting to allow for accurate counting of somatic cells and to

eliminate background noise.

Startup procedure, operation, shutdown

D. Systems operational check


procedure:
1. Start-up procedure:
a. Check for main power.
b.
c.

Check
Check

to see that air pressure

is

at

80

psi.

to see that reagents are in sufficient quantity for

use. If a container

is

used to collect waste,

it

one day's

should be emptied to pre-

vent overflow.
2.

Testing the samples:

Complete systems operational check and load the sample tray with standards (5) and then test first set of unknowns and do standards at end. If a
new rack is not placed in position, the ''START" button lamp will go out.
Repeat procedure for duplicate

set of

samples.

Shut-down procedure:

3.

After doing the


out and

all

last

sample, push the

"OFF"

functions stop. Air pressure and

button. All lamps should go

vacuum

are reduced to zero in

approximately 10 seconds. Clean the cups and rotating disc


E. Notes:
Contributions to the count by materials other than

which partly degenerated

if

needed.

cells, e.g., bacterial

be found, can be considered


of no significance in practice. Individual and intact bacteria will not be dyed
and consequently, will not contribute to the count. Dead and partly degenerated bacteria will react with the dye provided that the DNA molecule is
intact, but the signals arising from these are so small that they are absorbed
into the background noise without being counted.
Therefore, any inaccuracies in the count are most likely due to the follow-

clusters in

cells are to

ing:
1.

Error

pipetting of the sample.

in

The somatic

cells have lost their ability to absorb dye.


Degeneration of the somatic cells.
a. This error can arise since there is a certain tolerance in the volume
of sample taken and because the somatic cells might not be uniformly

2.
3.

distributed.
b.

Normally,

this ability

cells

do not

lose their ability to absorb

dye even though

diminishes as the age of the sample increases. However, use

of certain preserving agents, eg., formalin, results


nuclei of cells until they

become hard

particles.

in

hardening of the
is unable to

The dye

penetrate these hard particles with the result that the

DNA

molecule

B12.1

399

Ash

cannot absorb any dye. Consequently, signals from cells in a sample


preserved with formalin will be smaller than those cells in unpreserved
samples. This is the most dominating factor whether one is considering
effects of age on preserved or unpreserved samples or effects of other
handling. This means that if the cells have not degenerated for reasons
of prolonged or incorrect storage or any other treatment, it is possible to
obtain good results with the Fossomatic.
Factors which give rise to cell degeneration and thereby set limits on
the age of a sample are the following:
Bacterial growth in milk

High storage temperatures


High bacteria count when preserving agent

is

added

Vibration (cells are very fragile)

Freezing

particularly slow freezing which forms

crystals in the cells causing

them

ice

to rupture.

B11.2 References
1.

American Public Health Association. I%7. Standard Methods


Dairy Products. 12 Ed., pp. 287-291 and Chapter

12,

for the

Examination of

American Public Health Association,

New York
2.

Association of Official Analytical Chemists.


12th ed. Association of Official Analytical Chemists,

3.

Bremel, R.R., ScHULTZ, L.H., Gabler, F.R. and J. Peters.


samples by the membrane filter DNA procedure.

5.

1977. Estimating somatic

Food Prot. 40:32-38.


Grappin, R. and R. Jeunet. 1974. Premier essais de Tappareil "Fossomatic" pour la determination automatique du nombre de cellules du lait. Le Lait (Nov-Dec) 627-644.
Kelly, W.N. 1972. Collaborative study of an automated optical cell counting method for
raw milk. Associate referee report, presented 80th AOAC meeting, Oct 9-12. Association
cells in milk

4.

1975. Official methods of analysis.


Washington, D.C.

J.

of Official Analytical Chemists, Washington, D.C.


6.

Kelly, W.N.
matic

cell

1977.

Comparison of

count procedures

in

optical somatic cell count

and direct microscopic so-

a collaborative study on split milk samples. 91st

AOAC

meeting. Association of Official Analytical Chemists, Washington, D.C.


MocHRiE, R.D. and R.J. Monroe. 1977. Collaborative study of somatic

8.

cell counting
Fossomatic method. 91st meeting AOAC. Association of Official Analytical Chemists.
Washington, D.C.
Read, Jr., R.B., Reyes, A.L., Bradshaw, J.G.. and J.T. Peeler. 1%7. Electronic count-

9.

Schmidt-Madsen,

7.

ing of somatic cells in milk.

J.

Dairy Sci. 50:669-674.

D. 1975. Fluoro-opto-electronic cell counting on milk.

J.

Dairy Res.

42:227-239.
10.

Ward, G.E., and L.H. Schultz. 1973. Estimation of somatic cells in milk by the
desoxyribonucleic acid method with indole. J. Dairy Sci. 56:1097-1011.

B12.1

Ashi2

B12.11

Cheese

A. Apparatus and reagents:


1.

Crucibles, porcelain, Coors

#1 or platinum

dish.

filter-

SUPPLEMENTAL CHEMICAL CONTROL METHODS

400

2.

Desiccator, charged with efficient desiccant such as calcium chloride

or sulfate, alumina, etc.


3.

Analytical balance.

4.

Muffle furnace, thermostatically controlled.

5.

Atmospheric oven, gravity convection or forced

6.

Muffle tongs.

7.

Marking

8.

Iron tripod.

9.

Aqua

ink,

10.

permanent-type wax pencil for crucibles.

regia solution:

part technical grade

Steam bath

air.

HNO3

Mix three
and

parts of technical grade

HCl with one

dilute mixture 1:1 with distilled water.

(optional).

B. Preparation of crucibles:
Heat side of crucible to be

marked over low flame for a few minutes.


Apply marking ink while crucible is warm. Allow to dry. Fire over burner at
dull red heat.

Submerge crucible in bottle of aqua regia. Allow to remain for several


Remove, rinse in tap water, then in distilled water.
Dry crucible in atmospheric oven at 100 C. When dry, ignite in muffle
furnace or over burner to dull redness. Cool at room temperature for 5 minhours.

utes. Place in desiccator until ready to use. Crucibles should

aqua

regia, dried

and ignited

in this

manner

after

be cleaned

in

each use.

C. Procedure:

Weigh accurately about 10 g of cheese into a prepared, tared crucible.


Evaporate to dryness in an atmospheric oven at 100 C (or place on steam
hour).
bath and dry for approximately
Transfer to hood and place on iron tripod over burner. Heat cautiously to
prevent spattering, remove burner if fat ignites. Continue heating until all
1

volatile matter

is

smoked

off.

C (dull red heat). Incinerate sample


from carbon and a light gray or white ash remains.
Remove crucible from furnace. Cool at room temperature for 5 minutes.
Place in desiccator until ambient temperature is assured. Reweigh.
D. Calculations:
Place in muffle furnace at 550-660

until

it

is

free

^
%

,
=
Ash
,

Weight
of residue x 100
^
Weight of sample

E. Note:

This method

is

applicable for

all

cheeses and processed cheese products

including imitation cheeses with low or high fat content.

B12.12 Fluid Milk, Ice Cream Mixes, and Whey


A. Apparatus and reagents:
Same as B 12. 11(A) except item 8 (iron tripod)

to

be excluded.

B12.2 References

401

B. Preparation of crucibles:
Same as B12. 11(B).
C. Procedure:

Weigh accurately, approximately

10 g of sample into a prepared tared cruB12. 11(C) except the flame treatment on the tripod can
be omitted as these products are low in fat content and need no volatilizing
cible.

Proceed as

in

of fat as a pretreatment.

Whey

B1 2. 1 3 Dry Milk and

Products, Malted Milk and Animal Feeds

A. Apparatus and reagents:


Same as B 12. 11(A) except items 5-atmospheric oven and 8-iron tripod
can be excluded.
B. Preparation of crucibles:

Same

as B12. 11(B).

C. Procedure:

Weigh accurately

2 g of

sample into a prepared, tared crucible and pro-

ceed as in B12. 11(C) eliminating the atmospheric oven predrying and the
flame treatment on the tripod as products are low in moisture and fat content, and need no pretreatment.

B12.2 References
1.

Association of Official Analytical Chemists.

1975. Official

methods of

12th ed. Association of Official Analytical Chemists. Washington, D.C.


2.

Bianco,

L.J. Kraft Inc., Chicago, Illinois.

analysis,

Index
Allowable

standard deviation of difference in milkfat determinations


(Table 19:11), 251

Abnormal Milk, Screening and


Confirmatory Methods for the
Detection of (Chapter

8),

15-140

Alternate Viable Count Methods


(Chapter 21), 311-324

Acetonitrile partitioning, 262-263

Acid degree value (ADV), 380-381


Acidity, potentiometric pH, 358-361
Acidity titratable, for dairy products,

355-357
Acidophilus milk, microbiological

American Chemical Society, Committee


on Analytical Reagents, 286
American Public Health Association
(APHA),
grading charts, 210

test-

pipet specifications, 79, 175

ing, 163

Acid orange method, protein determination, 274-277


Acinetobacter, 110
Added water in milk,
thermistor cryoscope, 231-233
vapor pressure osmometer, 233- 234

Adenovirus infections, 23

ADV

(acid degree value), 380-381

Aflatoxins, 22-23

Agar contact (RODAC) method, surface


sampling, 200-203

Agar

layer, quantity of

medium

required

to insure uniform thickness, (Table

SMA, 69
Standard Methods for the Examination of Dairy Products, 65, 67
Standard Methods for the Examination of Water and Wastewater,
51,67, 203
American Society of Medical Technologists, reagent grade water, 62
Amido black method, protein determination, 269-273
Animal feeds, ash in, 401
Anthrax, 13
Antibiotic detection, 142-149, 331-344
Antibiotic medium, 55, 142, 145-146

9:1), 143

Antibiotic Residues in Milk and


Dairy Products, Detection of

Agar, preparation, 55-58


sterilization

reference standards for

and storage, 58-60

(Chapter

9),

141-150

Agitators, 38-39

AOAC (Association of Official AnalytiAir, Equipment, Containers and


cal Chemists), 7, 48, 261
Water, Microbiological Tests APHA (American Public Health Associ197-205
for (Chapter

Air,

16),

ation), 51, 65, 67, 69, 79, 175,

microbiological quality tests, 204,

349^351

327- 354

Alcaligenes, 110

403

210

Appendix A, Supplemental MicroBiological Control Methods,

404

INDEX

Appendix B, Supplemental Chemical Control Methods, 355-401

Chem-

Bacterial infections and intoxications


{Cont'd)
Pasteurella, 19-20
Shigellosis, 20
Staphylococcal enterotoxin, 20-21
Streptococcal infections, 21-22
Bacteriophage lactic culture, detection

Atmospheric oven method, moisture and


solids determination, 255-256
Auramine O, 18
Aureomycin-rose-bengal agar method,
detection of yeasts and molds, 327-

and enumeration, 353-354


Barium- 140, in milk, 283
recovery by gamma spectrometry,
304-308
BHC residue, in milk and dairy products, 261-269

Ash, alkalinity

of,

361-363

products, 399-401
Aspergillus flavus, 22
parasiticus, 22-23
in dairy

Association of Official Analytical


ists (AOAC), 7, 48, 261

329
Autoclave (steam

Biological
sterilization),

58-60

Automated plate loop count, 353


Automated turbidometry, for milkfat

de-

termination, 249-251

Automatic milk- vending machine,

12

Available chlorine, thiosulfate titration,

235-236

Stain

Commission

certifica-

tion

methylene blue chloride, 178


methylene blue thiocyanate, 189
resazurin tablets, 193
Botulism, 14

BR

Foss

test, inhibitory substances in


milk, 341-344

Brilliant green lactose bile broth

2%,

55,

96, 100

B
Babcock

fat determinations,
(Table 19:111), 259
Babcock fat test modified,
for milk and cream, 236-239
for milk products, 369-375

values

Bacillus, 107, 110


Bacillus anthracis, 13
B. cereus, 13-14

B. megaterium, 330
B. stearothermophilus 332-344
B. subtilis, 142-145
Bacitracin, in dairy products, 149
,

Bacteria, Direct Microscopic Meth-

od FOR (Chapter

14),

169-186

Bacterial

counts, interpretation direct


microscopic, 171-172
Bacterial estimates, direct microscopic
counts, 170-171
Bacterial infections and intoxications

Anthrax,

13

Bacillus cereus, 13-14

Botulism, 14
Brucellosis, 14-15
Clostridium perfringens, 15-16
Diphtheria, 16-17
Leptospirosis, 17
Listeriosis, 17

Mycobacteria, 17-19

Brucella abortus, 14
B. suis, 14
B. melitensis, 14
Brucella, in cheese, 12
in milk, 12

Brucellosis, 14-15

Buffer solutions:
phosphate, 62

magnesium sulfate, 62
Bulgarian buttermilk testing, 163
Bulk samples, 42
Bulk tanks, 36
Butter and margarine, moisture determination, 252-256
Butter, Brucella infections from, 14

Kohman

modified test for

fat,

mois-

ture and salt, 376-378

microbiological methods, 157-159

phosphatase

test, 219, 221,

226

potentiometric pH, 360-361


sampling, 44

Butter, Margarine and Related


Products,
Microbiological
Methods for (Chapter 11), 157159
Buttermilk
Bulgarian, 163
dry alkalinity of ash, 361-363
fat, Pennsylvania modification, 374
moisture determination, 255-256

405

INDEX
Buttermilk (Cont'd)

phosphatase

tests, 219, 221,

titratable acidity,

224

355-357

Chocolate milk and drink, fat test


Pennsylvania modification, 373
phosphatase test, 224

Chromatography determination,
ticides,

Calcium alginate swabs, 351-352


Calcium and strontium, recovery from a
single precipitation (Table 20:1), 293

Calibration, of gamma spectrometer, 306

of microscope, 180-181
of turbidometer for milkfat, 250-251
California mastitis test (CMT), 118-120
grading and interpretation (Table 8:1),
121

procedural steps (Fig. 8:2), 119


Casein powders, moisture and solids determination, 252-254
Casein, sampling, 43-44
Casein soy peptone agar, 55, 58
Cesium-137, in milk, 283
recovery by gamma ray spectrometry,
304-308
Cheese and cheese products, moisture
and solids determination, 252-266

Cheese and Other Cultured Products, Microbiological Methods


FOR (Chapter 12), 161-164
Cheese, ash in, 399-400
Babcock modified fat test, 371-372
extraneous matter in, 367-368
lactose in, 381-385
microbial flora, 161
microbiological testing, 162-163
microwave method for moisture and
solids in, 385-386
pathogens isolated from, 14, 16, 20
phosphatase test, 219, 221, 225
potentiometric pH method, 358-361
sampling, 45-46
Cheese food preparations, dry dairy
products use in, 153
Cheeses, Volhard, 365-366
Chemical control methods, supplemental
(Appendix B), 355^01
Chemical Methods (Chapter 19), 231282
Chemical methods, sampling for, 52-53
Chlorides, in dairy products, 363-365
Chlorine available, thiosulfate titration,
235-236

Chlortetracycline, in dairy products, 149


Chocolate, in frozen dairy products, 165

CIP

for pes-

264-268

(cleaned-in-place) equipment, 50

Citrate Azide Agar, 66

Citrate-azide agar method, enterococci


in butter,

328-329

Cleaned-in-place (CIP), equipment, 50


Cleaning, microscope slides, 172-173
sterilizing

and sanitizing equipment,

35
Clostridium, 110
Clostridium botulinum, 14

C. butyricum, 15
C. perfringens, 15
C. sporogenes, 15

CMT

(California mastitis test), 118-120


Coagulase-positive staphylococci in dry
milk, 154
Cocoa in frozen dairy products, 165
Coffee creamers, sampling, 40-41

CoLiFORM Bacteria (Chapter

6),

95-

105

Coliform determinations, butter and


margarine, 157-159, 328
cheese and other culutred products,
161, 163-164

condensed products, 153


containers and equipment, 200
dry dairy products, 153, 156
equipment for, 97
false counts, 165
frozen dairy products, 165, 168
ice cream, 165-168
liquid medium for, 99-100
MPN tables for, 102-104
plate and tube methods for, 98-99
solid media for, 99-100
Coliform organisms, 7-8, 16, 95-105
Collaborative studies, 3-4
Colony count, oval tube, 312-315
plate loop, 315-317
spiral plate, 317-324
Standard Plate, 88-92
Color standards (phenol) for phosphatase testing (Table 18:11), 223
Color standards, preparation, 223-224
Coloring materials, in frozen dairy products, 165

microbiological testing, 167

INDEX

406

Completed coliform tests, 96, 100


Composite schemes, two milk (Table
4:1),

70

Composites, milk, 69-70


Compound microscope, 176-177
Computing Standard Plate Count (Table
5:1), 90
Computing Standard Plate Count, 92-93
Concentrated dairy products. Standard
Plate Count, 85-86

Concentrated Milk and Dry Milk,


Microbiological Methods for
(Chapter 10), 151-156
Concentrated milk, microbiological testing, 152-153
phosphatase, 226
sampling, 43
Condensed milk sweetened, microbiological testing, 152-153
moisture determination, 254-255
Condensed whey, moisture determination, 254-255
Confirmatory procedures,
conforms, 96, 99^100
detection of abnormal milk, 125-139
microscopic, 125-134
Confirmed test for coliforms, 96, 99-100

Containers, Equipment, Water and


Air, Microbiological Tests for
(Chapter 16), 197-205
Containers, microbiological tests, 197202
Conversion chart, metric-U.S. system,
53
Cornell phosphatase test, 222-225

Corynebacterium diphtheriae
Coryneform group, 107

16

Cottage cheese, chlorides in, 364-365


dry dairy products in, 153
microbial growth, 161
microbiological examination, 163
phosphatase tests, 219, 221
Coulter counter, for somatic cell counting, 136-140
Counting colonies. Standard Plate
Count, 88-92
Counting colonies. Standard Plate Count
(Table 5:1), 90
Coxiella burnetii, 25-26
Cream, chlorides in, 363-364
cultured, 161, 163-164
in frozen dairy products, 165
phosphatase tests, 219, 221, 224

Cryoscope, thermistor, 231-232

Culture Media and Preparation


(Chapter 4), 55-75
Culture media, formulas and instructions, 56-61
listing,

55

Cultured dairy products, microbial

flora,

161

sampling, 45-46

Standard Plate Count, 85-86


Cultured milk, microbiological examination, 163

Cylinder count method, pasteurized milk


products, 329-330
Cylinder plate assay, 145-149

Dacron (polyester) swabs, 351-352


Dairy and Related Products, Sampling (Chapter 3), 33-54
Dairy animal feeds, moisture and solids
determination, 252-254
DDE, determination in milk and dairy
products, 261-269
DDT, in milk and dairy products, 261269
Dehydrated culture media, 56
Delvotest P, penicillin detection in raw
milk, 340-342

Detection of Abnormal Milk,


Screening and Confirmatory
Methods (Chapter 8), 115-140
Detection of Antibiotic Residues in
Milk and Dairy Products (Chapter 9), 141-150
Detergent residues, determination, 6465
Dieldrin, in milk and dairy products,
261-269
Dihydrostreptomycin, in dairy products,

149
Dilution water, for concentrated and dry

milk products, 151


SPC, 82
phosphate-buffered preparation,

for

62,

198
Dilutions, measuring, 86
Dilutions, preparing (Fig. 5:2), 84
Diphtheria, 16
Direct microscopic count (DMC),
bacterial estimates by, 170
confirmatory tests for abnormal milk,
125-134

407

INDEX
Direct microscopic count (DMC) (Cont'd)
dry dairy products, 153-156
interpretation, 171-172

Emulsifiers and stabilizers, sampling, 48


Emulsifiers in frozen foods, 165, 167
Encephalitis, tickbome, 24-25
Endotoxin in dried milk, 13

methods, 48
pasteurized milk, 169
raw milk, 169
sources of error, 170

Enterobacter {Aerobacter), 95
Enterococcus counts, butter and margarine, 158, 328-329
Enteropathogenic Escherichia coU, 16-

dry milk, 170

Direct Microscopic Method for


Bacteria (Chapter

14),

169-186

Disc assay technique, penicillin detection, 142-145, 331-338


Discs, sediment, 207, 209-211
Disintegration method, microbial content paper container materials, 344-

347
Dispenser, sampling, 40-41
Distilled water, 61
(direct microscopic count), 109,
126, 153-156, 16^186
(direct microscopic leucocyte
count), 126
DMSCC (direct microscopic somatic cell
count), 126-135
Dried egg products, moisture and solids
determination, 252-254
Dry dairy products, microbiological
tests, 153-156
moisture and solids determinations,

17

Enterotoxins, from Bacillus cereus, 1314

Enterovirus infections, 24
Eosin Methylene Blue Agar, 66
Equipment, abnormal milk tests, 116,
118-120, 122, 127-128, 136
antibiotic determination, 142-149
collecting milk and related products,
33-35, 43-46, 48-52

DMC

direct microscopic test, 172

DMLC

phosphatase

Dry milk, ash determination, 401


direct microscopic examination, 170

phosphatase determination, 226


sampling, 43-44

Milk

Products,

Micro-

biological Methods for Con-

centrated Milk and (Chapter


10),

151-156

(Chapter

titratable acidity,

16),

197-205

Errors, in direct microscopic methods,

170
personal

in

Erythromycin

SPC, 93
in

dairy products, 149


somatic cell count),

(electronic

134-140
Escherichia 95
Escherichia coli, 95
enteropathogenic, 12, 16
ESPC (estimated Standard Plate Count),
,

91

Estimated Standard Plate Count (ESPC),


91

Dry whey, ash determination, 401


355-358

Dry whip topping, sampling, 43


Dye-binding methods for protein

deter-

mination, 269-277

Eggs and egg products, sampling, 48


Eggs in frozen dairy foods, 165
Electrometric procedure for pH, 58
Electron capture gas chromatography,
pesticide determination, 264-269
Electronic somatic cell counting, 134140

reduction tests, 189, 193

Equipment, Containers, Water and


Air, Microbiological Tests for

ESCC

38^387

Dry

microbiological tests, 49-50, 197-203


tests, 217, 220, 222

Estimates, bacterial direct microscopic,


170-171
Evaporated milk, microbiological examination, 151-152

sampling, 43
Extracts in frozen dairy foods, 165, 167
Extraneous matter in cheeses, 367-368
Food and Drug Administration (FDA),
grading charts, 210
Pesticide Analytical Manual, 261, 265
split-sample program, 6
Foodbome diseases, 16-17

Foodbome

epidemics, 166

Food poisoning outbreaks, 16-17, 153154, 166

INDEX

408

Freezing point determinations on milk,


231-235
Frozen dairy foods, 165-168
Frozen desserts, fat Gerber method,
375-376
moisture determination, 254-255
sampling, 46-48
Fruits in frozen dairy foods, 165
Fungi, aflatoxins from, 22-23
pathogenic, 22

"A" Pasteurized Milk Ordinance,


Standard Plate Count, 77, 93
temperature requirements for milk
samples, 41
Grading reactions, modified Whiteside
test, 116-118

Grade

H
Hepatitis infectious, via milk, 24
High temperature short-time (HTST),

108-109

Farm and plant inspection, 6


Farm bulk milk hauler, 36
Farm bulk tanks, 36, 95
Fat,

Hot-air sterilization, 35, 60, 82-83


Hydrolytic rancidity, in raw milk, 379381

automated turbidometry, 249-252

Babcock modified methods, 236-239

l-J

cream, acidity titratable, 357-358


fat, 245-249, 373-374
moisture determination, 254-255
sampling, 46-48
solids, 255-260
specific antimicrobial cylinder meth-

Gerber, 239-245
modified methods, 369-378
Roese-Gottlieb, 245-249
Film-drying box (DMC) (Fig. 14:3), 176
Filter holder. Teflon (Fig. 20:2), 287
Filter paper disc method, 141
Filtration rings and discs (Fig. 20:3), 288

Ice

Firm-walled capped and uncapped

Ice

od, 149

items, rinsing, 49, 198-199

Flavobacterium ,110
Flavoring extracts,

in

frozen dairy prod-

ucts, 165

microbiological testing, 167


Flavorings and colorings, sampling, 47
Flexible-walled items, rinsing, 49-50,

199
Florisil

chromatography, 267

column clean-up, 263


Fluid counting procedure (confirmatory
Florisil

DMSCC), 129-130
Fluorescent dye, automated Foss for somatic cell count, 396-399
Food additives, in frozen dairy products,

Ice

Cream and Related Food Products, Microbiological Methods


FOR (Chapter 13), 165-168
cream mixes, ash, 400-401

dry dairy products in, 153


Pennsylvania modification, 373374
phosphatase test, 224
Ice cream powder mixes, moisture and
solids determination, 252-254
Ice cream toppings and syrup, moisture
determination, 254-255
fat,

Illumination, critical, 179

165

Kohler, 178
Incubation, Standard Plate Count, 88
Incubator and incubator rooms, 81-82
Infectious hepatitis via milk, 24
Infrared milk analyzer (IRMA), 277
Infrared spectrophotometry, for fat, protein, lactose, 277-281

Gamma

Inhibitory substances in dairy products,

ray spectrometry, recovery of


radionuclides from milk, 284, 304308
Gelatins, moisture and solids determination,

252-254

fat tests, 239-245, 375-376


Glassware, cleaning, 64-65

Gerber

detection, 141-150
In-plant control methods,

Gerber fat test for frozen desserts,


375-376
Roccal procedure for milk and milk
mixes, 376-377

INDEX

409

Interfering substances, phosphatase test

control, 216

International Dairy Federation, electronic

somatic

cell

Leptospirosis, 17

Leucocytes in milk, 114


Levowitz-Weber modification,

man-Lampert

counting procedure,

stain for

Line samples, 39-^0, 50


Liquid impingement test,

Intersociety Council on Standard Meth-

Listeria

Products, 3
Interstate Milk Shippers Agreement,
Interval plating procedure, 62-64
(Fig. 4:1), 63
Iodine- 131, in milk, 283

recovery by

gamma

air quality,

204

17

Listeriosis, 17

Low-temperature long-time (LTLT), 109

LPC, laboratory pasteurization count,


108

LTLT

(low-temperature long-time), 109

spectrometry,

phosphatase, 213
Keeping-quality test, Moseley, 347
test,

Klebsiella, 95

M
Malted milk, ash

in,

401

Manual of Recommended Methods

for

Sampling and Microbiological Testing of Single Service Food Packages


and Their Components, 344

Margarine, Microbiological Methods FOR Butter and Related


Products (Chapter 11), 157-159
Margarine,

Kohler illumination, 178-180


Kohler illumination (Fig. 14:5), 178
Kohman modified method, for fat, moisture and salt in butter and margarine, 376-378

Laboratory accidents, reporting, 92


Laboratory pasteurization count (LPC),

Kohman

Laboratory, receiving samples, responsibility, 41-42


Lactic culture bacteriophage, detection
and enumeration, 353-354
Lactic culture-free butter and margarine,
SPC, 158-159

modification for

ture,

and

salt in,

fat,

mois-

376-378

microbiological methods for, 157-159


potentiometric (pH) method for, 360361
sampling, 44
Maslilis, Pasteurella, 19
Mastitis tests, 115
Media, culture, 5, 56-61
Mellorine,

in

Membrane

108

frozen dairy foods, 165


procedures, 197, 200,

filter

203, 388-392

Methods,

specific,

7-8

standard. Chapters 1-21

supplementary. Appendixes A, B 327354; 355-401


Methylene blue reduction method, 187-

Lactobacillus. 107

Lactometer readings,

monocytogenes,

Long-life products, procedure, 352-353


1

304-308
Ion-exchange determination of **'*Sr and
^oSr in milk, 284-295
Ion-exchange system (Fig. 20:1), 286
IRMA (infrared milk analyzer), 277

Kay-Graham

183-

184

134
Interpupillary distance, binocular microscope, 129, 180-181

ods for the Examination of Dairy

New-

DMC,

191

values for

(Table 19:IV), 260


for (Table 19:V), 260

SV.S values

Lactometric method, solids in milk, 257260


Lactose broth, 64
Lactose in cheese, 381-385
Leptospira interrogans var. pomona, 17

MF

(microscopic factor), 181


MF-Endo Broth, 66-67
with corresponding field diameters
and calculated
for
(Table 8:11), 131
Mg-O Celite column clean-up, 263

MF

WF

Microbacterium, 107
Microbial flora of cheese, 161

DMCC

INDEX

410
Microbial phosphatase, 216
Microbiological control methods, supplemental (Appendix A), 327-354

Microbiological Methods for But-

ter AND Margarine and Re-

lated Products (Chapter

11),

157-159

Microbiological Methods for


Cheese and Other Cultured
Products (Chapter 12), 161-164
Microbiological Methods for Concentrated AND Dry Milk Products (Chapter 10), 151-156
Microbiological Methods for Ice
Cream and Related Food Products (Chapter 13), 165-168
Microbiological Tests for Equipment, Containers, Water and
Air (Chapter 16), 197-205
Microbiologically stable milk products,
testing, 352-353
Micrococcus, 107
Microcoulometric chromatography, pesticide determination, 264-269
Microscope calibration, DMC, 180-181

DMSCC,

129, 134

Milk Composite Schemes (Table 4:1), 70


Milk containers retail, screening, 349
Milk Marketing Board (United Kingdom), electronic somatic cell counting procedure, 134
Milk Ordinance, Grade A Pasteurized, 7,
11

Milkoscan, for fat, protein, and lactose


determinations, 277-281
Milk, Radionuclides in (Chapter 20),
283-310
Milk raw, preliminary incubation treatment, 347-348
standards USDA, 185
Milk samples, from udder, 41
Milk, Sediment in (Chapter 17), 207212
Mixed-sample method, for sediment in
milk, 51, 208-211
Mobile tank trucks, 38
Modified Kohman method, fat, moisture
and salt, 376-378
Modified spectrophotometric phosphatase method, 220-222
Modified
Whiteside
screening
test

(MWT),

for

abnormal milk,

15-1 18

Microscope slides, 127, 172-173


Microscope, use in direct microscopic

Moisture and solids determinations, 252266, 376-378, 385-387

count, 125-134, 176-182


Microscopic Factor (MF), DMC calcu-

Molds and

lation, 181

DMSCC,

128-132
Factors, suggested when
making
(Table 14:1), 171
Microwave oven method, for moisture
and solids in cheese, 385-386
Milk and milk mixes, in-plant production
control (Roccal), 375
Milk and milk products, protein determination, 269-277
sampling for SPC, 85
Milk cans and weigh tanks, 38
Milk, added water, 231-235
chlorides in, 363-365
conforms, 95-104
fat, 236-243, 245-251, 369-370, 372378
in frozen dairy foods, 165
microbiological examination, 151-156
pathogens in, 11-32
phosphatase tests, 224
sediment in, 51-52, 207-212
titratable acidity, 357-358

Microscopic

DMC

medium

yeasts, 7

327-328
349
MPN (Tables 6:1, 6:11), 102-104
MPN, for conforms, 102-104
for water supplies, 203
Mueller-Hinton agar, 55
(modified Whiteside test), 115-1 18
Mycobacteria 17-19
for detection of,

Moseley keeping quality

test,

MWT

Mycobacterium tuberculosis,

Mycoplasma

18

infections, 25

Mycotoxins, 22-23

N
National Bureau of Standards, buffers,
58
thermometer certification, 33, 78
National Conference on Interstate Milk
Shipments, 5-6
Neomycin in dairy products, 149
Non-fat dry milk, detection of penicillin,
145

Novobiocin,

in

dairy products, 149

Nutrient agar, 55

INDEX

411

Nutrient broth, 55
Nuts, in frozen dairy foods, 165

OfiF-bottom

method

for sediment in milk,

208-211

One-hour

test,

resazurin reduction, 192-

194

Organochlorine pesticide residues

in

milk, 261-269

OSCC-II Technicon,

optical for somatic


count, 392-396
Osmometer, vapor pressure, 233-235
OTC (oval tube count), 311-314
cell

Oval tube count (OTC), 311-314


Oxidation-reduction indicators, 187,
191-192
Oxytetracycline,

in

dairy products, 149

Phosphatase, controls {Cont'd)


determination, 214-216
precautions, 214
Rutgers test, 225-226
sampling, 52
Scharer rapid test, 217-220
spectrophotometric, modified, 220222
Phosphatase Methods (Chapter 18),
213-229
Phosphatase reactivation in HTST,
UPM, and UHT methods, 226-228
Phosphate-buflFered dilution water, 62
Physical standards, for Standard Methods Agar, 68
Pipets, bacteriological transfer, specifi-

cations, 79, 175


Pipets, bacteriological transfer (Fig. 5:1),

80

Photopeak and photon energy range of

Packaged products, sampling, 40-41, 47


Paper container materials, disintegration
method, 344-347
Parevine, in frozen dairy foods, 165
Pasteurella multocida, 19
P. haemolytica, 19
Pasteurellosis, 19-20
Pasteurization, 7
Pasteurized milk, DMC, 169
safety, 11

Pasteurized milk products, cylinder


count method, 329-330
Pastries, combined with frozen dairy
foods, 165

radionuclides (Table 20:11), 306


and tube methods for coliforms,
relative value, 98-99
Plate cylinder count (PCC), for pasteurized milk products, 329-330
Plate loop count (PLC), 311, 315-317
automated, 353
Plate loop count (Table 20:1), 213
Plate

Plating, for Standard Plate

PLC

(plate loop count),

Count, 87
311, 315-317,

353
Polyester (Dacron) swabs, 351-352
Polymyxin, in dairy products, 149
Potato glucose agar, acidified, 55, 158,
163

Pathogenic bacteria, 7, 13-26


Pathogenic fungi, 22-23

Potentiometric method for pH, 358-361


Preliminary Incubation (PI), for raw

Pathogens

milk, 347-348
Preparation of culture media, basic

in Dairy Products, Significant (Chapter 2), 11-32

Penicillin

detection,

141-149,

331-344,

Penicillin use in dairy cattle, 141

Pennsylvania modification
375

fat test,

373-

Pesticides, organochlorine, 261-269

pH, adjustment of culture media, 57-59


butter and margarine, 360-361
cheese, 358-360
Phenol color standards, 218, 221, 223
Phenol color standards (Table 18:11), 223
Phenol standards, preparation of (Table
18:1), 218
Phosphatase, controls, 215-216
Cornell test, 222-225

steps, 56-58
Prepared culture media care, 56
Pressurized containers, sampling, 40-41
Presumptive test, for coliforms, 96
Procedures, adoption of new, 3
uniformity, 4-5

standard, 3

Process samples, 47
Productivity tests for SMA, 69-74
Protein determination, in dairy products,

269-277
Proteolytic bacterial count, butter and

margarine, 158-159
containers and equipment, 200

412

INDEX

Protozoa, 7

Replicate organism direct agar contact

Protozoan infections, 26

(RODAC), 200-203
Resazurin reduction method, 191-194
Residual and reactivated phosphatase,
227-228
Residual bacterial count (RBC), for con-

Pseudomonas, 110
Pseudomonas in milk,

13

Psychrophilic (see psychrotrophic)


Psychrotrophic bacteria, 6-8, 110-112
Psychrotrophic bacteria detection, 151159, 164, 168, 200

tainers, 199

Rickettsia, 7, 25
Rinse solution method, for

equipment

and containers, 197-200


Rinse test procedures, 49-51
Roccal, in-plant production control, 375
RODAC (replicate organism direct agar
contact), 200-203
Rose Bengal Agar with chlortetracycline
hydrochloride, 67
Rutgers phosphatase test, 225-226

Q-fever, 25-26
Quality control program, 8-9
Quality, measuring sanitary, 6
Quality Tests (Chapter 1), 1-10

Radioactive substances in milk, 283


Radioassay of milk, 283
Radioisotope precipitation test (RPT),
for Q fever, 26
Radionuclides in Milk (Chapter 20),

283-310

mycobacteria, 18
protozoa, 26
rickettsia, 25
viruses, 23-24
Rayon swabs, 353-354
(residual bacterial count), for con-

tainers, 199

rickettsial

23-26
Salmonella,
204

in

and other diseases,

dry milk products, 154,

outbreaks, 12
variability, 13

S. typhi, 12, 19
S.

HTST

and UHT pasteurized milk and


cream, 226-228
Recording, colony counts, 88-92
Reduction methods, methylene blue,
187-195
resazurin, 191-194

Reduction Methods (Chapter

15),

187-197
Reduction times, methylene blue, 187189
resazurin, 191-194

viral,

Salmonella dublin, 12, 19-20


S. heidleberg, 19^20
5. livingstone, 20
S. new brunswick, 19
S. newport, 19
S. paratyphi B, 19

Listeria, 17

Reactivation of phosphatase in

brucellosis, 14
leptospirosis, 17

Rancidity, hydrolytic, 379-381


Raw milk, direct microscopic count, 169
reduction tests, 187-192
sampling, 36-40
Raw milk and milk products, Brucella,
14

RBC

Safety procedures mandatory,


anthrax, 13

typhimurium 20
,

Salmonellosis, 19-20
Sample breaker, reentrant cross section
(Fig. 20:4),

Sample

290

size, thiosulfate titration for

chlorine (Table 19:1), 236

Samples, butter, 44
casein, 43-44
cheese, 4546
concentrated milk, 43

condensed milk, 43
cream, 33^2
cultured products, 45-46

INDEX

413
Split-sample tests, 5-6
Spore counts, frozen dessert ingredients,

Samples (Cont'd)
dry milk, 43-44
dry whip topping, 43^4
evaporated milk, 43
fluid milk, 33-42
frozen products, 46-48
ice cream, 46-48
margarine, 44
whey, 43-44

165
Stabilizers

microbiological testing, 167


Stains,

Sampling Dairy and Related Products (Chapter 3), 33-54


Sampling, frozen dairy foods, 166
plans, 42
Sanitation, equipment tests, 197-203
Saponification clean-up, 264
Sarcina liitea cylinder plate method, for
penicillin detection, 145-149
Scharer rapid phosphatase test, 217-220
modified spectrophotometric phosphatase test, 220-222

Screening and Confirmatory Methods FOR THE Detection of Abnormal Milk (Chapter 8), 115-140
Screening tests, microbiological quality
of air, 351-353
Sediment discs, 207, 209-210
Sediment in Milk (Chapter 17), 207212
Sediment in fluid milk, 51-52, 207-212
Sherbets, titratable acidity, 357-358
Shigella sonnet, 20
Shigellosis, 20

Significant Pathogens in Dairy


Products (Chapter 2), 11-32
Skim milk, Pennsylvania modification
test,

fat

374

DMC

(Fig. 14:1), 172


Solids in milk, lactometric method, 257-

Slide for

260
Somatic cell counts, 388-389
Spectrometry infrared, for protein, fat
and lactose, 277-281
Spectrophotometric phosphatase test
modified, 220-222
Spiral plate counting procedure (SPCL),
311-312, 317-324
Spiral plate counts, determination of vol-

ume

constants (Table 20:11), 322


Spiral plater (Fig. 20:2), 319
Spiral plates (Fig. 20:3), 320
(Fig. 20:4), 321

and emulsifiers, sampling, 48

Stabilizers, in frozen dairy products, 165

Levowitz-Weber

modification,

183-184
Standard discs, photographs, 210
Standard Methods Agar, 67
physical standards for, 68
productivity tests, 69
Standard Methods for the Examination
of Dairy Products Standard Methods Agar, 65, 67
Standard Methods for the Examination
of Water and Wastewater, plate
count agar, 67
sampling, 51
water supplies testing, 203
Standard Methods, functions of, for
,

quality control, 1-2

procedures

for,

2-5

collaborative studies of, 3-4

Standard Plate Count, acceptance,


butter, 157-158

condensed milk, 153


diluting samples, 85

discussion, 77
dry dairy products, 154-155

enumeration of psychrotrophs. 111


enumeration of thermodurics, 108
enumeration of thermophiles, 109
equipment and supplies for, 78-82
evaporated milk, 151-152
examination of samples for, 83
frozen dessert ingredients, 165-168
margarine, 157-159
materials for, 82-83
preparation of samples for, 84-85
vs alternate viable counts, 311

Standard Plate Count Method


(Chapter 5), 77-94
Standards USDA, manufacturing milk,
189, 193

raw milk, 185


Staphylococcal enterotoxin, 20-21
in dry,milk products,
153-154
in food poisoning, 12
Staphylococcus aureus, 20-21

Staphylococci,

INDEX

414
State Milk Sanitation Rating Authorities,
certification of containers, 34

analyses, for collaborative


studies, 4
productivity tests of SMA, 71-74
Statistical analyses for productivity tests
Statistical

of

SMA

(Table

74

4:11),

Statistical protocol, alternate viable


count vs SPC, 311-312

Statistical protocol alternate viable


count vs SPC (Table 20:1), 313

87-88
equipment, 87-88
medium, 87-88
Sterilized or microbiologically stable
milk products, testing, 352-353
Streptococcal infections, 21-22
Streptococcus, 107, 110
Streptococcus agalactiae 21-22
S. dysgalactiae, 21-22
Sterility controls, dilutions,

Tests by function {Cont'd)

added water
air, 5

S. equisimilis, 22

pyogenes, 22

5. uteris, 22

zooepidemicus 22
Streptomycin in dairy products, 149
Strip equivalents (SE) values of C^ and
C4 (Table 8:111), 135
S.

Strip

reticule

counting procedures

(DMSCC), 126-134
Strontium-89, in milk, 283-295
Strontium-90, in milk, 283-295
Strontium and calcium recovery from a
single precipitation (Table 20:1), 293
Sulfa drug, detection in milk, 330- 331
Sulfonamides, use in dairy cattle, 141

Supplemental Chemical control


Methods (Appendix B), 355-401
Supplemental Microbiological
Control Methods (Appendix A),
327-354
Surface-contact
methods,
microbiological examination of containers
and equipment, 200-203
Swab test procedure, 51, 200, 351-352
Sweetening agents, sampling, 48

TDE,

in milk and dairy products, 261269


Tests by function, for
abnormal milk detection, 115-138
acidity in dry whey powder, 355-356
acidity in liquid whey concentrate,
357-358

in milk,

231-235

204

alternate viable count, 311-324

and milk products,


141-149, 330-344
ash, in dairy products and feeds, 399401
available chlorine, 235-236
bacterial count, 158-159
bacterial toxins, 15, 20-21
antibiotics in milk

butter and margarine,

157-159, 360-

361, 376-378

buttermilk, 355-356, 361-363, 374-375


cheese and cheese products, 161-164,
252-257, 358-360, 364-368, 371372, 381-386
chocolate milk and drink, 247, 256,
273, 277, 370, 373

5.

concentrated and condensed milk


products, 152-153, 247, 255-256
dry milk, 153-156, 357, 361-363
dry whey, 355-356
enterococci, 158, 328-329

enumeration lactic culture bacteriophage, 353-354


equipment sanitation, 197-203
extraction of fat and pesticides, 261262
236-245, 249-252, 369-378
fluid whey, 357-358
hydrolytic rancidity, 379-381
ice cream and frozen products, 149,
165-168, 245-249, 255-260, 357-

fat,

358, 373-376
inhibitory substances in milk, 141-150

lactose in cheese, 381-385


microbial content of paper container
materials, 344-347
microbiologically stable milk products, 352-353
moisture, 385-388
molds and yeast detection, 153, 156159, 161, 163, 165, 168, 200, 327-

328
mycotoxins, 22-23
organochlorine pesticide residues,
261-269
pasteurized milk products, 329-330
pathogenic bacteria, 13-22
phosphatase, 214-220, 226
protein in milk, 269-281
proteolytic bacteria, 159

protozoa, 26

415

INDEX
Tests by function {Cont'd)

psychrotrophic bacteria, 110-112, 156,


159, 164, 168, 200
quality, 1-10
radionuclides in milk, 283-310
raw milk, 379-381
rickettsia, 25-26
rinsing equipment, 49-51
sherbet, 357-358
solids, 252-260
sulfa drugs in milk, 330-331
thermoduric enumeration, 107-109
toxicity of water, 62-64
viable counts of raw milk, 311-324
virus, 23-24
water, 51, 203-204
Tests by title,
Acetonitrile partitioning, 262-263
Acid orange protein, 274-277
Acidity, potentiometric pH, 358-361
Acidity, titratable, 355-358
Alkalinity of ash, 361-363
Amido black protein, 269

AOAC

pesticide residue, 261

Atmospheric oven, 255-256

Automated fluorescent dye, 396-399


Automated plate loop, 353

Tests by

title

Membrane

{Cont'd)
filter,

200

Methylene blue reduction, 187-191


MF-DNA, 390-394
Microbiological, air, 51, 204, 349-351
Microbiological, butter, margarine and
related products, 157-159
Microbiological, cheese, 161-164
Microbiological, concentrated and dry
milks, 151-156
49-51,
Microbiological, containers,

197-203
Microbiological,
197-203

equipment,

49-51,

Microbiological, ice cream and related


products, 165-168
Milko-tester, 250

Mixed-sample, 208-210
Modified IDF disc, 335-338
Modified Kohman, 376-378
Modified Sarcina lute a cylinder plate,
145-146
Modified spectrophotometric phosphatase, 220-222
Modified Whiteside, 115-118
Moseley keeping quality, 347

BR-Foss, 341-344

Off-bottom, 52, 208-209, 211


One-hour test, 192-194
OSCC-II Technicon, 392-396
Oval tube count, 311-314

California mastitis, 118-120

Overnight incubation (Method A),

Chlorides, 363-367

143-144
Phosphatase, 213-219
Plate loop, 311, 315-317
Potentiometric, pH, 358-361
Productivity, SM agar, 69-74
Psychrotrophic bacterial, 109
Resazurin reduction, 191-194
ROD AC plate, 202-203
Roese-Gottlieb, 245-249
Rutgers phosphatase, 225-226
Sanitation of equipment, 197-203
Saponification cleanup, 264
Scharer rapid phosphatase, 215, 217220
Screening, air, 349-351
Screening, retail milk containers, 349
Sediment, 51, 207-211
Short incubation (Method B), pen-

Babcock, modified, 236-251


Bacillus subtilis disc assay, 142-145

Chromatography, 264-269
Coliform, 95-105, 153, 156, 158, 163,
168, 200
Cornell phosphatase, 222-225
Delvotest, 338-341
Direct microscopic (DMC), 155, 169187

somatic

cell

count (DMSCC), 125-

134

Disc assay for penicillin, 331-338


Disintegration, 344-347
Dye-binding, 269-277
Dye reduction, 187-194
Electronic somatic cell counting, 134138

Extraneous matter, 367-369


Gerber fat, 239-245, 375-376
Interval plating, 62-64
Ion-exchange for strontium, 284-295

Kay Graham,

213

Laboratory pasteurization, 107-108


Lactometric, 257-260

icillin,

144

Short incubation (Method C), penicillin,

Somatic

144

cell

count, 388-399

Spiral plating, 311, 317-324

INDEX

416
Tests by title (Cont'd)
Split sample, 5

Standard Plate Count, 77-94, 151-155,

Volhard

157-158, 166, 199


Standard, water, 203-204
Surface contact, 200

Swab

Swabbing, 351
Thermistor cryoscope, 231-233
chlorine

rinse

Vapor pressure osmometer, 233-235


Volhard, chlorides, 365-366
Wisconsin mastitis, 120-125
Tetracycline, in dairy products, 149
in

365-366

Water, Equipment, Container and

TCA, 295-308

use

test,

contact, 200-202

Thiosulfate titration,
waters, 235-236

Violet Red Bile Agar, 68


Viral diseases, 23-25
Viruses, 7

diary products, 141

Thermistor cryoscope, freezing point determination on milk, 231-233

Thermoduric, Thermophilic, and


PsYCHROTROPHic BACTERIA (Chapter 7), 107-113

Thermodurics, 7, 107-109
from containers and equipment, 200
from dry dairy products, 156
Thermophilic bacteria, 7, 109, 156
Thiosulfate titration, available chlorine,

Air, Microbiological Tests for


(Chapter 16), 197-205
Water, for microbiological tests, 61-62
Water supplies, standard tests, 203-204
Whey, ash in, 400-^01
fat, Pennsylvania modification, 374
phosphatase test, 224
Whey concentrate products, moisture
determination, 254-255
refractometer for moisture and solids
in, 387-388
titratabie acidity, 355-357
Whey fluid, titratabie acidity, 357-358
Whey, sampling, 43-^4
Whiteside test, modified, 114-118
Whiteside test (modified), appearance of
scoring reactions (Fig. 8:1), 117
test, 120-125

Wisconsin mastitis
Wisconsin mastitis

test,

adding reagent

(Fig. 8:30), 123

235-236
Thin-layer chromatography, for pesticide determination, 264-269
Titration, thiosulfate, available chlorine,

235-236
Toxoplasma, 26
Toxoplasmosis, 26
Transfer instruments, for
Transfer instruments,

DMC,

DMC

8:6), 126
8:4), 124

timing inversion (Fig. 8:5), 125


direct microscopic
count, 181-182
direct microscopic cell count, 130-131

Working factor (WF),

182-183

(Fig.

X- Y-Z

14:2),

174
Trichloracetic acid (TCA), recovery of
Sr-89 and Sr-90, 295-304
Triple-reading test, resazurin reduction,

192-194
Tritium-3, in milk, 293

U
Udder, milk samples, 41
USDA, grading charts, 210
USDA, standards raw milk, 185, 189
US Pharmacopoeia, culture media specifications, 67

Vapor pressure osmometer, 233-235


Viable Count Methods, Alternate
(Chapter 21), 311-324

measuring fluid (Fig.


mixing reagent (Fig.

Yeasts and molds, 7


aureomycin-rose bengal agar method,
327-328
butter and margarine, 157-159
cheese and other cultured products,
161, 163

condensed milk, 153-154, 156


containers and equipment, 200
dry dairy products, 156
frozen desserts, 165, 168
Yersinia enterocolitica, 22
Yersiniosis, 22
Yogurt, dry dairy products in, 153
microbiological testing, 163
titratabie acidity, 357-358

Youden and

Steiner, statistical method,

3-4
Zinc-65, in milk, 283

i^f /

f"-^

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