SF 253 .A55
1978
/'y5"3ISSUED TO
SF 253 .A55
1978
^1
standard
Methods
I3mty JPtoduts
14th edition
standard
Methods
naitp Products
14th edition
Publication Office:
Fourteenth Edition
Copyright
1978
No
Inc.
may be reproduced,
graphical-
lOM 10/78
Number: 78-72892
Book Number: 0-87553-084-2
Printed
MA
Sci-
Warren
J.
Hausler,
Jr.,
Ph.D.
Iowa
City,
Iowa 52242
Representative: American Public Health Association (Editor, 13th Edition)
'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
W. Ullmann,^ Ph.D.
Bamum
Ave., Strat-
Howard
L. Bodily, Ph.D.
sociation, P.O.
Box
Project Officer
Ralston B. Read,
Jr.,
Ph.D.
^Dr. Reinbold
was with
the
Street,
Uiimann was with the Connecticut State Board of Health and with Diamond-Shamrock
CONTRIBUTORS
J.
Babel, Ph.D.
West
Virginia 25303
Inc.,
Box
Kraft,
Inc.,
CONTRIBUTORS
vi
ministration, 200
Street,
Alabama 36830
University, Auburn,
Warren
Whey
Products
Inc.,
614 McKinley
Director, Food,
15
W. Mont-
Oak
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
New
Claude Harper,
Jr.,
B.S.
S. State St.,
Chicago,
Illinois
Regions, Beatrice
60605
vania 191 18
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
CONTRIBUTORS
viij
New
Inc., P.O.
Box
504,
York 12524
University of Missouri
Professor, Department of
versity, 2121 Fyflfe
State Uni-
biology,
nati,
F.
Norman
F. Olson, Ph.D.
Professor, Department of
CONTRIBUTORS
ix
Professor
of
Food
Microbiology,
Illinois,
55108
John
J. Redys, B.S.
Director, Laboratory Division, Connecticut State Department of
Health, 10 Clinton St., Hartford, Connecticut 06101
Blakeley
St., Seattle,
Washington 98105
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
New York
14853
CONTRIBUTORS
Irvin S.
26506
Donald
I.
Thompson, M.S.
Water Laboratory Evaluation Program, Wisconsin
Laboratory of Hygiene, 465 Henry Mall, Madison, Wisconsin
53706
William
W. Ullmann, Ph.D.
Bamum
Ave., Strat-
A& M
Microlife
Technics,
1833-57th
St.,
Richard
W. Webber,
Mayo
Bldg., Uni-
Minnesota 55455
B.S.
PDQD, FSQS,
U.S.
Farms,
Inc.,
Tennessee 37219
Quality Assurance.
Ave.. Rockford.
Illinois
61101
CONTRIBUTORS
Albert F.
xi
Zimmerman, B.S.
Edmund
A. Zottola, Ph.D.
Professor, Department of
St.
Nutrition, University of
PREFACE
The
Methods for
ucts
cil.
is
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
in the
The
first
edition of
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
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
pouch
sure freezing point of milk, acid injury in coliform bacteria, automated colo-
Changes
in the
fications to
Bacteria"; Chapter 15, "Reduction Methods"; Chapter 16. "Microbiological Tests for Equipment. Supplies, and Water"; and Chapter 20, "Radionuclides
in
Milk".
Major changes
in the
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
expanded
now
contain
MgS04
PREFACE
xvi
dure has replaced the distilled water suitability test, c.) the expression
"microbiologically suitable water (MSW)"' is introduced, and d.) formulae
for
clarified.
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
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.
ameter, of
test
according to Chapter
17,
much
milk products.
Acknowledgments
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.
in
PREFACE
xvii
Dr. Elizabeth D. Robinton served as copy editor and prepared the index for
this
book. Her
lications for
Phyllis, for
efiforts
J.
APHA,
ress.
Madison, Wisconsin
October, 1978
for
TABLE OF CONTENTS
Intersociety Council
iii
Contributors
Preface
1.
xiii
Quality Tests
Introduction
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.
Cream Samples
33
Sam-
4.
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
ences 74
Medium
691 Refer-
XX
5.
Table of Contents
77
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
Solid Medium 991 Coliform Test with a Liquid Medium 991 Confirmed Test from a Solid Medium 991 Completed Test from a Liquid
Medium
MPN
Coliforms
7.
107
Thermoduric Bacteria 1071 Thermophilic Bacteria 1091 Psychrotrophic Bacteria IIOI References 112
8.
9.
in
Introduction 1411 Bacillus suhtUis Disc Assay 1421 Modified Sarcina lutea Cylinder Plate
fat
Method
NonMethods 1491
References 149
10.
Table of Contents
11.
xxi
Related Products
157
Introduction 1571 Microbiological Methods 1571 Sampling Procedure 1581 Bacterial Counts 1581 Yeast and Mold Counts 1591 References 159
12.
13.
Raw
169
Use of
Stain 1831
185
15.
Reduction Methods
187
Method
16.
Table of Contents
xxii
17.
Sediment
IN
Milk
207
18.
Phosphatase Methods
213
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
19.
Chemical Methods
Introduction 2311
231
Added Water
in
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
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
311
324
Spiral Plate
Table of Contents
xxiii
Appendixes
A.
327
Raw
ucts 352/
B.
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
where food
is
many
aesthetic considerations.
Food must be
attractively
presented, have desirable odor and flavor characteristics, and possess other
qualities that
have
little
to
is
free
from patho-
practices
is
needed.
to provide a product in
appearance
have been preand
affect the
QUALITY TESTS
diction within
in
proce-
is
also desirable.
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,
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
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
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
tions.
.5
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
tration range
should be
unknown
to the collaborator.
ax =
Vo-'l
a'is
0-5
where,
cr't
= component
cr^
als
= component of variance
of variance
among
analysts (laboratories)
replicates
x sample
inter-
action
computed
ctx
in
^^
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
a committee to standardize
and,
in turn,
much
is
determinations
gate
much
a constant need to
made
in
work
to dealers
who employ
ties.
these
facili-
in
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
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
it
becomes
relatively
is
approved, representative of
all
types of
QUALITY TESTS
is
1.8 Integration of
Control
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
inter-
They can
tell
when shown
whether a product
is
falls just
(a)
come
grossly contaminated.
The
cardinal
is
to classify
1.10
many
Association
of Otficial
fourteenth edition,
Analytical
method.-'
tests differs
product
may be
fore,
Decisions as to which
test
different products,
products, frequency of testing, and values considered satisfactory for compliance must be
made by
ents, as
determined
in
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
QUALITY TESTS
refrigerated.
may
Numbers
For
this reason,
monitor radionuclides
in
in
optimum form,
methods
to
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-
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
methods given
1.11
Quality Control
Decisions
made by
in
the Laboratory
a regulatory agency or a
company
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-
ing
suitability of the
References
1.12
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.
Black,
J.
war areas
in the
1956. Split sample evaluations of milk laboratories. Pub. Health Lab. 14:94.
Black, L.A.
Black, L.A.
J. .Milk
Food
Technol. 21:187-191.
5.
Blac
K,
in certification
of milk laboratories.
J.
nol. 23:13-18.
6.
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.
split
J.
23:315-319.
10.
Donnelly, C.B.,
J.
J.T.,
in-
14.
15.
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.
Inc.,
19.
New
method
F.J.
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.
C.
Vanderzant.
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.
J.
8.
23.
Nelson. F.E.
1943. Factors
Peeler.
J.T..
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.
26.
27.
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.
29.
30.
U.S.
USPHS
Youden,
milk.
J.
Milk
ordinance
31.
& Welfare.
in
1965.
Service.
Official Analytical
AOAC,
Association of
CHAPTER
SIGNIFICANT PATHOGENS
I.S.
2.1
IN
DAIRY PRODUCTS
Introduction
in
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,
to
complacency
in
the
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
McCoy "
Salmonella
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.
London
'"-
raw milk
'''''
^'^^
in
"'-
'^-
way
of their importance.
No
techniques
will
may
calorimetry
2.2
13
load.
tive tests
in dried
milk
^^
^^
^^'
portance
in
,'^'^
E.
coli
'"^
and Bacillus
in the
The report of
the Salmonella
again emphasizes
by both E.
coli
^^' ^^^
and
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.-*'^''^'^-'^'''
'^
and Intoxications
'"'
is
possibility.
Isolation of 5. anthracis
is
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
^^^'^
SIGNIFICANT PATHOGENS
14
is
is
became
is
ill.
easily accomplished.''^'*'*'
C. Botulism:
is
1 1
reported.''*'-
normally found
in soil.
may be contaminated
D. Brucellosis:
Caution
Infection of
man
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
human
infection.
^^
be a reservoir for Brucella organisms.
from Spain.
'''
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
Bacterial, Mycotic,
Clostridium perfringens
is
it
is
pres-
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
meat products.
Isolation of C. perfringens
is
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:
This organism
is
is
in
'* ^*^
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-
E. coli at a
Isolation of E. coli
may
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-
However,
appli-
cation of this procedure to examination of milk and milk products has not
Tulloch et
2.2
Bacterial Infections
and Intoxications
17
in
is
properties
of invasiveness or toxin production has cast doubt on the significance of
serotyping as an indicator of enteropathogenicity. Not all strains of entero-
A number
genicity.
H. Leptospirosis:
Caution Specialized Safety procedures are mandatory.
Many
in
dairy cattle in
all
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:
'""'
and
1.3
L.
still
contained
organisms/g.
monocytogenes
specimens;
is
more
to techniques
and awareness
SIGNIFICANT PATHOGENS
18
human
been reported
in the
udder
in
in
South Africa.'""
only a small percentage of
found
in
market milk.
Kells and Lear^** have stated that the present pasteurization standards pro-
(161
F),
strains.
Of
351 samples of raw milk tested, 175 were positive for atypical acid-fast bacil-
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
''
'*''
whose
man
is
not clearly
defined.
tests
may be
required to de-
it
2.2
Bacterial Infections
and Intoxications
19
in
''*''
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
^'
''
''--'
or
compromised patient."
the only
method
to detect
may be made
if
desired.
its
is
mem-
''"''
L. Salmonellosis:
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
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.
may
create a whole
new problem
area.
The
specific
method-
is
imperative
in the
M.
Shigellosis:
pri-
humans and
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
may
for
Streptococci
commonly
'^^-'^^
(Lance-
SIGNIFICANT PATHOGENS
22
field's
that milk
some
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:
It
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
route of con-
2.3
Mycotoxins
"
but
However, considerable
interest
2.4
Viral, Rickettsial
23
is
in
'
'''
Caution
A. Adenovirus infections:
Kaplan
is
serological evi-
cattle
SIGNIFICANT PATHOGENS
24
will not
survive
B. Enterovirus infections:
Enteroviruses
mals.
may be
isolated
As human pathogens,
from the
intestinal tract of
man and
ani-
those viruses causing diarrhea in infants are the ones of greatest importance.
Several bovine enteroviruses have been isolated," but no evidence exists
way
into milk
may be
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
was inactivated a
million-fold
of milk used in making the cheese. Potter'^ points out that current methods
which show
that poliovirus
persist in cottage
and
Isolation
identification of these
human
.'^'-
and
Rickettsial Infec-
^^^-^
found in
improved method for recovery of enteroviruses from food.
C. Infectious hepatitis:
may
is
excreted
in
a small outbreak
in
Georgia
in 1945.
*^^
D. Tickhorne encephalitis:
Evidence for the role of milk from goats, sheep and cows
in
man
The
virus
is
et
al.,'''
transmission
Group B
Blaskovic,''^
ar-
and
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
known
.^'^
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.
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%
study
of the
in
cows
California
in
showed
that
82%
of the dairy
cows
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
Human
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.-'*^
The Q-fever
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.
titers
15 sec
30 min or 74.3
''^
An
additional 2.7
(to a
temperature of 65.7
for
Protozoan Infections
A. Toxoplasmosis:
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
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
were seropositive.'"'
27
2.6
References
2.6
References
1.
Allcroft,
toxin in
2.
human food
Carnaghan.
1963.
Groundnut
products from animals fed toxic groundnut meal. Vet. Rec. 75:259-263.
Allcroft,
aflatoxin B, intake by
3.
4.
5.
Barnum, D.A.
Comp. Med.
J.
6.
1954.
J.
A.,
southern California.
7.
67:691-692.
8.
CM.,
10.
Biberstein, E.L., Behymer, D.E., Bushnell, R., Grenshaw. G., Riemann, H.P.,
and C.E. Franti. 1975. A survey of Q fever (Coxiella burnetii) in California dairy cows.
from dried
WHO
Bull.
24:771-784.
12.
Blaskovic, D.
cephalitis.
Press,
13.
New
1962.
Some
Complex. Academic
York.
Bodily, H.L., Updyke, E.L., and J.O. Mason, Eds. 1970. Diagnostic Procedures for
American Public Health Association,
New
14.
15.
16.
York.
Braun, J.L. 1962. The epidemiology of Q fever in Iowa. Pub. Health Rep. 77:171-176.
Brewington, C.R., and J.L. Weihrauch. 1970. Survey of commercial milk samples for
aflatoxin M. J. Dairy Sci. 53:1509-1510.
Brown, G.L., Colwell, D.C, and W.L. Hooper. 1968. An outbreak of Q fever in
Straffordshire.
17.
18.
J.
Hyg. 66:649-655.
19.
20.
1975.
from
21.
22.
Cliver, D.O. 1973. Cheddar cheese as a vehicle for viruses. J. Dairy Sci. 56:1329-1331.
Cliver, D.O., and E.H. Bohl. 1962. Isolation of enteroviruses from a herd of dairy
cattle. J.
23.
24.
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.
Collins, R.N., Treger, M.D., Goldsby, J.B., Boring 111, J.R., Cohoon, D.B., and
R.N. Bark. 1968. Interstate outbreak of Salmonella new brunswick infection traced to
powdered milk. J. Am. Med. Assoc. 203:838-844.
SIGNIFICANT PATHOGENS
28
25.
Dack, G.M.
1952.
1095.
26.
27.
28.
1956. Food Poisoning. 3rd ed. University of Chicago Press, Chicago, 111.
Davis, R.D., Dulbecco, R., Eisen, H.N., Ginsberg, H.S., and W.B. Wood. 1973. Microbiology, 2nd ed., Harper & Row, N.Y.
Davison, I. 1%1. A set of bacteriophages for typing bovine staphylococci. Res. Vet. Sci.
Dack, G.M.
2:396-407.
29.
related
com-
pounds.
30.
Deibel, R.H., and J.H. Silliker. 1963. Food-poisoning potential of the enterococci.
J.
Bacteriol. 85:827-832.
31.
USPHS
32.
1968. Laboratory
methods
in
anaerobic bacteria.
on bulk milk
collection:
pipeline milking plants and bulk milk tanks as sources of bacterial contamination of milk
a review.
33.
J.
EcKMAN, M.R.
Mexican cheese.
J.
232:636-
637.
34.
Enright, J.B.
Enright,
36.
37.
J.B.
burnetii
EsTOLA,
and
its
T.,
29:179-185.
41.
J. P., Morrissey, R.A., Rose, N.J., Schurrenberger, R., GavE.W., and G.W. Meyerholz. 1973. Epidemiological investigation of Q fever in a
major milkshed region of the United States of America. J. Hyg. Epidemiol. Microbiol.
Immunol. 17:375-384.
George, J.T., Wallace, J.G., Morrison, H.R., and J.F. Harbourne. 1972. Paratyphoid in man and cattle. British Med. J. 3, 208-211.
Goldblatt, L.A. 1969. Aflatoxin, Scientific Background, Control and Implications. Academic Press, N.Y.
Gorbach, S.L., and CM. Khurana. 1972. Toxigenic Escherichia coli; a cause of infan-
42.
Gray, M.L.
38.
39.
40.
tile
diarrhea in Chicago.
New
Eng.
J.
Med. 287:791-795.
Amer.
J.
563.
43.
of Domestic Animals,
4th ed.
44.
F.J.
Babel.
N.Y.
45.
Hammond, D.M.,
and P.L. Long. 1973. The Coccidia. University Park Press, Baltimore,
Md.
46.
Harbourne,
paratyphi
J.F.,
Randall,
C.J.,
I.
J.G.
Wallace.
1972. Salmonella
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.
isolated
C.J.,
J.
Brownlie.
1973.
Hyg. 71:163-170.
in cattle. J.
2.6
50.
References
29
Hutchinson,
R.I.
Med.
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5381:479-480.
51.
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.
Jellison. W.L., Huebner, R.J.. Beck, M.D., Parker, R.R., and E.J. Bell. 1948. Q
fever studies in southern California. VIII. Recovery of Co.xiella burnetii from butter made
from naturally infected and unpasteurized milk. Pub. Health Rep. 63:1712-1713.
53.
54.
55.
JuFFS, H.S. 1973. Identification of Pseudomonas spp. isolated from milk produced
South Eastern Queensland. J. Appl. Bacteriol. 36:585-598.
56.
Jung, M.
icol.
57.
coli in retail
1974.
Raw
antibi-
in
in
12:131-138.
Kells, H.R., and S.A. Lear. 1960. Thermal death time curve of Mycobacterium tuberculosis var. bovis in artificially infected milk. Appl. Microbiol. 8:234-236.
59.
Keogh,
pathogens
60.
in
Kostemba, K.D.
1973. Filtration
survival of
The
J.
methods
1963.
Hyg. 61:175-185.
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Microbiol. 26:149-154.
62.
Q fever in
the
Milwaukee
area:
II.
Con-
Lackman, D.B.,
Pickens. 1967.
64.
Philip, R.N., Casper, E.A., Bell, J.F., Enright, J.B., and E.G.
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F.P.,
N.J.
Truant, Eds.
J.T.
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Manual of
Clinical
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69.
Lennette, E.H., Clark, W.H., Ahinanti, M.M., Brunetti, O., and J.M. Covert.
1952. Q fever studies. XIII. The eflFect of pasteurization on Coxiella burnetii in naturally
infected milk. Amer. J. Hyg. 55:246-253.
Levine, J., and F.B. Bang. 1968. Clottable protein in Limulus: Its localization and kinetics of its coagulation by endotoxin. Thromb. Diath. Haemorrh. 19:186-197.
Lie, J.L., and E.H. Marth. 1967. Formation of aflatoxin in Cheddar cheese hy Aspergillus flavus
70.
Marth.
J.
LuoTO,
pH
values.
Q fever infections
in cattle.
Pub.
Lynch, G.P.
1972.
Mycotoxins
in feedstuflFs
and
their eflFect
on dairy
cattle. J.
Dairy Sci.
55:1243-1255.
73.
MacNamee,
J.K. 1963.
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in
Med.
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22:437-440.
74.
Marier,
An
R.,
Wells,
J.G.,
coli
I.J.
Mehlman.
to
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imported
SIGNIFICANT PATHOGENS
30
75.
Marth, E.H.
review.
J.
1969. Salmonella
76.
Matsievskii, V.A., Logacher, A.U., Fedorina, A. P., and A.S. Pislova. 1971. Outbreak of food poisoning caused by Escherichia coli 0124: K72(B17). Zhurnal. Mikrobiogii,
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77.
McCoy.
J.
H. 1975. Trends
in
in
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Hyg. 74:271-282.
78.
McKiNNEY,
79.
in
81.
82.
Miller, W.R. 1964. Occurrence and geographic distribution of Q fever antibodies in Alabama dairy cattle. Pub. Health Rep. 79:83(^838.
Minor, T.E., and E.H. Marth. 1971. Staphylococcus aureus and staphylococcal food
intoxications. A review. I. The staphylococci: characteristics, isolation, and behavior in
artificial media. J. Milk Food Technol. 34:557-564.
Minor, T.E., and E. H. Marth. 1972. Staphylococcus aureus and staphylococcal fdod
intoxications. A review. II. Enterotoxins and epidemiology. J. Milk Food Technol.
35:21-29.
83.
review.
III.
Staphylococci
in
dairy foods.
J.
38.
84.
intoxications.
in
J.
Montie, T.C, Kadis, S., and S.J. Ajl. 1970. Microbial Toxins. Vol. III. Bacterial ProAcademic Press, N.Y.
Morris, G.K., Merson, M.M., Sack. D.A., Wells, J.G., Martin, W.T., DeWitt,
W.E. Feeley, J.C, Sack, R.B., and D.M. Bessudo. 1976. Laboratory investigation of
tein Toxins.
86.
Escherichia
87.
Murphy,
coli. J. Clin.
Microbiol. 3:486-495.
88.
Naguib, K.
Work.
1946.
J.
1972. Identification
in
raw milk
in
Egypt.
J.
Appl.
Bacteriol. 35:525-530.
89.
90.
Nelson, C.I.
Olsen, CD.
mal milk.
91.
Am.
Park, H.S., Marth, E.H., and N.F. Olson. 1973. Fate of enteropathogenic strains of
Escherichia coli during the manufacture and ripening of Camembert cheese. J. Milk Food
Technol. 36:543-546.
92.
Phillips,
its
role in the
American
diet. J.
Dairy
Sci. 58:1751-63.
93.
Pogodina, V.V.
94.
95.
96.
1961. Epidemiology
borne encephalitis.
J.
tick-
I.E., Steyn, M., Rinsma, R., and R.C. Tugtin. 1972. Reduction of the aflacontent of milk by processing. Food Cosmet. Toxicol. 10:383-387.
98. Purchase, I.E., and L.J. Vooster. 1968. Aflatoxins in commercial milk samples. S. Afr.
97.
Purchase,
toxin
Med.
J.
42:219-221.
2.6
99.
References
31
Raska, K.
1966.
J.
nol. 10:413-428.
100.
101.
RiEMANN. H. 1969. Food Borne Infections and Intoxications. Academic Press. N.Y.
RiEMANN. H.P.. Meyer, M.E., Theis, J.H., Kelso. G.. and D.E. Behymer. 1975. Toxoplasmosis
in
J.
Pediatrics 87:573-576.
102.
103.
Rose, N.A. and H.R. Friedman, Eds. 1976. Manual of Clinical Immunology. American Society for Microbiology, Washington. D.C.
104.
Rude,
I.
1974.
A.,
1961.
106.
ScHULTZ, G. 1%7. Studies on the occurrence oi Listeria in raw milk. Monatshefte fijr Vet.
Med. 22:766-768.
Sebald, M., Jouglard, J., and G. Gilles. 1974. Botulism in man due to cheese. Ann.
Microbiol. (Paris) 125A:349-357.
107.
in
J.
47:216-218.
108.
SiNNHUBER. R.O.. Lee. D.J.. Wai es. J.H., Landers. M.K.. and A.C. Keyl. 1970. AflaM. a potent liver carcinogen for rainbow trout. Fed. Proc. 29:568.
Sipka. M.. Zakui a. S.. Kovincic, I., and B. Stajner. 1974. Secretion of Listeria mono-
toxin
109.
cytogenes
110.
abortion
111.
in
its
XIX
International Dairy
Stark.
in a
cow.
J.
55:416-421.
113.
R..
Sullivan.
Sullivan.
R..
12, reovirus 1,
117.
118.
J.
J.
for the
Assoc. Off.
J.
Jr. 1968.
Method
R.. Gassolitis.
milk. (Abstr.)
116.
methods
dairy products.
Chem. 57:852-857.
Sullivan.
in
from raw
Tierny, J.T., and R.B. Read, Jr. 1969. Thermal destruction of adenovirus
and Herpes simplex in milk. J. Dairy Sci. 52:897. (Abstr.)
Svartz, N.
1972.
agent exists
in milk.
482.
119.
Taylor,
Jr., A..
Santiago. A.. Gonzalez-Cortes. A., and E.J. Gangarosa. 1974. Outin Trinidad in 1971 traced to a commercial ice cream product. Am.
120.
Epidemiol. 100:150-157.
R.C
and
CM.
Eklund.
121.
122.
Tucker, E.A.
related to
cows.
380.
II.
Powassan
virus from
A natural
SIGNIFICANT PATHOGENS
32
124.
Tucker, C.B., Fulkerson, G.C, and R.M. Neudecker. 1954. A milkborne outbreak of
shigellosis in Madison County, Tenn. Pub. Health Rep. 69:432-436.
TuiNSTRA, D.G., and J.M. Bronsgeest. 1975. Determination of aflatoxin M, in milk at
125.
TuLLOCH,
123.
Chromatog. 111:448-451.
K.J., Formal, S.B., and F.A. Franklin. 1973. Invasive
enteropathic E^c/jer/c/j/a coli dysentery. Ann. Int. Med. 79:13-17.
U.S. Department of Health, Education and Welfare. 1965. Grade "A" Pasteurized Milk Ordinance. Recommendations of the United States Public Health Service.
the parts per trillion level.
126.
Jr.. E.F.,
J.
Ryan,
Washington, D.C.
127.
U.S.
Program
1969. Procedures
Governing
129.
Health Service.
U.S. Public
ing.
Health Service.
U.S. Public
1971.
130.
131.
132.
Health Service.
U.S. Public
U.S. Public
Health Service.
Weekly Rep.
135.
134.
Buenos
25:187.
136.
137.
Vlad, a.
Diergeneesk. 89:1082-1086.
1972.
Parazitologia, Epi-
WiENEKE, A. A.
141.
142.
human
beings.
J.
Young,
staphylococcal enterotoxin A.
J.
CHAPTER
Fluid Milk
3.1
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
When
a dial type
at least
might be broken.
B.
Sample
transfer instruments:
Individually
wrapped
sterile
or pre-
may be
line
the burden of
dem-
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-
*Use of metric
found
at the
fitted to
units:
end of
this chapter.
33
measurement
is
for temperatures
and volumes
is
34
line fitting.*'
Collection of an
initial
All
Sample containers:
1.)
liters)
minimum
of
3.)
4.) Single-service,
non-sterile leak-proof
vials
^^
for samples of
are non-toxic
tamination of the
lip
rigid
plastic bag.
E. Shipping case:
When
common
carrier, the
shipping case must be provided with a tamper-proof sealing and locking device,
Up" on
top of
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
ment or mechanical
"
'
shipping case
'"
[3.1 1(E)].
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
pling devices
instruments
to collect
in
is
at all
all
detergent solution. Avoid excessive exposure to strong alkali and, immediately after treatment, rinse thoroughly to
remove detergent
residues.
be steam-sterilized
ppm) of
all
times to
at least
100
mg
per
liter
ment
36
when samples
payment or
to
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
first
first
Do
it
exactly like
all
When
samples are
to
B.
Raw
1.
milk:
milk:
milk marketing.
When
is
critically
impor-
becomes a
pling operation.
3.13
37
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
ther-
Sani-
into milk.
at
least
once a
Identification
b.
(1)
locations.
Record time
(2)
in
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)
When
(2)
drain,
(3)
Do
(4)
in
pockets
may be used
to
(5)
needed.
Do
(6)
not hold or
fill
ferring sample.
Fill
(7)
ing of
full to
air
when
folding or whirl-
(8)
Sample
ply.
(9)
ature.
38
Rinse dipper
(11)
in
to sanitizing solution,
d.
Use
(1)
Keep
ice level in
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.
(3)
somatic
cell
[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
it
is
homogeneous
is
deter-
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
which
at full
Adequate
0.1%
as determined by an Official
AOAC
When
agiin
more than
the appro-
3.13
39
installation or truck,
record and use this time as the guide for proper agitation time for taking
samples.
Studies
show
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
motor.
b.
driven motor and placed on the manhole with the agitator suspended
in
the milk.
An
'
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.
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
An approved
automatic
it
[3.11(B)].
line
drip-sampling device
may be used
to provide
at
fat,
the tank
for milk-
at
0-4.4 C, should
accompany
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
in
at
some other
40
processing step [3.13(B.5)].
When
collecting samples of
the sampling cock in the storage tank door, use proper sanitization proce-
for
needle of which
stainless
steel
is
may be
suit-
special line-sam-
The nipple can be clamped on or perany desired spot. Cool samples and deliver promptly to
nipple [3.11(B)].
manently located
at
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
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
(container of product)
is
cream
sample
dures, randomly select sufficient samples for testing requirements and also
to represent a lot [3.2].
unopened containers
a.
bag
original
to the laboratory.
in
3.14
41
may be
tamination.
Do
transferred to the
at all
ized containers.
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
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
and 4.4
all
[3.13(A)].
recommended:
When
is
42
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
When
3.1]
Unfortunately,
it
is
may
in
a single sample
lot,
all
set of physical
con-
literature.^
'*-
taken
all at
once
(single sampling), or in
3.22
43
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
production or
Sometimes an
if
3.21
is
When
official
number or
all
tag corresponding
C and
deliver to
eration
when
appropriate.
[3.11-3.12].
3.
Field sampling of
unopened containers:
When
necessary to obtain
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.
44
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
trier if
it
is
off),
in
is
70%
it
wiped
alcohol
into the
For
between each
trier
sample.
2.
Wrap
them
in
before sterilization.
3.
Sample
trier,
remove
at least 120 g
serting trier
including,
when
mm
spoon, transfer butter to sterile sample container. Use a small portion of the
3.24
45
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
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
print,
laboratory.
knife or spatula to
the
covered
in
[3.12(C, D)and3.23(A.l)].
2.
clean,
Sample container:
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
[3.24(A.l)]. Sub-
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.
When
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.
trier
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
horizon-
faces,
toward
center of cheese, d.) with cheese transported in barrels, boxes or other bulk
containers, or cheese which
is
formed
into large
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).
in
pled by taking the entire cheese. Crumbled, grated, and dehydrated cheese
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
A Equipment and
.
supplies: 13.11-3.12].
50 ml. For frozen ingredients, use a sanitized dry knife, spoon, butter
trier,
3.25
ers,
Ice
may
such as a
47
drill.
Where
sterile,
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
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,
48
0^.4 C and
plugs or
separately.
6.
less than 30 g of
sweetener to
sterile
Sampling
Direct Microscopic
Procedures
Count Methods
To
collect
scopic examination, follow the general procedures for milk and cream
[3.11-3.12 and 3.13(B)1.
Tubes,
vials,
mm
x 15-18
mm
lips,
in plastic
3.33
49
2.
To avoid
until
they are to be
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
reaching the
filler
70%
alcohol,
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
rinse solution.
Where
70%
With sanitized
tents. Lift
fill
50
ing
in
at
0-4.4
until solu-
be
cumbersome and
difficult to refrigerate
and transport
Swab
may
to the laboratory,
and
in
fill
16.31.
a control sample.
CIP
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
The
sanitary condi-
3.34
Sediment
in Fluid
51
Milk
transport to laboratory.
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.
where product
is filled
into containers,
will
air
and
3.) in
is
standardized
at
3.34 Sediment
in
Fluid Milk
52
filtering disc
Trier:
sterile for
sampling solid
0-4.4
tablet for
made
is
objectionable in physical or
in addition to the
determination of milkfat.
53
References
3.4
3.37 Radionuclides
in
Milk
CONVERSION CHART
The following metric measurements used in Chapter 3
the U.S. System for the reader's convenience:
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.
2.
45
180
F
F
F
F
14th ed.
J.
54
3.
1951.
The
J.
on preserved milk
14:105-108.
4.
1959.
Sons, N.Y.
5.
in Industrial Statistics.
Inc.,
6.
L.J.,
8.
9.
10.
11.
Levine, B.S. 1950. Effect of formaldehyde on the direct microscopic count of raw milk.
Publ. Health Rep. 65:931-38.
12.
R.T.,
Methods
for the
J.
37:159.
13.
NMC,
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
4.1
P.
Bell
Introduction
testing of
employed
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
Jr.
55
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
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
The
medium, whether
in
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
C.
rod.
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
may cause
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.
if
necessary. The
pH
of agar
within the
medium
or area of
medium
equilibration of temperature
when placed
in a
waterbath).
Sterilize at 121
recommended
(149.8 F) for 15
[4.3].
excess of
3-4%
If
larger
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.
58
25 C.'"
to equilibrate
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
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
pH
4.4 Sterilization
is
and Storage
medium may
medium
pH
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
medium
test
into plates.
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
if
After sterilization, gradually reduce pressure within the autoclave (no less
than 15 min
recommended)
can be
lost
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,
some degree
they retain
is
indicated
when
itself,
however,
is
not an indication of
sterility.
B. Hot-air sterilization:
Sterilize
in hot-air sterilizers
so that materials at
the center of the load are heated to not less than 170
than
hour
city,
at 170 C).
is
To
in-
loaded to capa-
Spore
doubtful situations.
is
in itself
a discussion of
in
standard-
ized procedures.
dures that
dairy
lication.'^
medium
indicating
when
it
4.6
is
Suitability of
Water
iSl
F.
and
tion
sure,
it
sterilization.
medium
When
to the
minimum necessary
to insure solu-
Prolonged storage
in a
beaker.
in
a sterile
emergency.
/.
Moving
Any
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
if
in
the apparatus
is
available. Generally,
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
make
the deter-
mination.
Water and
pH
If
7.2 with
N NaOH
solution,
and make up to
Magnesium
2.
MgS04
make
liter
7H2O
sulfate:
To
growth
liter
for 15 min,
in the buffer
and store
in
before use.
of solution.
To prepare
and
Some
data
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
To determine any
toxicity in prepared
"
done rou-
4.7
63
Dilution Water
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
agar
64
1.
Procedure:
a.
The
treated water to be
examined
is
amounts
made
is
by
99-ml
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:
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
for Detergent
Residues
phosphates, carbonates, or
ic
type (quaternary
cidal
65
4.9
A. Cleaning glassware:
in
which
resist
it
in a
distilled
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
are then rinsed four times in tap water and six times in distilled water
before sterilizing.
is
dipped
in the
presently-used
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.
66
^'^
Yeast extract
Casitone or trypticase
(USP pancreatic
digest of casein)
Sodium citrate
Sodium azide
10
20
0.4
Agar
make
0.01 g
15
To
10
g
liter
D.
MF-Endo
Broth:""
results,
the
67
4.9
it
may be
Peptone
Glucose
Potassium dihydrogen phosphate (KH2PO4)
Magnesium
sulfate
(MgS04
10
g
g
THjO)
0.5
Agar
20
Distilled
chlortetracycline hydrochloride:.5
E.
in
water to make
g
g
liter
1% Rose Bengal
in distilled
water. Sterilize at
100
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
cial Analytical
liter
5.0
Yeast extract
2.5
Glucose
Agar
1-0
15.0
make
Final
pH
in-
of medium:
(a
25
after sterilization at
liter
121
for
15 minutes.
Methods Agar
shall
con-
in
the
744. 2"
given
68
Agar
4.
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
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,
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
at 121
for 5 minutes.
The mixture
is
is
rotated to
poured into
C and
35
not be
G. Violet
more than
Red
Bile
total
2,
should
Agar:
Bile
In-
should be boiled for 2 min, then used immediately as a plating mediafter cooling to 45 C.
stead,
um
50.
it
It
shall
shall
Methods Agar
be prepared from ingredients which meet specifications for inMethods Agar medi-
um
It
shall
C.
It
shall
claved at 121
F.
56
It
for 15 minutes.
shall not
at
50 to
for 2 hr after autoclaving that could interfere with the Standard Plate
Count.
4.1
69
Productivity Tests
4.11
Productivity of each
lot
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;
official testing is
not permissible.
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
121
at
for 15 minutes.
Do a preliminary
dilution to use
If
tested on
that which
a preliminary count
is
if
will yield
3.
If
a preliminary count
testing, undiluted
preparation.
Eighteenth Street,
NW,
is
Washington,
DC
20036.
70
made
medium more
Plate
than once.
two consecutive
dilutions,
if
Use
its
wall.
E. Plating pattern:
Plating shall be
1
done according
Number plates
Table
4:1.
COMPOSITE]
in
Table
4:1.
4.11
71
Productivity Tests
6.
Invert
all
plates; incubate at 32
for 48 hours.
X =
and
medium,
let
for reference
and
media:
x=
^^
N
Then
test
media:
c.v. =
(ijioo.
When
72
value
is
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
and
St
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
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
numbers of
Nr = number
Nj = number
IXr
=
(N + Nt)
NrNt
distributed as Student's
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
5%
coefficient of varia-
small. Accordingly, a
1%
(a
following table.
is
D =
is
computed. This
ratio de-
er
A
o-
2.
Assume
^S ^
=
.10
Xr
medium and
.50.
5%
and that a
5%
difference
difference, and
is
to be detected
between
10%
.10
cients of variation of
the test
10%
coefficient of variation
and a
X^ uofuev of D imt
ctte
mmTt?"
v?f
-sriicices -^smursu:
;C&^i|irilM>lT4
*5
onr.irrc
"^'
..A.
"^r
T'
-^
-rencrilm
i.
it
-unr
"tai -tfw
rv
otuipcai uanini-
.-.,
>
^-:
rwsr -~...
-
^u/"*.
Jvparaiwm jf cfteiitft.
-n
vwater^MwiyMs.
luwi
'llism-
CHAPTER
5.1
Introduction
Historically, the
main
is
or per
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
Jr.
77
78
5.2
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
^')
[see A.8].
B. Storage space:
0-4.4
D. Thermometers:
(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
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
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.
5.2
Vigorous
stirring of the
79
water
in
the bath
is
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.
'^'
'**
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
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
to
remove
toxic
are submerged in
82-C (180 F) water containing a
suitable detergent.
when new,
may
Volume delivered
in
4-sec
maximum,
last
at
least
85-mm
inside diameter,
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,
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
/.
81
Containers of stainless steel or aluminum, with covers, preferred. Charpaper may be used. Singleservice petri dishes may be stored in their original containers.
Balance:
Balance should be sensitive to 0.1
J.
g,
single-pan balance
preferred.
K. Mechanical shaker:
A shaker as described in the 11th edition of this manual
may be
used.
Water bath:
L.
N.
and
visibility.
Tally:
An
incubator or incubator room must be properly constructed and conTemperature within the incubator must not vary more than 1 C
trolled.
setting.^
tor or incubator
room
peratures.
82
trolling
in
periodically
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
is
may be
maintained and
re-
5.3 Materials
in
Chapter
4.
(MS) water:
MS
MS
[4.7J.
Make
tests at least
[4.6].
Examination of Samples
5.5
83
Fill
will
contain 99
When
utes.
less
2 ml.
When
bulk-sterilized diluent
5.4 Sterilization
A. Steam:
[See 5.2(Q) and 4.4(A)]
Hot
B.
air:
temperature,
each
lot
minimum temperature
pose.
Examination of Samples
5.5
A. Temperature:
After collection of samples, refrigerate and expeditiously transport to the
testing laboratory.
Upon
separate pilot container treated exactly as the samples; record the temperature.
Do
B. Collection, storage,
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
0-4.4
C.
If
Chemical
tests:
analysis [5.6(B)].
first
remove portions
for microbiological
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
all
5.7
Diluting
85
Samples
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
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
the pipet
is
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
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
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
:
86
dilution blank.
Weigh
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
um;
this
B. Pouring
Select the
to
be plated
in
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
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
88
B. Additional controls:
at least
5.10 Incubation
Incubate plates
Plates
at
32 1
for 48
in the
trolling ventilation
15% of
its
and
dif-
7.
^^
air circulation.
Agar
in plates
medium by con-
5.11
on materials
on the report
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
light.
Avoid
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
5.11
particles
air
in
large
fingerprints,
B.
One
films
off"
petri
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
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.
E.
No
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
90
from
plates
all
cable.
5:1,
Sample No.
1007.
Table
5:1.
from
all
Examples
dilutions of
for
Computing Standard
Gram
Sample
Plate
Count per
Milliliter or
per
91
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.
For example,
dilution.
if
number of
portions of the plate that are representative of colony distribution and calculate the
If there are
few-
if
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
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,
If
pressed-glass or single-use
diameters than 91
mm
tory personnel should determine the actual diameter and use a correction
factor for multiplication.
Do
crowded
plates
numerous
/.
to
Spreaders:
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
92
first
type
is
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
Do
to originate
from
when
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.
is
itors" (GI).
Count
as
"Growth
Inhib-
substances
in the
5.12
To compute
number
(if
same
number of
colo-
by the reciprocal of the dilution used. Record dilutions used and number of
colonies on each plate counted or estimated.
5.15
93
References
5.13 Reporting
Plate
(I).
Avoid inaccuracies
in
own
5%
5.15 References
1.
Methods
1960. Standard
for the
Examination of
Archambault,
J., J.
1937.
ditions for counting plates (with suggestions for a standard colony counter).
Health 27:809-812.
3.
Babel,
F.J.,
Jr., J.C.
Peters,
I.I.,
Speck. 1955. The standard plate count of milk as affected by the temperature of incubation.
J. Dairy Sci. 38:499-503.
4.
5.
J.
L. D.
Witter.
on
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.
Courtney,
J.L. 1956.
The
Donnnelly,
sis
J.
employee
C.B., Harris, E.K., Black, L.A., and K.H. Lewis. 1960. Statistical analysplit with state laboratories. J. Milk Food Tech-
nol. 23:315-319.
10.
11.
plastic pipettes. J.
Fowler,
Clark,
in
J.L.,
13.
W.S. Foster,
J.F.,
Dairy Products.
12.
Jr.,
J.
Food
in
Prot. 41:4-7.
Jr.
and automatic
procedures and plastic versus glass petri dishes for enumerating bacteria
Milk Food Technol. 33:400-404.
in
raw milk.
J.
94
14.
holding dilutions
15.
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
J.
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.
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.
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.
Introduction
6.1
all
organisms
is
at
32
within 48 hours.
One source
of these
members
and Klebsiella.
ed
in
A few
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
95
COLIFORM BACTERIA
96
in
10), butter, margaand related products (Chapter 11), cheese and other cultured products
(Chapter 12), and ice cream and related products (Chapter 13).
6.2 Definitions
cream
is
indicated when:
at least 0.5
Agar
C on
a solid plating
milk and
measuring
in
incubation at 32
Bile
1.)
''-
'^' *^
medium
'-
'-
'^'
^'^
2 hr of
2%
Brilliant
3 hr
Green
incubation
[6.9, 6.11].
confirmed
Agar
test
plates
Since results of determinations of total number of bacteria in dairy products by the agar plate and direct microscopic
liliter
is
milliliter" (or
laboratory personnel are usually familiar with reports based on such quan-
tities.
which
97
General Procedure
6.6
medium
used.**-* Progress
6.4
being
made
in increasing
Sampling
Sampling
6.5
is
'"
is
done
in
in 3.13.
[5.2],
Media
6.6
Nutrient Agar.
General Procedure
Examine samples of
media
[6.8, 6.9].
COLIFORM BACTERIA
98
pasteurizer. Therefore, a completed test
and
official
ucts
is
When
Green Lactose
Bile Broth.
in
samples,
Green Lactose
Brilliant
16.11].
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
When
plants,
it
sample.
is
If
average
per
are that
1%
if
each
may
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
is
low
6.8
is
obtained.
Transfer
Add
to
recorded as directed
in 6.12.
Medium
to obtain at least
at32C
for 48
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
6.1
liquid
medium
[6.9, 6.10],
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
closely
conform
"n"
number
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
Record
results, indicating
each, and
number of
''''-"
if
into
three of five
tubes with 10-ml portions in each have gas and none of five tubes with
1-ml portions
in
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
volume inoculated
number
listed in
if
the greatest
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,
if
the greatest
volume
in-
by
1,000.
When more
than three different volumes are tested, use results from only
Use results of the smallest volume having a
reading of 5/5 and results obtained with the next two smaller volumes.
Significant results in the following examples are in boldface type; values
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
6.15
3.
105
References
BusTA, F.F.
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.
Hartman,
p. a.,
Hartman.
P.S.
and
W.W. Lanz.
1975.
VRB-2
coli-
1.
12.
Maxcy, R.B.
tests
by
fer-
13.
14.
Dis. 17:183-212.
15.
McCrady, M.H.
Health
16.
J.
(Toronto) 9:201-220.
Archambault.
zation control.
18.
J.
Cerny.
J.
1953.
The
cubation upon bacterial counts of market milk and related products, particularly after hold-
20.
21.
22.
23.
24.
How
25.
Woodward,
26.
R.L. 1957.
probable
is
in ice
cream,
number?
J.
Amer. Water
and im-
Cream Manufac-
turers.
27.
Yale, M.W.
1937.
Comparison of
in
J.
CHAPTER
Thermoduric Bacteria
7.1
7.11
Introduction
To
3)
essing plant.
107
108
7.13 Equipment
A. Test tubes,
sterile:
Screw-capped, 20x125
mm;
plastic liner.
in the
bath
without a drop
7.14 Procedure
tubes
ing period in the ice bath, as well as during pasteurization. Place rack of
tubes
in
or they
may be immersed
short-time
When
within 5 minutes.
(HTST)
is
approximately
The temperature of
Do
V2 in.
above
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.
7.15 Interpretation
Presence
in individual
contamination from
7.16 Reporting
7.2
109
Thermophilic Bacteria
7.17 Limitations
(LTLT)
tions obtainable in
in the
modem HTST
The
cover
plating
all
medium and
may
may
not re-
as an indicator of
^^'
'^'^
Thermophilic Bacteria
7.2
7.21
Introduction
bacteria
which grow
in
including pasteurization
(LTLT), 62.8
or higher),
C.^'**
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).
110
7.3
7.31
Psychrotrophic Bacteria
Introduction
be included
From
in the
psychrotrophic group.
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.
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
tion contamination.
The Standard
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,
'^
conjunction with the oxidase test'' does not involve any change
tory procedures,
SPC and
in labora-
be obtained on the same plate. The keeping quality test of Moseley (Appenif the currently recommended
5- or 7-day
initial
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
medium
112
may grow,
results.
To
evaluate
/7/flA7r
test (see
Appen-
7.33 Reporting
After determining the colony count, report as "Psychrotophic Bacterial
Count per
Baumann,
organisms
2.
D.P., and
Buchanan,
of psychrophilic micro-
The Williams
&
Claydon.
PBC/ml (PBC/g).
in dairy products. J.
per gram); or
References
7.4
1.
milliliter'' (or
DiCKERSON,
Jr.,
J.
Jr. 1968.
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,
Cliffs,
J.C.
Olson,
Jr. 1957.
N.J.
preliminary report.
J.
27:304-307.
8.
9.
10.
11.
12.
13.
New
Hankin,
nisms
in
F.J.
L., and
W.F. Dillman.
1968.
Hii
EMAN,
J.
John Wiley
&
Sons,
in
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.
J.
Johns,
CK.
Annual
1969.
Con-
J.
23:1375141.
17.
18.
among Enterohacteridceae
Olson,
(Abstr).
H.C
1960.
The
isolated
from food.
J.
by thermoduric psychrophiles.
J.
7.4
19.
20.
113
References
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.
24.
cies of Bacillus
Jr. 1969.
Two
Thomas,
J.
Thomas,
S.B.,
G.J.,
and time
Milk
in milk. J.
1966. Effect of
in milk. J.
pH
of plating
medium
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.
CHAPTER
Introduction
8.1
shall
it
shall
may
be practically free of
This includes milk from cows with mastitis, milk from cows treated with
medicinal agents which
may be
to be prejudicial to
Concentrations of leucocytes
ally
in
milliliter are
rion,
By
gener-
this crite-
cows
latent
of leucocytes.
in milk.
Although polymorphonuclear
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)
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
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:
1(X)
ml with
distilled
water; store
in flint glass
if it
becomes cloudy.
size. Use
5.
Sample containers:
6.
Sterile [3.11(C)].
B. Procedure:
All samples
must be stored
at
0-4.4
C from
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
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
test is
^Available from photographer C. Hadley Smith, Carey Building, Ithaca, N.Y. 14850 ($1.50).
8.31
117
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
cell
count
is
usually between
clumping will be
and 2 million per
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
is
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
results:
cells in place
The
The
CMT
purple).
is
a measure of the
pH
indicator (bromcresol
DNA
number of somatic
(deoxyribonucleic
At
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,
2.
Cornwall continuous-pipetting
Cornwall 2-ml syringe.
outfit.
119
120
3.
4.
5.
6.
CMT
7.
Sink and cold water faucet or bucket, for waste and rinse water.
reagent,'
produced under
license.'^
1.
36 hr after sampling.
2.
Mix sample(s)
3.
as specified in 5.6(B).
5.
^'^*-
^^'
Do
'"
in
val-
tion categories.
D. Precautions:
If
1.
milk
is
Weaker
ing starts.
Because of the
4.
critical
time factor
in
reading the
make
is
CMT,
use of paddles
not recommended.
It is
5 seconds.
5.
It is
important to use
It
is
of reactions.
8.33 Wisconsin Mastitis Test
The
principle of
samples was
first
raw milk
the California Mastitis Test. Subsequently, methods
used
in
Box
288,
to
NASCO,
121
Table
Symbol
8:1.
Suggested
Visible Reaction
Meaning
Negative
(cells
per ml)
0-200,000
of precipitate formation
Trace
slight precipitate
150,000-500,000
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
Distinct
positive
may
disappear.
mixture tends to
Strong
positive
Generally
>
5,000,000
Alkaline
milk
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
122
for
WMT
mm
mm
ID,
located about
mm
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
dial
circular dial
markings.
WMT
11.
WMT
may
reagent
12.
at 35
test.
(95 F) during
the test.
^Available from Dr. Z. D. Roundy, 725 South. 600 West, Orem. Utah 84057; or Dairy Re-
Box
288,
SAvailable under Catalog No. 5638-01930 from Belico Glass, Inc, Vineland, NJ, 08360; or
Me
04096
123
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.
124
3.
test.
right side
up
in
4.
rack.
Mix by
placing a 10-ml
Mohr
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
NOTE
5.
hold tubes
ings in millimeters.
125
WMT
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.
this
126
(DMC) and
enumerate bacterial
sequently, Levowitz
test tube.
(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)
tors.''-
**
analyst
among
Variations
the
three
in results
confirmatory
ately or later.
may be
2.
Stained films
3.
may be made
Expensive equipment
is
required
(i.e.,
microscope).
during exami-
8.41
127
Microscopic Procedures
2.
(0.01 ml)
Preparation of stain and staining of slides calls for great care. Proper
performance of the
test requires
sults.
2.
3.
drafts.
4.
Microscope
slides: (clean),
cm^ marked on
cording to
6.
of
APHA
Bent-point needle:
Of 30-gauge
7.
and
cm^.
(104-113 F).
8.
9.
Slide-storage boxes.
11.
Compound
12.
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.
Inc.,
Box
288,
128
single-Strip
single horizontal
is
facilitate
20.
modifica-
21.
tion:
hood or
in
ventilated
a well-ventilated atmosphere.
22.
Immersion
23.
oil:
(68 F).
and
Make
touch-oflF.
marked
level surface
fix
them by drying
it
is
at
is
area.
Wipe needle
40-45
within 5 min on a
dry.
prepared.
CAU-
from the
slide
min-
utes.
Submerge
Remove
slide
and stand
it
in near-vertical position
on absorbent paper,
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.
is
advisable to conduct
final
all
procedures
in
drying, in a ventilated
fume hood or
in
well-ventilated atmosphere.
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
NOTE:
8.41
Microscopic Procedures
129
lows: For polymorphonucleated cells, count as a cell unit those cells having
per
milliliter.
mm)
distance (65
are to be
made by an
from
ies substantially
When
whose
estimates
set up,
use
observed.
To
field
changes
tri-
cali-
in interpupillary setting.
to provide
maximal
microscope lamp
To determine
the micro-
proceed as follows:
Measure the
(1)
e.g.,
0.146
(2)
field
diameter
in
mm.
To determine
(r
Vi the field
To convert
area in
(4)
mm- by
100.
To determine
the
number of such
fields in
cm-, divide
cm^ by
Since only 0.01 ml of milk was spread over a 1-cm^ area, multiply
the
number of
per
milliliter
The
in
final
fields
fields
of milk.
value obtained
each instance
is
is
dependent on a
(MF) and
and a
definite
130
MF
Working
b.
When
factor (WF):
MF
3.1416 X
the
r^
by the number of
fields
counted
to
is condetermine the
WF.
Performance of
c.
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
of fields to be examined
is
10. If
MF of 500,000,
must
is
is
made by averaging
first
40 cells were so
milliliter.
lies in
the 300,000-
full
count per
number of required
milliliter is
fields
computed according
cell
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.
croscopic
somatic cells x
No. of
or
Round
per ml
it
'.
of the microscopic
field for
8.41
Microscopic Procedures
Table
131
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
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
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
field
is
mm).
Area of
single strip
mm^
11.28
mm
(in
mm)
by divid-
No. of
single strips
100
mm^ ^
SSF = No.
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
132
Performance of the
b.
Focus on the
single-strip count:
film
edge
in
computed
as follows:
T^,o^^
DMSCC
per ml
EXAMPLE.
If
assuming
computed
off the
answer
per ml
to
two
count
is
as follows:
DMSCC
Round
= 200 x
6,250
1,250,000
milliliter.
The
unit area
examined
is
strip of milk film, defined in length by the fixed diameter of the circular milk
film [8.41(C.4)]
and
in
critical
concentration.
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
width x 11.28)
expressed
in the
following equation:
Strip
^ reticle factor
(SRF) = r^rx
-r^
rr-r-^
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
allel lines
alignment
if
move
control,
133
its
following rules:
Count somatic
(1)
field to
Count
Count
(2)
(3)
cells as
all
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
EXAMPLE.
DMSCC/ml =
Round
off to
two
72
+ 80 + 78 + 69
^^
SRF
of 15,160,
1,130,000
DMSCC
would be
number of
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
is
a modification
134
is
the count
is
lows:
_ Maximum
SE
EXAMPLE.
matic cells per
In
reticle
The SRF
is
cell
concentration
SRF
milliliter.
proper eyepiece
acceptable somatic
legal
is in
maximum number
is
when
the
^^ =
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
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
cells
limit.
slide in
marks on
boxes for
slides, since
"
'*'-
persion of fat
is in
'''
is utilized.'-
^' ^'
'2-
''^'
'''
Table
Gl
8:111.
135
136
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:
1.
100-/>tl
fitted
manometer.
requirements.
Glass medicine
4.
vials:^^ 7
dram, 66
mm
height, 25
mm outside diame-
snap-cap.
5.
6.
7.
Timer: 10 minutes.
Vortex Jr. Mixer.
8.
Racks
9.
(2):
To accept medicine
vials
vials.
ity.
10.
in
deionized water.
in
pH
An
84.5
12.5
12.
W. 20
St.,
^^Demuth Glass Works, Div. of Brockway Glass Co., Inc., Parkersburg, W.Va. 26101.
"Obtainable from Rohm & Haas, Philadelphia, Pa. 19104.
137
D. Procedure:
1. Fixing milk samples.
a. Hold all milk samples
refrigerated at 4
before fixing.
b.
c.
e.
up time and
min hold
time).
Time
for 2
a.
set the
lower threshold
Somaton solution do
at
five or six
blank counts on the Coulter Counter. The averIf it is not, clean the glassware used,
must be
at
b.
Mix each
c.
d.
(1)
(2)
on the Vortex
Jr.
mixer).
the edge
Expel milk and diluent from diluter into an accuvette, being careful to
down
Do
not mix
in
accuvette.
step
f.
surface.
g.
Time
Cool samples
in ice
water
at
to 4
for 3
to
room temper-
E. Calibration:
1.
particles of
known
size
138
in
is
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
If not, dilute
may be
re-
quired.
week
is
sufficient.
F. Precautions:
2.
before use.
3.
Samples should be
at
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
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
manometer and
diluter used.
8.5
References
8.5
1.
CuLLEN, G.A.
milk. Vet.
2.
139
References
1965.
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.
11,
Levowitz, D.
J.
16:260-264.
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,
Seminar
11.
Newbould, F.H.S.
12.
13.
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.
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-
Read,
Jr., R.B.,
Bradshaw,
J.
J.G.,
and J.T. Peeler. 1971. Collaborative study of conin milk. J. Milk Food Technol. 34:285-288.
Read,
Jr., R.B.,
J.
J.T.
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.
1956.
The
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.,
test. J.
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
9.1
Introduction
Widespread use of
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
Use
of standardized
materials will
reduce variability
in results
of
'' '^- ^^
assay
can be
all
reliable assays
ANTIBIOTIC RESIDUES
142
Assay
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
of the stock
with
known
standards.
paper
expiration date.
Diameter
Vi inch (1.25
cm), non-
discs
may be used
if
sterile discs.
NOTE.
All discs
used
in testing
size
and absorbance to
fine points.
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
To
petri dishes
Use of
the
solidify
amount of medium
in
Table
center of a
[9.21(D)]
Table
9:1.
Quantity of
Medium Required
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
water bath
at
9.22(A).
until
growth beOp-
tionally, hold
in 9.24.
9.23 Interpretation
Examine
Any
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).
the
same
plate. Alternatively
145
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
It
does not
9.24 Reporting
in
To
If tests in
If tests in
9.23(A) were
use reference discs [9.21(D)] as a guide. Report absence of inhibas Not Found or Negative.
penicillin,
itor
Method
for Detection of
A. Agar:
Beef Agar).
mm,
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
D.
1% phosphate
and dilute to
buffer,
optimum amount of
liter
for
<
weeks.
pH 6.0
monobasic powa-
ANTIBIOTIC RESIDUES
146
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.
/.
Glass heads: Sterile, for spreading suspension over the agar surface
J.
the
10
Roux
USP Sodium
K.
in
bottle.
Penicillin
by dissolving an
may be used
for 2 days.
M.
0-4.4 C.
9.32 Procedure
A. Assay plate:
Prepare a suitable amount of Antibiotic
(Chapter
4),
and cool
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
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,
is
0.05 unit/ml.
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-
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
9.32 Procedure
3.
147
at
30
invert plates to
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
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
is
20
mm,
the correction
is
mm.
mm.
+0.02
same
If the
plates
17.0
mm,
the cor-
is
17.2
tions:
3a
L
Where L and
+ 2b +
-e
..
H _
3e
+ 2d~ +
-a
(0.0125 and 0.2 unit/ml) concentrations for the standard response line;
c
eters for each of the other concentrations used for the standard re-
sponse
line.
Controls:
may produce
is
limit of detectability.
will
On
occasion, the
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
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
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.
Interpretation:
inhibitor
4.
To
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)]
final
concentration
in
149
References
9.5
terms of units/gram.
If additional dilution
final
potency.
9.33 Reporting
penicillin. Report the absence of
Samples found positive for nonspecific
must be assayed by other methods before reporting.
inhibitors
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
is
necessary,
should be consulted.
References
9.5
1.
and
'^
Abraham,
E.P.,
Chain,
E.,
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.
New York
10.
11.
State.
J.
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,
Insulin Analysis.
1945.
J.M., Savage,
plate
method.
J.
Bacteriol. 50:701-709.
13.
F.J.,
in milk. J.
ANTIBIOTIC RESIDUES
150
14.
Methods of Analysis.
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.
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
J.
Assoc.
OflF.
CHAPTER
10
Clark,
Jr., J.L.
Dizikes,
and C.K.Johns
10.1
Evaporated Milk
The
resulting prod-
is
ing, or
When
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:
(MS) water
[4.6].
10 dilution. If the
Jr.
151
Where needed,
sample
is
pre-
coagulated or
152
MS
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'^
48
3 hr.
3 hr
for
C. Counting plates:
Count plates
[5.11].
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
is
milk.
It
is
pasteurized, but
153
may be used
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
45%
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
When
10.1(B).
and counting
plates:
For
plating, incubating
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].
E.
Yeast and
mold count:
in tote
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
re-
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
numbers
in
dairy products
is
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.
is
in 3.21.
1.
and flame.
2.
3.
Aluminum
into pieces
4.
and
foil
or paper: Standard
foil
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
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.'^
Whenever
possible,
155
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
1 1
foil
transfer contents aseptically to the initial dilution blank. Dilutions are easily
made
in
initial
appropriate dilutions.
may
colonies accurately.
the
number and
count. ^
If
An
make
is
it
difficult to
count
reported to reduce
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
[14.4]
of value.
dium
citrate
Do
10 dilution.
it
when
single-
precipi-
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
It
must be recognized
that in
156
may be
'^
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.
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
10.4 References
1.
2.
3.
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.,
J.L.,
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.
B.
1957.
Macy, H.
1928.
Some
J.
Dairy Sci.
11:516-526.
10.
Marth, E.H.
J.
11.
Miller,
J.J.
richia coli.
12.
J.
Esche-
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
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-
CHAPTER
11
Jr.,
Jim
L.
Dizikes,
and
Vernal S. Packard
11.1
Introduction
process and
if
may
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
158
chrotrophic and proteolytic bacteria and b.) the microscopic method for
esti-
do coliform bacteria.
Plating methods to detect proteolytic and psychrotrophic bacteria are useful in
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
"^
11.3
a collaborative study.
in
Sampling Procedure
11.4 Bacterial
A. Procedure:
Before plating,
[3.23].
Counts
warm
dilution blanks
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
fer 11 g of
warm
(40 C) dilution
Divide 5 ml of
1
2 dilution).
For
10,
two
because
it
is
unsatisfac-
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:
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
11.5 Yeast
only to the
for
10%
tartaric acid.
Add
acid
will
Pour plates
[5.8]
and incubate
at 21
2 C.
third
mold colonies are nuday and count again on the fifth day, if
If
possible.
11.6 References
1.
1975. Official
Methods of Analysis.
BucHBiNDER,
in dairy
products.
3.
in dairy
products.
4.
5.
6.
L.,
7.
Shadwick, G.W.,
Jr.
1938.
study of comparative methods and media used in microI. Yeast and mold counts. Food Res. 3:287-298.
CHAPTER
12
12.1
F.
Olson, Robert
F.
Anderson, and
Robert Sellars
Introduction
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
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
dard
tests. ^'
162
12.2 Collection
and Preparation
of
Samples
3.
imately equal portions, each consisting of not less than 5 g taken from
dif-
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.
accommodate
0.1 g
and readability of
0.1
gram.
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-
tained.
The
As an
alternative method,
sterilized
transfer
it
*NASCO.
177-ml
(6 oz)
to a flat surface
Fort Atkinson,
10
mg
Wl
is
Whirl-Pak bag* or
53538
its
12.3 Microbiological
Analyses
X 125-mm
rolling a 15
163
test
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,
this
analysis of
States. 12
sterile spatula,
closed plastic sample pouch, gently knead and mix the enclosed curd.
Aseptically
remove
Add
99 ml of
warmed
it
upside
down on
the
described in 12.2(C) to disperse and dilute the cottage cheese curd. Proceed
and
semifluid products:
until a
amounts of
homogeneous dispersion
11
be dispersed
in
2% sodium
citrate with a
12.2(D).
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
amounts of the
container or a shaken dilution tl2.2(C,D,E)], into sterile petri plates and add
164
Violet
Red
Bile Agar.
'^
MPN
Coliform
in 7.32.
12.4 References
1.
2.
cream.
3.
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.
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
III.
Staphylococci
in
dairy foods.
A review.
J.
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.
studies on
the preparation of hard cheese samples for microbiological assay using a plastic
technique.
J.
pouch
CHAPTER
13
13.1
Introduction
Methods
are given in other chapters as follows: evaporated milk, condensed milk, dry
1 1
and
fluid
5.
Microbiological methods described here are also applicable to frozen dairy foods in
which vegetable
fats
Mellorine and Parevine and to water ices, pops, and non-dairy creamers.
fruits, nuts,
to the partially
may be
late,
fiers
important
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
166
coliforms present in fruit ice cream which give positive results with the coH-
form
test
in inter-
In a
Any
ingredient of a frozen
The
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
more uniform
B. Dilution blanks:
in
may
facilitate dis-
13.3
Sampling Procedures
ice
cream and
related
Samples for
13.4 Preparation of
Plating
A. Unmelted samples:
When
dition,
at
aseptically weighing an
B. Melted samples:
If
sis,
the sample
hold
it
at
is
room temperature
still
for not
Count
167
until
of sterile
C. Coloring, flavoring,
Where necessary
not more than 40 C
to 40
1 1
g) of
to ensure
for
1:10 dilution.
Weigh
blanks
1 1
g (or
g)
when preparing
initial
dilutions.
Where
than 20 min;
if
a longer period
is
it
g into
prevent lumping, swirl con-
room temperature
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
Where
the stabilizer
is
at a
Count
Proceed with general instructions for diluting and plating samples and for
incubating and counting plates [5.6-5.14].
168
Because
it
initial
number of samples
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
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
13.9 References
1.
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
fruit ice
Food
CHAPTER
14
14.1
Thompson,
Lyie Eckberg
Introduction
Raw
Milk to be Pasteurized
tent of bacterial
contamination
in
their stainability to
170
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
in
Method
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
faulty preparation
the minute
amount of milk
actually
examined
in counting;'"^
When
may
be recognized easily.
14.7 Bacterial
Counts or Grades
171
Dependent values,
MF
milliliter
172
enough apart,
if
Take samples
preservatives
'^'
Samples
in sterile, preferably
'"
only as needed
in the
sample to a
14.9
slide [14.9(A)]
tests, refer to
Chapter
5 for
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
Use
is
not acceptable.
slides.
free
ly
New
may be soaked
in
14.9
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
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
'"*
is
to store slides in
95%
alco-
tities
Where
certification
is
may be encountered
To determine
'*^
or pipet,^
first
Withdraw a representative
parts from one syringe with parts of another. Handle parts carefully
Box
288,
Amboy,
when
N.J. 08861,
174
'^^^-:::^
APPROX20mm
6-7mm
PLUNGER
(P^
r^
BARREL
SPRING
PISTON
MEASURING
TUBE
0.01ml
14.9
pH
7.0.
first
175
use.
make
removed from
the instrument.
To
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
frequently
2.
Pipet:
APHA
mm
40-60
tip.
The
tip is
mark
is
NOTE: When
not
in use,
in
When
filled
with
preparing for
use, rinse the bore and exterior thoroughly in clean water until free of the
C
A
Bent-point needle:
bent-point needle
is
D. Diying surface:
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.
E. Forceps
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.
176
H. Compound microscope:
A
tive,
binocular microscope
is
preferred, with
monocular or paired
who wear
eyeglasses.
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
14.9
lines.
is
required for a binocular microscope. Before using such a disc, determine the
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.
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
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.
M. Hand tally:
See 5.2(N) (double hand
tally preferred).
0.01-mm
divisions.
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
14.10 Materials
A. Chemical reagents (as required):
Chemical reagents
fied.
shall
tightly closed.
Dry
stain certified
satisfac-
tory for the purpose intended; or, liquid stains prepared from dry stains so
certified shall
be used.
C. Immersion
oil:
Immersion
type
is
14.11
used.
oil
Type B (medium
viscosity)
is
C and
of a nondrying
preferred.
Illumination Adjustment
When
to secure
maximum
brightness of field.
Use
is
reduce glare.
14:5).
However, mi-
14.11 Illumination
Adjustment
179
"CRITICAL" ILLUMINATION
LD
(Redrawn from
original of the
acceptable.'^
To
follows:
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
is
it
will
very small.)
180
in
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
to obtain
spot of
even
field
of view and
field in
Remove
F.
spot in the
scope condenser.
E. While looking at the
diaphragm
light
light
Do
light.
to center the
fills
left
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
down drawtube,
for use.
If light is
/.
or
more neutral-density or
scope, usually
in
the
filter
insertion of one
between the lamp and micro-
may be reduced by
filters
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
When
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
field is to
milliliter.
mm)
made by an
from
common
whose
interpupillary distance
When
estimates are to be
was
181
phragms
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.
micrometer
fol-
lows:
A. Measure
diameter
field
To determine
B.
area of
square radius
field,
(r
decimal, as 0.146
mm.
Vz field diameter)
and
multiply by 3.1416.
C.
To convert
area of one
field in
fields in
cm^
divide
cm^ by the
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
fields
of milk.
The
field
obtained
is
viewed
is
Use of
is in
millimeters, obviates the need for conversion of area into square cen-
timeters, determination of
plication
by 100
MF
10,000
,,^
3.1416 X
or
clumps per
^
MF
r^
milliliter
multi-
of milk.
40,000
3.1416 x d^
stant, a
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
of fields counted.
\\"F. as
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\
To avoid
bacterial
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:
'^'^
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.
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)].
tip slightly
in
clean
183
tip
0.01 -ml
pipe:
ma\ be expected.
holders must be sealable and maintained free from residues that might subsequentl) contaminate the staining solution
in
uhich
Use
Make
Newman-Lampert
stain.
^^
Avoid
use of liquid reagents containing suspended foreign matter. After stain has
CAUTION Prepare
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
in stain solution.
Repeated use of
stain in
an
184
Proceed as above
in
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.
may
in a relatively
refrigerate.
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.
(Table
B. Count
all
14:1).
clumps seen
in
strips
running com-
pletely across the film. This procedure requires less time than the
examine a
When
it
and
is
first
to
less fatiguing.^*
is
maximum
number of
185
14.20 References
may be preserved
solvent, slides
when
punitive action
reports.
oil
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.
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.
J.
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.
Levine, B.S. 1950. Effect of formaldehyde on the direct microscopic count of raw milk.
Pub. Health Rep. 65:931-938.
11.
p.
Levowitz, D.
scopic count.
13.
Levowitz,
An
1957.
J.
methods
direct micro-
D., and
M. Weber.
J.
Milk Food
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.
Newman, R.W.
1952.
in the
J.
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.
Richards, O. 1956. The effective use and care of the microscope. American Optical
pany, Buffalo, N.Y.
Com-
186
19.
(June 26).
20.
1969.
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
A. Introduction:
number of
bacteria in milk in terms of the time interval required, after starting in-
become
oxygen dissolved
in the
when incubation
mixture, which
in
is
started, to
To measure this
oxygen depletion,
dem-
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
is
Numerous comparisons
initial bacterial
con-
total
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
"
REDUCTION METHODS
188
the
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,
bacterial content
is
is
maintained and
of bacterial populations
1.
sample
may
result
incubated
at
36
when
the
factors:
species
many
other
by reduction
common
tests. ^^
milk contaminants
This
is
psychrotrophic bacteria.'"2.
Failure of
medium and
3.
some
at the
into
is
"acceptable."
When
cir-
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:
F.
Proceed as
in 3.32(B).
Tube
4.
tubes; or
flat
closures:
Rubber or other
(metal) plates, slightly larger than tube rack tops, having one
means
to
allow inversion of tubes in racks without loss of contents and without contamination of adjacent tubes.
5.
a.)
36
volume of water
with
within 10 min after placing samples therein; c.) to maintain water level
slightly
above
and
d.) to protect
samples from
9.
Amber
mm)
Interval timer.
REDUCTION METHODS
190
/.
Precautions:
compounds
Do
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
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
Use
solution in proportion of
solutions weekly.
Mark
in
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
test portions
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.
Method
191
L. Recording
and reading
tests:
at
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.
Because of uneven disappearance of blue color that may be noted as retest, record at each reading any sample as
when
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
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
is
REDUCTION METHODS
192
full
pink, at which
is
reversible. ^^ Reduction of
original
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.
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
is
^^-
This sensitivity
is
regarded as advanta-
and
2.)
is
dormant bacteria
**
''*
'*'
^^
'^
further incubation.
'^^'*
The
triple
reading test
*^
is
D. Standards:
The
provided for observing color and inthe end of three successive hourly intervals and classifica-
verting tubes at
'''
193
Method
USDA
standards
in
recommended standards
in
-^
classify
provide dual
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
associations.-*'
E.
Provide equipment as
in 3.32(A).
Proceed as
in 3.32(B).
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
7/4 (blue); 10
H. Precautions:
Proceed as
/.
in 15.1(1).
Prepare solutions as
tablets.
Testing samples:
Proceed as
in 15.1(K).
MD
21218.
REDUCTION METHODS
194
not shake) each rack of tubes three times and then incubate [15.1(H.5)].
Avoid making
rapidly.^
may be
whether by daylight
or by
artificial light
lamp against
background
a neutral gray
^- '^
accurately.
L. Resazurin triple reading test:^^
Proceed as
Adhere
in
15.2(E-K).
to the following
Incubate
at
36
[15.2(G.3)].
2.
After the
initial
incubation period of
once,
whether or not a color comparison has been made. Then continue incubation
at
3.
Make
final
M. Resazurin one-hour
test:
15.3 References
1.
Abele, C.A.
1945.
its
test as a
means of estimating
the bacterial
3.
Dabbah,
R.,
Inc.
ADMI
1970.
Recommended
sanitary/quality standards
III.
Olson,
Jr.
resazurin reduction one-hour test for grading milk intended for manufacturing purposes.
J.
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
Newland.
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
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. 21:227-237.
13.
in
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.
after pasteurization.
18.
19.
20.
22.
on the dye
more accurate
Johns, C.K. and R.K. Howson. 1940. Potentiometric studies with resazurin and methylene blue
21.
J.
in milk. J.
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.
A. 1951. The effect of penicillin on the methylene blue reduction time and
25.
Rowlands,
26.
Thornton, H.R.
test.
Canad.
J.
Pub. Health
24:192-196.
27.
CHAPTER
16
Introduction
16.1
16.2
Equipment
6.31
membrane
filter
techniques.'
3.
4.
[4.1].
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.
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.
MS
liter; filter;
8.
9.
lOOg of Na2S203-5H20
in
sterile.
B. Rinsing containers:
in
fol-
lows:
1.
line
without touching
valves.
lips
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
conveyor
in
reaching the
2.
inject
70%
alcohol, aseptically
fill
DE
19839.
NY
11377.
199
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,
swab an area of
Where tubes
are sealed,
liner
rinse solution
assembly.
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
present, record as
b.
When
If
no colonies are
200
Numbers
rinse solution
Membrane
filter
procedures
may
membrane
samples taken
yield
(if
filter
at the
procedure
'
for analysis.
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.
in
Chapter
3)
may be
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.
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
Swah
''^
contact method:
swab moistened in an
The swab is then rinsed
thoroughly
difficult to clean.
polished, large or accessible are easier to clean than those which are irregu-
lar,
ly
Methods
201
reached.
If
radii, or less
convenient-
these latter areas are not properly cleaned, residues will accu-
when chemical
solutions are
applied.
1.
Equipment and
See Chapter
supplies:
5,
modifications:
a. Screw-capped
screw-capped vials
vials
(7 to 10
cm
in small
claved
in
MS
is
away from
water
(5
etc) containing
1%
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.
lution of
Glenwood, IL 60425.
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
plete cycles of 15
cm
at the
end
manually; or
treats cotton
swab heads
of each cycle.
Groups of
vials
similarly to the
2-, 1- and/
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
laborative studies
'^
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
surfaces.
ternary
ammonium compounds.
^Falcon Plastics, 550 West 83rd Street, l.os Angeles, CA. 90052.
203
It
may be
used, or prefera-
dehydration.
2.
Procedure:
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
RODAC
plate or
Water
for milkhouse
shall
To be
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.
204
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.
'^'-'^
16.6 References
1.
2.
3.
swab and
Baldock.
terial
5.
RODAC
methods
steel surfaces.
contamination of surfaces.
Barnes, J.M.
1952.
J.
Methods
to assess bac-
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.
BucHBiNDER,
L.,
BucK,
Jr.,
8.
swab
10.
J.
alginate soluble
utensils.
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.
&
1970.
universal neutralizing
medium
for antimicrobial
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.
1964.
Measurement of
on
bacterial contamination
sur-
1976. Dairy
p. 545.
The Avi
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.
Marshall,
J.
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.
N.Y.
Tredinnick,
1948.
J.
test-
Committee
J.E., and
J.
Tucker.
1951.
swabbing
24.
surfaces.
J.
Witter, L.D.
J.
field
bacteria A review.
J.
CHAPTER
SEDIMENT
E.O. Wright,
W.L
IN
17
MILK
Jr.,
17.1
Introduction
milk with
known amounts
for
different
may
reveal
Jr.
207
SEDIMENT
208
at
IN
MILK
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
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;
tests
may
milk
d.) the
technique of
may work
allow enough time to draw the gun across the bottom of the can)
fast to
too
may
vary.
sample
is
sample.**-^
Methods
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
A. Tester:*
The
able between testing of samples to permit sanitary removal of the used disc
Company,
Company,
1512
III.
Inc..
17.3
209
NOTE
disc.
of results (see
17.3(C)). Milk or
disc or be
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).
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
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.
as applicable.
The
cm) or 0.10
in.
(0.25
cm)
that
filter 12
mg
ture (60-ml aliquot) through the disc, using a clean flask to catch the
trate.'-^
Transfer the
filtrate to
fil-
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
Food
Filters Corp.,
SEDIMENT
210
IN
MILK
E.
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
standard discs.
is
as follows:
first
When
disc, read
it
sediment
in
a sample
first
is
equal to
disc. If the
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.)
*These grading charts may be purchased from the Standardization Branch, Dairy Division,
Department of Agriculture. Washington, D.C. 20250.
211
17.6 References
Remove
disc
from
tester
still
waxed envelope.
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
its full
length.
Remove
sample method.
ing diameter:
cm)
0.20-in.
0.14-in. (0.35 cm)
0.10-in. (0.25 cm)
(0.51
filtering
filtering
filtering
in 17.
5A
diameter
diameter
diameter
17.6 References
Methods
1.
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.
4.
1967. Standard
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
in
J.
in
J.
36:87-88, 310-315.
7.
1960.
Farm
J.
Milk Food
holding tanks.
9.
10.
Watson, N.E.
Watson, N.E.
J.
1952.
sediment
pump
J.
J.
CHAPTER
18
PHOSPHATASE METHODS
F.J. Babel, T.L.
18.1
Introduction
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
pumps
of
known
is
The
test is
simple and
is
The 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,
ty
be used
in
been
21,22
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
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
Gum
in
containers of butyl
alcohol.
C. Reagents:
Keep
all
tamination.
215
18.3 Controls
nell test;
liter
Remove
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
CQC
preparing
CQC
decompose with
week.
powder
to
When
opening the bottle to avoid moisture condensation on the reagent. RefrigCQC solution and discard it when it deteriorates (turns brown).
erate the
Phosphatase Procedures
from
A.
Negative control:
negative control should be used with each type of dairy product being
show no
color.
B. Positive control:
As
0.1
Amboy.
N.J.. 08861
PHOSPHATASE METHODS
216
done on
this
When
at
90
for
one min,
structures
may be
pres-
may be
obtained
when
the substrate
is
disodium phenyl
same amount of
buffer solution.
mono-
is
in the
interfering-substances con-
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:
reported as negative.
E. Collection of samples (Chapter 3):
tested.
217
Phosphatase Test
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.
3.
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)
dium bicarbonate
2.
in
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
Phax
dissolve
4.
Phos-Phax
CQC
tablet in 25
reagent: Dissolve 30
ml of water.
mg
of crystalline
CQC
in 10
ml of methyl
if it
turns brown.
PHOSPHATASE METHODS
218
5.
SHoO) in 100 ml
copper
can be omitted
CQC.
6. A2-Butyl
as follows:
Add
0.1
N NaOH
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.
Standards:
phenol, transfer to a
stable for several
2.
1
liter
months
in the refrigerator.
Solution
a. just
milliliter
Table
Phenol Solution
18:1.
Phosphatase Test
4.
219
[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.
tubes.
a.
Buttermilk, sour cream, and creamed cottage cheese are best pre-
test tube.
When
When
4.
40
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
milliliter
is
is
Phosphatase
IVIethod
^^
^^
A. Apparatus:
1.
mm
or 16 x 150
mm
glass-
stoppered.
2.
3.
Pipets:
4.
6.
7.
Centrifuge: Safety head, or equivalent, that will hold six 15-ml tubes
5.
at
Same
as 18.4(A.3).
accommodate
16
x 150-mm glass-stop-
9.
lent.
mm.
B. Reagents:
in this
method.
Phsophatase Method
221
C. Standards:
2.
[18.4(C.3)].
D. Controls:
See, section 18.4(D) for controls applicable to this
method.
E. Procedure:
in
Section 18.4(E).
1.
16 X 125
mm
test tube.
gum
mix by inversion.
2.
Incubate the
at
40
for
hr in a
water bath.
3.
CQC
reagent
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
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
of sample.
is
Any
of improper pasteurization.
18.6 Cornell
Phosphatase Test
^^-^
A. Apparatus^:
1.
2.
3.
5.
milliliter.
10.
11.
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.
Company,
may be
18.6 Cornell
223
Phosphatase Test
4.
of copper sulfate
CQC
5.
make up
to
liter.
CQC
in
25 ml of absolute
6.
Bp
117.5 C;
do not
neutralize.
1.
make up
to
liter.
Keep
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
Make
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
Table
18:11.
Buffer 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.
Remove approximately
ml of alco-
hol from the extracted standards [18.6(C.4)] with a pipet and place in clean,
ed from the
Read
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
in
On
negative controls
may
fla-
interpretation.
E. Procedure:
whey,
ice
tracted butyl alcohol solution from each test tube with a clean pipet into a
mm
in
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:
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
ed by addition of
alkali.
The color
is
compared
is
bore.
Tubes
15
2.
Pipets:
3.
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.
The pH
3.
is
10.15 at 25 C.
NaOH
in
100 ml.
4.
addition of approximately
N NaOH
and make up to
if
pH
by
liter.
refrigerated.
C. Procedure:
The procedure
the
AOAC
is
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
to differentiate
1.
^Reagents
Institute,
Am-
PHOSPHATASE METHODS
226
glass rod for 2
paper.
b.
min and
immediately through
filter
Melt
sample
Butter
at
2.
3.
Add
filter
testing.
c.
Whatman No.
origi-
for testing.
drop
Add
drop (0.04 ml) of color developer to each tube, mix and com-
pare visually.
D. Interpretation:
If
shows
ly
pasteurized.
is
/xg
ml of pasteurized
dard
in the
which
18.8
is
same milk as
very important
in visual
same hue
comparisons.
The phosphatase
test
(88.3
for
sec to
227
Phosphatase
is
in
products
reactivation behavior.^''
Mg
salts.
:^''^^
A. Differentiation of reactivated from residual phosphatase
results
phosphatase,
compare
To differentiate reactivated from residual
obtained on testing a
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
Warm
in 25 ml of water.
H2O
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.
re-
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.
same sample
0.1-ml
2.
while
Mohr
in
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
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
if
is
phosphatase.
al
D. Precautions:
A false-positive
phosphatase
test
about 16 C or 2 hr at about 20 C.
Reactivated phosphatase in an unrefrigerated product
al phosphatase.
may be
positive test
However,
reactivated phosphatase.
if
is
allowed to stand
at
it
may be
necessary to
18.9 References
1.
2.
1975. OflRcial
Methods of Analysis,
Washington, D.C.
3.
AOAC,
Offic.
S.J.
J.
Agr.
in
BuRGWALD, L.H.
1939.
The phosphatase
test.
its
application
J.
Dairy Sci.
22:853-873.
5.
Campbell,
McFarren.
J.
Assoc.
Offic. Agr.
Chem. 44:444-
448.
6.
F.J.
Babel.
Fram, H.
cream.
8.
J.
1957.
Phosphatase reactivation
in
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.
10.
J.
Henningson, R.W.
bicarbonate buffer.
11.
J.
1960.
13.
Chemical
in milk. J.
Assoc.
Assoc.
stability
Jr. 1933.
effect of
by mi-
new
alkaline phosphatase
Chem. 51:802-807.
Offic. Anal.
Chem. 53:869-871.
18.9 References
14.
229
KosiKOWSKi, F.V.
1949.
Science
test.
110:480-481.
15.
KosiKowsKi, F.V.
1951.
The
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.
phosphatase method.
19.
and cream.
20.
J.
Assoc.
Offic. Agr.
Chem. 43:414-426.
1710.
21.
22.
23.
O'Brien,
Scharer, H.
1938.
Improvements
Scharer, H.
its
in the rapid
products.
J.
phosphatase
improper
J.
Milk
Technol. 2:16-20.
26.
Scharer, H.
Amer.
J.
Pub. Health
35:358-360.
27.
Scharer, H.
1953.
test. J.
88.
28.
29.
J.
Tramer.
II. J.
III. J.
J.
Tramer.
treat-
treat-
treat-
31.
J.
30.
I.
J.
Tramer.
J.
Tramer.
J.
CHAPTER
19
CHEMICAL METHODS
L.J.
J.A. Burke,
19.1
Introduction
Appendix B.
19.2
Added Water
19.21
Milk
in
Thermistor Cryoscope
In 1956 Shipe
-^
in 1958'-** as
AOAC,
scope.
**
'^
concentration of solutes
in
the solvent.
As applied
to milk
when water
is
CHEMICAL METHODS
232
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
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
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.
Bath, 2 to 3
9.
Pipets, clean
Use
in.
grade, each
number of samples
tested.
19.22
233
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.
2.
to
-7 C) and
level of solution.
3.
2-ml sample).
4. Adjustment of
D. Procedure:
It is
tests,
stirrer
and freezer-vibrator.
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:
By common
1%
Where T = base
usage,
-^
100
(T - T')
true
-0.542 - (-0.520)
ro"^7
,^^
, ^^
or =
^ ^^^ " 4.06
4%
^*^
CHEMICAL METHODS
234
(SMEDP
13,
taut
4.
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.
B. Standardization:
1.
Install
instrument
in
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.
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.
*Wescor,
Inc.
if
churned
UT
84321.
it
in
may
fat interferes.
manner
require a
235
19.3
D, Calculation:
Calculate the per cent added water as follows:
% added
H2O = ^ ~
x 100
K.
are
E. Precautions:
in
^^
in
5.
6.
Analytical balance.
2.
3.
4.
Mohr
8.
overnight.
add
.25
solution.
12.
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
10
swirl to mix.
over
50
25
chlorine.
3.
color
is
4.
least
reached.
Add
ml of starch and
30 seconds.
If
color, immediately
is
in
source of contamination.
Calculation:
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
a.
TC
AOAC
specifica-
tions.
Milk
b.
mm).
test
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.
g.
Trunnion
h.
for acid
60 C.
Reading
light
to
bottle,
for measuring
e.
i.
fat
column.
bottle.
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.
Sponge.
n.
2.
Procedure:
homogeneous by pouring
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)
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.
tests with a
all
"Diam
tabulation below.
of wheel"
is
the distance
DiamofwheeKin.)
(cm)
No. ofrpm
10
12
25.4
30.48
1,074
980
18
20
50.8
800
759
14
909
16
848
22
24
55.88 60.96
724
693
B. Cream^:
1.
Cream
(15.24
meeting
b.
test bottles
AOAC
Pipet
shortneck 6
g,
in.
to
50%
specifications.
Mohr,
10 ml.
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
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
starting with
19.42
Fat:
239
Gerber
Whichever method
tained at 55 to 60
is
in
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.
The
19.42 Gerber
19- 20
This method
is
A. Milk:
1.
a.
test bottle,
8% Clear,
transparent, color-
Total length
190
cylindrical
Neck,
Neck
15
ID
11.5
2.5
1.0
mm
mm
mm
0.5
diam
total
not
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
mm
diam
graduated length
mm
etched, perpendicular to
flat
mm
mm
tube
pigment contrasting
sharply with color of fat; width of
mm
0.10 to 0.20
lines
in the flat
tube at 20
1.000 ml
is
is
15.0
13.546
mm
gHg).
It
length
1%
the 0.
side
lines,
and the
.0%
lines,
lines
mm on each
flat
face.
to the
8%
line
name
is
where required
to attest
specifications.
b.
To hold bottles
in
and a
solid
clamp-on"
Milk
test pipet
To
contain
11
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:
19.42
Fat:
241
Gerber
Wall thickness
Total length
340
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, O D
Graduation to bulb
40
6.5
mm
mm
mm
0.5
15
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
closed contain-
ers.
by weight, stored
in tightly
132
(it
in tightly
closed contain-
ers, in
water, and
test bottle.
Shake and
centri-
fuge bottles as required in the test for fat in milk [19.42(A.2)]. If oily
both with
and a single
new and
Determinations should
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
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
is
of rubber or
molded
key: rim and channel are reinforced by metal, plastic, or other firm binding or insert.
The key
is
to rotate at 1.100
Standard
style,
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.
er (Item b), expedites washing. After reading, return bottles to the rack,
bottles as a imit.
2.
Procedure:
Measure
10 ml of
H.SO4
0.2 ml at 15.5-21.1
at
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
then
at first, to
Add
it
(CAUTION HOT)
until the
at
curd
at
the
tip.
prevent reaction
full 3
slowly,
is
it
at least four
19.42
Fat:
243
Gerber
shaking of
filled,
in
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
remove the
at least 5
fit
min
in
the bath,
lock stopper, optionally using a desk reader [19.42(A.l)k]. Gently push the
the nearest
column
to tester
rate readings.
Proper
columns contain no
fat
light or
pale to strong yellow in color, depending on the season of the year and
When
on
determine adherence to standards or provide the basis of payment, test samples in duplicate, repeating the tests when duplicate determinations vary
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
mm.
CHEMICAL METHODS
244
Graduated volume
at
length
20
in the flat
tube
is
lines are
flat
less than 3
the
0.5%
mm long, the
lines,
1.0%
lines
extending
"Cream,
50%
name
or symbol of the
tube
is
Graduated volume
at
20
in the
centered on the
flat
mm
mm
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
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
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
Read
0.5%
This method
is
1.
Same
ing:
a.
cream
lent
Procedure:
2.
Measure
10
0.2
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
19.43 Roese-Gottlieb
3'
32
A. Milk, cream, ice cream and frozen desserts, nonfat dry milk and dry
a.
b.
Aluminum
dishes*
Cork stoppers,
to
fit
d.
Crucible tongs.
e.
Analytical balance.
f.
g.
h.
i.
j.
flask
mm.
#6.
Interval timer.
Mohr
Hot plate.
Water bath, maintained
at 100 C.
CHEMICAL METHODS
246
k.
1.
Stirring rods.
Hood, or
volatile solvent
m. Automatic shaker
exhaust apparatus.
optional.
n.
Desiccator,
o.
p.
Wash
filled
flasks.
bottle.
q.
r.
Alcohol
Ammonium
s.
t.
u.
2.
a.
extraction flask.
3.
Add
Procedure:
25 ml of ethyl ether to sample, stopper with cork and shake very
if
is
is
practically clear.
As stoppers
are
in
flask peri-
rpm or
let
suit-
with equal parts of the two ethers and add washings to dish. Repeat extrac-
19.43
247
Fat: Roese-Gottlieb
if
product.)
container
is equal to
Notes:
4.
a.
Correct
agents used.
fat
If
fat.
blank
is
re-
or purified.
b.
fat
Animal feeds
Cheeses
Cream
(regular, sour
Dairy puddings
5.
Equipment
"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
fat
of analysis.
a.
Turn on the
hr before begin-
Add
b.
7 ml of distilled water at 60
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
in the
is
hydroxide.
Add
ml of
and shaking
d.
Add
in
if
one
is
it.
While
Remove
unit).
it
carefully
flask
lit is
Remove
flask
for 30 seconds.
plate
Remove
down over
the sample.
19.44
Automated Turbidometry
249
flask
7.
Calculation
% Fat
gam
in
^^
weight or sample
'.
1.
Same
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:
with
HCI using
dicator.
in
is
necessary since
.".
test
The sample
is first
light
homogenized
to
CHEMICAL METHODS
250
produce
fat
is
it
(EDTA)
to
light scattering,
proval.
and
3.
4.
Distilled water.
5.
EDTA
sodium
EDTA
EDTA.
0.5-1 ml of Triton
salt, 'Hiu
NaOH
EDTA,
in
EDTA,
Na^
10
C.
ml of polysorbate
(The di-
can be used
in
III unit.)
B. Calibration of instrument:
Each instrument
in triplicate
is
Wc
to
6%
in fat
content.
The
calibration milk
S(D2)
Sd =
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.
The Milko-tester
will
same samples.
and preparation of sample:
C. Collection
1.
2.
Samples
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.
Table
19:11.
Reference method
^^^^
cow samples
Mojonnier or ether
0.04
0.06
0.06
CHEMICAL METHODS
252
19.5 Moisture
19.51
and Solids
Vacuum Oven
Crucible tongs.
5.
Analytical balance.
slip-in
flat-bot-
cover,
Cenco
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.
taining a
in.
8.
Steam bath.
9.
10.
B. Collection
distillation
26
(65
P.
Natural cheese:
253
Vacuum Oven
19.51
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).
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
should be
at
it,
the
E. Calculations:
^
%
^,
Moisture =
Solids
100
Moisture
F. Notes:
1.
The 100
C vacuum oven
Casein powders
Cheeses
Cheeses
Gelatins
Ice
CHEMICAL METHODS
254
to the following
products:
Sugars.
2.
vacuum oven with door closed for 15 min at atmospheric pressure before
proceeding to turn on vacuum normally used in test procedure.
cheeses.
19.52
x 60
mm.
Atmospheric oven.
10.
11.
P.
rinsing
2.
night to dry.
3.
Remove
C. Procedure:
1
Weigh about
3 to 5
balance.
Add sample
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
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
D. Calculations:
Loss
^
=
Moisture
Solids
...
100
x 100
p-
weight
in
.
Weight^
oi^
(g)
sample
(g)
Moisture
E. Note:
The 100
C vacuum oven
following products:'
Condensed whey
Frozen desserts
Ice cream
Ice cream toppings and syrups
Sweetened condensed milk
Whey
concentrates
Yogurts.
19.53 Atmospheric
Oven
7.
Steam
8.
bath.
B. Collection
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:
to center,
Before taking sample for analysis, the wedge shaped segment should be
cut very finely with the aid of a knife or spatula
all
fine slices
about V32
in.
flat
and mixed
(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
avoided as
fat
is
to be
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
at 100
washed thoroughly,
rinsed,
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.
liquid
cocoa, cottage cheese, liquid creams, egg albumin liquid and powders,
milks liquid, condensed whole and skim milk, sweetened condensed skim,
Product item
Buttermilk, liquid
Dry on
steam bath
Oven temp
C 2
Hours in
oven
19.54
Solids
in Milk:
19.54 Solids
in Milk:
Inspectors in 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
'^'^
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
ml of
liquid.
The
scale
is
0.60
0.05
0.1
WALL
THICKNESS
3.40
0.05
0.2
WALL
THICKNESS
SCALE
BALLAST
FIRST QUALITY
LEDGER PAPER
Figure 19:2. Dimensions of 39
**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
known accuracy
at
15.5
two sp gr
(60 F) at
levels.
flow tube or
samples.
4.
Lactometer cylinder: ID
movement
diameter
'
for
AOAC
Babcock
test
Official
Methods of Analysis,
equipment.
B. Procedure:
1.
Lactometer
test:
39
C by immersing
at intervals 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
bath.
thermometer
2.
Fat
at
test:
Amounts should be
lent.
3.
its
equiva-
General suggestions:
large
with only negligible change in specific gravity, provided that the milk
is
hr
gent-
ly stirred
19.54
259
Method
Calculations:
Milk,
total solids
where
F=
1.33
fat
F+
273
Milk,
Tables
19:111,
milk,
total solids
nonfat solids
'
^"^^
L = lactometer reading in
Skim
-JT^^
0.33
1.33
F+
273
F+
in
degrees.
273
L +
L
1000
0.40
when one
calculates the per cent of total solids. Table 19:111 gives values of 1.33
Ffor
Babcock
fat
from 25.5 to 34.9 covering the range for whole milk. Table 19:V gives values
Table
19:111.
CHEMICAL METHODS
260
Table 19:IV.
19.6
in
may be contaminated
261
Milk
Residues
in
Milk (Examination
of
Official Analytical
of
Na
or
Add
CHEMICAL METHODS
262
for
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
fat
filter.
2 g of
Na
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
CH3CN
NaCl
into a separator
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
a 22 X 50
mm
in
263
Milk
into a
500-ml Kuderna-Danish
concentrator. Rinse the separator and then the column with three portions of
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
CHEMICAL METHODS
264
E. Saponification cleanup:
This procedure
eluate
umn
when
is
additional cleanup
[19.6(D)]
if
is
thin-layer or paper
15%
15%
Florisil
chromatography
is
used.
F. Determination:
1.
^' ^
chromatography
ppm
heptachlor epoxide
its
in milkfat.
down from
may be
sample
(10%
re-
(Lim-
that for
NOTE:
desired.
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
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
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.
3.
Filter tubes:
1.
mm.
Approximately 22
mm
deliv-
621400, or equivalent).
5.
6.
Micro-Snyder column:
2-ball type
or equivalent).
7.
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
enough
in
stopper that
it
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).
Generally, vapors from such lots will turn moistened red litmus paper blue
when
odor
10.
mouth of
pronounced amine
detectable.
CH3CN
(item
Alcohol: Ethanol,
USP,
reagent-grade; or
MeOH, ACS
specifica-
tions.
12.
NaOH
or
KOH
in
Must be
free of elec-
tron-capturing substances.
14.
Eluting solvent,
6%: Dilute 60 ml of
liter
ethyl ether.
16.
contain
2%
at
34-35
C and
below
Florisil:
PR
60/100
is
obtained
in
(1250 F).
When
ing to pint-size (500 ml) glass jars or bottles with g-s or foil-lined screwtops
and store
130
130
C
C
in
in g-s bottles
after 2 days.
18.
or
in a
See
>
5 hr at 130
desiccator
at
reheat at
and standardization.
suitability; re-
Magnesium
jar.
20.
22.
MgO
-I-
Sodium
sulfate,
anhydrous, granular.
WV
25411.
suit-
19.6
H.
in
267
Milk
Suitability
^^
Solvents
in all-glass
apparatus.
Purity test:
1.
test.
Place 300 ml of
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
and
159?^
its
in
the
6%
eluate.
It
also possible that dieldrin and endrin will be incompletely eluted by the
15%
is
that separation of
endrin
[19.6(1.2)]
may be used
to ad-
1.
a.
A.
b.
c.
d.
e.
(pellets,
''^Suitable
&
NaOH
Jackson Laboratories.
Inc.
to 500 ml
Muskegon,
CHEMICAL METHODS
268
f.
CP quality,
500
g.
Transfer
ml = 20 mg).
Phenolphthalein indicator
and dilute to
milliliters (1
Dissolve
in
milliliters.
h.
Ethyl
alcohol
USP-grade
or absolute,
neutralized
to
phenol-
phthalein.
Hexane
i.
Reagent-grade,
Standardization:
2.
a.
b.
Weigh 100-200 mg of
Add
(about 10
3.
mg
per
NaOH
milliliter).
Procedure:
Calculation:
mg/g
Florisil
= 200 -
Evaluation:
5.
is
J.
Evaporation techniques:
To concentrate
Evaporate to about
the concentrator and
ml as
fit
in
above.
Remove
19.71
Protein:
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.
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
A.
used
in the
Amido
1.
MK
c.
Plastic bottle
d.
Plastic
e.
Hot
f.
g.
h.
Plastic
i.
j.
Plastic syringe 2
k.
1.
Forceps."'
Kimwipes, or equivalent,
capacity.
beakers
m. Glass
fiber filters.
n.
Stainless steel
o.
p.
Distilled water.
(2)
3-liter
#PRM504
(This solu-
Inc.
Chem-
CHEMICAL METHODS
270
2.
Solutions:
a.
Working O-liquid
Carefully measure 10
solution).
See 19.71(A.6)a.
of tap water at
the
When
dilution
is
complete, transfer solution to the 10-liter bottle rinswhich the chemicals were heated with approximate-
plastic syringe,
and on
b.
book,
Should be made
in suf-
made
in log
is
bottle.
Ammonia
Preparation by volume:
distilled water.
2% to 2
solution,
lot.
of distilled water.
3.
Initial calibrations:
a.
Syringe calibration
continual use.
To
will
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.
maximum
in
the
g.
When
records
show
It
should not
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.
271
Dye
delivery
It
is
at all
An
times.
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
Replace
filter
and
clean
tube
and
check
the
"0-liquid"
rest
CHEMICAL METHODS
272
two solutions
the
The
If
major adjustment
is
required,
Determine
sample. Carefully
fill
on an homogenized milk
first
fill
after
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
is filtering,
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
replace
filter
Use same
tissue to
19.71
Protein:
273
Release pressure and turn stopper away from the mixing tube.
tube with distilled water and turn stopper
tube.
Apply pressure
in
Fill
let
strument
b.
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:
The dye
a.
drifting to
mine
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.
Creams
Cultured buttermilk
274
CHEMICAL METHODS
1.
Model
Color analyzer Model
Protein analyzer
a.
ing accessories:
101.
b.
f.
g.
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
1.
n.
tacle.
p.
q.
Calibration test
o.
kit
liters)
dispensing bottle.
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.
10-ml.
w. Beaker
X.
y.
liter
aa.
Cleaner
for cuvette.
with instructions (used for preparing calibra-
tion curves)
SL
151
1.
may be
PO Box
148, Boulder,
CO
of dye per
it
is
80302
ready
milliliter.
mg
19.71
Protein:
Dye Binding
Acid Orange
275
(#SL-1212) and 50
liters
(1)
Fill
full
of distilled water.
Add
(3)
Warm
(4)
is no
Complete the
the solution
(5)
2.
hr. or until
Preparation of colorimeter:
a.
to
warm up
is left
on
at all
times. 24 hr a
in
color analyzer,
it
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.
When
e.
3.
the analyzer
b.
d.
Fill
the cuvette, in turn, with each of the calibration test kit solu-
tions.
obtained.
CHEMICAL METHODS
276
4.
(1)
(2)
Attach the automatic 40-ml pipet to the stand and dispensing vessel, using the tubing and clamps provided.
(3)
Fill
dispensing
tip
is
The automatic
pipet
automatically refilled as
it
each
fill
now
is
is
being
(4)
lytical
.05 g of reagent
dye.
b.
Syringe
eries.
5.
Procedure:
a.
at
26
into a 2-oz
Add 40 ml
b.
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
Slowly squeeze a
sufficient
amount of
drops
will
be
is
filter
paper
in the
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
Calculation:
vc Protein
52
mg of dye/ml)
7.0512
(42.24 X
cali-
19.8
277
Spectrophotometry
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:
a.
Method
is
ucts such as
Creams
Cultured buttermilk
On
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
ses have
in
'^
B. Milkoscan:^
a.
b.
Ca propionate
nate
c.
and
H2O
stock
solution
Dissolve
Dissolve
15
g of
(fat,
protein
Ca
propio-
liter.
50 g of lactose
H2O
in
water
liter.
Box
in
dilute to
(fat
is
Company
(Ltd),
PO
CHEMICAL METHODS
278
2.
Principle of method:
groups
in
is
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
is
high
in fat
before analysis,
may
or
may
phenomenon
is
maximum
resulting
in results.
Scattering
absorption to a slightly
from the
the fat absorption band. Strategic location of the transmission of the fat
midway between
sample
filter
at
temper-
99%
at a level
of
or better.
c. Proper adjustment of
ponent concentration.
com-
19.8
279
(J.
mogenizer.
e.
composition.
Maintaining a low water vapor content
f.
in
strument.
3.
Preparation of samples:
Incorporation of air
fat.
in the
a.
teurized
Warm
b.
to 40
alternate pairs,
place
in
in
the order of
c.
d.
W,,
C and
first
first
If M,, M-,.
and second results of alternate
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
checking linearity of
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
in
the in-
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
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.
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.
Either plot the instrument readings (vertical axis) against the standard
values, or do a least squares regression analysis, using the instrument
Reduce
281
References
19.9
its
prevailing reading
h-
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
fat
may be achieved
in the
same way
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
19.9 References
1.
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.
1975. Official
Methods of Analysis,
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.
for multiple residues of organochlorine pesticides in foods and feeds. Residue Rev. 34:5990.
8.
Carr, R.L.
dues
9.
in fatty foods. J.
method
Chem. 46:1043-1049.
1963.
J.
Assoc.
OflF.
CHEMICAL METHODS
282
10.
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
I,
MK
12.
13.
GiNN, R.E.
Pro-Milk
II.
1974.
related to
its
use.
J.
37:218-221.
14.
Heinemann,
1954.
B.J.,
total solids in
J. J.,
J.
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.
Sherbon, J.W.
dye binding.
J.
J.
Sherbon, J.W.
1975.
method
Sherbon. J.W.
1977.
Does protein
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.
29.
Shipe. W.F. 1956. The use of thermistors for freezing point determinations.
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.
at 102 F.
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-
Chapter 20
RADIONUCLIDES
IN
MILK
Introduction
20.1
is
result of fallout
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
found
served
limit
(ti/2
in
also
milk
site,
is
its
level
is
how-
may be
(ti,2 = 245
12.3 years)
in the milk.
may
days)
(ti/2
(^H) ob-
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
''*
'^'I
tested.
at
pCi per
liter
of milk level
is
^^
RADIONUCLIDES
284
IN
MILK
20.2 Sampling
Sampling of milk for analysis depends on whether samples are intended
by farm practices, b.) in the
To study
power generating
on radionuclides
in
consumer
level.
average sales
in the
community served.
least
one gallon
preserved with
HCHO
110 ml of
40%
HCHO
with analysis of
^'''I,
'*Ba,
and
'^^Cs
enzyme
is
then shipping the column to the laboratory for further processing.^" This
presupposes that
20.4
all
Determination of
'Sr
and
is in
-"*Sr In
Milk
By
Ion
Exchange
in fluid
A. Principle:
liters)
of milk
[10 ml of 40%
at 4
HCHO
C, for up to
285
and ^Sr
oxalate, which
is
^'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:
counting
total
is
determined by
radiostrontium for
*^
^'^Sr
determination,
^'^Sr
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
in Fig. 20: 1.
tory funnel; a
1.9-cm-diam separatory
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
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
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
"
8.
9.
10.
Analytical centrifuge.
11.
Analytical balance.
12.
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
I.
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
Water
the determination.
Ammonium
tate
(NH4CoH;jOo)
cial acetic
1
liter
2.
3.
acetate buffer,
in
its
pH
5.0:
pH
Ammonium
Ammonium
NH,OH
to
liter
with water.
4.
5.
trate,
water.
6.
size.
Cation-exchange
The commercially
resin:
available
RADIONUCLIDES
288
IN
MILK
0.950"
0.005"
0.040"
0.9325"
0.0025-
column and
rinsing
chloride-free
when
to
8.
Diethyl ether.
10.
Formaldehyde
1 1
pH
to
37%.
solution,
Add
liter.
Hydrochloric acid, 2N: Add 167 ml of cone HCl to water and dilute
liter.
21N: sp gr 1.48
13.
14.
15.
liter.
liter.
liter
Nitric acid,
16.
to
HNOu
to
Add
6.25 ml of cone
HNO.j
to
with water.
18.
warm
90% HNO,.
70% HNO,.
i.e.,
i.e..
17.
Sodium
20.
brown
in
liter.
19.
g of silver nitrate
in
bottle.
NaCl
in
liter.
21.
lute to
22.
to
is
NaOH.
9.
12.
7.
to
it
in
water and
di-
liter.
liter.
81
gof Na.,CrO.,
in
and
289
*<'Sr
23.
each of
six
Sr,
24. Tributyl
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
meter. Digest
in
pH
to 1.5 with
6N
fluid.
Process
is
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.
1 1
18,
RADIONUCLIDES
290
IN
MILK
0~
3.5
liter level
254mm
47mm
89mm
11
1mm
4.
Add
170 ml of
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
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:
and 5Sr
291
natant
fluid.
4.
in
If
known
filter
to
solution through
filter
filter
filter
in
20.4(F.11,19).
If
5.
HNO3
to the
TBP.
6.
Shake
draw
off
and
dis-
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.
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
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.
of the precipitate
Wash
14.
ter,
is
on the
filter.
warm wa-
three times with 959c ethyl alcohol, and three times with diethyl ether.
An
warm
room temperature,
Then
Disperse the precipitate uniformly over the dish bottom. Dry un-
der infrared
light to
in
an internal proportional
method chosen
for the
unknown.
liter
of
4N NaCl
that order
Wash
293
,_,^
HNO3
Sr Recovery
Ca Recovery
^^
14N
16N
18N
81
98
100
move from
1.4
11
1.7
51
4.0
2.6
0.9
2
clear
fluid.
6.
7.
of
6N
fluid.
5.
No. 541
filter
with 4 ml of
paper.
Add
21N HNO:j
20 ml of
Add
ml of water and
HNO3,
RECORD
stir,
90 ingrowth.
13.
Dissolve precipitate
8.5-9.0 with
in
6N NH4OH. Add
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
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
RECORD
H. Calculation:
I.
cpm
Srac.ivky.pCi/li.er
r^e^d,I,,V
cpms
Ls
+ bkgr
= Standard
^P^^tJkgr
+ bkgr
'^'^Y,
l-bkgr
R,
E,
D,
ly
= ingrowth
um
correction factor
e"'''
V =
sample volume,
in liters.
1
2.
^''Y
E.D.
cpmg
rTv
cr
Cs(AsEs
Eyly)
20.5
295
where Es = counter
Ds
efficiency for
correction factor
e"*^'
*^^Sr
as SrCOg, cpnVpCi,
a =
cpm^hkgr
ts
where
is
to the time of
counting
19)].
= Standard
'^P"^hkgr
+ bkgr
IbRgr
a self-ab-
SrCOa
in the
**^Sr
Precision
and accuracy:
""Sr determinations
showed
little
bias.
and
^^Sr in Milk
by
TCA
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
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
C. Apparatus
and equipment:
1.
with short cone bottom and spout. Also bottles, 250-ml capacity.
Pipets: 1-ml, volumetric,
2.
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
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
wide
in rolls.
Glass-fiber
9.
Analytical balance.
10.
filter
paper^*:
Low-background
/3-counter, thin
end-window ^-counter, or
inter-
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
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
in
in
water, add
7.
8.
1
to
liter.
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)
HNO3
to
liter.
12.
0.
g of methyl orange in
100 ml of water.
13.
Mixed
phthalein in 100 ml of
50%
^'^Available as Manufacturer's
**H.
0.1 g of phenol-
No. 50A, E.
I.
NJ
07105.
RADIONUCLIDES
298
MILK
14.
warm
IN
liter.
15.
100 ml of
50%
in
16.
(Na2C03)
17.
(NaOH)
in
in water, cool,
and
dilute to
liter.
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
oven
if
is
used.
TCA, 50%
(CCI3COOH)
in
[20.4(D.25)].
Cool
in
precipitate in
tared
stainless
20.5
TCA:
^'Sr
and ^Sr
299
E. Procedure to
1.
One
liter
a 3-liter beaker.
2.
Add
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,
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
in 5
Add 3 ml of water and warm to dis6N NH4OH, using 1-2 drops of mixed in-
solve. Adjust
dicator. If
pH
to 8.5-9.0 with
minimum
HNO3).
RADIONUCLIDES
300
5.
IN
MILK
retaining
for 20.5(F.7).
it
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.
fluid.
HCl and
heat in a
with water.
NH4OH
Add
6N
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
*Sr
301
indicator end point. Digest in a hot water bath for 5 min, cool in an ice bath
to
fluid into
RE-
Contin-
water.
decay.
5.
Add
is
dry under an infrared lamp, cool, weigh, and count beta ac-
of SrCOg.
7.
An
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
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.
sample
a function of counter
is
solids, T; i.e.,
geome-
is
is
con-
ute.
2.
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,
formulae
3.
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
ly
= ingrowth
correction factor
'"'Y
20.5
TCA:
^Ssr
and ^"Sr
303
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
= correction
from
sample collection
cpms
+ hkgr
Is
SrCOa, cpm/pCi,
where
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
t(5i4gr
120.5(6.6 or
V =
7)],
sample volume
in liters
correction factor
e~^' for
/.
Precision
time
7)].
and accuracy:
In a collaborative study'^ of
known
is
two
*^^Sr
and
^"Sr.
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
products
in dairy
dioiodine C^'I),
(^*'K) is
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-
gamma
Gamma
of a multichannel
spectrometer.
in selected
*^' ^"
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
in
the
method
gamma
present
in
dairy
radionuclides in
and radiocesium
137, with
B. Interference:
Any gamma
ray-emitting radionuclide, other than those under examinaor as a contaminant of the sample or sample vessel has
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
20.6
Gamma
These
Ray Spectroscopy:
fission
products
'^M, ^"^Ba,
may cause
and "^Cs
305
Compton-continuum counts.
may be minimized by
In
helpful.
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
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.
D. Reagents:
1.
salt in distilled
NJ
and various
07400.
plastic
RADIONUCLIDES
306
IN
MILK
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
&
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
Transfer 3.5
2.
liters
beaker, place beaker over detector, and count the standard for a time
tilled
Repeat
Repeat
3.
4.
5.
this
Calibrate
quently
6.
this
if
gamma
gamma-ray
of dis-
fre-
resolution changes.
liters
room temperature
liters
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
20.6
Gamma
Ray Spectroscopy:
^^ii,
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,
particular range,
=
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.
to
e.
f.
g.
h.
in
'^oBa
equations
mode
to the four
elements
in
the matrix.
RADIONUCLIDES
308
that a
computer be used
through
h.
ming counts
in
MILK
IN
done
readily
in
''"I.
the absence of a
net
cpm/EV
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:
4.
A =
where
A -
A,)= activity at
t
=
=
Ae-^'
some
interval of time
(the
radionuclide.
t,
original time,
between
decay constant)
A,,
and A, and
=-^
the half
where T,
life
must be
.,
in
the half
is
the
same
life
of the
units, ie,
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
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.
1,
USDHEW,
Baratta,
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.
Gamma-Ray
Spectrometry, Vol
2.
Pergamon
Press,
York.
FAO/WHO
Series
7.
ton,
8.
DC
ton,
9.
DC
Heath, R.L.
2.
11.
13.
for the
development of
radia-
gamma
Goldin.
1960. Determination of
'^'I,
'"Cs, and
gamma
&
Common
1968.
USDHEW, PHS
12.
Background material
1961.
20402.
10.
20402.
2,
laboratory
in-
gamma
of iodine-131
in
milk.
J.
Agr.
USDHEW,
69-MKAP-l,
Street, Winchester,
14.
15.
nuclide analysis.
16.
ashing
17.
J.
strontium 90 determination
18.
19.
J.E.
protein-bound iodine
in
MuRTHY, G.K.
formaldehyde.
20.
J.
Campbell.
1960.
A method
in milk. J.
Goldin.
1959.
A method
Campbell.
milk by formaldehyde.
J.
USPHS Pub
1960. Matrices.
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
andD.Y.C. Fung
Introduction
21.1
(PLC) procedures
are
applicable specifically for examination of raw milk. Although both are single
medium
in
each
loop counts are satisfactorily precise within the range of 30-300 colonies.
Limitations of the plate loop method,
been
reported.*^
The
spiral plate
is
pasteurized milk and milk products, and has been applied to other foods.
The SPLPC
is
a mechanical
manpower,
Count (SPC) pro-
less skilled
cedure.
21
.2
21
.3
and
in
in
5.5.
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
312
viable count
milk.
until 25
log,,,
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
21.4 Oval
for both
how
to evaluate results
Tube Method ^
-mm
neck (Fisher
Scientific
mm,
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,
its
to
tilt
when
21.3 Statistical
e
3
o
U
>
S
u
S
3
O
o
o
Comparison Table
313
314
5.
6.
tally,
work
area, stor-
B. Materials:
C. Procedure:
1.
it
to
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
wire rack (with agar adhering to upper side of the container) for 48
32
in
3 hr at
C.
milliliter"
(EOTC/ml).
If
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 ^
made
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.
ly
allow
it
to cool 15 sec or
316
outfit,
showing
vertical
removal of loop
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.
5.
32
12-15
hr at
C.
EPLC/ml.
21.6 Spiral Plate Count
Method ^^^
Spiral Plater
2.
Spiral
3.
Vacuum
act as a
vacuum
'
reservoir and a
4.
5.
^Available
land 20760.
to
5 ml.
x 15
mm.
318
6.
7.
mended.
Polyethylene bags, about 30 cm x 20 cm x 40 cm.
solution,
hypochlorite
sodium
Commercial
8.
9.
5% NaOCl
10.
11.
12.
approximately
(bleach).
An
sterile
delivery system
is
recommended
is
to
reproducible
a suggested
1.
The following
plates:
40-45 ml) of
2.
sterile agar, at
Allow agar
to solidify
on a
petri dish.
Place the solidified agar plates in polyethylene bags, close with ties
C. Preparation of samples:
Shake samples as described
in 5.6
inoculation.
and 21.4(C).
The
Spiral Plate
minimum
is
alignment
line
Method
319
ul.L!l|liP!|ll|p
Figure 21:2. Spiral plater dispensing crystal violet dye onto surface of
15-cm agar
plate.
number of
colonies
in
have shown the method proficient not only with milk analysis but also with
other foods.'E. Plating procedure:
Check
plane
is
parallel at
about
necessary. (Use
vacuum
tip; if
tip is
moved through
to
the cover
if
is
vacuum.
sec using sodium
the system by
sec before
320
million.
is
closed.
An
agar plate
is
motor
32
started.
C.
F. Sterility controls:
Check
sterility
Method
321
subtilis
77,000/ml.
Two
G. Cautions:
322
150-mm Plates
Method
323
deposited on that particular grid area. The following formula should be used:
,,
Volume
,
r,
Spiral colonies
counted
in
area
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
may be
One example
given
in
Table
is
21:11.
1.
in this
all
the segments
Any count
wedge, then
number of
all
If
the
in the
bacteria
ume contained
is
irregularities in
same segments
in
is
in all the
segments counted.
75
in
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
50 colonies,
e.g., if
the
first
324
From Table
21
33
volume suggested
77,000
is
0.0007 ml.
SPLPC/ml
0.0007
When
SPLPC
dent (LA).
4.
If
When
when
Compute SPLPC,
stances
in
5.11(1.3).
highest
unless restricted by detection of inhibitory subsample, excessive spreader growth or laboratory accidents, see
first
two left-hand
left is five
next
or greater and
21.7 References
1.
Campbell,
J.E.,
and J.E. Gilchrist. 1973. Spiral plate method for counting bacteria
in
Donnelly,
Campbell.
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
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
325
APPENDIX A
A1
.1
An
acidified
medium
to
making
Molds
medium
to Detect
in the presence of
There are several disadvantages to the
difficult to distinguish
particles,
and
-^
of
soil fungi. ^
fortified
with chlor-
1.
Count
2.
3.
328
B. Procedure:
1.
distilled water,
which
g of chlortetracycline in 99 ml of
in the refrigerator.
with a
then stored
tion to sterile
is
It is
pH
antibiotic solu-
of chlortet-
/xg
of this medium.
Plating and incubation: Prepare and plate samples as for the Stan-
seven days
^-^
for five or
1].
A1.2 References
1.
2.
3.
J. P.
Martin.
estimating
4.
5.
1950.
The use of
1968.
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.
A2.1
to be
in
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.
1.
Count
2.
Plating
medium:
Citrate Azide
Agar of Reinbold
et al.^-
B. Procedure:
A3.1
329
and significance:
of butter
is
An enterococcus
attainable and
is
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.,
Jr. 1965.
in butter. J.
The
relationship between
249.
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.
1.
Sampling cylinder:
A 2.8-mm
cylinder
is
fashioned
at the distal
mm
slit
in
end
the
15
minutes.
3.
Dilution blanks.
4.
Petri dishes.
5.
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
sterile
A3. 2 References
1.
products.
2.
J.E.,
Jr.
J.
Thompson,
J.
L. A.
Black.
1960.
A4.1
This method
cillus
'
is
megateriitm
plate loop
method
for deter-
in
Milk
ATCC
is
commonly used
in mastitis
therapy.
ATCC
1.
2.
3.
AK
4.
Sporulation
9855.
Medium #2.
6.
9.
in.
from the
Centrifuge: Refrigerated.
10.
Refrigerator.
11.
Water
bath:
surface of sterile
AK
sporulation
ATCC
medium #2
in a
3.
After incubation,
MS
at
an
RCF
at
35 C.
from surface of
of 5,000 for 15 min
cells
Assay
331
A5.1
at 3
C. Testing procedure:
1.
it
liquid in a
water bath
concentration of about 5 x
Add
3.
10^
spores per
milliliter
at
final
of agar.
4.
formation.
Pipet 4 ml of inoculated
5.
medium
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
(
overcome
least 5
/Ltg
aqueous solution
to
at
PABA
in
40-44 C.
A4.2 Reference
Read,
1.
Jr. R.B.,
Bradshaw,
This procedure
the
0.0025 unit/ml
skimmed
is
qualitative
in
1971. De-
A5.1
for
J.G.,
and antibiotics
Technique-
skimmed
milk,
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.
Agar medium:
15 g of agar,
The
final
final
pH
pH
0.
Sterilize at 120
g of glucose,
for 15 minutes.
of 8.0 0.1.
Distilled water:
5.
Petri dishes:
Good
water
stoppered
penicillin
Use solution
C when
in a refrigerator at 5
Follow the
sterile bottle.
not
in use.
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.
1 C.
var. calidolactis
organism:
stearothermophilus
Test
Use Bacillus
maintain
the culstrain C953 (Netherlands Institute for Dairy Research). To
11.
12.
ture, streak
in
Water
at 5
C.
C.
150-ml Erlenmeyer
flask.
A5.1
2.
Assay
333
medium
in the flask
NOTE. When
is
more than
at 55
C and
use
in
the liquid culture should have a viable colony count of 50-100 x 10^/ml
when incubated
at 55
for 48 hr
if it
it
should be discarded
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
thickness of 0.8
they
.0
mm thick of the
mm.
5.
6.
Preferably use assay plates on the day they are prepared; however,
may be
to solidify
on a
level surface.
proximately 10 ml to a suitable
2.
Add about
0.4
sterile
334
F. Test procedure:
Mark
1.
off
mm
wax
2.
than 5
in
the meantime. If
it
is
at
not
more
-15 C)
to
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.
Repeat steps
and 4
in
Repeat steps
and 4
in duplicate,
in-
When
random
10.
all
nine discs have been placed on the assay plate, [A5. 1(C)] in
it
at
55
in front
of a suitable light
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
If
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
If
is
not present.
are smaller in average diameter than clear zones around discs containing the
335
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
is
by
penicillin,
milk.
A5.2 References
1.
2.
A6.1
Milk^^
ation Procedure
lactis as the test
in
is
is
sensitive to as
little
var. calido-
as 0.002 unit of
penicillin/ml of milk.
1.
2.
extract, 5 g of
The
final
pH
g of meat
distilled water.
5.
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
Store at 0-4.4 C.
9.
10.
not use
11.
12.
Incubator: 55
13.
C.
5).
controlled.
Do
336
in
Incubate for 18 hr
55
C and
refrigerate at 0-4.4
Prepare and
justed to a final
sterilize, in
pH
of 8.0
0.
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
tions of 0.004
is
for not
ml with buffer.
Make
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.
A6.1
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.
the
marked
6.
side
flat
section.
8.
in front
F. Penicillinase control:
1.
Add
in
A6.1(E).
2.
Mix
3.
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
Touch
the
flat
side
down on
6.
trol
trol
7.
8.
in duplicate,
in
con-
When
all
in
random fashion,
338
H. Interpretation of results:
1. Clear zones around discs containing the
/.
1.
2.
To
[A6.1(D.2)] as a guide.
3.
If
A6.2 References
1.
2.
A7.1
A
in
DelvotestP
in
some
laboratories.^'^
cose, 2.0
mg of non-fat
mg
mg
of tryptone, 5.0
mg
of glu-
room temperature.
3.
4.
tips:
Delvotest P
A7.1
5.
339
at
0-4.4 C.
Water
B. Controls:
As
-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
When
line
medium.
sodium or potassium
penicillin
[A7.1(A.9)] in sufficient
known concentration
1% phosphate
This solution
may be
this solution,
tubes [A7.1(A.12)].
be stored
When
at
-10 C
Cap and
freeze at
-IOC.
ml into
15
x l(X)-mm
at
may
room temperature.
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.
legibly
1)]
indelibly.
each
ampule.
5.
0.1
tip for
340
ferred.
to
dispensing.
two and a
in
for
half hours.
7.
8.
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).
Mix
3.
well.
Incubate as
in
[A7.1(C. 6)].
E. Interpretation of results:
1.
medium
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
NOTE If
the
penicillin, a false-
medium
of the am-
interpretation.
F. Reporting:
2.
A7.2 References
1.
penicillin in milk.
J.
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
Food
A8.1
in milk. J.
Doodewaard,
BR Foss
Test*
3-
method
to detect
low
penicillin
Prot. 40:23-24.
T.,
in milk.
4 5
3.
4.
vial.
Store at
1.
room temperature.
2.
Hot
Medium No.
plate:
stirrer.
5.
Glass beaker:
6.
Thermometer: 0-100 C.
7.
8.
9.
liter.
10.
11.
Bacterial culture:
agita-
tion.
lidolactis
C953 containing
10
mg
room temper-
ature.
12.
Test plates: With small cells each having a volume of 0.2 ml.
13.
Aluminum
14.
Microliter syringe:
foil:
Manufacturing) or equivalent.
15. Penicillin: Crystalline sodium or potassium
Do
penicillin
G. Store at -10 C.
Store at
Penicillinase
0^.4 C.
1% phosphate
cone:
Available
pH
from
biological
0.1: Dissolve
supply
houses.
8.0 g of
monobasic
in distilled
17.
buffer,
6.0
*System Enterotox
^NR. 5531 Baierdorf AG., Hamburg or equivalent.
342
1.
in
2.
at 121
Cool the
3.
sterile
medium
foil
in flask.
to 55
stir
in
the agar.
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.
8.
plates in
downwards
at
aluminum
foil.
As
-negative control samples must be included with each series of samples tested
buff'er
[A8.1(A.17)] to give a
This solution
may be
this solution,
sterilized antibiotic-free
15
X 100
control
mm
tubes [A8.1(A.19)].
may be
temperature.
stored at
When
-10 C
-10 C. This
positive
at
room
medium.
D. Screening
test
procedure:
1.
Remove
the
aluminum
2.
3.
Peel
off"
freeze at
later use.
Cap and
foil
wrapping of the
test plate.
A8.1
BR Foss
4.
343
Test
1(
A.
14)]
add
0.
After
filling,
is
hr in a refrigerator
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.
10.
Incubate for 2 hr
in
removed
in
A8.1(D.3).
a waterbath at 60
agita-
tion.
1
1.
12.
medium
no change
in
of each well.
milk sample wells indicates the presence of substances inhibitory to the test
organism.
13.
of the solid
as in A8.1(E).
E. Confirmatory test procedure:
1.
Add
Mix
3.
treated,
4.
well.
F. Interpretation of results:
1.
portion of the
same sample
is
medium
medium containing
NOTE:
If
the
penicillin, a false-neg-
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 an inhibitory
G. Reporting:
1. Report the presence of penicillin when demonstrated.
344
If tests
2.
3.
A8.2 References
DiCKES. G.J. 1973. The disappearance of residual penicillin
1.
in
milk.
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.
MuLLER,
in
4.
F.J.
1973:
6 pp.
Shraudy.
5.
E..
in
A9.1
The
test.
method
is
where the
The method is
wet-waxed paplastic or metal
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.^
1.
is
mm
pulp.
2.
3.
Plating
4.
5.
6.
Forceps,
7.
Balance.
8.
sterile.
in transit
to laboratory.
9.
Disintegrator:
Sterile
interior,
high-speed,
electrically
operated,
in
a 1,000-ml cup.
A9.1
345
two methods
is
accept-
able.
a.
roll,
with a
minimum
radial
in.
(2.54
shall
346
C.
Sample preparation:
With
sterile scalpel or
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-
Resume
that
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.
Keep
sample.
E. Results, reporting
sum
and standards:
Multiply the
A11.1
Raw
347
Milk
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
The procedure
to be
used
is
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.
A10.1
Agar
A. Procedure:
After making a Standard Plate Count, store containers of freshly proc-
Large increases
in
a refrigerator at 7
in bacterial
A10.2 Reference
1.
L.J.,
Al 1 .1 Preliminary Incubation
(PI) for
Raw
Milk
The Standard
Plate
'^-
348
dling the milk.
is
incubated 18 hr
at 12.8
before deter-
B. Procedure:
The sample
to
a given producer.
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
them to4 C
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
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.
2.
J.
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.
A13.1
349
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.
A12.1
12, 1973.
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)
cubate containers
at
32
(89.6 F);
space
if
is
not, incubate
them
at
room temper-
ature. After 48 hr, note the percentage of containers with turbid broth
and
in air
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
may
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,
350
The
viability
in air
may be
influenced by
temperature, humidity and light rays. Air currents, the number of micro-
B.
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
bottom area
is
Connect impinger
to
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
in air. Pre-
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
Bacteriol 76:471^84.
crobiol. 10:181-184.
3.
and evalua-
6.
&
Sons, Inc.,
Ehrlich.
brane
R. 1955.
filters. J.
New
1969.
An
York.
Technique
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.
7.
Instr. 22:187.
plants.
10.
J.
1934.
Number
J. Sci. Inst.
1950.
The conifuge
size separating
sampling device
27:272.
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"*
10%
*^
sterilization.
Prepare a
tion.
III.
60425.
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..
1958.
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
4.
5.
6.
Products
been an appreciable increase
in the
number and
types of milk products offered for sale that are labeled "sterilized" or "longlife" or the equivalent.
uct
is
where they
may
be viable bacteria
product. Refrigeration
ilized;
however,
it
is
may
2.
may
where
of the
marked
ster-
or
life
or sterilized products:
in the original
two
petri dishes
and
at
32 C.
^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
A17.1
result of inadvertent
believed useful
in
3.
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.
A16.2 Reference
Donnelly, C.B., Leslie,
1.
Read,
Jr.
products.
J.E.,
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.
354
cycle, for
using standard dilution procedures and a plaque forming technique. Alternatively the effect of diluted
phage
filtrate
in sterile re-
rich
medium
phage
particle
numbers.
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
is
A17.2 References
Frazier,
W.C, Marth,
E.H., and R.H. Deibel. 1968. Laboratory manual for food micro-
New
.1.
Zealand
J.
1976.
The
1975.
Improved medium
and
APPENDIX B
J.A. Burke, D.
Acidity: Titratable
B1.1
B1
Lehman, W. Roth,
.11
Whey Concentrate
Products
titration.
Phenolphthalein. 1.0%.
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.
bottle
356
B. Procedure:
pend sample
umetric
Make up
flask.
stir
in solution.
to
well.
pipet 25 ml of
droxide to the
first
permanent (30
sec) color
to pink.
C. Calculations:
Acidity
is
expressed as
lactic acid (1
ml of
0.
N NaOH =
0.009 g of lactic
acid).
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,
whey concentrate =
-,
= ml
D. Note: The tabulation below
is
of O.IN
NaOH
x 0.30
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
2 Dry
Same
as
F/t
in.
B 1.1 1(A)
Electric
13.
mixer,*
diam x V4
Milk
in.
(replace
(4.34 x 0.63
of:
steel
cm)
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
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
Same
r^;
18 (weight of sample)
Ices,
and Yogurts
as Bl. 11(A).
B. Preparation of sample:
frozen, heat to 37
encountered.
C. Procedure:
43401.
358
Add
droxide to the
first
permanent
D. Calculations:
Acidity expressed as
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)
NaOH
20
ml of O.IN
NaOH
10
ml of
18
4.
milk:
When
When
18-g sample
9-g sample
is
is
used, rinse
distilled water.
^Ice
Cream and
ice
cream mixes
Same as
in
rated milk.
5.
6.
^Ycgu?ts
in
in
B1.2 References
1.
2.
Doan,
analysis.
fluid
milk products.
J.
Dairy
Sci. 40:1643-1644.
pH
Cheese
2.
pH
meter.
Beaker, 50 ml.
raspberry, cherry)
it
The
room temperature and proceed with
white yarn and boil for 10 min or until yarn has absorbed the color.
titration.
pH
359
3.
4.
Wash
5.
6.
in
1-C divisions.
bottle.
ACS
7.
Benzene,
8.
Buffer solutions,
grade.
pH
4.00 and
pH
trates).
indicator.
1
in
fill
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
360
to replace saturated
face of the glass electrode and insures against leakage. Storage in distilled
in
may cause
inefficiency
C. Standardization of
1.
pH meter:
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
the
4.0 and
meter manual.
D. Procedure:
1.
is
sample so that
covered.
end of day.
2.
operation.
When
used for at
procedure.
least
'/-^
hr,
it
is
Same
13.
Beakers, 30, 40 ml
14.
15.
Refrigerator
Same
as B2. 11(B)
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
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.
2.
mately
2 hr,
which
will
permit any
fat
remaining
in the
serum
to rise to the
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
2.
3.
Burette,
Mohr
5.
Wash
6.
7.
Watch
8.
Desiccator,
bottle.
glasses, 90
filled
mm.
with efficient desiccant such as Drierite (calcium
sulfate), 4
9.
1.
Bunsen burner.
12.
13.
14.
Analytical balance.
15.
16.
Steam bath.
Hot plate, thermostatically controlled.
17.
362
Hydrochloric acid,
Sodium hydroxide,
18.
19.
0.
IN.
0.
IN.
1.0%
B. Procedure:
Weigh accurately
part of
it
becomes
Place dish
in
white.)
for
hour.
(It is
very
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
may be
gently
Add
0.
stir,
10 drops
363
B4.1 Chlorides
C. Calculations:
All results are expressed as
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:
If the
is
is
used:
ml O.IN HCl/lOOg =
1000
Weight of sample
or
50
100
Weight of Sample
B3.2 Reference
1.
1975. Official
B4.1
B4.11
methods of analysis,
DC.
Chlorides
Milk
and Cream
ml divisions.
cream, volumetric, 9 ml.
2.
Pipet, standard
3.
Glass
4.
5. Pipet,
stirring rod.
USA
#1.
in 0.1 ml.
6.
Distilled water.
7.
8.
B. Procedure:
Pipet 9 ml of milk or
pipet with an equal
Add
If
sample
is
cream, rinse
dish.
ni-
364
C. Calculations:
^
%
^,
Chlorides
ml of
silver nitrate
Weight of sample
35.46 X 100
rzr^
, ^^
Factor 3.55 =
-,c
.^
where 35.46
is
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.
2.
3.
4.
5.
Electric mixer.
6.
Wash
7.
8.
9.
Filter paper,
bottle.
Whatman #2
10.
Erlenmeyer
11.
12.
13.
14.
Distilled water.
15.
16.
B. Procedure:
C. Calculations:
^
%
,.
,,
..
Sodium chloride =
ml of
silver nitrate
365
D. Notes:
c
T^
oc
58.45 X 100
1.
Factor 5.85
2.
dilution
CO .c
where 58.45
^^^
is
1.
2.
Beaker, 50 ml.
3.
4.
Bottle, wash.
5.
6.
7.
Filter paper,
8.
9.
Whatman #2
mm.
10.
Funnel, 75
11.
Graduate, 25 ml.
12.
Hood
13.
Hot
14.
15.
Pipet,
16.
plate.
T joint,* 5-ml
17.
Thermometer, 0-100 C.
18.
Tongs.
19.
20. Ferric
ammonium
ACS,
sulfate,
capacity.
divisions.
FeNH4(S02)212H20, ACS.
sp gr 1.42.
21.
Nitric acid,
22.
23.
24.
Silver nitrate,
25.
Sucrose.
ACS.
B. Solutions:
1.
2.
1-liter
3.
4.
Ferric
ammonium
NJ
distilled
07003.
water
in
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-
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.
lenmeyer
flask.
Wash
through folded
filter
D. Calculations:
Sodium chloride =
58.5
(ml X
silver nitrate)
(ml x
grams of sample
Chloride ion
=
35
(ml x
silver nitrate)
(ml x
grams of sample
E. Notes:
1.
added
nitrate
enough
to
sample
will
for
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.
1975. Official
Extraneous Matter
B5.1
Cheeses
A. Apparatus and reagents:
B5.11
1.
ical
Natural
2.
3.
Beaker, 2
liter
liter
mixing.
4.
Beaker, 2
#7904.
5.
6.
Bottle, glass-stoppered.
7.
Bottle, wash.
Cellophane envelopes,"
8.
Ys
in.
3 in. (4
9.
10.
13.
steel.
14.
Lintine disc,*
cm) diam.
15.
Microscope,
16.
Pipet,
17.
18.
Stirring
19.
Hot
Mohr,
!4 in. (3.1
minimum x
12 magnification.
0.5 ml.
multi-stirrer.
Suction
filtering
B. Preparation of sample:
soft natural
cheese into
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:
18th St.
'USDA
111.
60604.
No
CFR
58.2728.
368
1000 ml of
agitate for
10% sodium
hr at 55
1
C.
The
ideal
temperature
using
medium
(Should
filter
rest of
sample
in the
waffle
side up.
Remove
on the card the kinds of extraneous matter present, using the appropriate
standard.^
container.
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
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
temperatures;
c.) controlling
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
With refinements incorporated in the following Babcock methods, improved accuracy resulted as was shown by collaborative studies conducted
among
different laboratories.*
A. Homogenized milk
':
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.
to
60
(pref-
erably 57 C).
h.
i.
ature of 5=55.
j.
k.
370
1.
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
Hold the
item
Babcock,
B6.1i,
3.
in the
min or
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
fat
which
Same
a.
Milk
test bottles,
capacity 18
NBS
8%, 6V2-in.
(16.5
to
g.
**
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
brownish
2.
Procedure:
Proceed as
in
3, 2,
Ohio 45202.
it
becomes yellow
nati,
when
in color.
and 2
ml).
Fat: Modified
B6.1
Cream
C.
1.
371
Babcock
and spreads:^
a.
b.
Cream
NBS
(0-50%
in
0.5%
divisions),
f.
g.
h.
i.
Column meter,
test bottle.
ature at 55-60 C.
o.
p.
Weight, 9
q.
Sponge.
r.
g.
ACS
perature).
(red or blue reader), sp gr not to exceed 0.85 at 20 C.
s.
Glymol
t.
temperature of 60
or
above.
2.
Procedure:
addition.
Carefully transfer contents of the beaker to a fat test bottle and rotate
the bottle
B6.1, item
3.
distilled
372
rinse to the test bottle. Place bottle in mechanical shaker for about 5
until all traces
min or
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
Preparation of sample:
2.
Procedure:
3.
On
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
B6.11(C.2).
Because of the
(<
0.5 -
difficulty
Same
a.
tle
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
than 0.5%
fat
divisions.
b.
Roccal,
c.
Roccal solution
sulfuric acid.
tion will
II
Mix
to
keep about
will ap-
pear, indicating that the Roccal has dissipated and will not be eflfective
B6.1
Fat: Modified
in testing.
Babcock
373
when
it
becomes yellow
brownish
to
in
color.
Procedure:
2.
pipet.
F. Chocolate milk
This procedure
is
s.
Ammonium
t.
Normal
u.
which provide a
Same
and additions:
a. Milk
late.
1.
modification):^'
NBS
hydroxide
(NH4OH-28
to
29% NH3).
mix
3.5 parts of
H2SO4
2.
Preparation of sample:
butyl alcohol and mix sample and alcohol thoroughly by swirling the bottle.
Then add
"Hold
cream procedure
."
Note:
4.
Temper
test bottles in
less than 3
min
as specified in 19.41(B.2).
Same
374
a.
in.
Ice
cream
test bottle
20% NBS
9-g graduated in
V5%
divisions, 6V2
(16.5 cm),
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
and add 2 ml of
NH4OH
rotating
."
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
375
/.
1.
Same
2.
Mix
as B6. 11(A).
Procedure:
the sample thoroughly by shaking and swirling the bottle. Pipet
Do not
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
and add steadily in small portions (apand 4 ml), 17.6 ml of the prepared Roccal-sulfuric acid
3.
uncertain.
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.
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
'
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
B. Procedure:
fat
B6.2 References
1.
2.
its
products. Labora-
Newlander, J.A. and H.V. Atherton, 1977. The Chemistry and Testing of Dairy Products.
AVI
B7.1
Fat,
in
Kohman Method)
A. Apparatus and reagents:
1.
Analytical balance.
2.
4.
3.
5.
Crucible tongs.
6.
Hot
7.
Volumetric
8.
9.
10.
Erlenmeyer
250 ml.
11.
12.
377
13.
Distilled water.
14.
equivalent.
16.
17.
15.
(7 to 13
C)
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)
1.
the
,^^
x 100
aluminum beaker.
2. Use a glass rod
to
before removing
few
in
milliliters
com-
of petroleum ether
3.
Tilt the
378
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
thoroughly.
3.
The
salt will
flask.
4.
5.
Add
NaCI =
with standard
titrate
Do
not titrate to
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
B7.2 Reference
1.
1937. Laboratory
methods of
379
Hydrolytic Rancidity
B8.1
Hydrolytic Rancidity
B8.1
An
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
cream
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.
6.
Centrifuge:
that will
accommodate a
An unheated
cen-
trifuge is satisfactory.
Aqueous methyl
7.
and
alcohol: Equal
distilled water.
8.
Water
9.
10.
Erlenmeyer
flask:
(preferably 57 C).
cm) No.
19 needle.
50 ml.
absolute methanol.
KOH:
KOH
Alcoholic
er suitable standard.
14.
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
cream
in either
after 10
at least
in boiling
water
380
After the test bottle has been in boiling water for 15-20 min, remove bottle
and centrifuge
it
for
minute.
Add
sufficient
to bring
the fat column well within the graduated portion of the test bottle neck.
minute.
above
C. Titration procedure:
Transfer
Then,
g of milkfat
= ml of
milkfat x 0.90
ADV
(ml
KOH
for sample
- ml
of
KOH
Weight of
N
Titration blank
=normality of alcoholic
= titration
KOH
for blank)
x 100
fat
solution
in
ADV
0.75 X 0.90
^36
0.675
2.015
0.675
(ADV
example
just given.)
in
reporting
B9.1
Lactose
in
Cheese
381
E. Interpretation:
Express results
100 g of
fat).
in
= 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
7.
Watch
8.
9.
Wash
10.
mm.
glasses, 95
bottle.
Desiccator,
dicating, 4
4.25
mm.
6.
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
14.
CM
apparatus.
15.
Suction
16.
17.
Iron tripod.
filtering
18.
Bunsen or
19.
Terrill
burner or equivalent.
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,
382
29.
30.
Copper
28.
sulfate,
ACS
grade.
32.
33.
Nitric acid,
34.
Distilled water.
ACS
grade, sp gr 1.42.
B. Solutions:
Copper
1.
distilled
water
Make up
to
copper sulfate in
volume and mix
for
store
3.
tilled
in
Do
not
one part of
nitric acid
water.
Prepare a
asbestos.
1:4
Pour hot solution out offilter flask. Follow with 10-ml portions of
for 30 minutes.
Cool
in
To
reuse the asbestos mat, dissolve the cuprous oxide precipitate in the
Wash
thoroughly with
lytical
Gooch
Dry the
on an ana-
distilled water.
this
in
place of the
type of crucible
since the hot alkaline tartrate solution attacks the sintered glass disc.
is
The
col-
is
Lactose
B9.1
in
After each
there
is
Cheese
383
filtration, the
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:
from a
bottle.
Rinse beaker thoroughly and add rinsings to flask. Dilute soluml with distilled water and mix thoroughly.
Add
of alcohol. The
filtrate
should be blue;
sample so
if
it
that the 25
is
in
desiccator for
F. Calculations:
1.
Use
the
Munson Walker
table
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
384
0.2788 g X 1000
b.
The fourth
figure
is
278.8 milligrams
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
^-^^
190.06 which
2.
is
lactose
No. 2788.
is
in solution.
X 100
X 100
Explanation of factors:
3.
a.
(in
x 50 x 1000
= grams of sample
z^-r
milligrams)
b.
Because
385
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.
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
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
This method
1.
2.
Analytical balance.
3.
Pyrex
4.
Spatula.
5.
(digital
cm)
in
diameter.
B. Preparation of sample:
Natural cheese: to obtain a
1.
it
is
neces-
shall
be avoided as
fat separa-
tion occurs.)
"Apollo
Official
Illinois
60014.
386
2.
Process cheese:
blender as product
is
It is
may
sample on
paper
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
D. Calculation:
At the end of the
moisture.
After the
final
reading
is
is
oven.
E. Note:
It is
um oven method
to
make
is
being
maintained.
2.
3.
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
until
0% mark
is at
W 26th
St,
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
C. Calculation:
Read
directly
Solids
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.
being examined
is
Do
to
If
85%
or
the mate-
Distilled
rity.)
5.
Plastic
6.
B. Procedure:
Place about
to 2 drops of
warm sample
to
light.
field-dividing line
and
scale.
Make
the neces-
tometer.
C. Calculation:
Solids
[B10.13(D.6)
= Reading on
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.
388
3.
in-
This method
6.
(40 to 60%).
is
whey concentrate
with a
B1 0.2 References
2.
Bianco. L.J.
MiCKLE, J.B.
3.
1.
Methods of determining
Sta.
ucts.
AVI
PiEPER, H.,
4.
Stuart
Jr., S.A.,
Membrane Filter-DNA
1 .1 1
The
3.
io
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
4.
5.
Mastitis
3.
catalog
test
flasks.
membrane
#EMWP 02500).
MA
01730.
filter:
25-mm
diam
(Millipore
Somatic
B11.1
Cell
Count
389
6.
7.
Spectrophotometer: Digichrome/^^
Automatic diluter dispenser: capable of handling 22.5
Disposable test tubes: 13 x 100 mm.
8.
9.
10.
11.
steel.
ml.^^^
12.
Wash
13.
Two
bottle,
liter.
65
at
C, the other
at
90
with gable
cover.
14.
Heat-tempered storage
15.
graduated cylinder.
16.
17.
Amber
18.
Concentrated HCl.
-liter
bottles.
storage bottles.
19.
20.
Trisodium
ethylenediaminetetraacetic
acid
(EDTA)
(Millipore
Corp.).
21. Distilled or deionized water.
22. Triton
B. Solutions:
1.
Stock HCl
(1 liter):
Pour 584 ml of
HCl
Diluting agent
60
distilled
(1 liter):
make
Mix
liter
it
of solution.
liter
4.
Stock indole
(1 liter):
to
make
of solution.
made
in the
amber
pore Corporation.
WI
53562
following
from the
Milli-
390
C. Procedure:
The
MF-DNA
test
milk sample.
Periodically (at least once a week) include one high and one low Somatic
Cell
already trapped.
#MBOOO
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,
proper temperature.
2.
Mixing milk with diluting agent: Swirl the milk sample to ensure unitip of the dispensing probe from the dilutor into the
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
plate
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.
392
of milk.
for example, the reading
If,
is
1.04, record
it
milk.
E. Notes:
1
cell
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
393
Pipets: Disposable, 4
2.
HCHO
4.
of absolute methanol.
5. Diluent/wash solution:
liter
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
liter with
of
absolute
methanol, and dilute to
in
900
ml
NH2OH HCl
6.
methanol.
8.
to
EDTA,
liter
and
tetrasodium
filter
through
salt:
in
H2O,
dilute
Whatman No.
Add
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
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:
Pump sample
with air
pumped
at 0.32
at 0.32
ml/min.
Pump
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.
NY
10591.
394
C. Start-up procedure:
Place
all
pump
Check
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.
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,
at
20
or
hr at
55 C.
G. Procedure:
Place
H2O
in first
cells in
response), in
fifth
H2O cup
H2O
every
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
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 =
peak height
is
is
>2,000,000/ml.
If
following by sample with low cell count at a high sampling rate, reanalyze
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
rate of analysis), as
at
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.
Do
it
and
it
shaking
is
bottles, ini-
at
ft is
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
396
Wash any
spills
may be
cam must
be used.
is
scope.
2.
Standard solution:
a.
Dye
solution,
idium bromide
is
diluted in
liter
1.00 g of eth-
Triton X-100
distilled water.
stored in
Heating to 60
Working
a.
Dye
solution:
solution
liter
of
buff'er.
pure
b.
Diluting solution
c.
ammonia
buffer.
1%
25%
C. Analytical system:
The Fossomatic
is
started
by pressing the
"POWER ON"
5Manufactured by Foss Electric, HillerOd, Denmark, and available from Foss America,
Inc.,
P.O.
Box
504, Fishkill,
New York
12524.
"LINE FAILURE"
397
"LAMP IGNITION"
the bottom
is lit
is
DNA,
is
is
controlled by
means of a sensor
in the heating
coil circuit.
The rotary
table
now
is
stirred vigorously
to
keep somatic
formed
in the milk.
through a tube
in the sealing
tube, through a
charge
cells
may have
filter
cap.
is
The mixture
is
is
introduced
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
discharge jet to the periphery of the disc. This takes place at a constant
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,
398
When
by the blue
signal
is
light
and
emit red
will
light.
A discriminator is set
fied
at a
Check
Check
is
at
80
psi.
use. If a container
is
it
one day's
vent overflow.
2.
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.
all
last
"OFF"
vacuum
if
needed.
clusters in
cells are to
ing:
1.
Error
in
The somatic
2.
3.
distributed.
b.
Normally,
this ability
cells
do not
become hard
particles.
in
hardening of the
is unable to
The dye
DNA
molecule
B12.1
399
Ash
is
added
Freezing
them
ice
to rupture.
B11.2 References
1.
12,
for the
Examination of
New York
2.
3.
5.
4.
J.
Kelly, W.N.
matic
cell
1977.
Comparison of
count procedures
in
AOAC
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.
J.
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
#1 or platinum
dish.
filter-
400
2.
Analytical balance.
4.
5.
6.
Muffle tongs.
7.
Marking
8.
Iron tripod.
9.
Aqua
ink,
10.
regia solution:
Steam bath
air.
HNO3
Mix three
and
(optional).
B. Preparation of crucibles:
Heat side of crucible to be
aqua
regia, dried
and ignited
in this
manner
after
be cleaned
in
each use.
C. Procedure:
volatile matter
is
smoked
off.
until
it
is
free
^
%
,
=
Ash
,
Weight
of residue x 100
^
Weight of sample
E. Note:
This method
is
applicable for
all
to
be excluded.
B12.2 References
401
B. Preparation of crucibles:
Same as B12. 11(B).
C. Procedure:
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
Same
as B12. 11(B).
C. Procedure:
Weigh accurately
2 g of
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.
1975. Official
methods of
Bianco,
analysis,
Index
Allowable
8),
15-140
355-357
Acidophilus milk, microbiological
test-
ing, 163
Adenovirus infections, 23
ADV
Aflatoxins, 22-23
Agar
layer, quantity of
medium
required
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
(Chapter
9),
141-150
Agitators, 38-39
Air,
16),
349^351
327- 354
Alcaligenes, 110
403
210
404
INDEX
Chem-
Ash, alkalinity
of,
361-363
products, 399-401
Aspergillus flavus, 22
parasiticus, 22-23
in dairy
329
Autoclave (steam
Biological
sterilization),
58-60
de-
termination, 249-251
12
235-236
Stain
Commission
certifica-
tion
BR
Foss
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
B. megaterium, 330
B. stearothermophilus 332-344
B. subtilis, 142-145
Bacitracin, in dairy products, 149
,
od FOR (Chapter
14),
169-186
Bacterial
Anthrax,
13
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
fat,
mois-
phosphatase
226
405
INDEX
Buttermilk (Cont'd)
phosphatase
titratable acidity,
224
355-357
Chromatography determination,
ticides,
of microscope, 180-181
of turbidometer for milkfat, 250-251
California mastitis test (CMT), 118-120
grading and interpretation (Table 8:1),
121
CIP
for pes-
264-268
(cleaned-in-place) equipment, 50
328-329
35
Clostridium, 110
Clostridium botulinum, 14
C. butyricum, 15
C. perfringens, 15
C. sporogenes, 15
CMT
6),
95-
105
INDEX
406
70
Corynebacterium diphtheriae
Coryneform group, 107
16
55
flora,
161
sampling, 45-46
149
Dilution water, for concentrated and dry
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
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-
14),
169-186
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
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
DMLC
phosphatase
Milk
Products,
Micro-
151-156
(Chapter
titratable acidity,
16),
197-205
170
personal
in
Erythromycin
SPC, 93
in
(electronic
134-140
Escherichia 95
Escherichia coli, 95
enteropathogenic, 12, 16
ESPC (estimated Standard Plate Count),
,
91
deter-
mination, 269-277
ESCC
38^387
Dry
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
INDEX
408
Grade
H
Hepatitis infectious, via milk, 24
High temperature short-time (HTST),
108-109
l-J
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
Ice
od, 149
Flavobacterium ,110
Flavoring extracts,
in
ucts, 165
199
Florisil
chromatography, 267
DMSCC), 129-130
Fluorescent dye, automated Foss for somatic cell count, 396-399
Food additives, in frozen dairy products,
Ice
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
252-254
Gerber
detection, 141-150
In-plant control methods,
INDEX
409
control, 216
somatic
cell
Leptospirosis, 17
man-Lampert
counting procedure,
stain for
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
LTLT
spectrometry,
phosphatase, 213
Keeping-quality test, Moseley, 347
test,
Klebsiella, 95
M
Malted milk, ash
in,
401
for
Kohman
modification for
ture,
and
salt in,
fat,
mois-
376-378
in
Membrane
108
filter
203, 388-392
Methods,
specific,
7-8
Lactobacillus. 107
Lactometer readings,
monocytogenes,
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
New-
DMC,
191
values for
SV.S values
MF
MF
WF
Microbacterium, 107
Microbial flora of cheese, 161
DMCC
INDEX
410
Microbial phosphatase, 216
Microbiological control methods, supplemental (Appendix A), 327-354
11),
157-159
DMSCC,
129, 134
(MWT),
for
abnormal milk,
15-1 18
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,
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
Nutrient agar, 55
INDEX
411
Nutrient broth, 55
Nuts, in frozen dairy foods, 165
OfiF-bottom
method
208-211
One-hour
test,
194
in
milk, 261-269
OSCC-II Technicon,
in
80
PLC
Count, 87
311, 315-317,
353
Polyester (Dacron) swabs, 351-352
Polymyxin, in dairy products, 149
Potato glucose agar, acidified, 55, 158,
163
Pathogens
milk, 347-348
Preparation of culture media, basic
Penicillin
detection,
141-149,
331-344,
Pennsylvania modification
375
fat test,
373-
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
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
tainers, 199
Rickettsia, 7, 25
Rinse solution method, for
equipment
Q-fever, 25-26
Quality control program, 8-9
Quality, measuring sanitary, 6
Quality Tests (Chapter 1), 1-10
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
outbreaks, 12
variability, 13
S. typhi, 12, 19
S.
HTST
15),
187-197
Reduction times, methylene blue, 187189
resazurin, 191-194
viral,
Listeria, 17
Reactivation of phosphatase in
brucellosis, 14
leptospirosis, 17
RBC
typhimurium 20
,
Salmonellosis, 19-20
Sample breaker, reentrant cross section
(Fig. 20:4),
Sample
290
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
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
fat
374
DMC
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
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
,
procedures
for,
2-5
discussion, 77
dry dairy products, 154-155
Staphylococci,
INDEX
414
State Milk Sanitation Rating Authorities,
certification of containers, 34
of
SMA
(Table
74
4:11),
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,
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
TDE,
in milk,
231-235
204
157-159, 360-
361, 376-378
5.
fat,
358, 373-376
inhibitory substances in milk, 141-150
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)
AOAC
Tests by
title
Membrane
{Cont'd)
filter,
200
197-203
Microbiological,
197-203
equipment,
49-51,
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
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-
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
134
Kay Graham,
213
icillin,
144
Somatic
144
cell
count, 388-399
INDEX
416
Tests by title (Cont'd)
Split sample, 5
Volhard
Swab
Swabbing, 351
Thermistor cryoscope, 231-233
chlorine
rinse
365-366
TCA, 295-308
use
test,
contact, 200-202
Thiosulfate titration,
waters, 235-236
Thermodurics, 7, 107-109
from containers and equipment, 200
from dry dairy products, 156
Thermophilic bacteria, 7, 109, 156
Thiosulfate titration, available chlorine,
Wisconsin mastitis
Wisconsin mastitis
test,
adding reagent
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
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
Youden and
3-4
Zinc-65, in milk, 283
i^f /
f"-^
ISBN 0-875S34m-