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Enumeration of Coliforms, Faecal Coliforms and of E. Coli

Health Products and Food Branch - Ottawa
Proposed Official Method MFO-23
July 2002
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1. APPLICATION
This method is applicable to the enumeration of coliforms in pasteurized milk and cream
and other non-fermented dairy products, cheese derived from pasteurized milk, without
ripening, including fresh cheeses or lactic curd with a minimum of 50% moisture (e.g.
cottage cheese), frozen dairy products (ice cream and ice milk), fermented dairy
products, butter, and milk powders and other dairy product powders and the enumeration
of Escherichia coli in cheese made from a pasteurized or unpasteurized source to
determine compliance with the requirements of Section B.08.011 of the Regulations of
the Food and Drugs Act. This revised method replaces MFOs -2 and -4, dated November
30, 1981, and MFO-14 dated November 30, 1983.
2. DESCRIPTION
2.1 This MPN method has been shown to produce satisfactory results with
naturally-contaminated foods for the detection of coliforms, faecal coliforms and
E. coli (8.1-8.7).

2.2 Equivalent Methods

The methods MFHPB-17, MFHPB-19, MFHPB-26, MFHPB-27, MFHPB-31, MFHPB34, and MFHPB-35 are considered equivalent to the MPN method presented here,
can be used to confirm the presence of coliforms and/or E. coli and to determine
compliance with the Regulations of the Food and Drugs Act listed above and in
Table I of this method. All these methods are found in Volume 2 of the
Compendium of Analytical Methods.
3. PRINCIPLE
The presence of coliforms in a food usually indicates that it has been manufactured under
unsanitary conditions. The presence of faecal coliforms and specifically E. coli usually
indicates potential (post-processing) contamination of the product with faecal matter. This
test involves a multiple tube fermentation technique which estimates the "Most Probable
Number" (MPN) of total coliforms, of faecal coliforms and of E. coli.
4. DEFINITION OF TERMS
See Appendix A of Volume 1.
5. COLLECTION OF SAMPLES
See Appendix B of Volume 1.
6. MATERIALS AND SPECIAL EQUIPMENT
The following media (1 to 7) are commercially available and are to be prepared and
sterilized according to the manufacturer's instructions. See also Appendix G of Volume 1
and reference 8.3 for the formula of individual media.

Peptone Water (0.1%)

Aqueous Sodium Citrate (2.0%), tempered to 40-45C

Lauryl Sulfate Tryptose (LST) broth

Brilliant Green Lactose 2% Bile (BGLB) broth

Escherichia coli (EC) broth or EC broth with MUG (4-methylumbelliferyl--Dglucuronide)

Levine's Eosin Methylene Blue (L-EMB) agar or Endo agar

Nutrient Agar (NA)

Covered water bath, with circulating system to maintain temperature of 45.0 C.

Water level should be above the medium in immersed tubes

Thermometer, calibrated and certified

Incubator, 35C
NOTE: It is the responsibility of each laboratory to ensure that the temperature of
the incubators or water baths are maintained at the recommended temperatures.

Where 35C is recommended in the text of the method, the incubator may be at
35 +/-1.0 C. Similarly, lower temperatures of 30 or 25 may be +/- 1.0C.
However, where higher temperatures are recommended, such as 43 or 45.5C, it
is imperative that the incubators or water baths be maintained within 0.5C due
to potential lethality of higher temperatures on the microorganism being isolated.

Stomacher, blender or equivalent

Control cultures (use ATCC cultures or equivalent):
positive control: E. coli
negative control: Enterobacter aerogenes (EMB, GIMViC), Salmonella berta (MPN
broths)
NOTE: Some strains of E. aerogenes will give false-positive reactions in the MPN
broths (LST, BGLB and EC broths) by producing a small gas bubble. Therefore,
use S. berta for these broths and E. aerogenes for EMB agar and GIMViC tests.

pH meter or paper capable of distinguishing to 0.3 to 0.5 pH units within the

range of pH 5.0 to 8.0

Vortex mixer or equivalent

Supplies needed for confirmation (commercially available):
The following supplies may be needed for confirmation; use A or B (see 7.6). The
choice of further identification schemes (7.6.2) may require alternate media

A. IMViC media and reagents:

a. Tryptone (or tryptophane) broth
Indole reagents

b. Buffered Glucose broth

Voges-Proskauer test reagents
Methyl red solution

c. Simmon's Citrate (SC) agar

B. Rapid Identification Kits

Gram stain reagents

PROCEDURE
Each sample unit should be analyzed individually. Carry out the test in accordance with
the following instructions:

7.1 Handling of Samples Units

7.1.1 In the laboratory prior to analysis, except for shelf-stable foods,

keep sample units refrigerated (0-5C) or frozen, depending on the nature
of the product. Thaw frozen samples in a refrigerator, or under time and
temperature conditions which prevent microbial growth or death.

7.1.2 Analyze sample units as soon as possible after their receipt in the
laboratory.

7.2 Preparation for Analysis

7.2.1 Have ready sterile peptone water for the analysis of ice cream and
ice milk, and sterile sodium citrate (2%) tempered to 40-45C for the
analysis of all cheese.

7.2.2 Clean the surface of the working area with a suitable disinfectant.

7.3 Preparation of Sample

7.3.1 To ensure a truly representative analytical unit agitate liquids or free

flowing materials until the contents are homogeneous. If the sample unit
is a solid, obtain the analytical unit by taking a portion from several
locations within the sample unit.

7.3.2 Prepare a 1:10 dilution of the food by aseptically blending 11 (10) g

or mL (the analytical unit) into 99 (90) mL of the required diluent, as
indicated in Table II.

7.3.3 Shake dilutions 25 times through a 1-foot (30 cm) arc in

approximately 7 seconds. With products that require blending, blend or
stomach for the minimum time required to produce a homogeneous
suspension and to avoid overheating, blending time should not exceed 2.5
min.

7.3.4 With foods that tend to foam, use blender at low speed and remove
aliquot from below liquid/foam interface.

7.3.5 Check pH of the food suspension. If the pH is outside the range of

5.5-7.6, adjust pH to 7.0 with sterile 1N NaOH or 1N HCl. The food
homogenate (1:10 dilution) of dry foods should stand at room
temperature for 15 min. In all other instances, the analysis should be
continued as soon as possible.

7.3.6 Prepare succeeding decimal dilutions as required using a separate

sterile pipette for making each transfer.

7.3.7 Shake all dilutions (7.3.3) immediately prior to making transfers to

ensure uniform distribution of the microorganisms present.

7.4 Determination of Coliforms

7.4.1 Presumptive Tests

7.4.1.1 Use lauryl sulfate tryptose broth (LST). Dispense in 10 mL

volumes into tubes containing gas vials (inverted Durham tubes).

7.4.1.2 Arrange LST broth tubes in rows of five and mark them
identifying the sample unit and the dilution to be inoculated (Table
III).

7.4.1.3 Inoculate each of separate sets of five tubes of LST broth

with each dilution of food homogenate, according to the scheme in
Table III.

7.4.1.4 In order to verify growth conditions in the elevated

temperature water baths, inoculate a culture of E. coli known to
ferment lactose and produce gas at 45C and a culture of
Salmonella berta into tubes of LST broth as a positive and negative
control, respectively, for each bath used. Transfer into all media
used at different stages of the procedure. Set up an uninoculated
tube of medium corresponding to each step in the procedure as a
media control.

7.4.1.5 Mix inoculum and medium by gently shaking or rotating

the tubes, but avoid entrapping air in the gas vials.

7.4.1.6 Incubate the inoculated LST broth tubes at 35C for 24 2

h. Examine for gas formation (gas formation may be either a gas
bubble or effervescence), record results, and if required, begin on
the same day the confirmed and presumptive E. coli (faecal
coliform) tests for all gas-positive tubes (7.4.2 and 7.4.3).

7.4.1.7 Incubate gas-negative tubes for an additional 24 2 h,

examine, record the number of additional gas-positive tubes, add
to the result obtained in 7.4.1.6 and begin the confirmed and
presumptive E. coli (faecal coliform) tests for the additional gaspositive tubes.

7.4.1.8 The absence of gas in all of the tubes at the end of 48 4

h of incubation constitutes a negative presumptive test.

7.4.1.9 Compute the "MPN" of Presumptive Coliforms per g(mL) of

food by following the instructions in Appendix D to convert the
number of gas-positive tubes to MPN values. Record results.

7.4.2 Confirmed Test

7.4.2.1 Use BGLB broth dispensed in 10 mL volumes in tubes

containing gas vials.

7.4.2.2 Shake or rotate the positive LST broth tubes to mix the
contents and transfer one loopful from each tube to a tube of BGLB
broth (avoid transferring pellicle). Sterile wood applicator sticks
may be used for making the transfers. Do not discard the LST
broth tubes at this time.

7.4.2.3 Mix inoculum and medium by gently shaking or rotating

the tubes, but avoid entrapping air in the gas vials.

7.4.2.4 Incubate the inoculated BGLB broth tubes at 35C for 24

2 h. Examine for gas formation (gas bubble or effervescence) and
record results.

7.4.2.5 Incubate gas-negative tubes for an additional 24 2 h, reexamine, record the numbers of additional gas-positive tubes and
add to the result obtained in 7.4.2.4.

7.4.2.6 Formation of gas during 48 4 h incubation constitutes a

positive confirmed test.

7.4.2.7 Compute the "MPN" of Confirmed Coliforms per g (mL) of

food by following the instructions in Appendix D, to convert the
number of gas-positive tubes to MPN values. Record results.

7.4.3 Determination of Presumptive E. coli (Faecal Coliforms) in

Cheese

7.4.3.1 Use EC broth (with or without MUG), dispensed in 10 mL

volumes in tubes containing gas vials.

7.4.3.2 Shake or rotate the positive LST broth tubes (obtained in

7.4.1) to mix the contents and transfer one loopful from each tube
to a tube of EC broth (avoid transferring pellicles). This transfer
should be made simultaneously with 7.4.2.2 above.

7.4.3.3 Mix inoculum and medium by gently shaking or rotating

the tubes, but avoid entrapping air in the gas vials.

7.4.3.4 Incubate the inoculated EC broth tubes in a water bath at

45C for 24 2 h. Maintain the water level in the bath at least 1
cm above the level of the medium in the tubes.

7.4.3.5 Examine for gas production (gas bubble or effervescence),

record results, and begin on the same day E. coli identification for
all gas-positive tubes (7.5 below).

7.4.3.6 Incubate gas-negative tubes for an additional 24 2 h,

examine, record the number of additional gas-positive tubes, add
to the results obtained in 7.4.3.5 and begin the E. coli identification
for the additional gas-positive tubes.

7.4.3.7 Formation of gas during 48 4 h incubation constitutes a

positive presumptive E. coli (faecal coliform) test.

7.4.3.8 Tubes containing EC-MUG broth should also be examined

under UV light (366 nm) for glucuronidase activity. Blue-green
fluorescence indicates a positive presumptive E. coli (fecal
coliform) test.
Precautions: Follow safety precautions in the manufacturer's
instructions when using the UV light. Negative controls of the ECMUG broth should be also examined under the UV light to ensure
that the tubes do not fluoresce.

7.4.3.9 Compute "presumptive E. coli" MPN per g(mL) of food

following the instructions in Appendix D to convert the number of
gas-positive tubes to MPN values. Record results.

7.5 Identification of E. coli

7.5.1 Gently shake each gas-positive EC broth tube (7.4.3.5 and 7.4.3.6)
and streak a loopful of the culture onto a separate L-EMB or Endo agar
plate.

7.5.2 Incubate the plates at 35C for 18 to 24 h, and examine for colonies
which are non-mucoid, nucleated, with or without a metallic sheen.

7.5.3 If the colonies are well isolated on L-EMB or Endo agar plates, pick
two typical colonies and streak onto NA slants. Incubate at 35C for 18-24
h. Use these cultures for confirmation (7.6). If the colonies are not well
isolated on L-EMB or Endo agar plates, continue with 7.5.4.

7.5.4 Select two typical colonies from each plate and streak onto separate
NA plates to obtain discreet colonies.

7.5.5 Incubate the NA plates at 35C for 18-24 h, and from each of them
pick an isolated colony and streak onto a separate NA slant.

7.5.6 Incubate the slants at 35C for 18-24 h. Use these cultures for
confirmation.

7.6 Confirmation of E. coli

From the slant make a smear, then Gram strain and examine microscopically.
Record results. If the organisms are not Gram-negative, non-spore forming rods,
they are not considered to be E. coli. Further confirmation can be done by either
completing the GIMVIC tests (7.6.1) or by the use of a rapid identification kit
(7.6.2).

7.6.1 GIMVIC
From one of the two colonies picked and streaked onto NA slants (7.5.5
above), transfer inoculum into a separate tube of each of EC broth (G
medium) and the IMViC media. Collectively they are referred to as the
GIMViC media, where the "G"-medium is the secondary EC broth, "I"
medium is Tryptone broth, "M"- and "V"-medium is Buffered Glucose
broth, and "C"-medium is Simmon's Citrate agar. If GIMViC tests are not
carried out within 96 h of inoculating the agar slants from the nutrient
agar plates, prepare fresh NA slants prior to inoculating the GIMViC media.
Inoculate one tube of each of the GIMViC media for each of the isolates to
be identified. Inoculate E. coli and E. aerogenes into each of the GIMViC
media for positive and negative controls.

7.6.1.1 Gas Production at 45.0C (G)

Incubate inoculated tubes of G medium (EC broth) in a water bath
at 45.0C for 24 2 h. Examine for gas production. If no gas has

been produced, incubate for an additional 24 2 h and reexamine. Record results.

7.6.1.2 Indole (I)

Incubate inoculated tubes of Tryptone or tryptophane broth at
35C for 24 2 h. Add indole reagent (commercially available) to
each tube. Follow manufacturer's instructions for use. A dark red
colour in the alcohol layer indicates a positive test. An orange
colour probably indicates the presence of skatole and may be
reported as a reaction. A yellow colour would be considered
negative.

7.6.1.3 Methyl-Red Voges Proskauer Tests (MR & VP)

Inoculate 2 tubes of Buffered Glucose broth and incubate at 35C
for 48 2 h. Use MR and VP reagents (commercially available).
Follow manufacturer's instructions. The test is VP-positive if an
eosin pink colour develops after 5-10 minutes. The MR test is
positive if a red colour develops, and negative if a yellow colour
develops.

7.6.1.4 Simmon's Citrate Test (C)

In inoculating the slants of SC agar, use a straight needle and
apply a light inoculum. Use care to avoid transferring nutrients
together with inoculum as these nutrients (carbon) could lead to
the development of a blue colour and an incorrect interpretation.
Incubate the slants at 35C for 48 2 h and observe for growth.
Visible growth (positive reaction) is usually accompanied by a
change of colour from green to deep blue.

7.6.1.5 The characteristic GIMViC reaction pattern for E. coli is

given in Table IV. If necessary, commonly occurring coliforms may
be differentiated by using the data in Table V.

7.6.1.6 If, in the IMViC media, characteristic reactions for E. coli

are obtained irrespective of whether gas is produced or not in the
G-medium, the other isolate need not be further tested. However,
if the first isolate gives a non-characteristic IMViC pattern, test the
second isolate for its GIMViC reaction pattern. Repeat confirmation
steps. If both isolates fail to produce IMVIC reaction patterns
characteristic of E. coli, then E. coli is considered to be absent from
the tube of primary EC broth from which the isolates originated.

7.6.2 Rapid identification Kits

Rapid identification kits may be used to identify E. coli. Follow
manufacturer's instructions.

7.6.3 Calculation of MPNs

Compute the MPN of E. coli per g (mL) of food by following the instructions
in Appendix D of Volume 1, based on the number of tubes found to contain
Gram-negative, non-spore forming, rod-shaped bacteria producing GIMViC
reaction patterns characteristic of E. coli as given above or confirmed by
rapid identification kits as E. coli.

REFERENCES

8.1 American Public Health Association. 1992. Compendium of Methods for the
Microbiological Examination of Foods. Third Edition. C. Vanderzant and D.F.
Splittstoesser (eds.). American Public Health Association Inc., Washington, D.C.
20005.

8.2 American Public Health Association. 1992. Standard Methods for the
Examination of Dairy Products. 16th Edition. R.T. Marshall (ed.). American Public
Health Association Inc., Washington, D.C. 20005.

8.3 American Public Health Association. 1995. Standard Methods for the
Examination of Water and Waste Water. Nineteenth Edition. A.D. Eaton, L.S.
Clescen and A.E. Greenberg (eds.). American Public Health Association, Inc.,
Washington, D.C. 20005.

8.4 Atlas, R.M. 1997. Handbook of Microbiological Media. Second edition. L.C.
Parks (editor). CRC Press Inc.

8.5 International Commission on Microbiological Specifications for Foods. 1978.

Microorganisms in Foods; Their Significance and Method of Enumeration. Second
Edition. University of Toronto Press.

8.6 McGuire, O.E. 1964. Wood Applicators for the Confirmatory Test in
Bacteriological Analysis of Water. Public Health Reports. 79: 812-814.

8.7 Powers, E.M. and T.G. Latt. 1977. Simplified 48-Hour IMViC Test: an Agar
Plate Method. Appl. Environ. Microbiol. 34: 274-279.
TABLE I
Criteria and sampling plans for coliforms and E. coli in specific foods

Determination Food

Regulations
of the Food
and Drugs
Act

Criteria

No. of
Acceptance Concentration
Sample Number (c) of
Units
Microorganisms
(n)
(m)

Maximum
Concentration of
Microorganisms
(M)

Coliforms

pasteurized
B.08.011
milk and cream
and other nonfermented
dairy products

10

Coliforms

cheese derived B.08.011

from
pasteurized
milk, without

10

100

ripening,
including fresh
cheeses or
lactic curd with
a minimum of
50% moisture
(e.g. cottage
cheese), frozen
dairy products
(ice cream and
ice milk),
fermented
dairy products,
butter, milk
powders and
other dairy
product
powders
E. coli

cheese from
pasteurized
milk and
unpasteurized
source

B.08.011

100

1,000

Lot: A batch or production unit which may be identified by the same code. When there is no
code identification, a lot may be considered as (a) that quantity of product produced under
essentially the same conditions, at the same establishment and representing no more than one
day's production; or (b) the quantity of the same variety of product from one and the same
manufacturer available for sampling at a fixed location.
n: The number of sample units usually but not always selected at random from a lot and
examined in order to satisfy the requirements of a particular acceptance plan used. This is the
sample.
m: The numerical value of "m" represents acceptable concentrations of microorganisms, usually
per g or mL. In a 2-class plan, "m" separates sample units of acceptable and defective quality; in
a 3-class plan, "m" separates sample units of acceptable quality from those of marginally
acceptable quality. The "m" values listed in the table are based on levels achievable under GMP.
M: (Only in a 3-class plan), the numerical value of "M" represents unacceptable concentrations of
microorganisms, usually per g or mL, that indicate a (potential) health or injury hazard,
imminent spoilage or gross insanitation; "M" separates sample units of marginally acceptable
quality from those of defective quality. A value determined for any one sample unit of a sample
that is greater than that of "M" renders the pertaining lot unacceptable.
c: The maximum allowable number of marginally acceptable sample units. "c" is the acceptance
number of a plan. When this number is exceeded, the lot becomes unacceptable.

TABLE II. Preparation of the Initial Dilution

Type of Food Product

Preparation

Treatment

frozen dairy products (ice cream

pipette directly into LST and/or into peptone water diluent shake

and ice milk)

all cheese

weigh into previously (40-45C) 2% aqueous sodium

citrate (Na3C6H5O7.2H2O)

blend or
stomach

TABLE III. Marking and Inoculating Scheme

Tube
Marking*

Dilution

Volume of dilution inoculated into LST

broth tubes

Amount of product
represented per tube

undil. 100

1 mL of undiluted liquids into 10 mL single

strength medium

1 mL

undil. 100

10 mL of 10-1 dilution of solids into 10 mL of

double strength medium

1g

1:10 10-1

1 mL of 10-1 dilution into 10 mL single strength

medium

0.1 g or mL

1:100 10-2 1 mL of 10-2 dilution into 10 mL single strength

medium

0.01 g or mL

1:1000 10-3 1 mL of 10-3 dilution into 10 mL single strength

medium

0.001 g or mL

1:10000
10-4

1 mL of 10-4 dilution into 10 mL single strength

medium

0.0001 g or mL

Further dilutions of the food may be inoculated in the same manner, into single strength medium,
depending on the anticipated level of contamination of the food.

* Other marking schemes may be used.

Table IV
GIMVic Pattern for E. coli Biotypes
Gas at 45C

Indole

Methyl Red

Voges-Proskauer

Citrate

Type I

Type II (Anaerogenic)

TABLE V**
Differentiation of Commonly Occurring Coliforms
Gas in EC broth at
45C 0.2C

Indole
test

Methyl red
test

VogesProskauer test

Growth on
citrate

Escherichia coli
Type I (typical)
Type II (anaerogenic)

+
-

+
-

+
+

Intermediates
Type I
Type II

+
+

-*
-*

+
+

Enterobacter aerogenes
Type I
Type II

+
+

+
+

+
+

+
-

+
+
-

Enterobacter cloacae
Irregular
Type I
Type II
Type VI
Irregular
other types

Reactions variable

* Weak positive reactions are occasionally found.

** Reference 8.5.
The method described above, being comprised of 14 pages and identified as MFO-23 and dated
July 2002", is hereby designated the "Official Method" referred to in Section B.08.011 of the
Regulations of the Food and Drug Act for the microbiological examination of pasteurized milk and
cream and other non-fermented dairy products, cheese derived from pasteurized milk, without
ripening, including fresh cheeses or lactic curd with a minimum of 50% moisture (e.g. cottage
cheese), frozen dairy products (ice cream and ice milk), fermented dairy products, butter, and
milk powders and other dairy product powders and cheese made from a pasteurized or
unpasteurized source.

Assistant Deputy Minister

Date Modified: 2002-07-01
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