Professional Documents
Culture Documents
www.hc-sc.gc.ca
Skip to content | Skip to institutional links
Common menu bar links
Franais
Home
Contact Us
Help
Search
canada.gc.ca
Home > Food & Nutrition > Research Programs & Analytical Methods > Analytical Methods > Compendium of
Analytical Methods > Nutrient Methods
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).
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.
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.
PROCEDURE
Each sample unit should be analyzed individually. Carry out the test in accordance with
the following instructions:
7.1.2 Analyze sample units as soon as possible after their receipt in the
laboratory.
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.
7.3.4 With foods that tend to foam, use blender at low speed and remove
aliquot from below liquid/foam interface.
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).
7.4.2.2 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.
7.4.2.5 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.
7.5.1 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.
7.6.1 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.
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.
8.6 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
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.
Preparation
Treatment
pipette directly into LST and/or into peptone water diluent shake
blend or
stomach
Dilution
Amount of product
represented per tube
undil. 100
1 mL
undil. 100
1g
1:10 10-1
0.1 g or mL
0.01 g or mL
0.001 g or mL
1:10000
10-4
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.
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
Important Notices