Government of Canada Gouvernement du Canada

Official Method MFO-18

July 2002
Revised: September 2003

HEALTH PRODUCTS AND FOOD BRANCH

OTTAWA

ENUMERATION OF COLIFORMS, FAECAL COLIFORMS AND OF E. COLI

IN WATER IN SEALED CONTAINERS AND PREPACKAGED ICE USING THE MPN METHOD

1. APPLICATION

The Most Probable Number (MPN) method is applicable to the enumeration of coliforms, faecal
coliforms and aerogenic Escherichia coli in water in sealed containers (including mineral and
spring water) and prepackaged ice in accordance with Sections B12.001, B.12.004 and B.12.005
of the Regulations of the Food and Drugs Act. This method replaces MFO-9 and MFO-15 dated
November 30, 1981.

Note: This method is not intended to be used to isolate and enumerate E. coli serotypes associated
with human illness, particularly the enterohemorrhagic serotype O157:H7. Many of the
pathogenic serotypes do not give a positive faecal coliform reaction and therefore would not be
detected and recovered by this method.

2. DESCRIPTION

The MPN procedure involves a multiple tube fermentation technique where three or more decimal
dilutions of the sample are inoculated into tubes of broth medium and incubated at a specific
temperature and for a specific time. The method is progressive; i.e., first determining the presence
of coliforms in the tubes, then determining if these tubes also contain faecal coliforms, and then
confirming whether E. coli is present. Based on the number of tubes indicating the presence /
absence of the three groups of organisms, the most probable number present can be estimated
from a standard statistical MPN table.

This method has been shown to produce satisfactory results with naturally-contaminated and
artifically-contaminated water in sealed containers (including mineral and spring water) and
prepackaged ice in HPFB studies (unpublished data).

2.1 Equivalent Methods

The methods MFHPB-17, MFHPB-19 and MFHPB-26 are considered to be equivalent to

the method presented here and can be used to determine the presence of or to enumerate
coliforms and E.coli and to determine compliance with the Regulations of the Food and
Drugs Act listed above and in Table III of this method. These methods are found in
Volume 2 of the Compendium of Analytical Methods.

Published on the Food Directorate’s (Health Canada's) website at http://www.hc-sc.gc.ca/food-aliment.

 MFO-18
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Revised: September 2003

3. PRINCIPLE

The presence of coliforms, faecal coliforms and aerogenic E. coli in water may be determined by
means of the MPN procedure. Briefly, this method involves serially diluting out the target
organisms in the sample, in 5-replicate aliquots, to extinction (8.6). The probable level of the target
organisms is then statistically estimated from an MPN table.

Gas production is used as an indication of ability to ferment lactose from LST broth (presumptive
coliform test); gas production from BGLB broth is considered confirmation of coliform presence;
gas production at 45o C from EC broth is used as confirmation of faecal coliform presence; and
appearance of typical nucleated, dark-centred colonies with or without metallic sheen when
positive EC broths are streaked onto L-EMB agar are indicative of E. coli. The typical colonies on
L-EMB agar must be confirmed by further biochemical tests to prove the presence of E. coli.

4. DEFINITION OF TERMS

See Appendix A of Volume 1.

5. COLLECTION OF SAMPLES

5.1 See Appendix B of Volume 1

5.2 Each sample unit shall consist of at least 500 mL.

5.3 Do not allow sample units of prepackaged ice to thaw during shipment.

6. MATERIALS AND SPECIAL EQUIPMENT

NOTE: The Laboratory Supervisor must ensure that completion of the analysis, described in
this method, must be done in accordance with the International Standard referred to as
"ISO/IEC 17025:1999 General requirements for the competence of testing and
calibration laboratories".

The media listed below (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 8.7
for the formulae of individual media.

NOTE: If the analyst uses any variations of the media listed here (either product that is
commercially available or made from scratch), it is the responsibility of the analyst or
Laboratory Supervisor to ensure equivalency.

1) Peptone Water (0.1%)

2) Lauryl Sulfate Tryptose (LST) broth

3) Brilliant Green Lactose 2% Bile (BGLB) broth

4) Escherichia coli (EC) broth or

EC broth with MUG (4-methylumbelliferyl-$-D-glucuronide)

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

6) MacConkey agar
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Revised: September 2003

7) Nutrient Agar (NA) or other non-selective agar

8) Covered water baths, with circulating system to maintain temperature of 45/C. Water level
should be above the medium in immersed tubes.

9) Thermometer, calibrated and traceable

10) Incubator, 35/C.

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 35°C 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 +/- 1.0°C. However, where higher temperatures
are recommended, such as 43 or 44.5°C, it is imperative that the incubators be maintained
within 0.5°C and water baths be maintained within 0.2/C due to potential lethality of higher
temperatures on the microorganism being isolated.

11) Control cultures (use ATCC cultures or equivalent):

positive control(s): E. coli that is known to produce gas at 45o C and is

capable of fermenting lactose to produce typical reactions
on L-EMB agar; if using EC-MUG, a strain that is known
to produce $-glucuronidase

EMB / IMViC negative control: Enterobacter aerogenes or an equivalent gram negative

rod that does not produce “positive” reactions on EMB
and is indole-negative, methyl red -negative, Voges-
Proskauer-positive, and citrate positive.

MPN broths negative control: Salmonella berta or an equivalent gram negative rod that
is gas-negative in MPN broths and in the secondary EC
broth

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 or an
equivalent culture for these broths and E. aerogenes or an equivalent culture for EMB
agar and IMViC tests.

12) Supplies needed for confirmation (commercially available):

The following supplies may be needed for confirmation; use A or B (see 7.7). The choice
of further identification schemes (7.7.5) may require alternate media.

A. IMViC media and reagents:

a. Tryptone (or tryptophane) broth
Indole reagents (available commercially)

b. Buffered Glucose broth

Voges-Proskauer test reagents (available commercially)
Methyl red solution

c. Simmon's Citrate (SC) agar

B. Rapid Identification Kits or Systems (such as API, Vitek or equivalent)

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Revised: September 2003

7. PROCEDURE

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

7.1 Handling of Sample Units

7.1.1 Water in sealed containers

(a) Do not store sample units for more than 24 h before analysis. Store under
refrigeration (0-5oC) conditions.

7.1.2 Prepackaged Ice

(a) If sample units are prepackaged in leakproof containers, thaw them in the
containers under refrigeration (0-5oC) prior to analysis.

(b) If sample units are not in leakproof containers, transfer the ice aseptically
to sterile plastic bags or other suitable sterile containers. Seal containers
to prevent contamination, and thaw sample units under refrigeration (0-
5oC). Do not store thawed sample units for more than 6 h before analysis.

7.2 Preparation for Analysis

7.2.1 Have ready sterile peptone water.

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

7.2.3 Arrange LST broth tubes in rows of five and mark them identifying the sample unit
and the dilution to be inoculated.

7.3 Preparation of Sample and Initial Set-up

7.3.1 Inoculate each of separate sets of five tubes of LST broth with each dilution to be
tested, according to the scheme, as follows.

Inoculate each of the five tubes of 10 mL double strength LST broth (first row) with
10 mL of the undiluted water sample. Inoculate each of the five tubes of 10 mL
single strength LST broth (second row) with 1 mL undiluted water. Inoculate each
of the five tubes of 10 mL single strength LST broth (third row) with 0.1 mL of
undiluted water.

7.3.2 Follow incubation of LST and confirmation steps for coliforms, faecal coliforms and
E. coli as required, and record results as MPN per 100 mL of water, by following
the instructions in Appendix D.

7.4 Incubation of LST

7.4.1 In order to verify growth conditions in the elevated temperature water baths,
inoculate one LST broth tube with the the MPN broths positive control and one
LST broth tube with the MPN negative control, 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.
 MFO-18
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Revised: September 2003

7.4.2 Mix inoculum and medium by gently shaking or rotating the tubes, but avoid
entrapping air in the gas vials.

7.4.3 Incubate the inoculated LST broth tubes at 35°C for 24 ± 2 h. Examine for gas
formation (gas formation may be either a gas bubble or effervescence), record
results, and begin the confirmed coliform, faecal coliform, and E. coli tests for all
gas-positive tubes, as required.

7.4.4 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 7.4.3 and
begin the confirmed coliform, faecal coliform and E. coli tests for the additional
gas-positive tubes, as required.

7.4.5 The absence of gas in all of the tubes at the end of 48 ± 4 h of incubation
constitutes a negative presumptive test.

7.5 Confirmation Steps for Determination of Coliforms

7.5.1 Use BGLB broth dispensed in 10 mL volumes in tubes containing gas vials.

7.5.2 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 or other appropriate transfer devices may be used for
making the transfers.

7.5.3 Mix inoculum and medium by gently shaking or rotating the tubes, but avoid
entrapping air in the gas vials.

7.5.4 Incubate the inoculated BGLB broth tubes at 35°C for 24 ± 2 h. Examine for gas
formation (gas bubble or effervescence) and record results.

7.5.5 Incubate gas-negative tubes for an additional 24 ± 2 h, re-examine, record the

numbers of additional gas-positive tubes and add to the result obtained in 7.5.4.

7.5.6 Formation of gas during 48 ± 4 h incubation constitutes a positive confirmed test.

7.5.7 Compute the MPN of Confirmed Coliforms per 100 mL of water by following the
instructions in Appendix D of Volume 1 of this Compendium, to convert the number
of gas-positive tubes to MPN values. Record results.

7.6 Confirmation Steps for Determination of Faecal Coliforms

7.6.1 Use EC broth (with or without MUG), dispensed in 10 mL volumes in tubes

containing gas vials.

7.6.2 Shake or rotate the positive LST broth tubes (obtained in 7.4) to mix the contents
and transfer one loopful from each tube to a tube of EC broth (avoid transferring
pellicles). Sterile wood applicator sticks or other appropriate transfer devices may
be used for making the transfers. This transfer should be made simultaneously
with 7.5 above.

7.6.3 Mix inoculum and medium by gently shaking or rotating the tubes, but avoid
entrapping air in the gas vials.

7.6.4 Incubate the inoculated EC broth tubes in a water bath at 45°C for 24 ± 2 h.
Maintain the water level in the bath at least 1 cm above the level of the medium in
the tubes.
 MFO-18
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Revised: September 2003

7.6.5 Examine for gas production (gas bubble or effervescence), record results, and
begin on the same day E. coli identification for all gas-positive tubes (7.7).

7.6.6 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 7.6.5 and
begin the E. coli identification for the additional gas-positive tubes.

7.6.7 The absence of gas in all of the tubes at the end of 48 ± 4 h of incubation
constitutes a negative presumptive test.

7.6.8 Formation of gas during 48 ± 4 h incubation constitutes a positive faecal coliform

test.

7.6.9 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 test; these tubes may be used for further testing described in
7.7 to confirm presence of E. coli.

PRECAUTIONS: Follow safety precautions in the manufacturer’s instructions when using the UV
light. Negative controls of the EC-MUG broth should be also examined under
the UV light to ensure that the tubes do not fluoresce.

7.6.10 Compute faecal coliform MPN per 100 mL of water following the instructions in
Appendix D to convert the number of gas-positive tubes to MPN values. Record
results.

7.7 Confirmation Steps for Identification of E. coli

7.7.1 Gently shake each gas-positive EC broth tube or each fluorescing EC- MUG broth
tube (7.6.5 and 7.6.6) and streak a loopful of the culture onto a L-EMB or Endo
agar plate.

7.7.2 Incubate the plates at 35°C for 18 to 24 h, and examine for typical non-mucoid,
nucleated, dark-centred colonies with or without a metallic sheen which are
indicative of E. coli.

NOTE: It is up to the Laboratory Supervisor to determine which dilutions and sets of presumptive (gas-
positive) MPN tubes are to be confirmed (and, subsequently, the number of colonies picked per
plate) to adequately determine the final and confirmed MPN count.

7.7.3 If the colonies are well isolated on L-EMB or Endo agar plates, pick one typical
colony and streak onto a non-selective agar such as NA (EMB or MacConkey can
also be used). Circle one other typical colony on EMB before storing the plates at
4/C, to be taken to non-selective media if the initial colony does not confirm as E.
coli. Incubate at 35/C for 18-24 h. Use these cultures for further confirmation.

If the colonies are not well isolated on L-EMB or Endo agar plates, pick two typical
colonies and re-streak onto EMB to obtain discrete colonies. Select one well
isolated typical colony from one of the EMB plates and streak onto a non-selective
agar such as NA (EMB or MacConkey can also be used). Refrigerate the second
EMB plate in case it is needed at a later point. Incubate as above and use these
cultures for further confirmation.
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Revised: September 2003

NOTE: Confirmation can be done by either completing the GIMViC tests (7.7.4) or by the use of a rapid
identification kit (7.7.5).

7.7.4 GIMViC

From the streaked plates (NA, EMB or MacConkey), 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 non-selective agar, prepare fresh plates or 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 IMViC positive and negative controls into each of the IMViC
media and MPN positive and negative controls into secondary EC broth.
Alternatively, IMViC tests may be done using any commercially available testing
system.

Gas Production at 45.0°C (G)

Incubate inoculated tubes of G medium (EC broth) in a water bath at 45.0°C for 24
± 2 h. Examine for gas production. If no gas has been produced, incubate for an
additional 24 ± 2 h and re-examine. Record results.

Indole (I)

Incubate inoculated tubes of Tryptone or tryptophane broth at 35°C for 24 ± 2 h.

Add indole reagent (commercially available) to each tube following manufacturer’s
instructions. 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.

Methyl-Red (MR) and Voges-Proskauer (VP) Tests (MVi)

Inoculate 2 tubes of Buffered Glucose broth and incubate at 35°C for 48 ± 2 h.

Use MR and VP reagents (commercially available) following 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.

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 35°C 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.

Interpretation

The characteristic GIMViC reaction pattern for E. coli is given in Table I. If

necessary, commonly occurring coliforms may be differentiated by using the data
in Table II. If characteristic reactions for E. coli are obtained with GIMViC tests,
the other isolate need not be further tested. However, if the first isolate gives a
 MFO-18
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Revised: September 2003

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.

7.7.5 Rapid Identification Kits

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

instructions.

7.7.6 Calculation of MPNs

Compute the MPN of E. coli per 100 mL of water by following the instructions in
Appendix D of Volume 1, based on the number of tubes found to contain isolates that
produce GIMViC reaction patterns characteristic of E. coli as given above or confirmed
by rapid identification kits as E. coli.

Table I

GIMViC Pattern for E. coli Biotypes

Gas at 45°C Indole Methyl Red Voges-Proskauer Citrate

G I M V C
Type I + + + - -
Type II - - + - -
(Anaerogenic)
 MFO-18
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Revised: September 2003

TABLE II**

Differentiation of Commonly Occurring Coliforms

Gas in EC Indole test Methyl red Voges- Growth on

broth at 45°C test Proskauer citrate
test
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 8.3.
 MFO-18
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Revised: September 2003

8. REFERENCES

8.1 American Public Health Association. 2001. Compendium of Methods for the Microbiological
Examination of Foods; Fourth Edition. Frances P. Downes and Keith Ito (eds.). American
Public Health Association, Washington, D.C.

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.

8.3 International Commission on Microbiological Specifications for Foods. 1978. Microorganisms

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

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

8.5 Powers, E.M. and T.G. Latt. 1977. Simplified 48-Hour IMViC Test: an Agar Plate Method.
Appl. Environ. Microbiol. 34: 274-279.

8.6 American Public Health Association. 1998. Standard Methods for the Examination of Water
and Waste Water; Twentieth Edition. Lenore.S. Clesceri, A.E. Greenberg and A.D. Eaton,
(eds.). American Public Health Association, Inc., Washington, D.C.

8.7 Atlas, R.M. 1997. Handbook of Microbiological Media. Second edition. L.C. Parks (editor).
CRC Press Inc.
 MFO-18
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Revised: September 2003

9. INTERPRETATION

The tolerance as specified hereafter and representing the maximum probable incidence of coliform
bacteria (Coliforms) and E. coli in water in sealed containers and prepackaged ice, shall be applied in
determining whether the tested lot of the product complies with Sections B.12.001, B.12.004 and
B.12.005 of the Regulations of the Food and Drugs Act.

Coliform bacteria (Coliforms) shall be considered absent in a lot when not more than one of the 5
sample units taken from the lot is positive for Coliforms, and the MPN for that sample unit is not more
than 10 Coliforms per 100 mL of water in sealed containers and prepackaged ice.

E. coli shall be considered absent in a lot when none of the five sample units taken from the lot is
positive.

The tolerances are summarized in the following table:

TABLE III. Criteria and sampling plans for Coliforms and E. coli

Determination Food Section of the Criteria

Regulations of
the Food and
Drugs Act

No. of Sample Acceptance Concentration Maximum

Units Number of Concentration
Microorganisms of Microorganisms
(n) (c) (m) (M)

Coliforms Water in B.12.001, 5 1 0* 10

sealed B.12.004 and
containers B.12.005
and
Prepackaged
ice

E. coli Water in B. XXXX 5 0 0* -

sealed
containers
and
Prepackaged
ice

* means less than the Lower Limit of Detection (LLD) of the method, and, in reality, means <1.8 for
MPN methods and <1 for membrane filter methods.

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.
 MFO-18
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Revised: September 2003

m: The numerical value of “m” represents acceptable concentrations of microorganisms, usually

per g or mL. In a 2-class plan (as for Salmonella), “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.
 MFO-18
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Revised: September 2003

The method described above being comprised of 12 pages and identified as MFO-18 and dated July 2002,
Revised August 2003, hereby designated the "Official Method" referred to in Sections B.12.001, B.12.004 and
B.12.005 of the Regulations of the Food and Drugs Act for the microbiological examination of water in sealed
containers and prepackaged ice.

Assistant Deputy Minister