Enumeration of Faecal Coliforms in Foods by the Hydrophobic Grid-Membrane Filter (HGMF) Method

Laboratory Procedure MFLP-55
September 1998

Microbiological Methods Committee
Microbiology Evaluation Division
Bureau of Microbial Hazards, Food Directorate,
Health Products and Food Branch, Health Canada
Postal Locator: 2204A1
Ottawa, Ontario K1A 0L2

E-mail: Don_Warburton@hc-sc.gc.ca

1. APPLICATION

The method may be used for enumeration of faecal coliform organisms in foods to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act. The method has been shown to produce satisfactory results with fish, ground poultry meat, black pepper, cheese and nuts (9.2). On the basis of data obtained with the latter products the HGMF method was granted Official Final Action status by the Association of Official Analytical Chemists (9.1). There is no reason to believe that the method cannot be used successfully for the enumeration of faecal coliforms in other foods and food ingredients. This revised method replaces MFLP-55, dated September 1993.

2. PRINCIPLE

The enumeration of faecal coliforms depends on formation of blue growths inside HGMF grid cells, indicative of lactose fermentation, after incubation on m-FC agar at 44.5°C for 18-24 h. The HGMF analysis takes 24-26 hr and yields counts that are as high, but with less random error than the tube Most Probable Number method (8.3, 8.4). A single dilution accommodates a wide range of contamination levels. Counting precision may be better than on conventional plates or membrane filters because the HGMF reduces the effect of individual visual acuity on the count (8.3). If a low count is expected, the detection limit can be lowered by filtering more of the suspension.

The HGMF method is capable of detecting faecal coliforms that grow poorly or ferment lactose slowly in LST or BGLB media (8.3). If necessary stressed organisms may be resuscitated for 4 hours on a non-selective medium before being exposed to selective growth conditions.

3. DEFINITION OF TERMS

See Appendix A of Volume 3 (8.4).

4. COLLECTION OF SAMPLES

See Appendix B of Volume 3 (8.4).

5. MATERIALS AND SPECIAL EQUIPMENT

1. HGMF (1600 grid-cell, 0.45 :m pore size). (Available from Gelman Sciences, Montreal, Que.).
   
   
2. Membrane filter forceps (Millipore Corp.).
   
   
3. Peptone water, 0.1% (PEP) or Peptone/Tween 80 diluent (PT).
   
   
4. Tryptic soy-magnesium sulfate agar (TSAM), if a resuscitation step is required.
   
   
5. m-FC agar plates (Commercially available; Difco Laboratories)
   
   
6. Enzyme solutions (Appendix E of Volume 3; 8.4) as required for some food products.
   
   
7. Colworth Stomacher or equivalent.
   
   
8. Spreadfilter with funnel (available from Richard Brancker Research Ltd., 27 Monk St., Ottawa, Ont.) plus pipet-tip prefilters (Filtaflex Ltd., P.O. Box 1224, Almonte, Ont., or Kalyx Biosciences Inc, 20 Camelot Drive, Nepean, Ont.), or ISOGRID filtration unit (Gelman Sciences).
   
   
9. HGMF Interpreter (Model MI-200; Richard Brancker Research Ltd.) or, for manual counting, Linecounter (Gelman Sciences).
   
   
10. Water bath capable of maintaining 35-37°C.
    
    
11. Incubators capable of maintaining 25°, 35° and 44.5°±0.5°C

6. PROCEDURE

Analyze each sample unit individually.
Carry out the test in accordance with the following instructions:

6.1 Handling of Sample Units

6.1.1 In the laboratory prior to analysis, except for shelf-stable foods, keep sample units refrigerated (0-5°C) 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.

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

6.2 Preparation for Analysis

Peptone water is adequate for many foods, but if foods contain appreciable quantities of fat (cheese and other dairy products, ground beef, etc.) the PT diluent should be used to improve filterability. Use PT diluent if the Spreadfilter is to be used.

6.2.1 Have ready sterile peptone water (PEP) or peptone/Tween 80 (PT) diluent, tryptic soy-magnesium sulfate agar (TSAM) plates, m-FC agar plates and enzyme solutions needed for food product category if required (see Appendix E of Volume 3; 9.5).

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

6.2.3 Mark clearly the duplicate Petri plates identifying sample, sample unit, dilution, and date of inoculation.

6.3 Preparation of Dilutions

6.3.1 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.

6.3.2 Prepare a 1:10 dilution of the food by aseptically adding 10 g or mL (the analytical unit) into 90 mL of the PEP or PT diluent. If the Spreadfilter is to be used to inoculate the HGMF, use PT diluent. Stomaching is the preferred method of suspending the organisms, since it minimizes the quantity of suspended food debris. Transfer presentative portion from 1:10 dilution for higher serial dilutions as needed. Consult Appendix E of Volume 3 to see if enzyme treatment is required. Enzyme treatment is not necessary for samples filtered at a dilution of 1:100 or greater.

6.3.2.1 Stomach for 1 minute.

6.3.2.2 If the 1:10 dilution is to be mixed by shaking, shake the dilution bottle 25 times through a 30 cm arc in approximately 7 seconds.

6.3.3 Filtration through the HGMF will remove acids or other inhibitors; there is no need to check and adjust the pH of the suspension.

6.3.4 The HGMF will allow counts to be made from suspensions containing up to 5,000 organisms/mL. There normally should be no need to prepare further dilutions. If this is necessary, prepare succeeding decimal dilutions as required, using a separate sterile pipette for each transfer. Record the dilution (C) used for analysis.

6.3.5 If a low count is expected, filter more than 1.0 mL of the suspension. Filter the total volume to be filtered in one operation; do not attempt to filter successive 1 mL aliquots. Record the volume (V) filtered.

6.3.6 Shake all dilutions immediately prior to making transfers to ensure uniform distribution of the microorganisms present.

6.4 Filtration

6.4.1 Agitate each stomacher bag or dilution bottle to resuspend material that may have settled out.

6.4.2 Handle HGMF with sterile forceps.

6.4.3 Following the manufacturer's instructions for use of the filtration apparatus, aseptically pipette 1.0 mL of the required dilution and inoculate the HGMF. Open the filter valve until all liquid has passed through and aseptically remove the HGMF. Do in duplicate.

6.4.4 Repeat with subsequent dilutions as required.

6.4.5 Follow the manufacturer's instructions for cleaning the filtration apparatus.

6.5 Plating and Incubation

6.5.1 If organisms in the sample might have been stressed by freezing or by other processing, then first carry out step 6.5.2. Otherwise, continue at step 6.5.3.

6.5.2 Transfer the HGMF to the surface of a TSAM plate by rolling it onto the agar to avoid trapping air bubbles. Incubate plates in an inverted position in stacks of not more than three, at 25°C for 4 h for dry foods, and 35°C for 4 h for all other foods.

6.5.3 Transfer the HGMF to the surface of a m-FC plate by rolling it onto the agar to avoid trapping air bubbles. Incubate plates in an inverted position in stacks of not more than two, at 44.5±0.5°C for 24±2 h.

6.6 Counting and Scoring HGMF

6.6.1 The blue growth inside HGMF grid-cells is caused by lactose-fermenting organisms, and are enumerated as faecal coliforms.

6.6.2 For automated counting, use an HGMF Interpreter, following the manufacturer's instructions for its use. For manual counting, use a Linecounter.

6.6.3 HGMF will give accurate counts over a wider range than is possible with plates. For preference, count HGMF containing 20-1580 blue grid-cells. Treat with caution results from HGMF containing more than 1580 pink grid-cells.

6.6.3.1 Count 1 (one) for each grid-cell showing any shade of blue. (DO NOT count the individual colonies if a grid-cell contains more than one blue colony). If a rough estimate indicates fewer than 200 occupied grid-cells, count the whole HGMF.

6.6.3.2 For higher densities (up to 50% occupied grid-cells), rotate the HGMF so that the centre indicator lies either to the left or right. Count positive (blue) grid-cells in the 4 rows immediately below the centre and in the 4 rows immediately above the centre (8 rows). Multiply this partial HGMF count by 5 to estimate the score of only one-fifth of the HGMF was counted.

6.6.3.3 If the HGMF is so full that counting negative (not-blue) grid-cells appears easier, then do so as in (6.6.3.1) and (6.6.3.2). Subtract the HGMF negative count from 1600 or 320, as appropriate. Multiply by 5 to obtain the score if only one-fifth of the HGMF was counted.

6.6.3.4 Record as too numerous to count (TNTC) any HGMF for which all grid-cells are blue.

6.6.4 Record the scores of both of the duplicate HGMF. If there are no blue grid cells, record the score as zero.

6.7 Calculating the Most Probable Number of Faecal Coliforms

See Appendix C of Volume 3 (8.4).

7. REPORTING RESULTS

7.1 Report average MPNGU as calculated in 6.7 rounded-off to two significant figures (e.g., record 2850 as 2.9 x 10³).

7.2 If the lowest dilution plated shows no blue grid-cells, the recorded value will be the lowest average obtainable with a given volume plated onto a given set of replicate HGMF, preceded by a "less than" (<) sign, e.g., for 1.0 mL and a set of duplicate HGMF (1 mL per HGMF) the value is <0.5. This figure should be multiplied by the dilution factor of the inoculum on the HGMF.

8. REFERENCES

8.1 AOAC 1985. Official final action hydrophobic grid membrane filter method for detecting total coliforms, fecal coliforms and E. coli in foods. J. Assoc. Offic. Anal. Chem. 68:481.

8.2 Entis, P. 1984. Enumeration of total coliforms, fecal coliforms and Escherichia coli in foods by hydrophobic grid membrane filter: collaborative study. J. Assoc. Offic. Anal. Chem. 67:812-823.

8.3 Sharpe, A.N. and P.I. Peterkin. 1988. Membrane filter food microbiology. Research Studies Press Ltd., Taunton, Somerset, U.K.

8.4 Appendices A, B, C and E, Vol. 3, Compendium of Analytical Methods, Polyscience Publications Inc., Montreal, PQ.