Enumeration of Escherichia coli in Foods by the Direct Plating (DP) Method

HPB Method MFHPB-27
September 1997

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Health Protection Branch
Ottawa

ENUMERATION OF ESCHERICHIA COLI IN FOODS BY A DIRECT PLATING (DP) METHOD

R.A. Szabo and E.C.D. Todd
Microbiology Research Division
Bureau of Microbial Hazards, HPB
Postal Locator: 2204A2
Ottawa, Ont., K1A 0L2

1. APPLICATION

This method is applicable to the enumeration of Escherichia coli biotype 1 in foods and food ingredients to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act. Where an Official Method for certain products is specified, that method shall be followed. This revised method replaces MFHPB- 27, dated December 1995.

2. DESCRIPTION

It has been used successfully for the enumeration of E. coli in red meats, poultry, pork, sausages, carcass washings, fish, shellfish, milk, ice cream, cheese, apple and strawberry purées, flour, carrots, potatoes, swede, and green beans (see Section 8). With the exception of bean and alfalfa sprouts in which large numbers of Klebsiella spp. may preclude an accurate determination of E. coli , the DP method can be used successfully for the enumeration of E. coli in other foods and food ingredients.

3. PRINCIPLE

The conventional Most Probable Number (MPN) procedure for enumerating E. coli in foods takes 8 to 12 days to complete, whereas, the Direct Plating (DP) method takes 24 to 30 h. In previous studies comparing these two methods, the MPN procedure was shown to be less precise and yielded lower counts of E. coli in frozen meat samples. In addition, the MPN procedure is incapable of enumerating late lactose fermenters and anaerogenic E. coli which comprise approximately 10% of the Escherichia strains (2). The main disadvantage of the DP method is its inability to enumerate non-indole producing E. coli which represent 3 to 5% of the E. coli strains (2); this disadvantage is offset by the brevity of the method and its facility in enumerating anaerogenic E. coli .

4. DEFINTION OF TERMS

See Appendix A of Volume 2.

5. COLLECTION OF SAMPLES

See Appendix B of Volume 2.

6. MATERIALS AND SPECIAL EQUIPMENT

1) A supply of 0.45 mm membrane filters of 85 mm diameter (MF) (Available from Nuclepore Canada,

Inc., Toronto, Cat No. 145318).

2) Peptone water (0.1%). 3) Nutrient agar (NA).

4) Tryptone Bile Agar (TBA).

5) Indole reagent.

6) A water-jacketed incubator to maintain a temperature of 44.5 ° C ± 0.5 ° or a similarly accurate incubator.

7) Blender or Colworth Stomacher 400.

7. PROCEDURE

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

7.1 Handling of Sample Units in the Laboratory

7.1.1 During storage and transport, the following shall apply: with the exception of shelf-stable products, keep the sample units refrigerated (0-5 ° C). Sample units of frozen products shall be kept frozen.

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

7.2 Preparation for Analysis

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

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

7.2.3 Have ready sterile peptone dilution water.

7.3 Preparation of Samples

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 For solid samples, blend or stomach 11 (10) g (the analytical unit) with 99 (90) mL of peptone water for 2 min. Make decimal dilutions in peptone water. If a low count is expected, blend 11 (10) g with 44 (40) mL of peptone water (1:5). For liquid samples, shake 11 (10) mL with either 99 (90) mL (1:10) or 44 (40) mL of peptone water (1:5). Liquid samples may also be plated undiluted (see below).

Note: Weight or volume in brackets indicates alternate procedure for making dilutions.

7.3.3 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.

7.3.4 If organisms in the sample might have been stressed by freezing or by other processing, proceed as follows, otherwise go directly to 7.3.9. On the prepoured and dried surface of a NA plate, place an MF and flatten it against the agar with a sterile glass or plastic spreader. Avoid trapping air bubbles.

7.3.5 In duplicate, plate 0.5 mL of two consecutive decimal dilutions, or if low counts are expected, 1 mL of the 1.5 dilution, or 0.5 mL of the undiluted sample unit (liquid samples) on the MF overlaying the NA. With a glass or plastic spreader evenly spread the inoculum without spilling it over the edge of the MF.

7.3.6 After the inoculum has been absorbed, incubate the plates right side up in stacks of not more than three deep in a 35-37 ° C incubator for 4 h.

7.3.7 Remove the MF from the NA with sterile forceps and transfer it to the prepoured and dried surface of a TBA plate. Avoid trapping air bubbles.

7.3.8 Incubate the TBA plates right side up in stacks of not more than three deep in a 44.5 ° C ± 0.5 ° incubator for 18-24 h.

7.3.9 If the samples are neither frozen nor processed, the NA step is omitted. Proceed as follows:

On the prepoured and dried surface of a TBA plate, place an MF and flatten it against the agar with a sterile glass or plastic spreader. Avoid trapping bubbles.

In duplicate, plate as in 6.3.5 but on the MF overlaying the TBA. Incubate the plates right side up in stacks of not more than three deep at 44.5 ° C ± 0.5 ° for 20-24 h.

7.3.10 After the 44.5 ° C ± 0.5 ° incubation of the TBA plates, remove the Petri plate covers, wipe dry and place 2.0 mL of indole reagent (9.4) into each cover. Lift each of the MF off the TBA surface and place it in its respective cover so that the entire undersurface of the MF is soaked with the reagent. Leave at room temperature for 10-15 minutes then remove the MF by dragging it over the lip of the cover to get rid of excess reagent. Place the MF on a flat surface (e.g. another clean Petri plate cover) and dry it under a 'germicidal' UV lamp (e.g. Philips TUV, 15 W or GE G 30T8, 30W) for approximatley 20 minutes. This is best done in a fume hood, suitable laminar flow cabinet or biocontainment unit to avoid exposure to HC fumes given off during drying.

Note: If colonies are to be kept for any reason, they must be picked from the MF before staining.

7.3.11 The pink to red colonies appearing on the MF are indole producers and are enumerated as E. coli biotype I. Use the dilution factor to calculate the number of E. coli biotype I per g or mL and record.

8. REFERENCES

8.1 Anderson, J.M., and A.C. Baird-Parker. 1975. A rapid and direct plate method for enumerating Escherichia coli biotype I in food. J. Appl. Bacteriol. 39 : 111-117.

8.2 Ewing, W.H. 1972. Differentiation of enterobacteriacese by biochemical reactions. CDC Atlanta, U.S. Dept. of Health, Education and Welfare.

8.3 Holbrook, R., J.M. Anderson, and A.C. Baird-Parker. 1980. Modified direct plate method for counting Escherichia coli in foods. Food Technol (Ans.) 32: 78-83.

8.4 Rayman, M.K., and B. Aris. 1981. The Anderson-Baird-Parker direct plating method versus the most probable number procedure for enumerating Escherichia coli in meats. Can. J. Microbiol. 27 : 147-149.

8.5 Rayman, M.K., G.A. Jarvis, C.M. Davidson, S. Long, J.M. Allen, T. Tong, P. Dodsworth, S. McLaughlin, S. Greenberg, B.G. Shaw, H.J. Beckers, S. Qvist, P.M. Nottingham, and B.J. Stewart. 1979. ICMSF methods studies. XIII. An international comparative study of the MPN procedure and the Anderson-Baird-Parker direct plating method for the enumeration of Escherichia coli biotype I in raw meats. Can. J. Microbiol. 25 : 1321-1327.

8.6 Sharpe, A.N., P.I. Peterkin, and M.K. Rayman. 1981. Detection of Escherichia coli in foods: Indole staining methods for cellulosic and polysulfone membrane filters. Appl. Environ. Microbiol. 41 :1310-1315.

8.7 Sharpe, A.N., M.K. Rayman, D.M. Burgener, D. Conley, A. Loit, M. Milling, P.I. Peterkin, U. Purvis, and S. Malcolm, 1983. Collaborative study of the MPN, Anderson/Baird-Parker direct plating, and hydrophobic grid-membrane filter methods for the enumeration of Escherichia coli in foods. Can. J. Microbiol. 29 : 1247-1252.

8.8 Yoovidhya, T., and G.H. Fleet. 1981. An evaluation of the A-1 most probable number and the Anderson and Baird-Parker plate count methods for enumerating Escherichia coli in the Sydney rock oyster, Crassostrea commercialis . J. Appl. Bacteriol. 50 : 519-528.

9. PREPARATION OF MEDIA AND REAGENTS:

When steam sterilization is used it is essential to allow sufficient time for the load to reach the required temperature before the actual sterilizing period commences. This varies with the nature and size of the load. Thus, proper exposure times should be followed to ensure sterilization of solutions in flasks and heat stable culture media. Refer to the sterilizer manual.

9.1 NUTRIENT AGAR

Beef extract (Bacto or equivalent)	3 g
Peptone (Bacto or equivalent)	5 g
Agar	15 g
Distilled water	1000 mL

Dissolve ingredients and autoclave at 121 ° C for 15 min. Pour 15-20 mL per plate on a flat surface. Plates keep for 1 week at 4 ° C. Nutrient agar is commercially available.

9.2 TRYPTONE BILE AGAR

Tryptone (Difco or equivalent)	20 g
Bile salts No. 3 (Oxoid)	1.5 g
Agar	15 g
Distilled water	1000 mL

Dissolve ingredients and autoclave at 121 ° C for 15 min. Pour 15-20 mL per plate on a flat surface. Plates keep for 1 week at 4 °C. Tryptone bile agar is commercially available.

9.3 PEPTONE WATER

Peptone	1.0 g
Distilled water	1000 mL

Dissolve peptone in distilled water and dispense in dilution bottles and in test tubes to obtain 99 mL, 90 mL and 9.0 mL respectively after sterilization at 121 ° C for 15 minutes.

9.4 INDOLE REAGENT

p - Dimethylamino-benzaldehyde (DAB)	0.5 g
1N Hydrochloric acid (HCl)	100 mL

Dissolve the DAB in 1N HCl and store in the dark below 20 ° C. The reagent is stable for at least 2 weeks. New lots of DAB must first be checked against a known indole producing E. coli , before use with unknown samples.