🦠 Presumptive Escherichia coli in food — MPN (lauryl sulfate broth 37 °C → EC broth 44 °C → indole)

Microbiology PN-ISO 7251

Overview

ISO 7251:2005 gives general guidelines for the detection and the enumeration of presumptive Escherichia coli by the technique of culture in a liquid medium and the calculation of the most probable number (MPN) after incubation at 37 °C and then at 44 °C; it is applied to products intended for human consumption and for the feeding of animals and to environmental samples from the production and the distribution of food. Note: some pathogenic strains of E. coli do not grow at 44 °C. A limitation is the great variability of the method (clause 10). The third edition replaced ISO 7251:1993 as well as ISO 11866-1:1997 and IDF 170-1:1999; the main changes: a temperature of 44 °C ± 1 °C for the second incubation, the covering of both detection and enumeration, the allowance of five tubes per dilution for certain products, a note about milk products that make the collection of gas difficult, and the calculation of the MPN according to ISO 7218. Presumptive E. coli are defined as bacteria which at 44 °C ferment lactose with the production of gas and which at 44 °C produce indole from tryptophan. In Polish food laboratories this method (PN-ISO 7251:2006) is used alongside the plate methods of ISO 16649 for the determination of E. coli in products in which small numbers are expected; the teaching materials of the UWM describe the analogous MPN determination of Enterobacteriaceae and coliforms in the three-tube arrangement.

Method principle

Detection: a liquid selective enrichment broth is inoculated with a defined amount of the initial suspension and incubated at 37 °C for up to 48 h, gas being checked after 24 h and 48 h; where there is turbidity or gas it is subcultured into EC broth and incubated at 44 °C for up to 48 h; the tubes with gas are subcultured into peptone water free from indole and incubated for 48 h at 44 °C, after which the production of indole from tryptophan is examined; the tubes with gas in EC and with indole are regarded as containing presumptive E. coli (the result "present/absent in x g or x ml"). Enumeration: three tubes of double-strength and three of single-strength broth are inoculated with a defined amount of the initial suspension, further sets of three — with the decimal dilutions; the procedure is as above, and the MPN is read from a table (annex A) according to the number of tubes whose subcultures gave gas in EC and indole at 44 °C. Lauryl sulfate broth: hydrolysate of plant and animal tissues 20 g (40 g double-strength), lactose 5 g (10 g), K₂HPO₄ 2.75 g (5.5 g), KH₂PO₄ 2.75 g (5.5 g), NaCl 5 g (10 g), sodium lauryl sulfate 0.1 g (0.2 g)/l; pH 6.8 ± 0.2; 9 ml in tubes of 16 × 160 mm with a Durham tube (single-strength) and 10 ml in tubes of 18 × 180 or 20 × 200 mm with a Durham tube (double-strength); 121 °C for 15 min. EC broth: casein digest 20 g, lactose 5 g, bile salts No 3 1.5 g, K₂HPO₄ 4 g, KH₂PO₄ 1.5 g, NaCl 5 g/l; pH 6.8 ± 0.2; 10 ml with a Durham tube. Peptone water free from indole: casein digest 10 g, NaCl 5 g/l; pH 7.3 ± 0.2; 5–10 ml. Kovacs reagent: 4-dimethylaminobenzaldehyde 5 g, 2-methylbutan-1-ol or pentan-1-ol 75 ml, HCl (ρ 1.18–1.19) 25 ml; dissolution in a bath at 50–55 °C, storage at about 4 °C away from light; the colour light yellow to light brown.

Applications

• Detection and enumeration of small numbers of E. coli in food (meat, milk products, delicatessen products, ready-to-eat food)
• Environmental samples from the areas of the production and the distribution of food
• Feed and raw materials
• Milk products — with the note that they may make the collection of gas in the Durham tube difficult (five tubes per dilution are allowed)
• A complement to the plate methods (ISO 16649) where the expected level is below their limit of quantification

Key parameters

Standard

Standard number

PN-ISO 7251:2006 (ISO 7251:2005)

Title (PL)

Mikrobiologia żywności i pasz — Horyzontalna metoda wykrywania obecności i oznaczania liczby przypuszczalnych Escherichia coli — Metoda najbardziej prawdopodobnej liczby

Title (EN)

Microbiology of food and animal feeding stuffs — Horizontal method for the detection and enumeration of presumptive Escherichia coli — Most probable number technique

Step-by-step procedure

1. Sample

   Prepare the sample, the initial suspension and the decimal dilutions according to ISO 6887-1 to -4 (milk: ISO 8261) or to the standard for the product.

2. Inoculation of the lauryl sulfate broth

   Detection: a defined amount of the initial suspension into a tube of the broth. Enumeration: three tubes each of double-strength and of single-strength broth with a defined amount of the suspension, and three tubes each of single-strength broth with the successive decimal dilutions.

3. Incubation at 37 °C

   Incubate for up to 48 h at 37 °C; check the production of gas after 24 h and 48 h (with milk products the collection of gas may be difficult).

4. Subculture into EC broth

   From every double-strength tube showing turbidity or gas and from every single-strength tube with gas subculture with a loop into EC broth; incubate at 44 °C for up to 48 h, checking gas after 24 h and 48 h.

5. Indole test

   From the EC tubes with gas subculture into peptone water free from indole, incubate for 48 h at 44 °C and add Kovacs reagent — the production of indole (a colour) indicates presumptive E. coli.

6. Result

   Detection: presence/absence of presumptive E. coli in x g or x ml. Enumeration: from the number of single-strength and double-strength tubes whose subcultures gave gas in EC and indole, read the MPN from the table of annex A (calculation according to ISO 7218); in the test report take into account the variability of the method and any deviations.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Cultures of E. coli may include pathogenic strains — work in a microbiological laboratory, autoclaving of the waste
• Kovacs reagent contains concentrated HCl and amyl alcohols — work in a fume cupboard, protection of the eyes and of the hands
• Incubation at 44 °C — control of the temperature of the bath; open the tubes with gas carefully

Frequently asked questions