🦠 Coliforms in food — detection and MPN in lauryl tryptose broth

Microbiology PN-ISO 4831

Overview

ISO 4831:2006 gives general guidelines for the detection and enumeration of coliforms in products intended for human and animal consumption and in environmental samples from the production and the distribution of food; the number is determined as the most probable number (MPN) after incubation in a liquid medium at 30 °C or 37 °C (the temperature to be agreed; milk and milk products — 30 °C). The enumeration method is applicable where the expected number is 1–100 per ml or g. It is less precise than the colony-count technique of ISO 4832, but it allows a larger portion to be examined and smaller numbers of bacteria per gram to be detected; the definitions of "coliforms" in the two standards differ. Coliforms are defined here as bacteria which at the agreed temperature ferment lactose with the production of gas; some of the strains described as coliforms (including E. coli) do not produce enough gas for it to be detected in the Durham tube (anaerogenic strains), so the method will not detect all the "presumptive coliforms" (e.g. certain Citrobacter, Enterobacter, Klebsiella). A limitation is the great variability of the method (clause 11). In the UWM teaching materials the same method is described as the 3-tube determination of the MPN in a medium with sodium lauryl sulfate (30 °C/48 h) with confirmation in a medium with lactose, bile and brilliant green.

Method principle

Detection: a tube of selective enrichment broth is inoculated with a portion of the sample and incubated at 30 °C or 37 °C for 24 h or 48 h; from the tubes with turbidity or gas a subculture is made into the confirmation medium (24 h or 48 h); the presence of coliforms is confirmed by turbidity and gas. Determination of the MPN: three tubes of double-strength broth are inoculated with a specified amount of the liquid sample or of the initial suspension, three tubes of single-strength broth — with the same amount, and further single-strength tubes — with the decimal dilutions; the double-strength tubes are incubated for 24 h, the single-strength ones for 24 h or 48 h, after which they are examined for gas or for turbidity preventing its detection; from the double-strength cultures and from the single-strength ones with gas/turbidity a subculture is made into the confirmation broth, which is incubated for 24 h or 48 h, and from the number of tubes with gas the MPN is read from a table. Lauryl tryptose broth (LST): digest of milk and animal proteins 20 g (40 g in the double-strength broth), 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) per 1000 ml; pH 6.8 ± 0.2; 10 ml portions in tubes of 16 × 160 mm with Durham tubes (single-strength) or of 20 × 200 mm without tubes (double-strength); 121 °C for 15 min. The confirmation broth: casein digest 10 g, lactose 10 g, dried ox bile 20 g, brilliant green 0.0133 g/l; pH 7.2 ± 0.2; 10 ml with a Durham tube. After sterilisation the Durham tubes must not contain air bubbles.

Applications

• Detection of the presence of coliforms in a specified amount of product (e.g. 1 g/1 ml) — hygiene control in establishments
• Determination of small numbers of coliforms (1–100 cfu/g) in milk and milk products (30 °C), ice cream and delicatessen products
• Environmental samples from the areas of the production and the distribution of food
• Feeds and raw materials, where the colony-count technique is not sensitive enough
• Additional criteria in in-house standards and customers' specifications

Key parameters

Standard

Standard number

PN-ISO 4831:2007 (ISO 4831:2006)

Title (PL)

Mikrobiologia żywności i pasz — Horyzontalna metoda wykrywania i oznaczania liczby bakterii z grupy coli — Metoda najbardziej prawdopodobnej liczby

Title (EN)

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

Step-by-step procedure

1. Sample

   Prepare the sample, the initial suspension and the decimal dilutions in accordance with ISO 6887 (the appropriate part), ISO 8261 or the standard for the product.

2. Detection — inoculation

   Depending on the limit of detection required, transfer x ml of the sample or of the suspension into 10 ml of double-strength broth (1 ml < x < 10 ml) or of single-strength broth (x ≤ 1 ml); incubate for 24 h ± 2 h (double-strength) or for 24 h ± 2 h and, in the absence of gas/turbidity, a further 24 h ± 2 h (single-strength).

3. Detection — confirmation

   From the double-strength tube and from the single-strength tubes with gas or turbidity subculture with a loop into the confirmation broth; incubate for 24 h ± 2 h, and in the absence of gas up to 48 h ± 2 h; a tube with gas = positive (presence of coliforms in x g/ml).

4. MPN — inoculation

   Inoculate three tubes of double-strength broth and three of single-strength broth with a specified amount of the sample or of the initial suspension, and further sets of three single-strength tubes — with the decimal dilutions (at least three successive dilutions).

5. MPN — incubation and subculture

   Incubate in the same way as for detection: 24 h ± 2 h for the double-strength broth, and for the single-strength one 24 h ± 2 h and, in the absence of gas or turbidity, a further 24 h ± 2 h; from the double-strength cultures and from the single-strength ones with gas or with turbidity preventing the detection of gas subculture into the confirmation broth and incubate for 24 h or 48 h.

6. Reading of the MPN

   From the number of confirmation tubes with gas in each series read the MPN per ml or g from the table; take the variability of the method (clause 11) into account in the interpretation; in the test report state the temperature of incubation and any deviations.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• The cultures may contain pathogenic Enterobacteriaceae — work under the conditions of a microbiological laboratory, autoclaving of the waste
• Brilliant green and bile salts — avoid raising dust when weighing them out
• Durham tubes — take care when opening tubes containing gas

Frequently asked questions