🦠 Coliforms in food — colony-count technique (pour plates on VRBL agar)

Microbiology PN-ISO 4832

Overview

ISO 4832:2006 gives general guidelines for the enumeration of coliforms in products intended for human and animal consumption and in environmental samples from the production and the distribution of food by counting the colonies after incubation on a solid medium at 30 °C or 37 °C (the temperature to be agreed between the parties; for milk and milk products 30 °C). The technique is recommended where the expected number of colonies exceeds 100 per ml or g. It is more precise than the MPN method of ISO 4831, but it does not allow so large a portion to be examined, which is why it is preferred where the number of bacteria is high; the definitions of "coliforms" in the two standards differ, so the micro-organisms enumerated need not be the same. Coliforms are defined here as bacteria which at the agreed temperature form characteristic colonies on agar with crystal violet, neutral red, bile and lactose and which in the confirmation test ferment lactose with the production of gas; the method detects on average about 90 % of the strains described in other publications as "presumptive coliforms" (e.g. certain Citrobacter, Enterobacter, Klebsiella). Precision: with a Poisson distribution the confidence limits are ± 16 % to ± 52 %; in interlaboratory studies sr = 0.20 log, sR = 0.35 log. In academic teaching materials (UWM — University of Warmia and Mazury) this method figures as an "additional criterion" determined by food manufacturers: plating on VRBL agar, 37 °C/24 h, counting of the dark purple-red colonies surrounded by a red zone.

Method principle

Two plates are prepared by the pour-plate technique with a specified amount of the sample (a liquid product) or of the initial suspension, and further pairs of plates from the decimal dilutions; the plates are incubated for 24 h at 30 °C or 37 °C; the characteristic colonies are counted and, if necessary, the fermentation of lactose is confirmed; the number of bacteria per ml or g is calculated in accordance with ISO 7218. The VRBL medium (agar with crystal violet, neutral red, bile and lactose): enzymatic digest of animal tissues 7 g, yeast extract 3 g, lactose 10 g, NaCl 5 g, bile salts 1.5 g, neutral red 0.03 g, crystal violet 0.002 g, agar 12–18 g, water 1000 ml; pH after boiling 7.4 ± 0.2 at 25 °C; the medium is brought to the boil, boiled for 2 min, immediately cooled in a bath at 44–47 °C, is not autoclaved and not reheated, is used within 4 h, and its sterility is checked with a control plate. The confirmation medium — brilliant green bile lactose 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 portions in tubes with Durham tubes; 121 °C for 15 min). The time from the end of the preparation of the initial suspension to the pouring of the plates must not exceed 15 min.

Applications

• Control of the hygiene of food production (meat, milk products, delicatessen products) where the expected number of coliforms is > 100/g
• Milk and milk products — incubation at 30 °C
• Environmental samples from the areas of the production and the distribution of food
• Feeds and plant raw materials
• Testing under in-house standards and customers' specifications (an additional criterion beyond Regulation 2073/2005)

Key parameters

Standard

Standard number

PN-ISO 4832:2007 (ISO 4832:2006)

Title (PL)

Mikrobiologia żywności i pasz — Horyzontalna metoda oznaczania liczby bakterii z grupy coli — Metoda płytkowa

Title (EN)

Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of coliforms — Colony-count technique

Step-by-step procedure

1. Sample and dilutions

   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. Inoculation

   With a sterile pipette transfer 1 ml of the liquid product or of the chosen dilution into the centre of each of two dishes (a separate pipette for each dilution).

3. Pouring of the medium

   Into each dish pour about 15 ml of VRBL at a temperature of 44–47 °C (not later than 15 min after the preparation of the suspension), mix carefully and leave it to solidify on a cold horizontal surface; prepare a control plate with 15 ml of the medium to check the sterility.

4. Overlay

   After complete solidification add about 4 ml of VRBL (44–47 °C) to the surface and leave it to solidify.

5. Incubation

   Incubate the plates inverted for 24 h ± 2 h at 30 °C or 37 °C (as agreed).

6. Counting

   Select the plates with 10–150 colonies; count the purple-red colonies of a diameter of ≥ 0.5 mm (sometimes with a reddish halo of precipitated bile) — typical, without confirmation. Count and confirm the atypical colonies (e.g. smaller ones) and all the colonies from milk products containing sugars other than lactose.

7. Confirmation and result

   Inoculate five colonies of each atypical type into brilliant green bile lactose broth, incubate for 24 h ± 2 h at the same temperature; consider as coliforms the colonies which give gas in the Durham tube and include them in the calculation; the result in accordance with ISO 7218; in the test report state the temperature of incubation, the sampling method and any deviations.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Boiling of the VRBL medium and work with agar at 44–47 °C — protection against burns; do not reheat the medium
• The cultures may contain pathogenic Enterobacteriaceae — good microbiological practice, autoclaving of the waste
• Crystal violet and brilliant green — avoid contact with the skin and raising dust when weighing them out

Frequently asked questions