🦠 Total count of micro-organisms in food and feed — surface plating on PCA agar at 30 °C (72 h)

Microbiology PN-EN ISO 4833-2

Overview

ISO 4833-2:2013 specifies a horizontal method for the enumeration of micro-organisms capable of growing and forming colonies on the surface of a solid medium after aerobic incubation at 30 °C. It is applied to products intended for human consumption or for feed, and to environmental samples from the production and the distribution of food and feed. Surface plating is indicated for products containing heat-sensitive organisms that make up a substantial part of the flora (e.g. psychrotrophs in chilled and frozen food, dried food), for products with obligately aerobic bacteria (e.g. Pseudomonas spp.), for products with fine particles difficult to distinguish from colonies in pour plating, for products of an intense colour that makes the recognition of colonies difficult, and where the assessment of quality requires the types of colonies to be distinguished. Besides manual surface plating, the standard describes the use of a spiral plater — a rapid method of counting surface colonies (annex A).

Part 1 of the standard (ISO 4833-1) is pour plating at 30 °C — it is that part which Regulation (EC) No 2073/2005 indicates in the process hygiene criteria (aerobic colony count in carcases and minced meat). In Poland it is in force as PN-EN ISO 4833-2:2013-12 (Polish version of 2017; it replaced PN-EN ISO 4833:2004) with the amendment A1:2022-06. The scheme of the method in the materials of PIWet-PIB: test portion of x g or x ml → initial suspension in 9x of diluent → plating on PCA → incubation at 30 °C for 72 ± 3 h → reading → interpretation → presentation of the results.

Method principle

A defined amount of the sample (liquid product) or of the initial suspension (other products) is plated on the surface of a solid agar medium in Petri dishes; further plates are prepared under the same conditions from the decimal dilutions. With a sterile pipette 0.1 ml of the sample or of the initial suspension is transferred onto a plate, 0.1 ml of the 10⁻¹ dilution (liquid products) or of the 10⁻² dilution (other products) onto the next one with a new pipette, and if necessary further dilutions; the limit of quantification can be lowered tenfold by plating 1.0 ml of the sample or of the initial suspension. The inoculum is spread evenly and as quickly as possible over the surface of the agar, the plates are left with their lids on for about 15 min at ambient temperature for it to be absorbed, then inverted and incubated aerobically at 30 °C for 72 h. After the incubation the plates are chosen that contain, as far as possible, fewer than 300 colonies; spreading colonies are counted as single ones, and where they are expected the plates are inspected after 24 h or 48 h and the visible colonies are marked. The number of micro-organisms per gram or millilitre is calculated from the number of colonies on the plates with fewer than 300 colonies. The medium (plate count agar with yeast extract, PCA) has after sterilisation a pH of 7.0 ± 0.2 at 25 °C (autoclave 121 °C, 15 min); when milk products are examined, skimmed milk powder at 1.0 g/l is added; 15–20 ml of medium is poured into the plates, the plates may be stored for up to 4 weeks at (5 ± 3) °C, and before use their surface is dried (e.g. in a cabinet at 37–55 °C or in a laminar flow cabinet).

Applications

• Chilled and frozen food with a heat-sensitive psychrotrophic flora; dried food
• Products with obligately aerobic bacteria (e.g. Pseudomonas spp.)
• Products with fine particles difficult to distinguish from colonies or of an intense colour, where pour plating makes the reading difficult
• Assessment of quality requiring the types of colonies to be distinguished; feed and environmental samples
• High-throughput laboratories — spiral plating (annex A)

Key parameters

Standard

Standard number

PN-EN ISO 4833-2:2013-12 + A1:2022-06 (ISO 4833-2:2013 + Amd 1:2022)

Title (PL)

Mikrobiologia łańcucha żywnościowego — Horyzontalna metoda oznaczania liczby drobnoustrojów — Część 2: Oznaczanie liczby metodą posiewu powierzchniowego w temperaturze 30 °C

Title (EN)

Microbiology of the food chain — Horizontal method for the enumeration of microorganisms — Part 2: Colony count at 30 °C by the surface plating technique

Step-by-step procedure

1. Preparation of the plates

   Prepare the PCA (for milk products with 1.0 g/l of skimmed milk), adjust the pH so that after sterilisation (121 °C, 15 min) it is 7.0 ± 0.2, cool it in a bath at 47–50 °C, pour 15–20 ml into sterile plates and leave it to set (storage ≤ 4 weeks at (5 ± 3) °C). Dry the surface before plating.

2. Initial suspension and dilutions

   From a test portion of x g or x ml prepare an initial suspension in 9x of diluent and decimal dilutions.

3. Inoculation

   With a sterile pipette transfer 0.1 ml of the liquid sample or of the initial suspension onto a plate; with a new pipette 0.1 ml of the 10⁻¹ dilution (liquids) or of the 10⁻² dilution (other products) onto the next one; if necessary further dilutions. To lower the limit tenfold plate 1.0 ml.

4. Spreading

   Spread the inoculum evenly and as quickly as possible over the whole surface of the agar with a spreader; leave the plates with their lids on for about 15 min at ambient temperature for it to be absorbed.

5. Incubation

   Invert the plates and incubate them in the incubator at 30 °C for 72 h.

6. Counting

   Choose the plates with (as far as possible) fewer than 300 colonies. Count spreading colonies as single ones; where they are expected, inspect the plates after 24 h or 48 h and mark the visible colonies.

7. Result

   Calculate the number of micro-organisms per gram or millilitre of sample and present the result in accordance with the standard.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Cultures of a mixed flora from food — aseptic working, autoclaving of the used plates
• Molten agar at 47–50 °C and the autoclave — risk of burns
• Spiral plater — disinfection of the dispensing system in accordance with the manufacturer's instructions

Frequently asked questions