🧫 Bacillus cereus in food

Microbiology PN-EN ISO 7932

Overview

STATUS (as of 02.11.2026). PN-EN ISO 7932:2005 — Polish version, published on 16.12.2005, adopts EN ISO 7932:2004 and ISO 7932:2004 [IDT]; it replaced the English version of this standard and PN-A-86034-14:1993. AMENDMENT PN-EN ISO 7932:2005/A1:2020-09 (English version, published on 16.09.2020) adopts ISO 7932:2004/Amd 1:2020 “Inclusion of optional tests” (31.03.2020). ISO 7932:2004 (third edition, 16.06.2004) is current; it replaced the 1993 edition with its 1997 corrigendum — the former confirmation tests (MYP medium, glucose fermentation, Voges-Proskauer reaction, nitrate reduction) were replaced by the haemolysis reaction and MYP medium.

SCOPE (clause 1). Enumeration of viable presumptive B. cereus by a colony-count technique at 30 °C in products intended for human consumption and for the feeding of animals and in environmental samples in the area of food production and food handling. The confirmatory stage has been restricted to the typical appearance on MYP agar and the haemolysis test, so the result concerns “presumptive” B. cereus — it does not distinguish them from the less commonly encountered B. anthracis, B. thuringiensis, B. weihenstephanensis and B. mycoides; an additional motility test helps when B. anthracis is suspected. Amendment A1 adds a note on the large diversity of the B. cereus group — 7 phylogenetic groups and a growing number of species.

MYP MEDIUM (5.2). Base medium: beef extract 1.0 g, enzymatic digest of casein 10.0 g, D-mannitol 10.0 g, sodium chloride 10.0 g, phenol red 0.025 g, agar 12–18 g, water 900 ml; pH of the complete medium after sterilization 7.2 ± 0.2 at 25 °C. To 90 ml of base medium are added 1.0 ml of polymyxin B sulfate solution (10⁶ IU in 100 ml of water) and 10.0 ml of egg yolk emulsion (yolks with 4 volumes of sterile water, 2 h at 44–47 °C, then 18–24 h at (5 ± 3) °C; storage up to 72 h). Plates with 15–20 ml of medium are dried before use at 37–55 °C. According to the data sheet of the MYP medium and chapter 14 of the BAM manual (FDA), B. cereus does not ferment mannitol and produces lecithinase, so it forms pink colonies surrounded by a zone of precipitate, while polymyxin restricts the growth of Gram-negative bacteria.

SPORES (introduction, 0.2). The spores of most B. cereus strains germinate readily on the surface of enumeration media and a heat shock is usually not needed; when it is desirable — e.g. for spore counts or to inhibit vegetative cells — 10 min at 80 °C is recommended. According to chapter 14 of the BAM manual (FDA), B. cereus is an aerobic spore-forming bacterium commonly found in soil, on vegetables and in many raw and processed foods.

OPTIONAL TESTS (amendment A1, 9.5, Annexes C–F). For isolated B. cereus group strains, for complementary (e.g. epidemiological) investigations: PCR detecting the cytK-1 and cytK-2 variants of the cytotoxin K gene (cytK-1 identifies B. cytotoxicus), PCR of the ces gene of cereulide synthetase — the emetic toxin (cereulide quantification is described in ISO 18465), a motility test for screening B. anthracis and microscopic examination of the parasporal crystal of B. thuringiensis. The amendment uses the term “B. cereus group” instead of “presumptive B. cereus”. According to chapter 14 of the BAM manual (FDA), the food poisoning takes two forms: diarrhoeal (incubation period 4–16 h, symptoms lasting 12–24 h) and emetic (1–5 h after consumption); it occurs when foods are prepared and held for several hours without adequate refrigeration before serving.

REGULATIONS. Commission Regulation (EC) No 2073/2005 (consolidated text as of 01.07.2026), item 2.2.11: dried infant formulae and dried dietary foods for special medical purposes intended for infants below six months of age — presumptive Bacillus cereus, n = 5, c = 1, m = 50 cfu/g, M = 500 cfu/g, method EN/ISO 7932, end of the manufacturing process; 1 ml of inoculum is plated on a Petri dish of 140 mm diameter or on three Petri dishes of 90 mm diameter (footnote 10).

WHAT WE DO NOT GIVE HERE. The publicly available text of the standard ends in 9.3 (selection of plates for counting). We do not give the standard’s description of typical colonies, the number of colonies to be confirmed, the course of the haemolysis test (9.4), the calculation (clause 10, Annex A) or the precision data — the colony appearance and haemolysis are described according to chapter 14 of the BAM manual (FDA).

Method principle

A specified quantity of the liquid sample or the initial suspension and further decimal dilutions is surface plated on a solid selective medium (MYP) in Petri dishes and incubated aerobically at 30 °C for 18–48 h (4.1–4.2). The number of B. cereus per 1 g or 1 ml is calculated from the number of confirmed colonies on plates at dilution levels giving a significant result (4.3). Confirmation comprises the typical colony appearance on MYP and the haemolysis test on sheep blood agar (clause 1, foreword).

Applications

• Products intended for human consumption and for the feeding of animals (clause 1)
• Environmental samples in the area of food production and food handling (clause 1)
• Dried infant formulae and dried dietary foods for special medical purposes for infants below six months — process hygiene criterion (Regulation (EC) No 2073/2005, item 2.2.11)
• Complementary investigations of isolated B. cereus group strains: cytotoxin K and cereulide genes, B. anthracis screening, B. thuringiensis crystal (amendment A1, 9.5)

Key parameters

Standard

Standard number

PN-EN ISO 7932:2005 + A1:2020-09

Title (PL)

Mikrobiologia żywności i pasz — Horyzontalna metoda oznaczania liczby przypuszczalnych Bacillus cereus — Metoda liczenia kolonii w temperaturze 30 °C

Title (EN)

Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of presumptive Bacillus cereus — Colony-count technique at 30 degrees C

Step-by-step procedure

1. Sample and dilutions

   The test sample, initial suspension and dilutions are prepared according to ISO 6887-1 and the standard for the product concerned (clause 8, 9.1).

2. Inoculation

   With a sterile pipette transfer 0.1 ml of the liquid sample or initial suspension to each of two MYP plates; repeat with further dilutions. For low numbers of B. cereus — 1.0 ml on one 140 mm plate or on three 90 mm plates, in duplicate (9.2.1–9.2.2).

3. Spreading

   Spread the inoculum as quickly as possible without touching the sides of the dish, with a fresh sterile spreader for each plate; leave the plates with the lids on for about 15 min at ambient temperature for the inoculum to be absorbed (9.2.3).

4. Incubation

   Invert the plates and incubate for 18–24 h at 30 °C; if colonies are not clearly visible, incubate for a further 24 h (9.2.4).

   🌡 Temperature: 30 °C

5. Selection of plates

   Select plates, preferably at two successive dilutions, with fewer than 150 colonies (9.3).

6. Presumptive colonies

   B. cereus colonies on MYP are pink (mannitol not fermented) and surrounded by a zone of precipitate (lecithinase) — according to chapter 14 of the BAM manual (FDA); the description of typical colonies in the standard is in 9.3, beyond the available text.

7. Haemolysis test

   Colonies are confirmed by haemolysis on sheep blood agar (5.3, clause 1); according to chapter 14 of the BAM manual (FDA), B. cereus is usually strongly haemolytic, with a 2–4 mm zone of complete (β) haemolysis, while B. anthracis is usually non-haemolytic after 24 h. The number of colonies to be confirmed and the test conditions are given in 9.4, beyond the available text. According to BAM (chapter 14), at least 5 pink, lecithinase-positive colonies are taken for confirmation, and the haemolysis test is performed on trypticase soy–sheep blood agar (24 h, 35 °C).

8. Result

   The number of presumptive B. cereus per 1 g or 1 ml is calculated from the number of confirmed colonies (4.3, clause 10).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• The standard has no clause on safety — for laboratory practice it refers to ISO 7218 (clause 5)
• The confirmation does not distinguish B. anthracis; when its presence is suspected, the standard points to an additional motility test (clause 1, note; amendment A1, Annex E)

Frequently asked questions