🧫 Bacillus cereus in food
Enumeration of presumptive Bacillus cereus in food by surface plating on MYP (Mannitol-Egg Yolk-Polymyxin) agar at 30°C with hemolysis confirmation.
Overview
Bacillus cereus is a Gram-positive, aerobic, spore-forming bacterium, widely distributed in the environment — it occurs in soil, dust, plants and many food raw materials. B. cereus causes two types of food poisoning: emetic syndrome (emetic type, caused by cereulide toxin — incubation 1–5 h) and diarrheal syndrome (diarrheal type, caused by Hbl, Nhe, CytK enterotoxins — incubation 8–16 h).
The PN-EN ISO 7932 standard specifies a horizontal method for enumerating presumptive B. cereus by colony count on MYP (Mannitol-Egg Yolk-Polymyxin Agar, Mossel's medium). The medium is based on three differentiation mechanisms: inability of B. cereus to ferment mannitol (pink colonies on phenol red background), lecithinase production (white precipitate zone around colonies from egg yolk) and polymyxin B resistance (inhibition of Gram-negative bacteria).
The term "presumptive" arises because the method does not allow complete differentiation of B. cereus from closely related species of the B. cereus sensu lato group (B. thuringiensis, B. mycoides, B. weihenstephanensis, B. anthracis). Confirmation is based on hemolysis test on sheep blood agar.
The method is required in quality control of rice, pasta, sauces, spices, dairy products, infant foods and ready-to-eat meals. B. cereus poses a particular threat in catering and gastronomy, where errors in cooling and reheating favor bacterial multiplication.
Method principle
The method uses MYP medium, in which mannitol and phenol red constitute an indicator system (B. cereus does not ferment mannitol — pink colonies), egg yolk emulsion detects lecithinase (white precipitate zone), and polymyxin B inhibits Gram-negative bacteria growth. We surface plate the sample and incubate at 30 ± 1°C for 24 h. Typical B. cereus colonies are large (approx. 5 mm), pink, rough, dry, surrounded by white precipitate zone. Confirmation is performed by hemolysis test — B. cereus shows strong β-hemolysis on sheep blood agar.
Applications
- Quality control of rice, pasta and cereal products
- Testing sauces, spices and spice mixtures
- Dairy products control (milk powder, cheeses)
- Infant food and milk substitute formula testing
- Ready-to-eat and catering meal monitoring
- HACCP systems verification — storage temperature control
- Plant raw materials analysis (flour, starch, herbal teas)
Key parameters
| Parameter | Value |
|---|---|
| Incubation temperature | 30 ± 1°C |
| Incubation time | 18–24 h (MYP), 24 ± 2 h (hemolysis) |
| Medium | MYP Agar (Mannitol-Egg Yolk-Polymyxin, Mossel's medium) |
| Colony type | Large (~5 mm), pink, dry, with white precipitate |
| Plating method | Surface (spread plate), 0.1 mL per plate |
| Confirmation | Hemolysis test on sheep blood agar (β-hemolysis) |
Standard
- Standard number
- PN-EN ISO 7932:2005
- Title (PL)
- Mikrobiologia żywności i pasz — Horyzontalna metoda oznaczania liczby domniemanego 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 preparation
Weigh 10 g sample into stomacher bag, add 90 mL BPW, homogenize for 60–120 s. Prepare decimal dilutions.
2. MYP medium preparation
Dissolve 46 g MYP Agar Base in 900 mL distilled water. Sterilize in autoclave (121°C/15 min). Cool to 45–50°C, add aseptically 100 mL egg yolk emulsion and polymyxin B solution. Pour 15–20 mL per plate, leave to solidify and dry.
3. Surface plating
Apply 0.1 mL of each dilution to MYP plate surface. Spread with Drigalski spatula. Leave until inoculum is absorbed (approx. 15 min).
4. Incubation
Invert plates and incubate at 30 ± 1°C for 18–24 h under aerobic conditions.
5. Colony reading on MYP
Count typical B. cereus colonies: large (~5 mm), pink (mannitol-negative), surrounded by white precipitate zone (lecithinase+). Also count atypical colonies (pink without precipitate).
6. Hemolysis test (confirmation)
Select min. 5 typical and 5 atypical colonies. Plate on sheep blood agar, separating colonies to enable hemolysis assessment. Incubate 24 ± 2 h at 30°C.
7. Hemolysis assessment
B. cereus shows strong β-hemolysis (clearing around colony). Calculate percentage of confirmed isolates.
8. Calculations and reporting
Calculate number of presumptive B. cereus taking into account percentage of confirmed colonies. Express result in CFU/g. Report result as "presumptive Bacillus cereus".
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Laboratory incubator 30°C | Binder BD 56, Memmert INB 200, POL-EKO CLN 53 | 5,000–18,000 PLN |
| Class II laminar flow cabinet | Thermo Scientific MSC-Advantage, ESCO Airstream AC2 | 25,000–60,000 PLN |
| Stomacher / homogenizer | Seward Stomacher 400 Circulator, BagMixer 400 | 12,000–25,000 PLN |
| Water bath 44–47°C | Memmert WNB 14, Julabo TW 12 | 3,000–10,000 PLN |
| Laboratory autoclave | Tuttnauer 2540M, Systec VX-65 | 15,000–45,000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| MYP Agar Base (Mossel's medium) | 69-65-8 | Peptone, meat extract, mannitol, sodium chloride, phenol red, agar; 500 g package |
| Egg yolk emulsion (Egg Yolk Emulsion 50%) | — | Supplement for MYP — detects lecithinase activity, 100 mL to 900 mL medium |
| Polymyxin B (selective supplement) | 1405-20-5 | Antibiotic inhibiting Gram-negative bacteria; working concentration 100 IU/mL; 10 vials package |
| Sheep blood agar (Columbia Blood Agar) | — | For hemolysis test — B. cereus confirmation (β-hemolysis); ready plates |
| D-Mannitol | 69-65-8 | Sugar alcohol — indicator system in MYP (B. cereus mannitol-negative) |
| Phenol red (Phenol Red) | 143-74-8 | pH indicator in MYP medium — color change upon mannitol fermentation |
| Buffered peptone water (BPW) | — | For sample dilutions, 500 g package |
Health and safety (OHS)
- B. cereus — BSL-2 pathogen capable of toxin production, use PPE
- B. cereus spores survive autoclaving — exercise special caution
- Polymyxin B — antibiotic, avoid skin contact and inhalation
- Work in class II laminar flow cabinet mandatory
- Autoclave used materials (121°C/30 min) before disposal
- Surface disinfection with sporicidal agent (e.g., 3% hydrogen peroxide)