GB 4789.14-2014National food safety standard - Food Contact Materials and Articles - Food Microbiological Examination - Bacillus Cereus (English PDF)

3.1 Phosphate buffer solution (PBS). see A.1 of Annex A.

3.2 Mannitol yolk polymyxin (MYP) agar. see A.2 of Annex A.

3.3 Trypticase soy polymyxin broth. see A.3 of Annex A.

3.4 Nutrient agar medium. see A.4 of Annex A.

3.11 Motility medium. see A.11 of Annex A.

3.12 Sugar fermentation tube. see A.12 of Annex A.

3.13 V-P medium. see A.13 of Annex A.

4.1 Examination procedures

The examination procedures of the plate count method of bacillus cereus are as shown

in Figure 1.

4.2 Operating procedures

4.2.1 Sample treatment

Frozen samples shall be unfrozen not exceeding 15 min below 45°C and not exceeding

4.2.3 Sample dilution

Absorb 1 mL of the 1.10 sample homogeneous solution of 4.2.2 to add to a dilution

tube containing 9 mL of PBS or normal saline; fully mix up to make 1.100 sample

homogeneous solution. In accordance with the estimation of sample contamination

degree, operate as above and make into ten-fold incremental serial dilution sample

homogeneous solution in succession. After each dilution, replace one 1 mL sterile

pipette or tip.

4.2.5 Isolation and culture

4.2.5.1 Isolation

Under normal conditions, allow plates to stand for 10 min after applying. If sample

solution is difficult to absorb, it can be placed in an incubator to culture for 1 h at 30°C

± 1°C; turn over plates after sample homogeneous solution is evenly absorbed; place

them upside down in the incubator; culture for 24 h ± 2 h at 30°C ± 1°C. If the colonies

are not typical, continue culturing for 24 h ± 2 h before observing. On MYP agar plates,

typical colonies are of a faint pink colour (indicating unfermented mannitol) and there

are white to pale orchid pink precipitation ring (indicating the production of lecithinase).

4.3 Confirmatory appraisal

4.3.1 Dyeing microscopic examination

Pick single colonies of pure culture for Gram's microscopic examination. Bacillus

cereus is Gram positive bacillus of size (1 µm ~ 1.3 µm) ×(3 µm ~ 5 µm); the spore is

oval which is located at the centre or one end of thallus, not expanding on thallus; both

ends of thallus are flat, normally arranged in the shape of short chains or long chains.

4.3.2 Biochemical identification

4.3.2.4 Root growth test

Item Bacillus cereus Bacillus thuringiensis Bacillus mycoides Bacillus anthracis Bacillus megaterium

4.3.2.6 Protein toxin crystal test

4.4 Calculation of results

4.4.1 Count and confirmation of typical colonies

4.4.1.1 Select plates having typical bacillus cereus colonies and plates of a total

colony count between 20 CFU ~ 200 CFU on three plates of the same dilution degree;

count typical colonies. In case of the phenomena of a) ~ f), calculate in accordance

with Equation (1) of 4.4.2.1.In case of the phenomenon of g), calculate in accordance

with Equation (2) of 4.4.2.2.

5.1 Examination procedures

The examination procedures of the MPN count method of bacillus cereus is shown in

Figure 2.

5.2 Operating procedures

5.2.1 Sample treatment

As in 4.2.1.

5.2.2 Sample preparation

As in 4.2.2.

5.2.3 Sample dilution

As in 4.2.3.

5.2.5 Culture

Use an inoculating loop to transfer 1 loop from each tube; inoculate on MYP agar plates

by streaking; culture for 24 h ± 2 h at 30°C ± 1°C. If colonies are not typical, continue

to culture for 24 h ± 2 h before observation.

5.2.6 Confirmatory appraisal

Select 5 typical colonies from each plate (select all if less than 5); inoculate them on

nutrient agar plates for pure culture by streaking; culture for 24 h ± 2 h at 30°C ± 1°C;

carry out confirmatory test. See 4.3.

Annex A

Culture media and reagents

A.1 Phosphate buffer solution (PBS)

A.1.1 Composition

Potassium dihydrogen phosphate 34.0 g

Distilled water 500.0 mL

A.3 Trypticase soy polymyxin broth

A.4 Nutrient agar

A.6 Motility medium

A.7 Nitrate broth

A.7.1 Ingredients

Peptone 5.0 g

Potassium nitrate 0.2 g

Distilled water 1,000.0 mL

A.8 Casein agar

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