GB 4789.40-2024National food safety standard - Food Microbiological Testing - Cronobacter Testing (English PDF)

Foreword

This Standard serves as a replacement of GB 4789.40-2016 National Food Safety Standard -

Food Microbiological Examination - Examination of Cronobacter.

In comparison with GB 4789.40-2016, the main changes are as follows.

---The title of the Standard is modified;

---The scope of application of this Standard is expanded to supplementary foods for infants

and young children;

---PCR identification method is added as optional content;

---The yellow pigment-producing and amygdalin items in picking yellow colonies and

biochemical identification are deleted;

---The pH adjustment method for culture media and reagents is modified;

---The preheating, selective enrichment temperature and TSA plate incubation temperature

and time are modified;

---The culture temperature of biochemical reactions and the preparation of amino acid

culture medium are modified.

National Food Safety Standard - Food Microbiological

Examination - Examination of Cronobacter

1 Scope

This Standard specifies the examination method for Cronobacter spp. in food.

This Standard is applicable to the examination of cronobacter in formula foods and

supplementary foods for infants and young children, milk and dairy products and their raw

materials.

2 Equipment and Materials

In addition to the routine sterilization and culture equipment of the microbiology laboratory,

other equipment and materials are as follows.

3 Culture Media and Reagents

4 Examination Procedures

The examination procedures of cronobacter are shown in Figure 1.

5 Operating Steps

6 Result and Report

In accordance with the colony characteristics, confirmatory test (biochemical identification)

and / or PCR identification results, report whether or not cronobacter was detected in the 100

g (mL) of sample.

7 Operating Steps

8 Result and Report

Based on the colony characteristics, confirmation test (biochemical identification) or PCR

identification results, in accordance with the number of positive tubes, in which, cronobacter

was detected, check the MPN retrieval table, and report the MPN value of cronobacter in 100

g (mL) of sample (see Table C.1 in Appendix C).

Appendix A

Culture Media and Reagents

A.1 Buffered Peptone Water, BPW

A.1.1 Ingredients

Peptone 10.0 g

Sodium chloride 5.0 g

Disodium hydrogen phosphate 9.0 g

Potassium dihydrogen phosphate 1.5 g

Distilled water 1,000 mL

A.2.2 Vancomycin solution

A.2.2.1 Ingredients

Vancomycin 10.0 mg

Distilled water 10.0 mL

A.2.3 Modified lauryl sulfate tryptose broth-vancomycin medium, mLST-Vm

For each 10 mL of mLST, add 0.1 mL of vancomycin solution. The final concentration of

vancomycin in the mixed solution is 10 g/mL.

NOTE. mLST-Vm needs to be used within 24 hours.

A.3 Trypticase Soy Agar, TSA

A.3.1 Ingredients

Tryptone 15.0 g

Plant peptone 5.0 g

Sodium chloride 5.0 g

Agar 15.0 g

Distilled water 1,000 mL

A.4 Oxidase Reagent

A.4.1 Ingredients

N, N, N, N-tetramethyl-p-phenylenediamine hydrochloride 1.0 g

Distilled water 100 mL

A.4.2 Preparation method

Freshly prepare a small amount. Store in the refrigerator away from light and use within 7

days.

A.4.3 Test method

Use a glass rod or disposable inoculation needle to pick a single characteristic colony and

spread it on a filter paper plate moistened with oxidase reagent. If the filter paper does not turn

fuchsia, purple or dark blue within 10 seconds, then, the oxidase test is negative, otherwise,

the oxidase test is positive.

NOTE. DO NOT USE nickel / chromium materials in the experiments.

A.5 L-lysine decarboxylase medium

A.5.1 Ingredients

L-lysine monohydrochloride 5.0 g

Yeast extract 3.0 g

Glucose 1.0 g

Bromocresol purple 0.015 g

Distilled water 1,000 mL

Appendix B

Gene Enrichment Target Reference Sequence of Cronobacter

The gene enrichment target reference sequence of cronobacter is as follows.

NOTE. the bold part is the primer synthesis reference sequence.

Appendix C

Cronobacter Most Probable Number (MPN) Retrieval Table

The retrieval table of most probable number (MPN) of cronobacter in every 100 g (mL) of test

sample is shown in Table C.1.

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