GB 4789.40-2024 English PDF (GB4789.40-2024)

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. 2.1 Constant-temperature incubator: 36 ?C ? 1 ?C, 41.5 ?C ? 1 ?C. 2.2 Refrigerator: 2 ?C ~ 5 ?C, ?20 ?C. 2.3 Constant-temperature water bath: 41.5 ?C ? 1 ?C. 2.4 Balance: with a division value of 0.1 g and 0.01 g. 2.5 Oscillator. 2.6 Sterile pipette: 1 mL (with a scale of 0.01 mL), 10 mL (with a scale of 0.1 mL) or micropipette and tip. 2.7 Sterile container: with a capacity of 100 mL, 200 mL and 2,000 mL. 2.8 Sterile petri dish: with a diameter of 90 mm. 2.9 pH meter or pH colorimetric tube or precision pH test paper. 2.10 Microbial biochemical identification system. 2.11 PCR instrument. 2.12 Centrifuge: with a rotation speed ? 12,000 r/min. 2.13 Gel imaging system or UV detector. 2.14 Agarose horizontal electrophoresis instrument or capillary electrophoresis instrument. GB 4789.40-2024 www.ChineseStandard.net ? Buy True-PDF ? Auto-delivery. Page 5 of 21 2.15 PCR reaction tube. 2.16 1.5 mL centrifuge tube. 2.17 10 ?L inoculation loop. 3 Culture Media and Reagents 3.1 Buffered peptone water, BPW: see A.1. 3.2 Modified lauryl sulfate tryptose broth-vancomycin medium, mLST-Vm: see A.2. 3.3 Cronobacter chromogenic medium. 3.4 Trypticase soy agar, TSA: see A.3. 3.5 Biochemical identification kit. 3.6 Oxidase reagent: see A.4. 3.7 L-lysine decarboxylase medium: see A.5. 3.8 L-ornithine decarboxylase medium: see A.6. 3.9 L-arginine dihydrolase medium: see A.7. 3.10 Carbohydrate fermentation medium: see A.8. 3.11 Simon¡¯s citrate medium: see A.9. 3.12 The internal transcribed spacer (its) PCR primers are shown in Table 1. The gene enrichment target refer...

GB 4789.40-2024
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard - Food Microbiological
Examination - Examination of Cronobacter
ISSUED ON: FEBRUARY 8, 2024
IMPLEMENTED ON: AUGUST 8, 2024
Issued by: National Health Commission of the People’s Republic of China; State Administration for Market Regulation.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Equipment and Materials ... 4
3 Culture Media and Reagents ... 5
Method I Qualitative Examination of Cronobacter ... 6
4 Examination Procedures ... 6
5 Operating Steps ... 7
6 Result and Report ... 11
Method II Quantitative Examination of Cronobacter ... 11
7 Operating Steps ... 11
8 Result and Report ... 11
Appendix A Culture Media and Reagents ... 12
Appendix B Gene Enrichment Target Reference Sequence of Cronobacter ... 19 Appendix C Cronobacter Most Probable Number (MPN) Retrieval Table ... 20 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.
2.1 Constant-temperature incubator: 36 C  1 C, 41.5 C  1 C.
2.2 Refrigerator: 2 C ~ 5 C, 20 C.
2.3 Constant-temperature water bath: 41.5 C  1 C.
2.4 Balance: with a division value of 0.1 g and 0.01 g.
2.5 Oscillator.
2.6 Sterile pipette: 1 mL (with a scale of 0.01 mL), 10 mL (with a scale of 0.1 mL) or micropipette and tip.
2.7 Sterile container: with a capacity of 100 mL, 200 mL and 2,000 mL.
2.8 Sterile petri dish: with a diameter of 90 mm.
2.9 pH meter or pH colorimetric tube or precision pH test paper.
2.10 Microbial biochemical identification system.
2.11 PCR instrument.
2.12 Centrifuge: with a rotation speed  12,000 r/min.
2.13 Gel imaging system or UV detector.
2.14 Agarose horizontal electrophoresis instrument or capillary electrophoresis instrument. 2.15 PCR reaction tube.
2.16 1.5 mL centrifuge tube.
2.17 10 L inoculation loop.
3 Culture Media and Reagents
3.1 Buffered peptone water, BPW: see A.1.
3.2 Modified lauryl sulfate tryptose broth-vancomycin medium, mLST-Vm: see A.2. 3.3 Cronobacter chromogenic medium.
3.4 Trypticase soy agar, TSA: see A.3.
3.5 Biochemical identification kit.
3.6 Oxidase reagent: see A.4.
3.7 L-lysine decarboxylase medium: see A.5.
3.8 L-ornithine decarboxylase medium: see A.6.
3.9 L-arginine dihydrolase medium: see A.7.
3.10 Carbohydrate fermentation medium: see A.8.
3.11 Simon’s citrate medium: see A.9.
3.12 The internal transcribed spacer (its) PCR primers are shown in Table 1. The gene enrichment target reference sequence is shown in Appendix B.
3.13 5 U/L thermostable DNA polymerase.
3.14 2.5 mmol/dNTPs: dATP, dTTP, dCTP, dGTP.
3.15 25 mmol/L MgCl2.
3.16 10  PCR buffer: see A.10.
3.17 Cronobacter quality control strain: ATCC 29544 or equivalent strain provided by an organization with strain preservation qualifications.
3.18 Escherichia coli quality control strain: ATCC 25922 or equivalent strain provided by an organization with strain preservation qualifications.
3.19 DNA extraction reagent: bacterial genomic DNA extraction kit.
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.
Method II Quantitative Examination of Cronobacter
7 Operating Steps
7.1 Sample Dilution
Respectively take 3 portions of 100 g (mL), 10 g (mL) and 1 g (mL) of the test sample, place them in sterile containers, and respectively add 900 mL, 90 mL and 9 mL of BPW that have been pre-heated to 41 C  1 C. Use hand to slowly shake it, until the test sample is thoroughly dissolved, prepare a 1 : 10 homogeneous sample solution, and at 36 C  1 C, culture for 18 h  2 h. Gently shake and mix the cultured pre-enrichment solution, respectively transfer-take 1 mL into 10 mL of mLST-Vm broth, and at 41.5 C  1 C, culture it for 24 h  2 h. 7.2 Separation and Identification
Same as 5.2, 5.3 and 5.4.
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.1.2 Preparation method
Heat and stir, until it is dissolved. If necessary, adjust pH, then, at 121 C, autoclave for 15 minutes. The pH of the sterilized culture medium at 25 C shall be 7.2  0.2. A.2 Modified Lauryl Sulfate Tryptose Broth-vancomycin Medium, mLST-Vm
A.2.1 Modified lauryl sulfate tryptose (mLST) broth
A.2.1.1 Ingredients
Sodium chloride 34.0 g
Tryptone 20.0 g
Lactose 5.0 g
Potassium dihydrogen phosphate 2.75 g
Dipotassium hydrogen phosphate 2.75 g
Sodium lauryl sulfate 0.1 g
Distilled water 1,000 mL
A.2.1.2 Preparation method
Heat and stir, until it is dissolved. If necessary, adjust pH. Dispense it into sterile test tubes, with 10 mL in each tube. At 121 C, autoclave for 15 minutes. After sterilization, the pH of the culture medium at 25 C shall be 6.8  0.2.
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
A.5.2 Preparation method
Heat and dissolve the ingredients. If necessary, adjust pH. Dispense 5 mL into each tube, and dropwise add a layer of liquid paraffin. At 121 C, autoclave for 15 minutes. After sterilization, the pH of the culture medium at 25 C shall be 6.8  0.2.
A.5.3 Test method
Pick the culture and inoculate it under the liquid surface of L-lysine decarboxylase medium. At 36 C  1 C, incubate for 24 h  2 h and observe the results. If the L-lysine decarboxylase test is positive, the culture medium will turn purple; if it is negative, the culture medium will turn yellow; the blank control tube will turn purple.
A.6 L-ornithine decarboxylase medium
A.6.1 Ingredients
L-ornithine monohydrochloride 5.0 g
Yeast extract 3.0 g
Glucose 1.0 g
Bromocresol purple 0.015 g
Distilled water 1,000 mL
A.6.2 Preparation method
Heat and dissolve the ingredients. If necessary, adjust pH. Dispense 5 mL into each tube, and dropwise add a layer of liquid paraffin. At 121 C, autoclave for 15 minutes. After sterilization, the pH of the culture medium at 25 C shall be 6.8  0.2.
A.6.3 Test method
Pick the culture and inoculate it under the liquid surface of L-ornithine decarboxylase medium. At 36 C  1 C, incubate for 24 h  2 h and observe the results. If the L-ornithine decarboxylase test is positive, the culture medium will turn purple; if it is negative, the culture medium will turn yellow; the blank control tube will turn purple.
A.7 L-arginine dihydrolase medium
A.7.1 Ingredients
L-arginine monohydrochloride 5.0 g
Yeast extract 3.0 g
Glucose 1.0 g
Bromocresol purple 0.015 g
Distilled water 1,000 mL
A.7.2 Preparation method
Heat and dissolve the ingredients. If necessary, adjust pH. Dispense 5 mL into each tube, and dropwise add a layer of liquid paraffin. At 121 C, autoclave for 15 minutes. After sterilization, the pH of the culture medium at 25 C shall be 6.8  0.2.
A.7.3 Test method
Pick the culture and inoculate it under the liquid surface of L-arginine decarboxylase medium. At 36 C  1 C, incubate for 24 h  2 h and observe the results. If the L-arginine decarboxylase test is positive, the culture medium will turn purple; if it is negative, the culture medium will turn yellow; the blank control tube will turn purple.
A.8 Carbohydrate fermentation medium
A.8.1 Basal culture medium
A.8.1.1 Ingredients
Casein (enzyme digestion) 10.0 g
A.9.1 Ingredients
Sodium citrate 2.0 g
Sodium chloride 5.0 g
Dipotassium hydrogen phosphate 1.0 g
Ammonium dihydrogen phosphate 1.0 g
Magnesium sulfate 0.2 g
Bromothymol blue 0.08 g
Agar 8.0 g ~ 18.0 g
Distilled water 1,000 mL
A.9.2 Preparation method
Heat and dissolve the ingredients (if necessary, adjust pH), then, dispense it into test tubes, with 10 mL in each tube. At 121 C, autoclave for 15 minutes. After cooling, make it into a slant. After sterilization, the pH of the culture medium at 25 C shall be 6.8  0.2. A.9.3 Test method
Pick the culture and inoculate it onto the entire slant with the culture medium prepared in 9.2. At 36 C  1 C, incubate for 24 h  2 h and observe the results. If it is positive, the culture medium will turn blue; if it is negative, the culture medium will turn green; the blank control tube will turn green.
A.10 10  PCR Buffer
A.10.1 Ingredients
1 mol/L Tris-HCl (pH 8.5) 840 mL
Potassium chloride (KCl) 37.25 g
Sterilized deionized water 160 mL
A.10.2 Preparation method
Place potassium chloride in a little amount of 1 mol/L Tris-HCl (pH 8.5), thoroughly dissolve it and use 1 mol/L Tris-HCl (pH 8.5) to reach a constant volume of 1,000 mL. At 121 C, autoclave for 15 minutes. Dispense it, then, store it at 20 C.
A.11 50  TAE Electrophoresis Buffer
A.11.1 Ingredients