GB 4789.40-2024 National food safety standard- Food microbiological examination - Cronobacter spp.
1 Scope
This standard specifies the methods for examination of cronobacter spp. in food.
This standard is applicable to the examination of cronobacter spp. in formula foods for infant and young children, supplementary foods for infants and young children, milk and dairy products, as well as their raw materials.
2 Equipment and materials
In addition to the conventional sterilization and culture equipment in microbiological laboratory, other equipment and materials required are as follows.
2.1 Constant temperature incubator: 36℃±1℃, 41.5℃±1℃.
2.2 Refrigerator: 2℃~5℃, -20℃.
2.3 Constant temperature water bath: 41.5℃±1℃.
2.4 Balance: with a sensitivity of 0.1g and 0.01g.
2.5 Oscillator.
2.6 Aseptic pipette: 1mL (with scale division of 0.01mL) or 10mL (with scale division of 0.1mL), or micropipettor and sucker.
2.7 Aseptic containers: with a capacity of 100mL, 200mL and 2,000mL respectively.
2.8 Aseptic culture dish: with a diameter of 90mm.
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 rotating speed ≥12,000r/min.
2.13 Gel imaging system or ultraviolet detector.
2.14 Agarose horizontal electrophoresis apparatus or capillary electrophoresis apparatus.
2.15 PCR reaction tube.
2.16 1.5-mL centrifuge tube.
2.17 10-μL 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 spp. 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 Simmons citrate medium: See A.9.
3.12 Internal transcribed spacer (its) PCR primers (see Table 1) and gene amplification target reference sequence (see Annex B).
3.13 5U/μL heat-resistant DNA polymerase.
3.14 2.5mmol/dNTPs: dATP, dTTP, dCTP, dGTP.
3.15 25mmol/L MgCl2.
3.16 10×PCR buffer: See A.10.
3.17 Cronobacter spp. quality-control strain: ATCC 29544 or equivalent strain provided by a unit with strain preservation qualification.
3.18 Escherichia coli quality-control strain: ATCC 25922 or equivalent strain provided by a unit with strain preservation qualification.
3.19 DNA extraction reagent: Bacterial genomic DNA extraction kit.
Foreword I
1 Scope
2 Equipment and materials
3 Culture media and reagents
4 Examination procedures
5 Operation steps
6 Results and report
7 Operation steps
8 Results and report
Annex A Culture media and reagents
Annex B Reference sequences for cronobacter spp. gene amplification targets
Annex C Retrieval table for most probable number (MPN) of cronobacter spp.
