1 Scope
This standard specifies the method for examination of Cronobacter in food.
This standard is applicable to the examination of Cronobacter in formula food for infant and young children, milk and milk products as well as in the raw material.
2 Apparatus and Materials
In addition to the apparatus for conventional sterilization and cultivation in microbiological laboratory, other apparatus and materials are as follows:
2.1 Constant temperature incubator: 25℃±1℃, 36℃±1℃, 44℃±0.5℃.
2.2 Refrigerator: 2℃~5℃.
2.3 Thermostatic water bath: 44℃±0.5℃.
2.4 Balance: with a sensibility of 0.1g.
2.5 Homogenizer.
2.6 Oscillator.
2.7 Aseptic pipette: 1mL (with scale division of 0.01mL), 10mL (with scale division of 0.1mL) or micropipettor and pipette tip.
2.8 Aseptic conical flask: 100mL, 200mL, 2000mL.
2.9 Aseptic culture dish: 90mm in diameter.
2.10 PH meter or pH colorimetric tube or precise pH paper .
2.11 Full-automatic microbe biochemical identification system.
3 Media and Reagents
3.1 Buffer peptone water (BPW): see A.1.
3.2 Modified lauryl sulfate tryptose broth-vancomycin medium (mLST-Vm): see A.2.
3.3 Enterobacter sakazakii 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 double hydrolase medium: see A.7.
3.10 Carbohydrate fermentation medium: see A.8.
3.11 Simmons citrate agar: see A.9.
Method I Qualitative Examination of Cronobacter
4 Examination Procedure
See Figure 1 for the examination procedure of Cronobacter.
Figure 1 Cronobacter Examination Procedure
5 Operation Steps
5.1 Pre-enrichment and enrichment
Take 100g(mL) of sample into the sterilized conical flask; add 900mL of buffer peptone water preheated to 44℃; shake gently with hand to dissolve the solution sufficiently and then incubate for 18h±2h at 36℃±1℃. Transfer 1mL to inoculate in 10mL of mLST-Vm broth and incubate for 24h±2h at 44℃±0.5℃.
5.2 Isolation
5.2.1 Gently mix mLST-Vm broth culture well, and respectively inoculate one loopful of enrichment culture on each of two enterobacter sakazakii chromogenic medium plates by streaking; the chromogenic medium must meet the requirements of GB 4789.28; incubate for 24h±2h at 36℃±1℃ or under the condition required for the medium.
5.2.2 Pick at least 5 suspected colonies (or all suspected colonies if less than 5) to inoculate on TSA plate by streaking, and incubate for 48h±4h at 25℃±1℃.
5.3 Identification
Directly pick yellow suspected colonies from TSA plate, for biochemical identification. The major biochemical characteristics of Cronobacter are detailed in Table 1. Biochemical identification kit or full-automatic microbe biochemical identification system is optional.
Table 1 Major Biochemical Characteristics of Cronobacter
Biochemical test Characteristic
Generation of yellow pigment +
Oxidase -
L-lysine decarboxylase -
L-ornithine decarboxylase (+)
L-arginine double hydrolase +
Citric acid hydrolysis (+)
Fermentation D-sorbitol (-)
L-rhamnose +
D-sucrose +
D-melibiose +
Amygdalin +
Note: +>99%, positive; ->99%, negative; (+) 90%~99%, positive; (-) 90%~99%, negative.
6 Result and Report
Report as Cronobacter detected and not detected in 100g(mL) of sample, comprehensively considering the colony shape and biochemical characteristics.
Method II Cronobacter Counting
7 Operation Steps
7.1 Dilution of sample
7.1.1 Solid and semi-solid sample: weigh 100g, 10g and 1g of samples aseptically and respectively add them into 900mL, 90mL and 9mL of BPW preheated to 44℃; gently shake to dissolve the solutions sufficiently to prepare into 1:10 homogeneous sample solutions and incubate for 18h±2h at 36℃±1℃. Transfer 1mL from each solution to inoculate in 10mL of mLST-Vm broth and incubate for 24h±2h at 44℃±0.5℃.
7.1.2 Liquid sample: weigh 100mL, 10mL and 1mL of samples with aseptic pipette and respectively add them into 900mL, 90mL and 9mL of BPW preheated to 44℃; gently shake to dissolve the solutions sufficiently to prepare into 1:10 homogeneous sample solutions and incubate for 18h±2h at 36℃±1℃. Transfer 1mL from each solution to inoculate in 10mL of mLST-Vm broth and incubate for 24h±2h at 44℃±0.5℃.
7.2 Isolation and identification
See 5.2 and 5.3.
8 Result and Report
Report the MPN of Cronobacter (see Table B.1) in 100g(mL) of sample according to the number of confirmed Cronobacter-positive tubes by consulting MPN retrieval table, in combination with the colony shape and biochemical characteristics.
Foreword i
1 Scope
2 Apparatus and Materials
3 Medium and Reagent
4 Examination Procedure
5 Operation Steps
6 Result and Report
7 Operation Steps
8 Result and Report
Appendix A Media and Reagents
Appendix B Most Probable Number (MPN) of Cronobacter
1 Scope
This standard specifies the method for examination of Cronobacter in food.
This standard is applicable to the examination of Cronobacter in formula food for infant and young children, milk and milk products as well as in the raw material.
2 Apparatus and Materials
In addition to the apparatus for conventional sterilization and cultivation in microbiological laboratory, other apparatus and materials are as follows:
2.1 Constant temperature incubator: 25℃±1℃, 36℃±1℃, 44℃±0.5℃.
2.2 Refrigerator: 2℃~5℃.
2.3 Thermostatic water bath: 44℃±0.5℃.
2.4 Balance: with a sensibility of 0.1g.
2.5 Homogenizer.
2.6 Oscillator.
2.7 Aseptic pipette: 1mL (with scale division of 0.01mL), 10mL (with scale division of 0.1mL) or micropipettor and pipette tip.
2.8 Aseptic conical flask: 100mL, 200mL, 2000mL.
2.9 Aseptic culture dish: 90mm in diameter.
2.10 PH meter or pH colorimetric tube or precise pH paper .
2.11 Full-automatic microbe biochemical identification system.
3 Media and Reagents
3.1 Buffer peptone water (BPW): see A.1.
3.2 Modified lauryl sulfate tryptose broth-vancomycin medium (mLST-Vm): see A.2.
3.3 Enterobacter sakazakii 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 double hydrolase medium: see A.7.
3.10 Carbohydrate fermentation medium: see A.8.
3.11 Simmons citrate agar: see A.9.
Method I Qualitative Examination of Cronobacter
4 Examination Procedure
See Figure 1 for the examination procedure of Cronobacter.
Figure 1 Cronobacter Examination Procedure
5 Operation Steps
5.1 Pre-enrichment and enrichment
Take 100g(mL) of sample into the sterilized conical flask; add 900mL of buffer peptone water preheated to 44℃; shake gently with hand to dissolve the solution sufficiently and then incubate for 18h±2h at 36℃±1℃. Transfer 1mL to inoculate in 10mL of mLST-Vm broth and incubate for 24h±2h at 44℃±0.5℃.
5.2 Isolation
5.2.1 Gently mix mLST-Vm broth culture well, and respectively inoculate one loopful of enrichment culture on each of two enterobacter sakazakii chromogenic medium plates by streaking; the chromogenic medium must meet the requirements of GB 4789.28; incubate for 24h±2h at 36℃±1℃ or under the condition required for the medium.
5.2.2 Pick at least 5 suspected colonies (or all suspected colonies if less than 5) to inoculate on TSA plate by streaking, and incubate for 48h±4h at 25℃±1℃.
5.3 Identification
Directly pick yellow suspected colonies from TSA plate, for biochemical identification. The major biochemical characteristics of Cronobacter are detailed in Table 1. Biochemical identification kit or full-automatic microbe biochemical identification system is optional.
Table 1 Major Biochemical Characteristics of Cronobacter
Biochemical test Characteristic
Generation of yellow pigment +
Oxidase -
L-lysine decarboxylase -
L-ornithine decarboxylase (+)
L-arginine double hydrolase +
Citric acid hydrolysis (+)
Fermentation D-sorbitol (-)
L-rhamnose +
D-sucrose +
D-melibiose +
Amygdalin +
Note: +>99%, positive; ->99%, negative; (+) 90%~99%, positive; (-) 90%~99%, negative.
6 Result and Report
Report as Cronobacter detected and not detected in 100g(mL) of sample, comprehensively considering the colony shape and biochemical characteristics.
Method II Cronobacter Counting
7 Operation Steps
7.1 Dilution of sample
7.1.1 Solid and semi-solid sample: weigh 100g, 10g and 1g of samples aseptically and respectively add them into 900mL, 90mL and 9mL of BPW preheated to 44℃; gently shake to dissolve the solutions sufficiently to prepare into 1:10 homogeneous sample solutions and incubate for 18h±2h at 36℃±1℃. Transfer 1mL from each solution to inoculate in 10mL of mLST-Vm broth and incubate for 24h±2h at 44℃±0.5℃.
7.1.2 Liquid sample: weigh 100mL, 10mL and 1mL of samples with aseptic pipette and respectively add them into 900mL, 90mL and 9mL of BPW preheated to 44℃; gently shake to dissolve the solutions sufficiently to prepare into 1:10 homogeneous sample solutions and incubate for 18h±2h at 36℃±1℃. Transfer 1mL from each solution to inoculate in 10mL of mLST-Vm broth and incubate for 24h±2h at 44℃±0.5℃.
7.2 Isolation and identification
See 5.2 and 5.3.
8 Result and Report
Report the MPN of Cronobacter (see Table B.1) in 100g(mL) of sample according to the number of confirmed Cronobacter-positive tubes by consulting MPN retrieval table, in combination with the colony shape and biochemical characteristics.
Contents of GB 4789.40-2016
Foreword i
1 Scope
2 Apparatus and Materials
3 Medium and Reagent
4 Examination Procedure
5 Operation Steps
6 Result and Report
7 Operation Steps
8 Result and Report
Appendix A Media and Reagents
Appendix B Most Probable Number (MPN) of Cronobacter
