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GB 4789.40-2024English260 Add to Cart 0-9 seconds. Auto delivery. National Food Safety Standards--Food Microbiological Testing--Cronobacter Testing GB 4789.40-2024 Valid GB 4789.40-2024
GB 4789.40-2016English70 Add to Cart 0-9 seconds. Auto delivery. National Food Safety Standard -- Food Microbiological Examination -- Examination of Cronobacter (Enterobacter Sakazakii) GB 4789.40-2016 Valid GB 4789.40-2016
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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 ......


GB 4789.40-2016 GB NATIONAL STANDARD OF THE PEOPLE’S REPUBLIC OF CHINA National Food Safety Standard – Food Microbiological Examination – Examination of Cronobacter (Enterobacter Sakazakii) ISSUED ON. DECEMBER 23, 2016 IMPLEMENTED ON. JUNE 23, 2017 Issued by. National Health and Family Planning Commission of the People 's Republic of China; China Food and Drug Administration. 3. No action is required - Full-copy of this standard will be automatically & immediately delivered to your EMAIL address in 0~60 minutes. Table of Contents Foreword ... 3  1 Scope ... 4  2 Equipment and materials ... 4  3 Medium and reagent ... 5  Method One Qualitative test of Cronobacter ... 5  4 Examination procedures ... 5  5 Operation steps ... 6  6 Result and report ... 7  7 Operation steps ... 8  8 Result and report ... 8  Annex A Medium and reagent ... 9  Annex B The most probable number (MPN) search table for Cronobacter ... 14  Foreword This Standard replaces GB 4789.40-2010 National food safety standard Food microbiological examination. Enterobacter sakazakii, SN/T 1632.1-2013 Detection of enterobacter sakazakii from dehydrated powdered milk for export - Part 1. Isolation and enumeration. Compared with GB 4789.40-2010, the main changes in this Standard are as follows. - modified the standard’s name to “National Food Safety Standard - Food Microbiological Examination - Examination of Cronobacter (Enterobacter Sakazakii)”; - modified the number of suspicious colonies picked. National Food Safety Standard – Food Microbiological Examination – Examination of Cronobacter (Enterobacter Sakazakii) 1 Scope This Standard specifies the examination method for Cronobacter in food. This Standard applies to the examination of Cronobacter in infant formula, milk and dairy products and their raw materials. 2 Equipment and materials In addition to microbial laboratory routine sterilization and training equipment, other equipment and materials are as follows. 2.1 Thermostat incubator. 25°C ± 1°C, 36°C ± 1°C, 44°C ± 0.5°C 2.2 Refrigerator. 2°C ~ 5°C 2.3 Constant temperature water bath. 44°C ± 0.5°C 2.4 Balance. resolution of 0.1 g 2.5 Homogenizer 2.6 Oscillator 2.7 Sterile pipettes. 1 mL (with 0.01 mL scale), 10 mL (with 0.1 mL scale) or micro pipette and suction head 2.8 Sterile conical flask. capacity of 100 mL, 200 mL, 2000 mL 2.9 Sterile Petri dish. 90 m in diameter 2.10 pH meter or pH colorimetric tube or precision pH test paper 2.11 Automatic microbial biochemical identification system 3 Medium and reagent 3.1 Buffer peptone water. 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 bishydrolase medium. see A.7 3.10 Carbohydrate fermentation medium. see A.8 3.11 Citrus citrate medium. see A.9 Method One Qualitative test of Cronobacter 4 Examination procedures See Figure a for the examination procedures of Cronobacter. 7 Operation steps 7.1 Sample dilution 7.1.1 Solid and semi-solid samples. sterilely weigh 100 g, 10 g, 1g of samples, respectively add into 900 mL, 90 mL, 9 mL of BPW that have been preheated to 44°C. Gently shake to fully dissolved, make 1.10 sample homogenizing solution, culture at 36°C ± 1°C for 18h ± 2h. Respectively transfer 1 mL to inoculate in 10 mL LmST-Vm broth, culture at 44°C ± 0.5°C for 24h ± 2h. 7.1.2 Liquid sample. use a sterile pipette to take 100 mL, 10 mL, 1 mL of samples, respectively add into 900 mL, 90 mL, 9 mL of BPW that have been preheated to 44°C. Gently shake to fully dissolved, make 1.10 sample homogenizing solution, culture at 36°C ± 1°C for 18h ± 2h. Respectively transfer 1 mL to inoculate in 10 mL LmST-Vm broth, culture at 44°C ± 0.5°C for 24h ± 2h. 7.2 Separation, identification Same with 5.2 and 5.3. 8 Result and report Combining the colony morphology, biochemical characteristics and according to the confirmed number of positive tubes of Cronobacter, check MPN search table and report the MPN value of Cronobacter per 100 g (mL) of sample (see Table B.1). sterilization. The vancomycin solution can be stored at 0°C ~ 5°C for 15 d. A.2.3 Modified lauryl sulfate tryptose broth - vancomycin medium, mLST-Vm Add 0.1 mL of vancomycin solution into per 10 mL of mLST. The final concentration of vancomycin in the mixture is 10 μg/mL. NOTE. mLST-Vm must be used within 24h. A.3 Tryptone soy agar (T S A) A.3.1 Composition Tryptone 15.0 g Plant peptone 5.0 g Sodium chloride 5.0 g Agar 15.0 g Distilled water 1000 mL A.3.2 Preparation method Heating and stirring to dissolved. Boiling for 1 min. Adjust pH to 7.3 ± 0.2. Autoclave at 121°C for 15 min. A.4 Oxidase reagent A.4.1 Composition N, N, N ', N' - tetramethylphenylene diamine hydrochloride 1.0 g Distilled water 1000 mL A.4.2 Preparation method Make a small amount of fresh preparation. Store in the refrigerator from light. Use within 7d. A.4.3 Test method Use a glass rod or a disposable inoculation needle to pick up a single characteristic colony and coat it on a filter paper plate moistened with an oxidase reagent. If the filter paper does not become magenta, purple or dark blue within 10s, the oxidase test is negative, otherwise the oxidase test is positive. NOTE. Do not use nickel/chromium material in the experiment. A.5 L-lysine decarboxylase medium A.5.1 Composition A.7.2 Preparation method Heat and dissolve the components, and if necessary, adjust pH to 6.8 ± 0.2. Sub-packaging 5 mL for each tube. Autoclave at 121°C for 15 min. A.7.3 Experimental method Inoculate the culture into the L-arginine decarboxylase medium, just under the liquid level of the liquid medium. Culture at 30°C ± 1°C for 24h ± 2h. Observe the results. L-arginine decarboxylase test is positive; the medium is purple; the negative is yellow. A.8 Carbohydrate fermentation medium A.8.1 Basal medium A.8.1.1 Composition Casein (enzyme digestion) 10.0 g Sodium chloride 5.0 g Phenol red 0.02 g Distilled water 1000 mL A.8.1.2 Preparation method Heat and dissolve the components, and if necessary, adjust pH to 6.8 ± 0.2. Sub-packaging 5 mL for each tube. Autoclave at 121°C for 15 min. A.8.2 Sugar solution (D-sorbitol, L-rhamnose, D-sucrose, D-melibiose, amygdalin) A.8.2.1 Composition Sugar 8.0 g Distilled water 100 mL A.8.2.2 Preparation method Respectively weigh 8 g of D-sorbitol, L-rhamnose, D-sucrose, D-honey disaccharide, amygdalin and dissolve into 100 mL of distilled water. Filter for sterilization, make 80 mg/mL sugar solution. A.8.3 Complete medium A.8.3.1 Composition Basal medium 875 mL Sugar solution 125 mL A.8.3.2 Preparation method Aseptically add each saccharide solution to the basal medium and mix well. ......


GB 4789.40-2010 National food safety standard.Food microbiological examination. Enterobacter sakazakii National Standards of People's Republic of China National Food Safety Standard Microbiological examination of food inspection Enterobacter sakazakii National food safety standard Food microbiological examination. Enterobacter sakazakii People's Republic of China Ministry of Health issued Issued on. 2010-03-26 2010-06-01 implementation Foreword This standard replaces GB/T 4789.40-2008 "Microbiological examination of food hygiene inspection Enterobacter sakazakii." This standard compared with GB/T 4789.40-2008, main changes are as follows. - Modify the standard English name; - Removed Appendix A A.3. This standard Annex A, Annex B normative appendix. This standard replaces the standards previously issued as follows. --GB/T 4789.40-2008. National Food Safety Standard Microbiological examination of food inspection Enterobacter sakazakii 1 Scope This standard specifies the food Enterobacter sakazakii (Enterobacter sakazakii) test method. This standard applies to infant formula, milk and milk products and raw materials Enterobacter sakazakii test. 2 Equipment and Materials In addition to the microbiological laboratory conventional sterilization and cultivation equipment, other equipment and materials as follows. 2.1 incubator. 25 ℃ ± 1 ℃, 36 ℃ ± 1 ℃, 44 ℃ ± 0.5 ℃. 2.2 Refrigerator. 2 ℃ ~ 5 ℃. 2.3 constant temperature water bath. 44 ℃ ± 0.5 ℃. 2.4 Balance. a sense of the amount of 0.1 g. 2.5 homogenizer. 2.6 oscillator. 2.7 sterile pipette. 1 mL (0.01 mL with scale), 10 mL (0.1 mL with scale) or micro pipettes and tips. 2.8 sterile conical flask. capacity 100 mL, 200 mL, 2000 mL. 2.9 sterile Petri dish. a diameter of 90 mm. 2.10 pH meter or pH colorimetric tubes or precision pH test paper. 2.11 automatic biochemical microbial identification system. 3 media and reagents 3.1 buffered peptone water (buffer peptone water, BPW). See Appendix A, A.1. 3.2 Modified lauryl sulfate tryptone broth - vancomycin (modified lauryl sulfate tryptose broth-vancomycin medium, mLST-Vm). See Appendix A A.2. 3.3 Enterobacter sakazakii chromogenic medium. 3.4 tryptone soy agar (trypticase soy agar, TSA). See Appendix A A.3. 3.5 biochemical identification kits. 3.6 oxidase reagent. See Appendix A A.4. 3.7 L- lysine decarboxylase medium. See Appendix A, A.5. 3.8 L- ornithine decarboxylase medium. See Appendix A A.6. 3.9 L- Arginine double hydrolase medium. See Appendix A A.7. 3.10 carbohydrate fermentation medium. See Appendix A A.8. 3.11 Simon's citrate medium. See Appendix A A.9. 1 mL mLST-Vm 10 mL Suspected colonies were picked The first method of testing for E. sakazakii 4 inspection procedures Enterobacter sakazakii test procedure shown in Figure 1. 1 E. sakazakii testing procedures 5 steps 5.1 pre-enrichment and enrichment Take test sample 100 g (mL) was added preheated to 44 ℃ containing 900 mL buffered peptone water Erlenmeyer flask to shake his hand slowly Fully dissolved, 36 ℃ ± 1 ℃ cultured 18 h ± 2 h. Pipette 1 mL transferred species in 10 mL mLST-Vm broth, 44 ℃ ± 0.5 ℃ cultured 24 h ± 2 h. 5.2 Separation 5.2.1 Mix gently mLST-Vm broth culture enrichment culture depicting a ring were streaked two Enterobacter sakazakii chromogenic 36 ℃ ± 1 ℃, 18 h ± 2 h report TSA Biochemical identification Yellow colonies were picked Test sample 100 g (mL) BPW dilution of 900 mL 44 ℃ ± 0.5 ℃, 24 h ± 2 h Enterobacter sakazakii chromogenic medium 36 ℃ ± 1 ℃, 24 h ± 2 h 25 ℃ ± 1 ℃, 48 h ± 4 h Medium plate, 36 ℃ ± 1 ℃ cultured 24 h ± 2 h. 5.2.2 picked one to five suspect colonies streaked on TSA plates. 25 ℃ ± 1 ℃ cultured 48 h ± 4 h. 5.3 Identification Yellow suspected colonies were picked directly from the TSA plates, biochemical identification. The main biochemical characteristics of E. sakazakii in Table 1. Optional Optional biochemical identification kits or automated microbial and biochemical identification system. Table 1 Main biochemical characteristics of E. sakazakii Biochemical features Yellow pigment produced + Oxidase - L- lysine decarboxylase - L- ornithine decarboxylase (+) L- arginine hydrolase double + Citric acid hydrolysis (+) D- sorbitol (-) L- rhamnose + Sucrose + D- D- melibiose + Ferment Amygdalin + Note. +> 99% positive; -> 99% negative; (+) positive 90% -99%; (-) 90% -99% negative. 6 Results and reporting Comprehensive colony morphology and biochemical characteristics, report every 100 g (mL) sample is detected or not detected E. sakazakii. The second method of counting E. sakazakii 7 steps 7.1 sample dilution 7.1.1 solid and semi-solid samples. a sterile sample weighed 100 g, 10 g, 1 g each of the three, has added preheated to 44 ℃, respectively containing 900 mL, 90 mL, 9 mL BPW gently shaking so fully dissolved to prepare a uniform liquid sample 1.10, set 36 ℃ ± 1 ℃ cultured 18 h ± 2 h. respectively Pipette 1 mL transferred species in 10 mL mLST-Vm broth, 44 ℃ ± 0.5 ℃ cultured 24 h ± 2 h. 7.1.2 Liquid samples. samples were taken with a sterile pipette 100 mL, 10 mL, 1 mL in triplicate, adding preheated to 44 ℃, respectively Sheng There are 900 mL, 90 mL, 9 mL BPW gently shaking so thoroughly mixed to prepare a uniform liquid sample 1.10, set 36 ℃ ± 1 ℃ culture 18h ± 2h. Pipette 1 mL transferred species in 10 mL mLST-Vm broth, 44 ℃ ± 0.5 ℃ cultured 24 h ± 2 h, respectively. 7.2 Isolation, Identification With 5.2, 5.3. 8 Results and Reports Comprehensive colony morphology, biochemical characteristics, according to the number of confirmed positive for E. sakazakii tube, check MPN table retrieval, report every 100 g (mL) Samples E. sakazakii MPN value (see Appendix B, Table B.1). Appendix A (Normative) Media and reagents A.1 buffered peptone water (the BPW) A.1.1 ingredients Peptone 10.0g Sodium chloride 5.0 g Disodium hydrogen phosphate (Na2HPO4 · 12H2O) 9.0 g 1.5 g of potassium dihydrogen phosphate 1 000 mL of distilled water pH 7.2 A.1.2 Method Heating and stirring until dissolved, adjusting the pH, 121 ℃ autoclave 15 min. A.2 modified lauryl sulfate tryptone broth - vancomycin (Modified lauryl sulfate tryptose broth-vancomycin medium, mLST-Vm) A.2.1 modified lauryl sulfate tryptone (MLST) broth A.2.1.1 ingredient Sodium chloride 34.0 g 20.0 g tryptone Lactose 5.0 g 2.75 g of potassium dihydrogen phosphate Dipotassium hydrogen phosphate 2.75 g Sodium lauryl sulfate 0.1 g 1 000 mL of distilled water pH 6.8 ± 0.2 A.2.1.2 Method Heating and stirring until dissolved, adjusting the pH. Aliquot each tube 10 mL, 121 ℃ autoclaving 15 min. A.2.2 vancomycin solution A.2.2.1 ingredient Vancomycin 10.0 mg 10.0 mL of distilled water A.2.2.2 Method 10.0 mg vancomycin dissolved in 10.0 mL of distilled water, filter sterilized. Vancomycin solution can be stored at 0 ℃ ~ 5 ℃ 15 days. A.2.3 modified lauryl sulfate tryptone broth - vancomycin (Modified lauryl sulfate tryptose broth-vancomycin medium, mLST-Vm) Each 10 mL mLST vancomycin was added a solution of 0.1 mL, a mixture of vancomycin final concentration of 10 μg/mL. Note. mLST-Vm must be used within 24 h of. A.3 Tryptone Soya Agar (TSA) A.3.1 ingredients 15.0 g tryptone 5.0 g of plant peptone Sodium chloride 5.0 g Agar 15.0 g 1 000 mL of distilled water pH 7.3 ± 0.2 A.3.2 Method Heating and stirring until dissolved, boiled for 1 min, adjusting the pH, 121 ℃ high pressure 15 min. A.4 oxidase reagent A.4.1 ingredients N, N, N ', N'- tetramethyl-p-phenylenediamine hydrochloride 1.0 g 100 mL of distilled water A.4.2 Method A small amount of freshly prepared, stored in the dark in the refrigerator, use within 7 d of. A.4.3 Test Method With a glass rod or disposable inoculating needle single characteristic colonies were picked and plated oxidase reagent moistened filter paper plates. If the filter paper No changes within 10 s of fuchsia, purple or dark blue, oxidase test was negative, otherwise it is oxidase test positive. NOTE. Do not use the experiment nickel/chromium materials. A.5 L- lysine decarboxylase medium A.5.1 ingredients L- lysine hydrochloride (L-lysine monohydrochloride) 5.0 g Yeast extract 3.0 g Glucose 1.0 g Bromocresol purple 0.015 g 1 000 mL of distilled water pH 6.8 ± 0.2 A.5.2 Method The ingredients are heated and dissolved, adjusting the pH if necessary. Each tube dispensing 5 mL, 121 ℃ high-pressure 15 min. A.5.3 Experimental Method Were picked and inoculated into culture medium L- lysine decarboxylase, just below the surface of the liquid medium. 30 ℃ ± 1 ℃ cultured 24 h ± 2 h, Observations. L- lysine decarboxylase test positive, medium purple, yellow negative. A.6 L- ornithine decarboxylase medium A.6.1 ingredients L- ornithine hydrochloride (L-ornithine monohydrochloride) 5.0 g Yeast extract 3.0 g Glucose 1.0 g Bromocresol purple 0.015 g 1 000 mL of distilled water pH 6.8 ± 0.2 A.6.2 Method The ingredients are heated and dissolved, adjusting the pH if necessary. Each tube dispensing 5 mL. 121 ℃ high-pressure 15 min. A.6.3 Experimental Method Picked culture was inoculated in L- ornithine decarboxylase medium, just under the level of the liquid medium. 30 ℃ ± 1 ℃ cultured 24 h ± 2 h, Observations. L- ornithine decarboxylase test positive, medium purple, yellow negative. A.7 L- arginine hydrolase dual medium A.7.1 ingredients L- arginine hydrochloride (L-arginine monohydrochloride) 5.0 g Yeast extract 3.0 g Glucose 1.0 g Bromocresol purple 0.015 g 1 000 mL of distilled water pH 6.8 ± 0.2 A.7.2 Method The ingredients are heated and dissolved, adjusting the pH if necessary. Each tube dispensing 5 mL. 121 ℃ high-pressure 15 min. A.7.3 Experimental Method Picked culture was inoculated in L- arginine decarboxylase medium, just under the level of the liquid medium. 30 ℃ ± 1 ℃ cultured 24 h ± 2 h, Observations. L- arginine decarboxylase test positive, medium purple, yellow negative. A.8 carbohydrate fermentation medium A.8.1 basal medium A.8.1.1 ingredient Casein (enzymatic digestion) 10.0 g Sodium chloride 5.0 g Phenol red 0.02 g 1 000 mL of distilled water pH 6.8 ± 0.2 A.8.1.2 Method The ingredients are heated and dissolved, adjusting the pH if necessary. Each tube dispensing 5 mL. 121 ℃ high-pressure 15 min. A.8.2 sugar solution (D- sorbitol, L- rhamnose, D- sucrose, D- melibiose, amygdalin) A.8.2.1 ingredient Sugar 8.0 g 100 mL of distilled water A.8.2.2 Method Were weighed D- sorbitol, L- rhamnose, D- sucrose, D- melibiose, sugars such as amygdalin component for each 8 g, was dissolved in 100 mL distilled Distilled water, filter-sterilized to prepare 80 mg/mL solution of saccharide. A.8.3 complete medium A.8.3.1 ingredient Basal Medium 875 mL Sugar solution 125 mL A.8.3.2 Method Aseptic Each saccharide solution was added to a basal medium, and mix; dispensing into sterile tubes, each tube 10 mL. A.8.4 Experimental Method Picked culture was inoculated at various sugars fermentation medium, just under the level of the liquid medium. 30 ℃ ± 1 ℃ cultured 24 h ± 2 h, Observations. Sugar fermentation test positive, medium yellow, red negative. A.9 Simon's citrate medium A.9.1 ingredients 2.0 g of sodium citrate Sodium chloride 5.0 g Dipotassium hydrogen phosphate 1.0 g Ammonium dihydrogen phosphate 1.0 g 0.2 g of magnesium sulfate Bromine thymol blue 0.08 g Agar 8.0 g ~ 18.0 g 1 000 mL of distilled water pH 6.8 ± 0.2 A.9.2 Method The ingredients are heated and dissolved, adjusting the pH if necessary. Each tube Aliquot 10 mL, 121 ℃ high-pressure 15 min, beveled. A.9.3 Experimental Method Picked the whole culture was inoculated medium slant, 36 ℃ ± 1 ℃ cultured 24 h ± 2 h, observe the results. Positive medium blue. Appendix B (Normative) Enterobacter sakazakii most probable number (MPN) retrieval table B.1 Enterobacter sakazakii most probable number (MPN) retrieval table Per 100 g (mL) check Enterobacter sakazakii most probable number (MPN) to retrieve samples are shown in Table B.1. Table B.1 Enterobacter sakazakii most probable number (MPN) retrieval table Positive tubes 95% confidence limit of the number of positive tubes 95% confidence limits MPN Lower limit Upper limit 100 101 MPN The lower limit 000 < 0.3 - 0.95 0.45 4.2 2.1 220 001 0.3 0.015 0.96 2.8 221 0.87 9.4 010 0.3 1.1 222 3.5 0.015 0.87 9.4 011 230 1.8 0.61 0.12 2.9 0.87 9.4 020 231 1.8 0.62 0.12 3.6 0.87 9.4 030 0.94 0.36 3.8 300 0.46 2.3 9.4 100 0.36 0.017 1.8 301 3.8 0.87 11 101 0.72 0.13 1.8 302 6.4 1.7 18 102 0.36 1.1 3.8 310 4.3 0.9 18 110 0.74 0.13 2311 7.5 1.7 20 0.36 111 1.1 3.8 31 212 3.7 42 120 1.1 0.36 4.2 31,316,442 0.45 121 1.5 4.2 320 9.3 1.8 42 130 1.6 0.45 4.2 32 115 3.7 42 200 0.92 0.14 3.8 32,221,443 201 1.4 0.36 4.2 323,299,100 0.45 4.2 2022 33 024 4.2 100 210 1.5 0.37 4.2 331,469,200 2112 0.45 4.2 33,211,018,410 212 2.7 0.87 9.4 333> 11042-- Note 1. This table uses three test sample volume [100 g (mL), 10 g (mL) and 1 g (mL)], each test sample inoculated three. Note 2. The amount of test sample listed in the table, such as switching to 1 000 g (mL), 10 g (mL), and 1 g (mL), the figures in the table should be reduced 10 times; such as switching 10 g (mL), 1 g (mL), and 0.1 g (mL), then the number should be increased accordingly within the table 10 times, rest on. ......

BASIC DATA
Standard ID GB 4789.40-2024 (GB4789.40-2024)
Description (Translated English) National Food Safety Standards--Food Microbiological Testing--Cronobacter Testing
Sector / Industry National Standard
Word Count Estimation 16,169
Date of Issue 2024/2/8

BASIC DATA
Standard ID GB 4789.40-2016 (GB4789.40-2016)
Description (Translated English) National Food Safety Standard -- Food Microbiological Examination -- Examination of Cronobacter (Enterobacter Sakazakii)
Sector / Industry National Standard
Classification of Chinese Standard X09
Word Count Estimation 12,155
Date of Issue 2016-12-23
Date of Implementation 2017-06-23
Older Standard (superseded by this standard) GB 4789.40-2010; SN/T 1632.1-2013
Regulation (derived from) National Health and Family Planning Commission Notice No.17 of 2016

BASIC DATA
Standard ID GB 4789.40-2010 (GB4789.40-2010)
Description (Translated English) National food safety standard. Food microbiological examination: Enterobacter sakazakii
Sector / Industry National Standard
Classification of Chinese Standard C53
Classification of International Standard 07.100.30
Word Count Estimation 10,183
Date of Issue 2010-03-26
Date of Implementation 2010-06-01
Older Standard (superseded by this standard) GB/T 4789.40-2008
Regulation (derived from) Circular of the Ministry of Health (2010)7
Issuing agency(ies) Ministry of Health of People's Republic of China
Summary This Chinese standard specifies the food sakazakii (Enterobacter sakazakii) test methods. This standard applies to infant formula, milk and dairy products and raw materials inspection Enterobacter sakazakii.