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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.
......
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 | 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 | 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. |
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