GB 4789.40-2016

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GB 4789.40-2016	English	70	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

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.
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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.