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食品安全国家标准 食品微生物学检验 克罗诺杆菌属(阪崎肠杆菌)检验
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GB 4789.40-2016
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标准编号: GB 4789.40-2016 (GB4789.40-2016)
中文名称: 食品安全国家标准 食品微生物学检验 克罗诺杆菌属(阪崎肠杆菌)检验
英文名称: National Food Safety Standard -- Food Microbiological Examination -- Examination of Cronobacter (Enterobacter Sakazakii)
行业: 国家标准
中标分类: X09
字数估计: 12,189
发布日期: 2016-12-23
实施日期: 2017-06-23
旧标准 (被替代): GB 4789.40-2010; SN/T 1632.1-2013
标准依据: National Health and Family Planning Commission Notice No.17 of 2016
GB 4789.40-2016: 食品安全国家标准 食品微生物学检验 克罗诺杆菌属(阪崎肠杆菌)检验
GB 4789.40-2016 英文名称: National Food Safety Standard -- Food Microbiological Examination -- Examination of Cronobacter (Enterobacter Sakazakii)
1 范围
本标准规定了食品中克罗诺杆菌属(Cronobacter)的检验方法。
本标准适用于婴幼儿配方食品、乳和乳制品及其原料中克罗诺杆菌属的检验。
2 设备和材料
除微生物实验室常规灭菌及培养设备外,其他设备和材料如下:
2.1 恒温培养箱:25℃±1℃,36℃±1℃,44℃±0.5℃。
2.2 冰箱:2℃~5℃。
2.3 恒温水浴箱:44℃±0.5℃。
2.4 天平:感量0.1g。
2.5 均质器。
2.6 振荡器。
2.7 无菌吸管:1mL(具0.01mL刻度)、10mL(具0.1mL刻度)或微量移液器及吸头。
2.8 无菌锥形瓶:容量100mL、200mL、2000mL。
2.9 无菌培养皿:直径90mm。
2.10 pH计或pH比色管或精密pH试纸。
2.11 全自动微生物生化鉴定系统。
3 培养基和试剂
3.1 缓冲蛋白胨水:见A.1。
3.2 改良月桂基硫酸盐胰蛋白胨肉汤-万古霉素
3.3 阪崎肠杆菌显色培养基。
3.4 胰蛋白胨大豆琼脂(trypticasesoyagar,TSA):见A.3。
3.5 生化鉴定试剂盒。
3.6 氧化酶试剂:见A.4。
3.7 L-赖氨酸脱羧酶培养基:见A.5。
3.8 L-鸟氨酸脱羧酶培养基:见A.6。
3.9 L-精氨酸双水解酶培养基:见A.7。
3.10 糖类发酵培养基:见A.8。
3.11 西蒙氏柠檬酸盐培养基:见A.9。
第一法 克罗诺杆菌属定性检验
4 检验程序
克罗诺杆菌属检验程序见图1。
图1 克罗诺杆菌属检验程序
5 操作步骤
5.1 前增菌和增菌
取检样100g(mL)置灭菌锥形瓶中,加入900mL已预热至44℃的缓冲蛋白胨水,用手缓缓地摇
动至充分溶解,36℃±1℃培养18h±2h。移取1mL转种于10mLmLST-Vm肉汤,44℃±0.5℃
培养24h±2h。
5.2 分离
5.2.1 轻轻混匀mLST-Vm肉汤培养物,各取增菌培养物1环,分别划线接种于两个阪崎肠杆菌显色
培养基平板,显色培养基须符合GB 4789.28的要求,36℃±1℃培养24h±2h,或按培养基要求条件
培养。
5.2.2 挑取至少5个可疑菌落,不足5个时挑取全部可疑菌落,划线接种于TSA平板。25℃±1℃培
养48h±4h。
5.3 鉴定
自TSA平板上直接挑取黄色可疑菌落,进行生化鉴定。克罗诺杆菌属的主要生化特征见表1。可
选择生化鉴定试剂盒或全自动微生物生化鉴定系统。
6 结果与报告
综合菌落形态和生化特征,报告每100g(mL)样品中检出或未检出克罗诺杆菌属。
第二法 克罗诺杆菌属的计数
7 操作步骤
7.1 样品的稀释
7.1.1 固体和半固体样品:无菌称取样品100g、10g、1g各三份,分别加入900mL、90mL、9mL已预
热至44℃的BPW,轻轻振摇使充分溶解,制成1∶10样品匀液,置36℃±1℃培养18h±2h。分别移
取1mL转种于10mLmLST-Vm肉汤,44℃±0.5℃培养24h±2h。
7.1.2 液体样品:以无菌吸管分别取样品100mL、10mL、1mL各三份,分别加入900mL、90mL、
9mL已预热至44℃的BPW,轻轻振摇使充分混匀,制成1∶10样品匀液置36℃±1℃培养18h±
2h。分别移取1mL转种于10mLmLST-Vm肉汤,44℃±0.5℃培养24h±2h。
7.2 分离、鉴定
同5.2和5.3。
8 结果与报告
综合菌落形态、生化特征,根据证实为克罗诺杆菌属的阳性管数,查 MPN检索表,报告每100g
(mL)样品中克罗诺杆菌属的 MPN值(见表B.1)。
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
3.10 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
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.
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
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
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.
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
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
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
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
Aseptically add each saccharide solution to the basal medium and mix well.
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