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食品安全国家标准 食品微生物学检验 肠杆菌科检验
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GB 4789.41-2016
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标准编号: GB 4789.41-2016 (GB4789.41-2016)
中文名称: 食品安全国家标准 食品微生物学检验 肠杆菌科检验
英文名称: National Food Safety Standard -- Food Microbiological Examination -- Enterobacteriaceae
行业: 国家标准
中标分类: C53
字数估计: 14,158
发布日期: 2016-08-31
实施日期: 2017-03-01
标准依据: 国家卫生和计划生育委员会公告2016年第11号
GB 4789.41-2016: 食品安全国家标准 食品微生物学检验 肠杆菌科检验
GB 4789.41-2016 英文名称: National Food Safety Standard -- Food Microbiological Examination -- Enterobacteriaceae
1 范围
本标准第一法适用于肠杆菌科含量较高的食品中肠杆菌科的计数;第二法适用于肠杆菌科含量较
低食品中肠杆菌科的计数。
2 术语和定义
2.1
肠杆菌科
在给定条件下发酵葡萄糖产酸、氧化酶阴性的需氧或兼性厌氧革兰氏阴性无芽胞杆菌。
2.2
肠杆菌科计数
按本标准规定方法,对每克或每毫升检样中的肠杆菌科进行计数。
2.3
最可能数
基于泊松分布的一种间接计数方法。
3 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:
3.1 恒温培养箱:36℃±1℃。
3.2 冰箱:2℃~5℃。
3.3 水浴箱:46℃±1℃。
3.4 天平:感量0.1g。
3.5 显微镜:10倍~100倍。
3.6 均质器。
3.7 振荡器。
3.8 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。
3.9 无菌锥形瓶或等效容器:容量150mL、500mL。
3.10 无菌培养皿:直径90mm。
3.11 无菌试管:18mm×180mm、15mm×150mm。
3.12 pH计或pH比色管或精密pH试纸。
4 培养基和试剂
4.1 缓冲蛋白胨水(BPW):见B.1。
4.2 缓冲葡萄糖煌绿胆盐肉汤(EE肉汤):见B.2。
4.3 结晶紫中性红胆盐葡萄糖琼脂(VRBGA):见B.3。
4.4 营养琼脂(NA):见B.4。
4.5 葡萄糖琼脂:见B.5。
4.6 革兰氏染色液:见B.6。
4.7 氧化酶试剂:见B.7。
4.8 无菌1mol/LNaOH:见B.8。
4.9 无菌1mol/LHCl:见B.9。
第一法 肠杆菌科平板计数法
5 检验程序
肠杆菌科平板计数法检验程序见图1。
6 操作步骤
6.1 样品的稀释
6.1.1 固体和半固体样品:称取25g样品放入盛有225mLBPW 的无菌均质杯中,8000r/min~
10000r/min均质1min~2min,或放入盛有225mLBPW 的无菌均质袋中,用拍击式均质器拍打
1min~2min,制成1∶10的样品匀液。
6.1.2 液体样品:以无菌吸管吸取25mL样品放入盛有225mLBPW的无菌锥形瓶(瓶内预置适当数
量的无菌玻璃珠)中,充分混匀,制成1∶10的样品匀液。
6.1.3 用1mL无菌吸管或微量移液器吸取1∶10样品匀液1mL,沿管壁缓缓注入盛有9mLBPW的
无菌试管中(注意吸管或吸头尖端不应触及稀释液面),振摇试管或换用1支1mL无菌吸管反复吹打,
使其混合均匀,制成1∶100的样品匀液。
6.1.4 按6.1.3操作程序,依次制成10倍递增系列稀释样品匀液。每递增稀释1次,换用1支1mL无
菌吸管或吸头。从制备样品匀液至样品接种完毕,全过程不得超过15min。
6.2 倾注平板和培养
6.2.1 根据对样品污染状况的估计及相关限量要求,选择2个~3个适宜的连续稀释度的样品匀液(液
体样品可以选择原液),每个稀释度接种2个无菌平皿。同时,分别吸取1mLBPW 加入两个无菌平皿
内作为空白对照。
6.2.2 将10mL~15mL冷却至46℃的VRBGA(可放置于46℃±1℃恒温水浴箱中保温)倾注于每
个平皿中。小心旋转平皿,使样品匀液与培养基充分混匀。
6.2.3 待琼脂凝固后,倾注一薄层同样的培养基覆盖平板表层。防止蔓延生长并使菌落特征更为
明显。
6.2.4 待VRBGA平板上层琼脂凝固后翻转平板,36℃±1℃培养18h~24h。
6.3 典型菌落计数和确认
6.3.1 肠杆菌科典型菌落为有或无沉淀环的粉红色至红色或紫色菌落。选取典型菌落数在15CFU~
150CFU之间、无蔓延菌落生长的平板,只计数典型菌落数。菌落计数以菌落形成单位(colony-
formingunits,CFU)表示。
6.3.2 其中一个平板有较大片状菌落生长时,则不宜采用,而应以无片状菌落生长的平板作为该稀释
度的菌落数;若片状菌落不到平板的一半,而其余一半中菌落分布又很均匀,即可计算半个平板后乘以
2,代表一个平板菌落数。
6.3.3 当平板上出现菌落间无明显界线的链状生长时,则将每条单链作为一个菌落计数。
6.3.4 从每个平板上至少挑取5个(小于5个全选)典型菌落进行确认;如果有不同形态的典型菌落,则
每种形态分别至少挑取1个菌落进行确认。
6.3.5 典型菌落的确认:
a) 分别将所挑选的每一个菌落,划线于营养琼脂平板,36℃±1℃培养18h~24h,挑取平板上
的菌落进行革兰氏染色镜检、氧化酶试验及葡萄糖发酵试验。
b) 革兰氏染色镜检:肠杆菌科为革兰氏阴性杆菌,无芽孢,大小为(0.3~1.0)μm×(1.0~
6.0)μm。
c) 氧化酶试验:用铂/铱接种环或玻璃棒(不要用镍铬接种环)挑取单个菌落涂于浸湿氧化酶试剂
的滤纸上,滤纸的颜色在10s内变成蓝紫色,判为阳性反应。
1℃培养24h±2h,若试管内的内容物变为黄色,判为阳性反应。
6.4 结果的计算
6.4.1 一般原则
若有两个连续稀释度的平板典型菌落数在适宜计数范围内,按式(1)计算,示例见 A.1、A.2、
A.3、A.4。
6.4.2 低菌落数
若最低稀释度(包括液体样品原液)平板的典型菌落数均小于15CFU,具有确证的肠杆菌科菌落,
则以确证的菌落数乘以最低稀释倍数计算。
若最低稀释度(包括液体样品原液)平板均无菌落生长,或典型菌落数均小于15CFU,且无确证的
6.4.3 特殊情况
第一稀释度平板上的典型菌落数均大于150CFU,且有确证的肠杆菌科菌落,以及第二稀释度平
板上无确证的肠杆菌科菌落或典型菌落数不在15CFU~150CFU之间,则以确证的菌落数乘以第一
稀释倍数计算,示例见A.5。
若所有稀释度的平板上典型菌落数均不在适宜计数范围内,且无确证的肠杆菌科菌落,则以小于1
乘以最低稀释倍数计算。
7 报告
7.1 菌落数小于100时,以整数报告。
7.2 菌落数大于或等于100时,对第3位数字进行修约后,取前2位数字,后面用0代替位数;也可用
7.3 数字修约按“四舍五入”原则进行。
7.4 若所有平板上为蔓延菌落而无法计数,则报告菌落蔓延。
7.5 若空白对照上有菌落生长,则此次检测结果无效。
7.6 称重取样以CFU/g为单位报告,体积取样以CFU/mL为单位报告。
第二法 肠杆菌科 MPN计数法
8 检验程序
肠杆菌科 MPN计数法检验程序见图2。
9 操作步骤
9.1 样品的稀释
9.2 接种和培养
9.2.1 非选择性前增菌
根据对样品污染状况的估计及相关限量要求,选择3个适宜连续稀释度的样品匀液,每个稀释度
3管,共9管BPW于36℃ ±1℃培养18h±2h。
9.2.2 选择性增菌
从BPW各培养管中,分别移取1mL培养物,接种于10mL的EE肉汤中,36℃±1℃培养24h±
2h。
9.2.3 分离
用接种环从EE各肉汤管中分别取培养物1环,划线接种于VRBGA平板,36℃±1℃培养24h±
9.3 典型菌落的确认
典型菌落确认见6.3。
10 报告
肠杆菌科为革兰氏阴性无芽胞杆菌,发酵葡萄糖产酸、氧化酶阴性。只要有1个菌落确认为肠杆菌
科,其所代表的EE管即为肠杆菌科阳性,依据EE阳性管数查 MPN表(见附录C),报告每克(毫升)样
品中肠杆菌科的 MPN值。称重取样以 MPN/g为单位报告,体积取样以 MPN/mL为单位报告。
GB 4789.41-2016
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
Microbiological Examination of Food Hygiene -
Examination of Enterobacteriaceae
ISSUED ON. AUGUST 31, 2016
IMPLEMENTED ON. MARCH 1, 2017
Issued by. National Health and Family Planning Commission of the
People’s Republic of China
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
1 Application Scope ... 3
2 Terms and Definitions ... 3
3 Apparatus and Materials ... 3
4 Culture Media and Reagents ... 4
5 Test Procedures ... 5
6 Operating Procedures ... 5
7 Report ... 9
8 Test Procedures ... 9
9 Operating Procedures ... 11
10 Report ... 11
Annex A Examples for Calculation of Results ... 12
Annex B Culture Media and Reagents ... 14
Annex C Retrieval Table of Enterobacteriaceae Most Probable Numbers
(MPNs) ... 18
National Food Safety Standard -
Microbiological Examination of Food Hygiene -
Examination of Enterobacteriaceae
1 Application Scope
This Standard specifies the method for the test of Enterobacteriaceae in foods.
The first method of this Standard applies to the bacterial counts of Enterobacteriaceae
in the foods which has a high content of Enterobacteriaceae; and the second method
applies to the bacterial counts of Enterobacteriaceae in the foods which has a low
content of Enterobacteriaceae.
2 Terms and Definitions
2.1
Enterobacteriaceae
The oxidase-negative aerobic or facultative anaerobic Gram-negative non-spore-
bearing bacillus, which ferments glucose producing acid under given conditions.
2.2
Enumeration of Enterobacteriaceae
The enumeration of Enterobacteriaceae per gram or per millimeter in accordance with
the method specified in this Standard.
2.3
most probable number; MPN
An indirect enumeration method based on Poisson's distribution.
3 Apparatus and Materials
In addition to the conventional sterilization and culture apparatus in the microbiological
laboratories, the other apparatus and materials are as follows.
3.1 Thermostatic incubator. 36°C ± 1°C.
3.2 Refrigerator. 2°C ~ 5°C.
3.3 Water bath. 46°C ± 1°C.
3.4 Balance. sensitivity 0.1 g.
3.5 Microscope. 10X ~ 100X.
3.6 Homogenizer.
3.7 Shaker.
3.8 Sterile pipettes. 1 ml (having graduates of 0.01 ml), 10 ml (having 0.1 ml) or
micropipettors and tips.
3.9 Sterile conical flasks or equivalent vessels. capacities 150 ml and 500 ml.
3.10 Sterile culture dishes. diameter 90 mm.
3.11 Sterile test tubes. 18 mm × 180 mm and 15 mm × 150 mm.
3.12 pH meters or pH colorimetric tubes or precise pH test paper.
4 Culture Media and Reagents
4.1 Buffered peptone water (BPW). see B.1.
4.2 Buffered glucose brilliant green bile broth (EE broth). see B.2.
4.3 Crystal violet neutral red bile salt glucose agar (VRBGA). see B.3.
4.4 Nutrient agar (NA). see B.4.
4.6 Gram stain solution. see B.6.
4.7 Oxidase reagent. see B.7.
4.8 Sterile 1 mol/l NaOH. see B.8.
4.9 Sterile 1 mol/l HCl. see B.9.
Method I The Enterobacteriaceae Plate Count
Method
6.1.1 Solid and semi-solid specimens. take 25 g of specimen to place into a sterile
homogeneous cup containing 225 ml of BPW, homogenize for 1 min ~ 2 min at 8 000
r/min ~ 10 000 r/min, or place into a homogeneous bag containing 225 ml of BPW, use
specimen solutions of 1.10.
6.1.2 Liquid specimens. use a sterile pipette to absorb 25 ml of specimen to pour into
a sterile conical flask (in which an appropriate quantity of sterile glass beads are
provided in the flask in advance) containing 225 ml of BPW, mix up, and make
homogeneous specimen solutions of 1.10.
6.1.3 Use a 1-ml sterile pipette or micropipette to absorb 1 ml of specimen
homogeneous solution of 1.10, pour slowly along the wall of the sterile test tube
containing 9 ml of BPW (attention is drawn to that the pipette or the tip shall not contact
with the diluent surface), vibrate the test tube or replace one 1-ml sterile pipette to blow
6.1.4 In accordance with the operating procedures of 6.1.3, make tenfold increasing
serial diluted specimen homogeneous solutions. For each time of increasing dilution,
replace one 1-ml sterile pipette or tip. The whole process from the preparation of the
specimen homogeneous solutions to the completion of inoculation of specimens shall
not exceed 15 min.
6.2 Pout plate and culture
6.2.1 In accordance with the estimation of the contamination of specimens and
relevant limit requirements, select 2 ~ 3 homogeneous specimen solutions (liquid
specimens may be raw solutions) of appropriate continuous dilution and inoculate the
BPW each to add to two sterile plates as blank control.
6.2.2 Pour 10 ml ~ 15 ml of VRBGA (which may be placed in a thermostatic water
bath for heat preservation at 46°C ± 1°C) cooled to 46°C to pour on each plate. Rotate
the plates carefully to mix up the specimen homogeneous solutions and culture media.
6.2.3 After the agar solidifies, pour a thin layer of the same culture medium to cover
the surface of the plates. Prevent the spreading growth and make the colony
characteristics more obvious.
6.2.4 Turn over the plate after the upper-layer agar solidifies on the VRBGA plate and
culture for 18 h ~ 24 h at 36°C ± 1°C.
6.3.1 The typical colonies of Enterobacteriaceae are pink to red or violet colonies
with or without precipitation rings. Select the plates with typical colony count between
15 CFU ~ 150 CFU and without spreading growth of colonies and only enumerate the
typical colonies. The colony counting is expressed by the colony-forming unit, CFU.
9 Operating Procedures
9.1 Specimen dilution
As in 6.1.
9.2 Inoculation and culture
9.2.1 Nonselective pre-enrichment
requirements, select 3 homogeneous specimen solution of appropriate continuous
dilution degrees, 3 test tubes for each dilution degree and altogether 9 test tubes of
BPW, and culture for 18 h ± 2 h at 36°C ± 1°C.
9.2.2 Selective enrichment
Transfer 1 ml of culture from each culture tube of BPW, inoculate in 10 ml of EE broth,
and culture for 24 ± 2 h at 36°C ± 1°C.
9.2.3 Separation
Use an inoculating loop to take one loop of culture from all EE broths, conduct streak
inoculation on the BRBGA plate, culture at 24 ± 2 h at 36°C ± 1°C and observe whether
9.3 Confirmation of typical colonies
See 6.3 for the typical colonies.
10 Report
Enterobacteriaceae is Gram-negative non-spore-bearing bacillus, which ferments
glucose producing acid and is oxidase negative. Once one colony is confirmed
Enterobacteriaceae, the EE tube represented by it is Enterobacteriaceae positive.
Look up in the MPN table (see Annex C) in accordance with the EE positive tube counts
and report the MPN values per gram (millimeter) of specimen. Report the sampling by
weight in MPN/g and the sampling by volume in MPN/ml.
Culture Media and Reagents
B.1 Buffered peptone water (BPW)
B.1.1 Ingredients
Peptone 10.0 g
Sodium chloride 5.0 g
Disodium phosphate 3.5 g
Potassium dihydrogen phosphate 1.5 g
Distilled water 1 000.0 ml
B.1.2 Preparation
separately into 500-ml wide-mouthed bottles with 225 ml each bottle, and conduct
autoclaved sterilization for 15 min at 121°C.
B.2 Buffered glucose brilliant green bile broth (EE broth)
B.2.1 Ingredients
Peptone 10.0 g
Glucose 5.0 g
Disodium phosphate (anhydrous) 6.45 g
Potassium dihydrogen phosphate 2.0 g
Bile salt 20.0 g
Distilled water 1 000.0 ml
B.2.2 Preparation
Dissolve the ingredients in B.2.1 in water, heat to boiling for full dissolution (heating is
preferably less than 30 min), cool the culture medium rapidly, adjust the pH to 7.2 ±
0.2 at 25°C, and load separately in sterile test tubes of 18 mm × 180 mm with 10 ml
each tube. They require no autoclaved sterilization and may be stored for one month
at 5°C ± 3°C.
B.3 Crystal violet neutral red bile salt glucose agar (VRBGA)
B.3.1 Ingredients
Yeast extract 3.0 g
Bile salt No. 3 1.5 g
Glucose 10.0 g
B.6 Gram stain solutions
B.6.1 Crystal violet stain so...
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