1、SN/T 1896-2007 中华人民共和国出入境检验检疫行业标准SN/T 18962007食品中大肠菌群和大肠杆菌快速计数法PetrifilmTM 测试片法 Rapid Enumeration of Coliforms and Escherichia coli in Foods PetrifilmTMColiform Count Plate and PetrifilmTME.coli/Coliform Count Plate Method 2007-05-23 发布 2007-12-01 实施中华人民共和国国家质量监督检验检疫总局T 发 布 TSN/T 1896-2007 前 言 本标准包括
2、第一法 Petrifilm1）测试片直接计数法和第二法 Petrifilm 测试片MPN 法。 本标准第一法修改采用了美国分析化学协会（ AOAC）官 方方法 食品中大肠菌群和大肠杆菌Petrifilm测试片计数法 991.14（ AOACOfficial MethodSM991.14: Dry Rehydratable film for enumeration of total coliforms and Escherichia coli in foods） ; 肉、禽和海产品中大肠菌群 Petrifilm测试片计数法 998.08（AOACOfficial MethodSM998.08:
3、Dry Rehydratable Film Method for Enumeration Confirmed Escherichia coli in Poultry, Meats and Seafood） 。 本标准的附录A 是规范性附录。 本标准由国家认证认可监督管理委员会提出并归口。 本标准起草单位：中华人民共和国辽宁出入境检验检疫局、浙江出入境检验检疫局、天津出入境检验检疫局、 内蒙古出入境检验检疫局、山东出入境检验检疫局、大连启元科技发展有限公司、3M中国有限公司。 本标准起草人：卢行安、施伟良、赵宏、张宏伟、郑文杰、刘中学、王金玲、吴刚、雷质文、刘冉、周振亚、陆苏飙。 本标准系首次发
4、布的检验检疫行业标准。 1）为美国 3M公司产品的商品标志。 I SN/T 1896-2007 食品中大肠菌群和大肠杆菌快速计数法 Petrifilm测试片法 1 范围 本标准规定了食品中大肠菌群和大肠杆菌快速计数法PetrifilmTM测试片法。 本标准适用于食品和原料中大肠菌群和大肠杆菌的计数，也适用于表面的卫生检测。 2 规范性引用文件 下列文件中的条款通过本标准的引用而成为本标准的条款。凡是注日期的引用文件，其随后所有的修改单 （不包括勘误的内容）或修订版均不适用于本标准，然而，鼓励根据本标准达成协议的各方研究是否可使用这些文件的最新版本。凡是不注日期的引用文件，其最新版本适用于本标准
5、。 SN0169 出口食品中大肠菌群、粪大肠菌群和大肠杆菌检验方法 3 原理 3.1 PetrifilmTM大肠菌群测试片 ( PetrifilmTMColiform Count Plate) 是一种预先制备好的培养基系统，含有 VRB (Violet Red Bile) 培养基，冷水可溶性凝胶和氯化三苯四氮唑（TTC ）指示剂，可增强菌落计数效果。表面覆盖的胶膜，可截留发酵乳糖的大肠菌群产生的气体。培养结束后计数红点周围有气泡的菌落为大肠菌群数。 3.2 PetrifilmTM大肠杆菌/ 大肠菌群测试片 ( PetrifilmTME.coli/Coliform Count Plate) 是一
6、种预先制备好的培养基系统，含有 VRB 培 养基，冷水可溶性凝胶和葡萄糖苷酶指示剂，可增强菌落计数效果。绝大多数 E .coli（约占 97%）能产生- 葡萄糖苷酸酶与培养基中的指示剂反应，产生蓝色沉淀环绕在大肠杆菌菌落周围。表面覆盖的胶膜，可截留发酵乳糖的大肠菌群产生的气体。培养结束后计数蓝点带气泡的菌落即为大肠杆菌数，红点带气泡和蓝点带气泡的菌落之和为大肠菌群数。 4 设备和材料 4.1 恒温培养箱： 36C 1C。 4.2 均质器（旋刀式或拍击式）或等效的设备。 4.3 pH 计或精密 pH 试纸。 4.4 PetrifilmTM测试片压板。 4.5 放大镜或 /和菌落计数器或Petri
7、filmTM自动判读仪。 5 培养基和试剂 5.1 无菌生理盐水：称取 8.5氯化钠溶于 1000 mL 蒸馏水中，121高压灭菌 15min。 5.2 1 mol/L NaOH：称取 40g NaOH 溶于 1000 mL 蒸馏水中。 5.3 1 mol/L HCl：移取浓盐酸 90 mL，用蒸馏水稀释至 1000 mL。 5.4 PetrifilmTM大肠菌群测试片。 5.5 PetrifilmTM大肠杆菌/ 大肠菌群测试片。 1 SN/T 1896-2007 第一法 大肠菌群和大肠杆菌PetrifilmTM测试片直接计数法 6 检验程序 检验程序见图1 。 检 样 25g (mL) 样品
8、+ 225mL 稀释液 图1 PetrifilmTM测试片直接计数法检测食品中大肠菌群和大肠杆菌数 7 食品检测的操作步骤 7.1 样品制备 按照 SN0169方法进行样品制备。 制备的 1:10样品匀液，无菌操作调节样品匀液的pH 为6.5 7.5，酸性样液用1mol/L NaOH 调节，碱性样液用1 mol/L HCl 调节2）。 7.2 样品匀液的接种和培养 7.2.1 对上述样品匀液做 10 倍 系列梯度稀释， 根据样品的污染程度，选取适宜的 23 个连续稀释度，每个稀释度接种 2 张测试片。 7.2.2 接种：将 PetrifilmTM大肠菌群测试片或PetrifilmTM大肠杆菌/
9、 大肠菌群测试片置于平坦实验台面，揭开上层膜，用吸管吸取 1 mL样液垂直滴加在测试片的中央，将上层膜缓慢盖下，避免气泡产生和上层膜直接落下，把压板（平面底朝下）放置在上层膜中央，轻轻地压下，使样液均匀覆盖于圆形的培养面积上。拿起压板，静置至少 1 min以使培养基凝固。 2）或根据产品标准规定的酸碱溶液来调节pH 值。 接种 1mL样品匀液到每张PetrifilmTM大肠菌群测试片 计数红色带气泡的菌落 报告大肠菌群数 36C1C 只检测大肠菌群 检测大肠菌群、大肠杆菌或同时检测两项10 倍梯度稀释接种 1mL样品匀液到每张PetrifilmTM大肠杆菌/ 大肠菌群测试片 24h2h 计数蓝
10、色带气泡的菌落 36C1C 24h2h 或 48h2h 计数蓝色带气泡和红色带气泡的菌落报告大肠杆菌数报告大肠菌群数2 SN/T 1896-2007 7.2.3 培养：将测试片的透明面朝上水平置于培养箱内，堆叠片数不超过 20 片，培养温度为 36C1C。大肠菌群检测时培养时间为 24h 2h；大肠杆菌检测时，如果是肉、家禽和水产品培养时间为 24h 2h，如果是其它产品，培养时间为 48h 2h。 8 表面检测的操作步骤 8.1 培养基准备： 用 1mL无菌稀释液水化PetrifilmTM大肠菌群测试片或PetrifilmTM大肠杆菌/大肠菌群测试片。静置至少 1h，使胶体（培养基）固化。
11、8.2 表面采样： 提起上层膜，使胶体部分置于待测物表面。用手指摩擦按压，保证膜与表面充分接触，然后将上层膜掀起，使之与物体表面分离，最后将其与下层合上。 8.3 培养： 将测试片的透明面朝上水平置于培养箱内，堆叠片数不超过 20 片，培养温度为 36 C1C。大肠菌群检测时培养时间为 24h 2h，大肠杆菌检测时培养时间为 48h 2h。 9 结果计算与报告 9.1 判读 9.1.1 目视、用菌落计数器、放大镜或PetrifilmTM自动判读仪来计数。 9.1.2 在PetrifilmTM大肠菌群测试片上，红色有气泡的菌落确认为大肠菌群数。培养圆形面积边缘上及边缘以外的菌落不作计数。当培养区
12、域出现大量气泡，大量不明显小菌落或培养区呈暗红色三种情况，表明大肠菌群的浓度较高，进一步稀释样品可获得准确的读数。 9.1.3 在P etrifilmTM大肠杆菌/大肠菌群测试片上，蓝色有气泡的菌落确认为大肠杆菌。蓝色有气泡和红色有气泡的菌落数之和为大肠菌群数。培养圆形面积边缘上及边缘以外的菌落不做计数。出现大量气泡形成、不明显的小菌落，培养区呈蓝色或暗红时，进一步稀释样品可获得准确的读数。 9.2 菌落计数：选取目标菌落数在 15150 之间的测试片，计数菌落数，乘以相对应的稀释倍数报告之。如果所有稀释度测试片上的菌落数都小于 15，则计数稀释度最低的测试片上的菌落数乘以稀释倍数报告之；如果
13、所有稀释度的测试片上均无菌落生长，则以小于 1 乘以最低稀释倍数报告之；如果最高稀释度的菌落数大于 150 个时，计数最高稀释度的测试片上的菌落数乘以稀释倍数报告之；计数菌落数大于 150 个的测试片时，可计数一个或两个具有代表性的方格内的菌落数，换算成单个方格内的菌落数后乘以 20 即为测试片上估算的菌落数 （圆形生长面积为 20 cm2）。食品中最终菌落浓度的单位以 “cfu/g (mL)”表示，表面上菌落数以“cfu/cm2”表示。 第二法 大肠菌群和大肠杆菌PetrifilmTM测试片 MPN法 10 MPN 法检验程序 3 SN/T 1896-2007 检验程序见图2。 图2 Pet
14、rifilmTM测试片MPN法检验程序 11 操作步骤 11.1 样品制备 见7.1。 11.2 样品匀液的接种 分别在做 10 倍递增稀释的同时，选择适宜的 3 个连续稀释度的样品匀液（液体样品可包括原液），吸取样品匀液，以 1mL接种量加入到 3 张测试片（根据检测的目的采用PetrifilmTM大肠菌群测试片或PetrifilmTM大肠杆菌/大肠菌群测试片)。每个稀释度接种 3 张，接种方法见 7.2.2。 11.3 培养 见 7.2.3。 11.4 判读 11.4.1 大肠菌群判读 PetrifilmTM大肠菌群测试片上大肠菌群菌落的判读见9.1.2， PetrifilmTM大肠杆菌/
15、 大肠菌群测试片上大肠菌群菌落的判读见9.1.3 。如果最低稀释度的3 张测试片不都有大肠菌群菌落，可根据大肠菌群菌落的存在与否，对所有9 张测试片进行阳性或阴性的定性判定，而无须计数每张测试片上大肠菌群菌落数目。 如果最低稀释度的3 张测试片上 均有大肠菌 群菌落，可以按照上述的方法对每张测试片进行大肠菌群菌落定性判定，也可以采用测试片直接计数的方法，计算测试片上的大肠菌群菌落数目。 11.4.2 大肠杆菌判读 PetrifilmTM大肠杆菌/ 大肠菌群测试片上大肠杆菌菌落的判读见9.1.3。如果最低稀释度的3 张测试片不都有大肠杆菌菌落，可根据大肠杆菌菌落的存在与否，对所有9 张测试片进行
16、阳性或阴性的定性判定，而无须计数每张测试片上大肠杆菌菌落数目。 10 倍梯度稀释 选择 3 个适宜稀释度的样品匀液，吸取 1mL 匀液 检 样 25g(mL)+225mL 稀释液 每个稀释度样品匀液各接种 3 张测试片 判 读 36 24 或 48h2h1h2h 报告结果 直接计数 查 MPN 表4 SN/T 1896-2007 如果最低稀释度的3 张测试片上均有大肠杆菌菌落，可以按照上述的方法对每张测试片进行大肠杆菌菌落定性报告，也可以采用测试片直接计数的方法，计算测试片上的大肠杆菌菌落数目。 12 结果报告 根据大肠菌群和/或大肠杆菌菌落阳性测试片数，查MPN检 索 表（见附录A），报告每
17、g (mL)样品中大肠菌群和/或大肠杆菌的MPN值。如果可以直接计数的，结果报告见 9.2。 5 SN/T 1896-2007 附 录 A （规范性附录） 1g(mL)检样中最可能数（MPN）表 使用九张测试片法，接种量（相当于样品的量）分别为0.1，0.01， 0.001（mL）。 阳性测试片数 95%置信区间 阳性测试片数 95%置信区间0.10 0.01 0.001 MPN 低 高 0.10 0.01 0.001MPN 低 高 0 0 0 1100 420 - SN/T 1896-2007 参考文献 1 AOAC. Official Method 990.14. Coliforms an
18、d Escherichia coli Counts in foods Dry Rehydratable Methods (Petrifilm E.coli Count Plate and Petrifilm Coliform Count Plate) Methods. 2 AOAC Official Method 998.08. Confirmed Escherichia coli Counts in Poultry, Meats and Seafood Dry Rehydratable Film Method Petrifilm E.coli/Coliform Count Plate Met
19、hod. 3 AOAC Official Method 989.10. Bacterial and Coliform Counts in Dairy Products Dry Rehydratable Film Methods (Petrifilm Aerobic Count Plate and Petrifilm Coliform Count Plate) Methods. 4 AOAC Official Method 986.33. Bacterial and Coliform Counts in Milk Dry Rehydratable Film Methods (Petrifilm
20、Aerobic Count Plateand Petrifilm Coliform Count Plate) Methods. 5 AOAC Official Method 2000.15. Rapid Enumeration of Coliform Counts in Foods Dry Rehydratable Film Methods Petrifilm Rapid Coliform Count Plate Method. 6 AOAC Official Method 996.02. Coliform Counts in Dairy Products High Sensitivity D
21、ry Rehydratable Film Method. 7 USDA/FSIS Microbiology Laboratory Guidebook 3rdedition 1998 Chapter 3 Examination of Fresh, Refrigerated and frozen prepared meat, poultry and pasteurized egg products. 8 Health Products And Food Branch (Canada, MFHPB-35). 2001. Enumeration of coliform in food products
22、 and food ingredients using 3MTMPetrifilmTMColiform Count Plates. 9 Health Products And Food Branch (Canada, MFHPB-34). 2001. Enumeration of coliform and E.coli in food products and food ingredients using 3MTMPetrifilmTME.coli/Coliform Count Plates. 10Association Francaise de Normalisation (AFNOR):
23、Cetrificate No.: 3M 01/2-09/89. 3MTMPetrifilmTM Coliform Count Plate Enumeration of Total Coliforms Reading Gas Producing Coliform colonies. 11NordVal Validation Ref. No. 2005-30-5408-00045. Renewal of the NordVal certificate of 3M Petrifilm E.coli/Coliform Count Plate. 12Korea Code of Federal Regul
24、atory, KFDA 2004: Dry Rehydratable Film Method Petrifilm Coliform Count Plate Method (7.8.5.4), Petrifilm E.coli/Coliform Count Plate Method (7.8.6.3). 13食品卫生检查指南 微生物卷 日本食品卫生协会 2004。 14American Public Health Association. 2001. Compendium of Methods for the Microbiological Examination of Foods, 4thed
25、. APHA, Washington, DC. 15Campden Food and Drink Research Association and Leatherhead Food Research Association study. EMMAS assessment 3M Petrifilm E. coli/Coliform Count Plate, 1998, UK 16New Zealand Food Safety Authority. MAF Standards D115 (Petrifilm Coliform Count Plate Method). MAF Standards D
26、107.1(Petrifilm E.coli/Colifrom Count Plate Method). 17Australia Victorian Dairy Industry Authority. Certificate Number 9504. Coliform Counts in Milk and Dairy Product using Petrifilm Coliform Count Plate. 18USDA FSIS. (Code of Federal Regulations) 9 CFR Part 310.25. Beef, swine, sheep, goats, horse
27、s, mules and other equine carcass sampling: Petrifilm E.coli/Count Plate; 9 CFR Part 381.94. Poultry, ducks, geese and guinea carcass sampling: Petrifilm E.coli/Count Plate. - 7 SN/T 1896-2007 Foreword This standard includes the method one-Petrifilm1）Plate direct enumeration and method two Petrifilm
28、 plate MPN method。 The method one in this standard refers to AOACOfficial MethodSM991.14: Dry Rehydratable film for enumeration of total coliforms and Escherichia coli in foods; AOACOfficial MethodSM998.08: Dry Rehydratable Film Method for Enumeration Confirmed Escherichia coli in Poultry, Meats and
29、 Seafood. The annex A of this standard is normative annex. The standard was drafted by Liaoning Entry-exit Export Inspection and Quarantine Bureau of P.R.China, Zhejiang Entry-exit Export Inspection and Quarantine Bureau, Tianjin Entry-exit Export Inspection and Quarantine Bureau, Neimenggu Entry-ex
30、it Export Inspection and Quarantine Bureau, Shandong Entry-exit Export Inspection and Quarantine Bureau, Dalian New Era technology development ltd.,3M China Ltd.。 The main drafers of this standard are Lu Xing-an, Shi Weiliang, Zhao Hong, Zhang Hongwei, Zheng Wenjie, Liu Zhongxue, Wang Jinling, Wu Ga
31、ng, Lei Zhiwen, Liu Ran, Zhou Zhenya and Lu Subiao. This standard is a professional standard of entry-exit inspection and quarantine promulgated for the first time. 1）Petrifilm is the trademark of 3M company. 8 SN/T 1896-2007 Rapid Enumeration of Coliforms and Escherichia coli in Foods PetrifilmTMCo
32、liform Count Plate and PetrifilmTME.coli / Coliform Plate Method 1 Scope This standard defines the method for rapid enumeration of coliforms and Escherichia coli in foods - PetrifilmTMcount plate method. This standard is applied to both the enumeration of total coliforms and Escherichia coli in food
33、s or raw material and the sanitary quantitative detection of surface. 2 Normative reference The following standard contains provisions which, through reference in this text, constitute provisions of this standard。For dated references, the subsequent amendments (besides the incorrect content) or revi
34、sions are not applied to this standard. But the person who reach the agreement on this standard are encouraged to study if using the lastest edtion of these references.For undated references the lastest edition of the publication referred to applies. SN0169 Detection of the coliforms, fecal coliform
35、s and Escherichia coli in export foods 3 Principle 3.1 PetrifilmTMColiform Count Plate The 3M Petrifilm Coliform Count Plate is a ready-to-use culture medium system which contains modified Violet Red Bile (VRB) nutrients, a cold-water-soluble gelling agent, and a tetrazolium indicator that facilitat
36、es colony enumeration. The membrane on the plate surface can trap the gas produced by coliform lactose fermentation. After incubation the red colonies with bubbles are counted as coliforms. 3.2 PetrifilmTME.coli/Coliform Count Plate The 3M Petrifilm E. coli/Coliform Count (EC) Plate is a ready-made
37、culture medium system which contains Violet Red Bile (VRB) nutrients, a cold-water-soluble gelling agent, an indicator of glucuronidase activity (5-bromo-4-chloro-3-indolyl-D-glucuronide) (BCIG), and a tetrazolium indicator that facilitates colony enumeration. The membrane on the plate surface can t
38、rap the gas produced by coliform lactose fermentation. After incubation the blue colonies with bubbles are counted as E.coli, red colonies with bubbles are counted as coliforms. 4 Equipment and Material 4.1 Constant temperature incubator: 36C 1C. 4.2 Homogenizer(revolving knive style or spanking sty
39、le) or other equivalent. 4.3 pH meter or precise pH test paper 9 SN/T 1896-2007 4.4 3M PetrifilmTMPlate and spreader 4.5 Magnifier or / and colony counter or 3M PetrifilmTMPlate Reader 5 Medium and reagent (Supplement) 5.1 Sterile saline solution: Weigh 8.5g NaCl and dissolve into 1000ml distilled w
40、ater, and then autoclave for 15min at 121C。 5.2 1mol/L NaOH： Weigh 40 g NaOH and then dissolve it into 1000ml distilled water. 5.3 1 mol/L HCl: Pipe 90ml HCl, and then dilute it into 1000ml distilled water. 5.4 PetrifilmTMColiform Count Plate. 5.5 PetrifilmTME.coli/Coliform Count Plate. Method 1 Pet
41、rifilmTME.coli/Coliform Count Plate direct enumeration 6 Flow chart The flow chart is showed in fig.1. Sample 25 g (mL) sample + 225mL diluentsFig. 1 Enumeration of total coliforms and Escherichia coli in foods by PetrifilmTM count plate method. 7 Procedure for food detection 7.1 Sample preparation
42、Prepare the sample in accordance with SN0169. After the 1:10 dilution is prepared, the pH of the dilution should be adjusted to 6.57.5. 1mol/L NaOH is used to ad just acidic sample, and 1 mol/L PetrifilmTMColiform Count Plate 2 plates each dilution, inoculating 1mL Red colonies with bubbles Report c
43、oliform C1C 36Coliform Cliform and E. coli Decimal dilutions， choose 23 suitable dilutionsPetrifilmTME.coli/Coliform Count Plate 2 plates each dilution, inoculating 1mL 24 h2h 36C1C 24 h2h 或 48 h2h Report E.coliRed and blue colonies with bubbles Blue colonies with bubblesReport coliform 10 SN/T 1896
44、-2007 HCl is used to adjust alkaline sample, or do the pH adjustment according to the product specification3. 7.2 Inoculation and incubation 7.2.1 Dilution： Prepare the decimal dilutions base on the contamination level of the sample, and then select 23 continous dilutions. For each dilution ，2 plate
45、s are inoculated. 7.2.2 Inoculation： Place the PetrifilmTMcoliform count plate or PetrifilmTME.coli/coliform count plate on the flat surface. Lift top film and inoculate 1 mL test suspension onto center of film base by pipette or micropipette. Carefully place top film down on inoculum. Avoid the pro
46、duction of the gas bubble. Roll the top film onto the bottom film, and do not drop the top film down. Distribute suspension over prescribed growth area with downward pressure in center of plastic spreader device (flat side down). Leave plate undisturbed at least 1 min to permit gel to solidify. 7.2.
47、3 Incubation： In incubator, place plates in horizontal position, clear side up, in stacks not exceeding 20 units. The temperature is 36C 1C. For the coliform detection, the incubation duration is 24 h 2 h；For the E.coli detection, the incubation time is 24 h 2 h for meat, poultry and seafood product
48、s，and 48 h 2 h for other products. 8 Procedure for the surface detection 8.1 Plate preparation：Hydrate PetrifilmTMplates with 1 mL of appropriate sterile diluents. Allow the hydrated plates to remain closed for a minimum of 1 hour before use. 8.2 Surface sampling: Lift the top film, and allow the circular gel portion of the top film to the contact the surface, Gently rub fingers parallel to the surface over the outer film side of the gelled area to ensure good contact with surface. Separate the top film and the surface, and then rejoin the top and bottom film. 8.3 Incubation： In incubator, p
