1、 中华人民共和国出入境检验检疫行业标准SN/T 18972007食品中菌落总数的测定 PetrifilmTM测试片法 Aerobic Plate Count in Foods PetrifilmTM Aerobic 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 18972007 I 前 言 本标准主要参考AOAC 990.12 食品中菌落总数的测定PetrifilmTM1测试片法。 本标准的附录A和附录B是资料性附录。 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
2、人民共和国辽宁出入境检验检疫局、黑龙江出入境检验检疫局、深圳出入境检验检疫局、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、曹际娟、李苏龙、吴刚、谢昭聪、秦成、郑秋月、齐震玉、王玉萍、邱驰、刘冉、于春梅、周振亚、陆苏飙。 本标准系首次发布的检验检疫行业标准。 1PetrifilmTM是由 3M公司提供的产品的商品名。 SN/T 18972007 食品中菌落总数的测定PetrifilmTM 测试片法 1 范围 本标准规定了食品中菌落总数的测定- PetrifilmTM 测试片法。 本标准适用于食品及原料中菌落总数的测定，也可以用于与食品接触的容器、操作台和其他设备表面的卫生
3、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。 SN 0168 出口食品平板菌落计数 3 术语和定义 3.1 菌落总数 Aerobic plate count 指样品经过处理，在一定条件下培养后，所得1mL(g)检样或单位面积样品中所含菌落的总数。 3.2 菌落形成单位 colony-forming units, cfu 一个细菌在平板计数琼脂培养基上生长形成肉
4、眼可见的菌落。 4 原理 4.1 PetrifilmTM细菌总数测试片 Aerobic count plates 是一种预先制备好的培养基系统，含有标准的培养基，冷水可溶性的凝胶剂和氯化三苯四氮唑 (TTC)指示剂，菌落在测试片上呈红色或粉红色，这样可增强菌落计数效果。 4.2 3MTM快速涂抹棒 Quick Swab 是一种预先制备好的环境涂抹系统，可以用于食品、饮料工业中的表面采样程序。3M 快速涂抹棒可以和所有3M PetrifilmTM测试片一起使用。它包括5英寸长的人造纤维涂抹头和1.4mL的letheen肉汤，letheen肉汤可以中和残留于被测物表面的消毒剂，5英寸长的人造纤维涂
5、抹头可涂抹弯曲管道。Quick Swab涂抹棒的纤维涂抹头和letheen肉汤是分开的，适合于干、湿两种取样方式。 4.3 3MTMeSwab涂抹棒 是一种预先制备好的环境涂抹系统，可以用于食品、饮料工业中的表面采样程序。3M eSwab涂抹棒可以和大部分3M PetrifilmTM测试片一起使用。它包括人造纤维涂抹头和10mL 缓冲蛋白胨水溶液。3M eSwab涂抹棒的一侧标有刻度，根据刻度可以在测试片上滴加确定体积的溶液。 5 设备和材料 1 SN/T 18972007 5.1 温箱：361，301。 5.2 冰箱：25。 5.3 pH 计或精密 pH 试纸。 5.4 放大镜或菌落计数器或
6、 3MTMPetrifilmTM自动判读仪。 6 培养基和试剂 6.1 1 mol/L NaOH：称取 40g NaOH 溶于 1000 mL 蒸馏水中。 6.2 1 mol/L HCl：移取浓盐酸 90mL，用蒸馏水稀释至 1000 mL。 6.3 3MTMPetrifilmTM细菌总数测试片和压板。 6.4 3MTMQuick Swab快速涂抹棒或 3MTM eSwab涂抹棒。 7 检验程序 菌落总数的检验程序见图1。 选择 23 个适宜稀释度， 各取 1mL分别加入PetrifilmTM测试片 检 样 制备样品匀液 做成几个适当倍数的稀释液压板放置中央处，轻压 静置至少 1min 培 养
7、 计数各测试片菌落数 计算菌落总数结果 报 告 图1 菌落总数的检验程序 8 操作步骤 8.1 检验样品的制备 2 SN/T 18972007 8.1.1 样品制备按照 SN0168 菌落总数测定方法的样品制备方法进行。 8.1.2 制备样品匀液 按 SN0168 方法制备 1:10 的样品匀液后，无菌操作调节该样品匀液的 pH 为 6.67.2，酸性样液用 1mol/L NaOH 调节，碱性样液用 1 mol/L HCl 调节，或根据产品标准规定的酸碱溶液来调节 pH 值。 8.2 检样稀释 与SN0168 菌落总数测定方法检验程序相同，培养基为PetrifilmTM菌落总数测试片。 8.3
8、 接种 根据食品卫生标准要求或对标本污染情况的估计，选择 23 个适宜稀释度检验。将测试片置于平坦表面处，揭开上层膜，用吸管或微量移液器吸取某一稀释度的 1mL 样液，垂直滴加在测试片的中央处，将上层膜盖下，允许上层膜直接落下，但不要滚动上层膜，将压板（凹面底朝下）放置在上层膜中央处，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。每个稀释度接种两张测试片，每张 1mL。 8.4 表面取样的样品（如拭子）参照附录 A。 8.5 空气取样的样品参照附录 B。 8.6 培养 将测试片的透明面朝上水平置于培养箱内，可堆叠至 20 片，361条件下培养 4
9、8h2h（水产品 301培养 72h3h）；如有产品标准等特殊要求，则按相应的标准或要求进行。 8.7 菌落计数和记录 8.7.1 到培养时间后应立即计数， 如果不能立即进行计数，可以将测试片放在15条件以下，不超过 7 天。 8.7.2 计数红色菌落，菌落计数和记录见 SN0168。 8.8 菌落计数说明 8.8.1 不论菌落大小都应计数。 8.8.2 当细菌浓度很高时， 整个测试片会变成红色或粉红色，将结果记录为“多不可计” (too numerous to count, TNTC)。 8.8.3 有时细菌浓度很高时测试片中央可能没有可见菌落，但圆形培养面积的边缘有许多小的菌落，其结果也记
10、录为“多不可计”（TNTC）；可对样品进行进一步的稀释，以获得准确的计数。 8.8.4 一些微生物会液化凝胶， 造成局部扩散或菌落模糊的现象。如果液化现象干扰计数，可以计数未液化的面积来估算菌落浓度。 8.9 计数和记录数据见 SN0168。 9 结果的表述 菌落计算的规则同 SN 0168 菌落总数测定方法。 固体检样以cfu/g为单位报告，液体检样以cfu/mL为单位报告，表面涂抹则以cfu/cm2报告。 3 SN/T 18972007 附 录 A （资料性附录） 表面取样方法 测定与食品接触的容器、操作台、和其他设备表面上的微生物数，可以为生产过程中污染水平以及执行作为按照生产卫生部分的
11、消毒效果提供一个评价2）。 表面取样方法包括表面擦拭法和直接平板接触法。 A.1 表面擦拭法 采样面积的大小可根据法规、内部标准和(或)监控地点的不同来设定，例如考虑到较低的细菌数，对成品线应采用较大的采样面积。 A.1.1 3M Quick Swab 快速涂抹棒法 A.1.1.1 弯曲红色塞管，折断后将肉汤培养基挤入管体内，干法取样的无需折断。 A.1.1.2 取出涂抹棉签后取样。 A.1.1.3 用棉签涂抹物体表面，重复3次。 A.1.1.4 放回涂抹棉签后摇晃10秒钟。 A.1.1.5 将样品倒入3MTMPetrifilmTM菌落总数测试片。 A.1.2 3M eSwab抹棒法 A.1.
12、2.1 在涂抹棒容器上标记样品信息。 A.1.2.2 打开旋盖，取出涂抹棒。 A.1.2.3 涂抹一定的待检面积。 A.1.2.4 将涂抹棒放回容器， 扭紧旋盖， 水平摇晃容器以释放涂抹头上的微生物；使用未稀释的溶液作下一步的检测。 A.1.2.5 打开容器顶端的翻盖。 打开翻盖时， 不要接触出液口，以避免污染，不要挤压容器壁，以避免溶液从出液口喷出。 A.1.2.6 翻转容器，按刻度挤压1mL样品到3MTMPetrifilmTM菌落总数测试片。 A.2 直接平板接触法 将PetrifilmTM菌落总数纸片胶体部分扣压在被测试的表面。培养后，通过计数形成的菌落可以对被测表面得出一个相当可靠的细
13、菌数。 A.2.1 用1mL无菌稀释液水化PetrifilmTM菌落总数测试片。 A.2.2 静置至少1 h，使胶体固化。 A.2.3 提起上层膜，使胶体部分置于待测物表面。 A.2.4 用手指摩擦上层膜外侧，保证膜与表面充分接触。 A.2.5 使上层膜与目的表面分离，然后将其与培养基合上。 A.2.6 将测试片置于培养箱内培养。 2） 稳定性是在环境监控过程中获得有用信息的关键点，所以在采样过程中应使用相同的步骤。理想情况下，应使用相同类型的取样设备，模板面积，技术员和采样技术。 4 SN/T 18972007 附 录 B （资料性附录） 空气取样方法 A.1 直接沉降法 A.1.1 用1
14、mL无菌稀释液水化3MTMPetrifilmTM菌落总数测试片。 A.1.2 静置测试片至少1h，使胶体固化（水化好的测试片可以冷藏7天） 。 A.1.3 在3M 固定夹的两端粘上双面胶带。 A.1.4 提起上层膜并贴于胶带上固定，将测试片于空气中暴露15min。 A.1.5 合上上层膜后置于培养箱内按规定条件培养。 5 SN/T 18972007 6 参考文献 1 AOAC. 2000. Official Method 990.12. Aerobic Plate Count in Foods Rehydratable film (Petrifilm Aerobic Count Plate)
15、method, Official Methods of analysis of AOAC International. 17th Ed. AOAC International, Gaithersburg, MD. 2 AOAC 2000 Official Method 986.33 Bacterial and Coliform Counts in Milk Dry Rehydratable Film Methods (Petrifilm Aerobic Count Plate and Petrifilm Coliform Count Plate) Methods. 3 AOAC Officia
16、l 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 Aerobic Microorganisms. Enumeration at 30 in Foods by means of Petrifilm TMPlates NMKL NO.146 1993. 5 USDA/FSIS Microbiology Lab
17、oratory Guidebook 3rdedition 1998 Chapter 3 Examination of Fresh, Refrigerated and frozen prepared meat, poultry and pasteurized egg products. 6 Health Products And Food Branch (Canada, MFHPB-33). 2001. Enumeration of total aerobic bacteria in food products and food ingredients using 3MTMPetrifilmTM
18、Aerobic Count Plates. 7 Association Francaise de Normalisation (AFNOR): Cetrificate No.: 3M 01/1-09/89. 3M Petrifilm Total Aerobic Count. Tour Europe, 92049 Paris La Defense Cedex. 8 CCFRA Microbiological Methods Manual Standard Plate Count (Total Viable Count) - 3M PetrifilmTMAerobic Count Plate Me
19、thod Method 2.5: 1997. 9 食品卫生检查指南 微生物卷 日本食品卫生协会 2004。 10American Public Health Association. 1992. Standard Methods For the Examination of Dairy Products, 16th ed. APHA, Washington, DC. pp. 221-222, 231-232. 11American Public Health Association. 1992. Compendium of Methods for the Microbiological Exa
20、mination of Foods, 3rd ed. APHA, Washington, DC. pp. 61, 80-89. 12Betts, G. D., R.P. Betts and R. Taylor. 1994. Technical Memorandum N. 703: Evaluation of 3M Petrifilm for Aerobic Plate Count, Yeast and Mould Count and Escherichia coli Count. Campden Food & Drink Research Association, Chipping Campd
21、en Gloucestershire, UK. 13U.S. Department of Health and Human Services. 1999. Grade “A” Pasteurized milk Ordinance: Recommendations of the Public Health Service. Publication No. 229. 1999 Revision. U.S. Govt. Print. Off. Washington, DC. - SN/T 18972007 7 Foreword This standard mainly refers to AOAC
22、990.12 3M PetrifilmTM2Aerobic Count Plate Method Annex A and annex B are informative annex. This standard was proposed by Certification and Accreditation Adminstrator of the Peoples Republic of China (CNCA), and is under the charge of CNCA. The standard was drafted by Liaoning Entry-exit Export Insp
23、ection and Quarantine Bureau of P.R.China, Heilongjiang Entry-exit Export Inspection and Quarantine Bureau, Shenzheng 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, Cao Jijuan, Li Sulong,
24、 Wu Gang, Xie Zhaocong, Qin Cheng, Zheng Qiuyue, Qi Zhenyu, Wang Yuping, Qiu Chi, Liu Ran, Yu Chunmei, Zhou Zhenya and Lu Subiao. This standard is a professional standard of entry-exit inspection and quarantine promulgated for the first time. 2Petrifilm is the trademark of 3M company. SN/T 18972007
25、Aerobic Plate Count in Foods- PetrifilmTM Aerobic Count Plate Method 1 Scope The testing method of aerobic plate count in foods (PetrifilmTMplate method) is specified in this standard. This standard can be applied to the aerobic plate count in food and raw materials, as well as the food contact cont
26、ainer, operation table, and other surface of equipments. 2 Normative reference The following standard contains provisions which, through reference in this text, constitute provisions of this standard。For dated references, the s ubsequent amendments (besides the incorrect content) or revisions are no
27、t 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. SN 0168 Aerobic Plate Count in exported foods 3 Terms and def
28、initions 3.1 Aerobic plate count Aerobic plate count is the total number of bactiera in 1ml(g) sample or one unit area after sample was treated and then incubated under certain condition such as media component, culture temperature and time, pH and oxygen demands;Based on the assumption that a live
29、organism can reproduce to one countable colony under certain culture condition,the aerobic plate count is used to describe the amount of the bacteria in foods per unit. The result specified by this method includes only the mesophilic and aerobic bacteria on the plate count agar media. 3.2 Colony-for
30、ming units, cfu One bacterium grows on the plate count agar media and forms the visible colony. 4 Principle 4.1 3MTMPetrifilmTMAerobic count plates PetrifilmTMplate are a ready-to-use product containing the standard medium, a cold-water-soluble gelling agent and TTC indicator that can enhance the ef
31、fect of colony enumeration. 4.2 3MTMQuick Swab 8 SN/T 18972007 It is a ready-to-use environmental swab system which is applied to the sampling program on the surface in food and beverage industry. 3M Quick Swab can be used with al the 3M PetrifilmTMPlates. It consists of a five inch long Dacron tipp
32、ed swab that uses 1.4 ml letheen broth to facilitate the recovery of bacteria. Letheen broth can neutralize the residual sanitizers on the surface，5-inch artifical fibre can swab the curving pipe.Quick Swab can be used to the dry and wet sampling mode. 4.3 3MTMeSwab It is a ready-to-use environmenta
33、l swab system which is applied to the sampling program on the surface in food and beverage industry. The 3M eSwab is designed to be used with most 3MTMPetrifilmTMplate. It consists of a 10 mm long rayon swab head and contains 10 ml buffered peptone water or phosphate buffered saline.The 3M eSwab has
34、 volume scale on sides, and it can be used to release sample with proper volume onto the PetrifilmTMplates. 5 Equipment and materials 5.1 Incubator： 361 301 . 5.2 Refrigerator： 25. 5.3 pH meter or precise pH paper. 5.4 magnifier and/or colony counter or 3MTMPetrifilmTMplate reader. 6 Media and agent
35、 6.1 1mol/L NaOH： Weigh 40 g NaOH and then dissolve it into 1000ml distilled water. 6.2 1 mol/L HCl: Pipe 90ml HCl, and then dilute it into 1000ml distilled water. 6.3 3MTMPetrifilmTMAerobic Count Plate and spreader. 6.4 3MTMQuick Swab and 3MTM eSwab. 7 Flow chart 9 SN/T 18972007 The procedure of ae
36、robic plate count is indicated in fig.1. Select 23 个 suitable dilution， inoculate 1mL onto Petrifilm plate SamplePrepare samplePrepare serial suitable dilution Put spreader on center of the bottom film and leave at least 1 minIncubationCount colonies on platesCalculate the colony numberReport Fig.1
37、The procedure of the aerobic plate count 8 Procedure 8.1 Sampling 8.1.1 The sampling program is in accordance with the related provisions in SN0168. 8.1.2 Sample preparation After the 1:10 dilution is prepared, the pH of the dilution should be adjusted to 6.67.2. 1mol/L NaOH is used to adjust acidic
38、 sample, and 1 mol/L HCl is used to adjust alkaline sample, or do the pH adjustment according to the product specification. 8.2 Sample dilution This process is in accordance with the related provisions in SN0168, and 3MTMPetrifilmTMPlate is used instead of agar. 8.3 Inoculation According to food hyg
39、iene standard or the contamination level of sample, 2-3 suitable dilutions are selected. Place PetrifilmTMplate on flat surface. Lift top film and inoculate 1 mL test suspension onto 10 SN/T 18972007 center of film base by pipette or micropipette. Carefully place top film down on inoculum. Distribut
40、e suspension over prescribed growth area with downward pressure in center of plastic spreader device (recessed side down). Leave plate undisturbed at least 1 min to permit gel to solidify. Two plates were inoculated for each dilution, and 1ml for each plate. 8.4 Surface sampling (Refer to Annex A) 8
41、.5 Air sampling (Refer to Annex B) 8.6 Incubation In incubator, place plates in horizontal position, clear side up, in stacks not exceeding 20 units. The incubation condition is 48h2h for 36C1C (For seafood, the condition is 72h3h for 30C1C); In accordance to the corresponding standard or requiremen
42、t if there is the specific requirement or standard for the products. 8.7 Enumeration and record 8.7.1 Count plates promptly after incubation period. After incubation is complete, plates may be stored frozen (15C) up to 7 days. Avoid this as a routine practice. 8.7.2 Count red colony, and the calcula
43、tion and record is in accordance with SN0168. 8.8 Interpretation of results 8.8.1 Count the red colonies regardless of its size or intensity. 8.8.2 When there is high concentration of bacteria, the whole plate will turn red or pink. And the result will be counted as “TNTC” (too numerous to count). 8
44、.8.3 Occasionally, on overcrowded plates, the center may lack visible colonies but many small colonies will be seen on the edges. When this occurs, record results as TNTC; further dilution of the sample is required. 8.8.4 Some organisms can liquefy the gel, allowing them to spread out and obscure th
45、e presence of other colonies. If a liquefier interferes with counting, an estimated count should be made by counting the unaffected areas. 9 Reading result The colony enumeration rule of the colonies is accordance with SN0168. Solid sample is reported with the unit of “cfu/g”, and the liquid sample
46、is reported with the unit of “cfu/mL”，and surface sample is reported with the unit of “cfu/cm2”. 11 SN/T 18972007 Annex A (informative annex) Surface sampling methods The microbial detection on the surface of the food contact container, operation table and other equipment can be applied to evaluate
47、the contamination level in product process or sanitization effect of product hygiene2）。 Surface sampling methods include the surface swab method and direct plate contact method. A.1 Surface swab Method The size of the sampling area depends on the regulation, internal standard, and/or monitoring plac
48、e. For instance, considering the low number of bacteria, the bigger sampling area can be applied to the product line. A.1.1 3M Quick Swab A.1.1.1 Bend the red snap valve and release the letheen broth to the tube. Do not bend the red snap first when the swab is used for dry mode sampling. A.1.1.2 Tak
49、e out the swab from for sampling. A.1.1.3 Swab the targeted area using swab, and three time for same point. A.1.1.4 After the swab is put back into tube, snake the swab vigorously for 10 seconds. A.1.1.5 Release the sample onto the 3MTMPetrifilmTMAerobic Count Plate. A.1.2 3M eSwab A.1.2.1 Label the container with sample information. A.1.2.2 Unscrew
