1、 中华人民共和国出入境检验检疫行业标准SN/T 18952007食品中金黄色葡萄球菌的快速计数法 PetrifilmTM 测试片法 Rapid enumeration of Staphylococcus aureus in Foods PetrifilmTMStaph Express 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 18952007 前 言 本标准包括第一法 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
2、析化学协会（AOAC ）官方方法 特定预加工食品和加工食品（冷冻千层面、奶油冻、冷冻什锦蔬菜、冷冻洋芋饼和冷冻裹浆蘑菇）中金黄色葡萄球菌的计数 Petrifilm金黄色葡萄球菌测试片法 2003.07（ AOACOfficial MethodSM2003.07: Petrifilm Staph Express Count Plate Method for the Enumeration of Staphylococcus aureus in Selected Types of Processed and Prepared Foods (frozen lasagna, custard, froz
3、en mixed vegetables, frozen hash browns, and frozen batter coated mushrooms)） ; 特定乳制品（冰淇淋、原奶、酸奶、奶酪和乳清粉）中金黄色葡萄球菌的计数 Petrifilm金黄色葡萄球菌测试片法 2003.08（ AOACOfficial MethodSM2003.08: Petrifilm Staph Express Count Plate Method for the Enumeration of Staphylococcus aureus in Selected Dairy Foods. (strawberry
4、ice cream, raw milk, vanilla yogurt, whey powder, and mozzarella cheese)）; 特定肉、家禽和海产品（熟制和切割的鸡肉、汉堡、三文鱼和香肠）中金黄色葡萄球菌的计数 Petrifilm金黄色葡萄球菌测试片法 2003.11AOACOfficial MethodSM2003.11: Petrifilm Staph Express Count Plate Method for the enumeration of Staphylococcus aureus in Selected Meat, Seafood and Poultry
5、。 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、中国合格评定国家认可委员会、内蒙古出入境检验检疫局、 深圳出入境检验检疫局、大连市产品质量检验所、黑龙江出入境检验检疫局、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、刘中学、曹际娟、李宏、孙杰、朱海、谢昭聪、陈兆君、刘颜泓、李苏龙、周振亚、陆苏飙。 本标准系首次发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 18952007 食品中金黄色葡萄球菌快速计数法 Petrifilm测试片法 1 范围 本标准规定了食
6、品中金黄色葡萄球菌快速计数法PetrifilmTM测试片法。 本标准适用于食品和食物中毒样品中金黄色葡萄球菌的计数。既适用于金黄色葡萄球菌含量较高的食品也适用于金黄色葡萄球菌含量较低而杂菌含量较高的食品。 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 0172 出口食品中金黄色葡萄球菌测定方法 3 原理 3.1 PetrifilmTM金黄色葡萄球菌测试片 ( S
7、taph Express Count Plate, STX) 是一种预先制备好的快速检验系统。它含有具有显色功能并经改良的Baird-Parker培养基，对金黄色葡萄球菌具有很强的选择性， 并含有冷水可溶性凝胶。测试片上的紫红色菌落为金黄色葡萄球菌。当测试片上出现除紫红色以外的其它任何颜色(如黑色或蓝绿色)，则必须使用确认反应片。此确认反应片含有显色剂和脱氧核糖核酸(DNA)。金黄色葡萄球菌产生的脱氧核糖核酸酶(DNase)会和反应片中的显色剂形成粉红色晕圈。 4 设备和材料 4.1 恒温培养箱：361。 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
8、.4 放大镜或/和菌落计数器。 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 90mL，用蒸馏水稀释至 1000 mL。 5.4 PetrifilmTM金黄色葡萄球菌测试片。 5.5 PetrifilmTM金黄色葡萄球菌确认反应片。 第一法 PetrifilmTM测试片直接计数法 6 检验程序 检验程序见图1。 1 SN/T 18952007 紫红色菌落 计数金黄色葡萄球菌菌落报 告 其
9、它颜色的菌落 在测试片中置入确认反应片36C 1C 培养 1h3h 1 mL 接种量，检样匀液加入测试片计数有粉红色晕的菌落为金黄色葡萄球菌 判读 36C 1C 24h 2h 检 样 25g (mL)+225mL 生理盐水 无菌落 图1 PetrifilmTM测试片直接计数法检验程序 7 操作步骤 7.1 样品制备 按照SN 0172 方法进行样品制备。 制备的1:10样品 匀液后， 无菌操作调节样品匀液的pH为6.08.0，对酸性样液用1mol/L NaOH调节，碱性样液用1 mol/L HCl调节2）。 7.2 样品匀液的稀释、接种和培养 7.2.1 接种：做 10 倍递增稀释，选择适宜的
10、 23 个连续稀释度的样品匀液（液体样品可包括原液）接种PetrifilmTM测试片，每个稀释度接种 2 片，每片 1 mL。将测试片置于平坦表面处，揭开上层膜，用吸管吸取某一稀释度的 1 mL样液垂直滴加到一张测试片的中央处，然后将上层膜缓慢盖下，避免气泡产生，切勿使上层膜直接落下，再把PetrifilmTM金黄色葡萄球菌的压板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。 7.2.2 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，在 36 C 1 C条件下培养 24h2h。 7.2.3 确认反应：
11、 如果上述测 试片上没有菌落生长或菌落全部是紫红色（典型的金黄色葡萄球菌特征），无需进行确认；如果测试片上出现黑色、蓝绿色菌落或紫红色菌落不明显，需使用PetrifilmTM 确认反应片作进一步确认。 2）也可以根据产品标准规定的酸碱溶液来调节pH值。 2 SN/T 18952007 将上层膜掀起，将确认反应片置入测试片的培养范围内，再将上层膜放下覆盖在确认反应片上，用手指以滑动的方式轻轻将测试片与确认反应片压紧，包括确认反应片的边缘，此步骤可使测试片与PetrifilmTM 确认反应片紧密接触并除去气泡，最后把插入确认反应片的测试片放在 36 C1 C的培养箱内培养 1h3h。 8 结果计算
12、与报告 8.1 判读： 紫红色的菌落直接计数为金黄色葡萄球菌；需要使用确认反应片作确认时，计数有粉红色晕圈的菌落。没有粉红色晕圈的菌落不是金黄色葡萄球菌，不应被计数。如果整个培养面积呈粉红色而没有明显的晕圈，说明金黄色葡萄球菌大量存在，结果记录为“多不可计”。 8.2 菌落计数： 培养结束后立即计数，可目视或用菌落计数器来计数，放大镜可辅助计数；选取金黄色葡萄球菌菌落数在 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
13、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。报告单位 “cfu/g (mL)”表示。 第二法 PetrifilmTM测试片 MPN法 9 检验程序 检验程序见图2。 图2 金黄色葡萄球菌PetrifilmTM测试片MPN法检验程序 10 操作步骤 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 24h12h 报告结果直接计数 查 MPN 表3 SN/T 18952007 10.1 样品制备 见7.1。 10.2
14、 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 1 mL 接种量加入到 3 张测试片。每个稀释度接种 3 张，接种方法见 7.2.2。 10.3 培养和确认 见 7.2.3 和 7.2.4。 10.4 判读 金黄色葡萄球菌菌落判读见8.1，如果最低稀释度的3个纸片不都有确认的金黄色葡萄球菌菌落，可根据金黄色葡萄球菌菌落的存在与否， 对所有9张测试片进 行阳性或阴性的定性报告，而无须计数每张测试片上金黄色葡萄球菌菌落数目。 如果最低稀释度的3个测试片上均有确认的金黄色葡萄球菌菌落， 可以按照上述的方法对每张测试
15、片进行金黄色葡萄球菌定性报告，也可以采用平板直接计数的方法，计算测试片上的金黄色葡萄球菌菌落数目。 11 结果报告 根据金黄色葡萄球菌阳性纸片数，查MPN检索表（见附录A），报告每g (mL)样品中金黄色葡萄球菌的MPN值。如果可以直接计数的结果报告见 8.2。 4 SN/T 18952007 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 g(mL)，0.01 g(mL)，0.001 g(mL)。 阳性纸片数 95%置信区间 阳性纸片数 95%置信区间MPN MPN 0.10 0.01 0.001 0.10 0.01
16、 0.001低 高 低 高 0 0 0 1100 420 - 注：表内所列检样量如改用0.01g(mL)、0.001g(mL)、0.0001g(mL)时，则表内数字应相应增加10倍，其余类推。 - 5 SN/T 18952007 Foreword This standard contains the Petrifilm1）Staph Express Count Plate（STX）direct enumeration method (method 1) and Petrifilm STX MPN method (method 2). The method 1 in this standard
17、refers to AOACOfficial MethodSM2003.07: Petrifilm Staph Express Count Plate Method for the Enumeration of Staphylococcus aureus in Selected Types of Processed and Prepared Foods (frozen lasagna, custard, frozen mixed vegetables, frozen hash browns, and frozen batter coated mushrooms); AOACOfficial M
18、ethodSM2003.08: Petrifilm Staph Express Count Plate Method for the Enumeration of Staphylococcus aureus in Selected Dairy Foods. (strawberry ice cream, raw milk, vanilla yogurt, whey powder, and mozzarella cheese); AOACOfficial MethodSM2003.11: Petrifilm Staph Express Count Plate Method for the enum
19、eration of Staphylococcus aureus in Selected Meat, Seafood and Poultry。 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, China National Accreditation Service for Conformity Assessment (CNAS), Neimen
20、ggu Entry-exit Export Inspection and Quarantine Bureau, Shenzhen Entry-exit Export Inspection and Quarantine Bureau, Dalian Institute of testing on Product Quality, Heilongjiang Entry-exit Export Inspection and Quarantine Bureau, Dalian New Era technology development ltd.,3M China Ltd.。 The main dra
21、fers of this standard are Lu Xing-an, Liu Zhongxue, Cao Jijuan, Li Hong,Sun Jie, Zhu Hai, Xie Zhaocong, Chen Zhaojun, Liu Yanhong, Li Sulong, 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
22、 trademark of 3M company. 6 SN/T 18952007 Rapid enumeration of Staphylococcus aureus in Foods Petrifilm Staph Express Count Plate（STX）Method 1 Scope This standard specifies the rapid enumeration of Staphylococcus aureus in Foods3M PetrifilmTMStaph Express Count Plate（STX）method. This standard is app
23、lied to the enumeration of the staphylococcus aureus in food and toxic sample. It is applied to not only the sample at high concentration of S.aureus, but also the the sample at low concentration of S.aureus. 2 Normative reference The following standard contains provisions which, through reference i
24、n this text, constitute provisions of this standard。 For dated references, the subsequent amendments (besides the incorrect content) or revisions 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 ref
25、erences.For undated references the lastest edition of the publication referred to applies. SN 0172 Detection of Staphylococcus aureus in export products 3 Principle 3.1 3MTMPetrifilmTM Staph Express Count Plate, STX It is a ready-to-use culture medium system which contains a cold-water-soluble gelli
26、ng agent. The chromogenic, modified Baird-Parker medium in the plate is selective and differential for S. aureus. Red-violet colonies on the plate are S. aureus. If non-violet colonies occur such as black or blue-green colonies, the 3M Petrilm Staph Express Disk should be used to identify S. aureus
27、from all suspect colonies. Staph Express Disk contains a dye and deoxyribonucleic acid (DNA). S. aureus produces deoxyribonuclease (DNase) and the DNase reacts with the dye to form pink zones. 4 Equipment and materials 4.1 Thermostatic incubator： 36 1. 4.2 Stomacher, blender or equivalent. 4.3 pH me
28、ter or precise pH paper. 4.4 Magnifier and/or colony counter. 5 Medium and agent 5.1 Sterile saline solution: Weigh 8.5g NaCl and dissolve into 1000ml distilled water, 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
29、 HCl: Pipe 90ml HCl, and then dilute it into 1000ml distilled water. 7 SN/T 18952007 5.4 PetrifilmTMStaph Express Count Plate (STX). 5.5 PetrifilmTM STX Disk. Method 1. 3M PetrifilmTMStaph Express Count Plate（STX ） Direct Enumeration Method 6 Flow Chart The flow chart is showed in fig.1. Only violet
30、 red colony Count all the violet red colony as s.aureus Report Non-violet red colonies Insert PetrifilmTMSTX Disk, and incubate 1-3hrs Inoculate 1 mL suspension onto STX Count all the pink zone as s.aureus Interpretation 36C 1C 24h 2 h Sampling 25g (mL)+225mL Buffer water No suspect Colony fig.1. Th
31、e flow chart of 3M PetrifilmTMStaph Express Count Plate (STX)-direct enumeration method 7 Procedure 7.1 Sample preparation Prepare the sample according to the SN 0172 standard or other related standard. After preparing the 1:10 dilution, the pH of the dilution should be adjusted to 6.08.0 ，1mol/L Na
32、OH is used to adjust acidic sample, and 1 mol/L HCl is used to adjust alkaline sample2）. 7.2 Inoculation and incubation 2）pH can also be adjusted according to the product specification. 8 SN/T 18952007 7.2.1 Inoculation: For each sample, after preparing the decimal dilutions, select suitalbe 2-3 con
33、tinous dilutions (Fluid sample can be undiluted) to inoculate 1ml onto each PetrifilmTM STX plate, and two plates for each dilution. Lift top film and inoculate 1 mL test suspension onto center of film base by pipette. Carefully and gently roll top film down on inoculum to avoid the gas bubble. Do n
34、ot let the top film down directly. Distribute suspension over prescribed growth area with downward pressure in center of plastic spreader device. Leave plate undisturbed at least 1 min to permit gel to solidify. 7.2.2 Incubation： In incubator, place plates in horizontal position, clear side up, in s
35、tacks not exceeding 20 units. The incubation condition is 36C 1C, and 24 h2 h. 7.2.3 Confirm reaction： If there is no growth of any bacteria or all the colonies are violet red(typical characteristic s.aureus), the use of disk is unnecessary; If there is any black or blue-green colonies or non-obviou
36、s violet red colonies, the 3M PetrifilmTMSTX Disk should be used for further confirmation. Lift the top film, and then insert the disk into the well of the plate，rejoin the top film and cover the disk. Use finger to press the plate and disk tightly including the edge of the disk. The air bubbles bet
37、ween the 3M PetrifilmTMplate and disk can be removed in this step. Finally put the plate inserted disk into the incubator at 36C1C for 1 h3 h. 8 Result calculation and report 8.1 Interpretation： Count the violet red colonies as confirmed S.aureus without disk insert;When the colonies are confirmed b
38、y disk, count all the pink zone as S.aureus. The colonies with pink zone are not S.aureus, and cannot be counted. If the whole inoculated area turn pink and there is no obvious zone, it means that there is great amount of S.aureus, and report the result as “TNTC”(too numerous to count). Further dilu
39、tion and retest is needed for more accurate result. 8.2 Colony calculation： Count the colonies promptly after the incubation period by eyes or standard colony counter. The magnifier-illuminator may also be used to facilitate counting. Select the plate when the colonies are within the range from 15 t
40、o 150 to count the colonies, and then multiply the dilution factor to report； If the number of colonies on all the plates is lower than 15, count the colonies on the plate with the lowest dilution, and then multiply the dilution factor to report; If there is no growth on the plates with all the dilu
41、tion, then report result as lower than one multiply the lowest dilution factor; If the colonies on the plate with the highest dilution is greater than 150, count the colonies on plate with the highest dilution, and then multiply the dilution factor for report. Additonally, circular growth area is ap
42、proximately 30 cm2. Estimates can be made on plates containing more than 150 colonies by counting the number of colonies in one or more representative squares and determining the average number per square. Multiply the average number by 30 to determine total count per plate. “cfu/g(ml)” is used as t
43、he report unit. 9 SN/T 18952007 Method 2: 3M PetrifilmTM Staph Express Count Plate（STX ）MPN Method 9 Flow chart The flow chart is showed in fig.2. 25g(mL)+225mL dilutionSample Select 3 suitable dilution, and inoculate 1ml ofeach dilution to 3 STX plates Interpretation 36 124h2h Direct enumeration Ch
44、eck MPN Table Report Prepare decimal dilutions Fig.2. The flow chart of 3M PetrifilmTMStaph Express Count Plate（STX）MPN method 10 Procedure 10.1 Sample preparation Refer to 7.1. 10.2 Inoculation When preparing the decimal dilution, select 3 suitable dilutions, and inoculate 1ml of each dilution onto
45、 3 STX plates. 3 Plates were inoculated for each dilution. Refer to 7.2.2 for detailed inoculation. 10.3 Incubation and confirmation Refer to 7.2.3 and 7.2.4. 10.4 Interpretation Refer to 8.1 for the colony interpretation of S.aureus. If not all the 3 plates with the lowest dilution have the confirm
46、ed S.aureus, record the result of the 9 plates as negative or positive based on the existence of the S.aureus on plates. It is not necessary to count all the colonies of S.aureus on each plate. 10 SN/T 18952007 If all the three plates with the lowest dilution have the confirmed S.aureus, count the S
47、.aureus and report qualitatively according the instruction above. Or count the colonies on STX plates according to the direct plate count method. 11 Result report Check the MPN table according to the amount of the STX plates with positive result (see annex A), and report the MPN result of the S.aure
48、us in each g (ml) sample. For the direct count report, refer to 8.2. 11 SN/T 18952007 Annex A (Normative annex) MPN table for 1g (mL) sample When 9 plates are used， inoculum volume（equal to the sample qualtiy ） is respectively 0.1， 0.01，0.001（mL ）. 95%Confidence Interval 95%Confidence Interval Positive plates Positive plates MPN MPN 0.10 0.01 0.001 Low High 0.10 0.01 0.001 Low High0 0 0 1100 420 - - 12
