SN T 1538.1-2005 培养基制备指南.第1部分 实验室培养基制备质量保证通则.pdf

1、中华人民共和国出入境检验检疫行业标准SN/T 1538. 1-2005 培养基制备指南第1部分:实验室培养基制备质量保证通则Guidelines on preparation and production of culture media 一Part 1 : General guidelines on quality assurance for the preparation of culture media in the laboratory CISO/TS 11133-1: 2000 , Microbiology of food and animal feeding stuffs一Guid

2、elines on preparation and production of culture media Part 1: General guidelines on quality assurance for the preparation of culture media in the laboratory, MOD) 2005-02-17发布中华人民共和国国家质量监督检验检瘦总局2005-07-01实施发布SN/T 1538. 1-2005 目次前言引言. . II l 范围2 规范性引用文件-3 术语和定义3.1 概述.3.2 质量保证术语3. 3 培养基术语.2 3.4 测试菌株术语

3、.4 4 培养基质量控制.4 4. 1 证明文件.4 4. 2 贮存.4 4.3 培养基的实验室制备.54.4 培养基的使用. . . 6 4. 5 培养基的弃置.7 5 成品的质量控制.75.1 物理指标控制.75.2 微生物指标控制.7 附录A(资料性附录)食品和动物源性产品微生物分析标准中指定的培养基成分.9 附录B(资料性附录)质控菌株的保藏和使用. . 10 附录c(资料性附录)培养基的质量保证一一-常见问题解答. . 11 SN/T 1538. 1-2005 前言SN/T 1538(培养基制备指南分为两个部分z一一第1部分:实验室培养基制备质量保证通则一一第2部分:培养基性能测试实

4、用指南本部分为SN/T1538的第1部分，对应于ISO/TS11133-1 :(食品和动物饲料微生物学一培养基制备指南一一-实验室培养基制备质量保证通则)(2000年英文版)。本部分与ISO/TS11133-1的一致性程度为修改采用，主要差异如下:一一按照汉语习惯对一些编排格式进行了修改;一一将一些国际标准的表述方式改为适用于我国标准的表述方式;一一一对前言和引言进行了修改;一一一删除了原标准第3章术语和定义中的引用标准;一一在规范性引用文件部分用ISO19000取代了ISO8402，加入了标准GB/T6682; 将部分中提到的特定国际标准改为特定标准;一一在4.2.4条款中加入了特定的温度值

5、，如4.C、20.C等;一在4.3.3中增加了对称量脱水培养基的描述;一一在4.3.7.1中增加了对不同培养基灭菌的描述;一一在4.3.7.3中对过滤器的组装条件进行了修改;一一在4.5中增加了培养基弃置的注解;在5.2.5条款中加入了菌落计数技术;删除了原标准中的注解1)。本部分的附录A、附录B和附录C均为资料性附录。本部分由国家认证认可监督管理委员会提出并归口。本部分由中华人民共和国山西出入境检验检疫局、中国检验检疫科学研究院负责起草。本部分主要起草人z李卫华、赵贵明、张建军、廉慧锋、刘沛、巩红霞。本部分系首次发布的检验检疫行业标准。I SN/T 1538.1-2005 引微生物学实验室的

6、主要工作是对各种微生物进行保存、复苏、培养、检测和(或)计数，所用的培养基应对样品和被测微生物都具有特异性。微生物学分析过程则要求培养基符合标准或满足最低性能要求，并保证能够获得重现性结果，这样才能使分析结果准确、可靠。SN/T 1538 1000mL)的培养基灭菌时可能会造成过度加热。灭菌效果的控制是关键。加热后采用适当的方法冷却，以防加热过度。这对于肠道菌培养基和大容器中的培养基等的灭菌十分重要。4.3.7.3 过滤灭菌过滤灭菌可在真空负压或正压的条件下进行。使用孔径为o.22m的滤膜和过滤垫。过滤前先将滤膜和滤垫灭菌。过滤器于1210C灭菌15min(可以整体灭菌也可以拆卸后灭菌)，灭菌

7、后在无菌条件下组装。注z一些滤膜上附着有蛋白质(如抗生素)。为达到有效过滤，应事先将滤膜用元菌水润湿。4.3.7.4 监测应对经湿热或过滤灭菌的培养基进行监测，尤其要对pH、色泽、灭菌效果和均匀度等指标进行监测。4.3.8 添加成分的制备制备含有有毒物质的添加成分(尤其是抗生素)时应小心操作(必要时在通风柜中操作)，避免因粉末的扩散造成实验人员过敏或发生其他不良反应;制备溶液时应小心按产品使用说明操作;不要使用过期的试剂;抗生素工作榕液应现用现配;批量配制的抗生素榕液分装后冷冻贮存，但解冻后的贮存溶液不能再次冷冻;厂商应提供冷冻对抗生素活性影响的有关资料，也可由使用者自行测定。4.4 培养基的

8、使用4.4. 1 琼脂培养基的融化将培养基放到沸水浴中或采用有相同效果的方法(如高压锅中的蒸汽)使之融化。经过高压的培养基应尽量减少重加热时间，避免过度加热。培养基融化后放入470C士20C的恒温水浴锅中保温，直至使用。融化后的培养基应尽快使用，放置时间一般不应超过4h。4.4.2 培养基的脱气必要时，将培养基在使用前放到沸水浴或蒸汽洛中加热15min，加热时松开容器的盖子;加热后盖紧，并迅速冷却至使用温度。4.4.3 添加成分的加入对热不稳定的添加成分应在培养基冷却至47C:l:20C时再加入。灭菌的添加成分在加入之前，应先放置到室温，避免冷的液体造成琼脂凝结或形成片状物。将加入添加成分的培

9、养基缓慢充分混匀，尽快分装到待用的容器中。4.4.4 平板的制备和储存倾注融化的培养基到平皿中，使之在平皿中形成一个至少2mm厚的琼脂层(直径90mm的平皿6 SNjT 1538.1-2005 通常要加入15mL琼脂培养基)。将平皿盖好皿盖后放到水平平面使琼脂冷却凝固。注:在培养过程中，培养基会损失水分。当水分损失的量大于培养基总量的15%时，就会影响微生物的生长。造成培养基水分损失的因素很多，如培养基成分，平皿中培养基总量和培养箱的类型等(如使用带风扇的培养箱，培养箱中的湿度偏低，平板在培养箱中放置的位置靠近加热管，培养温度过高等)，操作时应注意避免。凝固后的培养基应立即使用或存放于暗处和或

10、)4.C12.C冰箱的密封袋中，最多存放一周或按厂商提供的标准执行。在平板底部做好标记，标记的内容包括名称、制备日期和(或)有效期。也可以使用适宜的培养基编码系统进行标记。将倒好的平板放在密封的袋子中冷藏保存可延长贮存期限。为了避免产生冷凝水，平板应冷却后再装入袋中。贮存前不要对培养基表面进行干燥处理。对于采用表面接种形式培养的固体培养基，应先对琼脂表面进行干燥:揭开平皿盖，将平板倒扣于烘箱/培养箱中(温度设为25C50C);或放在有对流风的无菌净化台中，直到培养基表面的水滴消失为止。注意不要过度干燥。商业化的平板琼脂培养基应按照厂商提供的说明使用。4.4.5 培养培养时每垛最多堆放六个平板，

11、平板间要留有空隙以保证空气流通，使培养物的温度尽快与培养箱温度达到一致;液体培养基温度与培养箱达到一致取决于很多因素，如体积、内容物量、容器类型、培养类型等;使用厌氧罐时，堆放的平板数可以超过六个。4.5 培养基的弃置所有污染和未使用的培养基的弃置应采用安全的方式，并且要符合相关法律法规的规定。注g应根据相关法律法规和标准的要求以及各实验室的实际情况，在所制定的药品试剂安全管理规范当中加入有关培养基弃置的具体要求，以确保所弃置培养基的安全性。5 成晶的质量控制5. 1 物理指标控制培养基的实验室测试至少应包括:一-20.C25.C的pH值p并应观察以下内容:一一加入培养基的量，琼脂层的厚度;一

12、一色泽p一一一透明度和(或)是否存在肉眼可见的杂质p一一凝胶稳定性、蒙古稠度和温度。5.2 微生物指标控制5.2.1 污染的控制从每批制备好的培养基中选取部分样品进行污染测试。5.2.2 测试菌株测试菌株是具有其代表种的稳定特性并能有效证明实验室特定培养基最佳性能的一套菌株。测试菌株主要购置于标准菌种保藏中心，也可以是实验室自己分离的具有良好特性的菌株。实验室应检测和记录标准储备菌株的特性;新复苏的菌株可能会有非特异性反应，使用时应引起注意;最好使用从食品中分离的菌株。每种培养基的测试菌株应包括:一一具典型反应特性的强阳性菌株;一一微弱生长的阳性菌株(对培养基中选择剂等试剂敏感性强的菌株);

13、一一非特异性菌株。如:产生不同发酵反应和荧光反应的菌株;一一-阴性菌株。7 SN/T 1538. 1-2005 注:国际食品微生物委员会CICFM)和培养基菌株卫生工作组CWPCM)介绍了培养基评估用测试菌株的有效收集方法。5.2.3 即用型培养基和试剂商品化即用型培养基的生产厂如果经过ISO9001体系认证或满足相应质量要求，应向使用方提供相应的资质证明。这样，使用者就不必再进行大量的培养基测试工作，但应保证培养基的贮存条件。5.2.4 商晶化合成脱水培养基制备的培养基对每批制备好的培养基除用标准菌株进行测试，还应用实际样品进行检测，以更好地保证培养基的质量。不含指示剂或选择剂的培养基，只需

14、用阳性菌株进行检测;含有指示剂或选择剂的培养基，应使用能证明其指示或选择作用的菌株进行试验;复合培养基(即需要加入添加成分的培养基)需要用具备5.2.2 性能的菌株逐批进行验证;实验室制备的需加入添加成分的即用型培养基本条款同样适用。5.2.5 备别成分制备的培养基除了按照5.2.4条款进行培养基菌株测试外，还应对各别成分制备的培养基用Miles&. Misra技术、螺旋平板技术或菌落总数技术进行测试，以监控基础材料的质量，培养基的性能和实验室内部的配制规范。8 注:以上是最低要求。如果食品中含有受损的微生物细胞，还应考虑培养基在受损微生物恢复方面的适用性。菌株保存、传代技术和标准菌株的收集参

15、见附录BoSN/T 1538. 1-2005 附录A(资料性附录)食晶和动物源性产晶微生物分析标准中指定的培养基成分A.1 概述为了统一对不同微生物标准中培养基成分的描述，ISO/TC34/SC 9食品和农产品-微生物技术委员会对不同的培养基成分进行了分类，详见A.2A. 50 A.2 蛋白臆一一酶解酷蛋白1;一一酶解大豆粉;一一一酶解动物组织2、一一心酶解物;一一酶解明胶;一一酶解动、植物组织。A.3 浸膏肉浸膏;一一脑心浸膏;一一酵母浸膏;一一细菌学牛胆盐;一一胆盐;一-3号胆盐。A.4 琼脂细菌学琼脂。A.5 其他一一卵黄乳液;一一脱脂奶粉;一一酸解酷蛋白。1)包括胃蛋白酶消化的酷蛋白和

16、膜蛋白酶消化的曾在蛋白和膜蛋白陈。2)包括肉炼，胃蛋白酶消化的肉，膜腺消化的肉。9 SN/T 1538. 1-2005 附录B(资料性附录)质控菌株的保藏和使用B. 1 概述冻干法、一70C玻璃珠保存法、液氮保存法等是食品微生物中常用的菌种保藏方法，但这些方法并不可能适用于所有菌株。参见图B.lB.2 商业途径获得的标准菌株从标准菌株保藏中心获得的真空保存的菌株:或至少定义到属或种水平的菌株，来源已知，佐能稳定.标准菌株在实验室经过一次传代获得的一组相同的菌株.由标准储备商株经一次传代得到的商株.固B.1对于从菌种保藏中心或经ISO9001认证的商业机构购买的标准菌株，要以原始的包装形式进行保

17、藏，复苏和使用应按照厂商提供的使用说明进行。B.3 实验室制备的标准菌株的保藏用于质量控制或性能测定的标准储备菌株在保存和使用时应注意避免交叉污染，减少菌株突变或发生典型的性能变化;标准储备菌株应制备多份，并采用超低温(-70.C)或冻干的形式保存F测试菌株在每种培养基上的生长特性应全部文件化。B.4 工作菌株工作菌株由冻干或超低温保存的标准菌株制备(B.3)。转接操作时要注意避免标准菌株可能发生的交叉污染和菌株的退化。制备工作菌株时，应将参考原株转接到多份非选择培养基中培养，得到稳定期的菌株。10 对于商品化的保藏菌株必须严格按照厂商的说明执行。注:工作菌株不能再传代。但工作菌株如果处理或贮

18、存得当C即不存在交叉污染和(或)在一周内不退化.则可以多次使用。SN/T 1538. 1-2005 附录C(资料性附录)培养基的质量保证-一常见问题解答异常现象可能原因培养基不能凝固制备过程中过度加热低pH值造成培养基酸解称量不正确琼脂未完全溶解培养基成分未充分混匀pH值不正确制备过程中过度加热水质不佳外部化学物质污染测定pH值时温度不正确pH计未正确校准脱水培养基质量差颜色异常制备过程中过度加热水质不佳脱水培养基质量差pH不正确外来污染产生沉淀制备过程中过度加热水质不佳脱水培养基质量差pH未正确控制培养基出现抑制/重复性差制备过程中过度加热培养基脱水质量差水质不佳使用成分不正确，如，成分称量

19、不准，添加物浓度不正确选择性差制备过程中过度加热脱水培养基质量差配方使用不对添加成分不正确。如加入添加成分时培养基过热或添加浓度错误11 SN/T 1538. 1-2005 CONTENTS Foreword . . 13 Introduction 14 1 Scope 15 2 Normative references 15 3 Terminology 15 3.1 General 15 3.2 Terminology of quality assurance 15 3.3 Terminology of culture media 16 3.4 Terminology for test or

20、ganisms 19 4 Practices for qualitntrol of culture media 20 4.1 Documentation 20 4.2 Storage 20 4. 3 Laboratory preparation of media 21 4.4 Preparation for use 23 4. 5 Disposal of media 25 5 Quality control of finished product 25 5. 1 Physical quality control 25 5.2 Microbiological quality control 25

21、 Annex A (informative) Designation of the components of the culture media in standards on micro biological analysis of food and animal feeding stuffs products 27 Annex B (informative) Guidance on preservation and maintenance of control strains 28 Annex C (informative) Quality assurance of culture me

22、dia - trouble shooting 30 12 SN/T 1538. 1-2005 Foreword 5N/T 1538 Guidelines on preparation and production of culture media consist of the following parts: 一Part1 : General guidelines on quality assurance for the preparation of culture media in the laborato W飞-Part 2: Practical Practical general gUi

23、delines on performance testing of culture media. This standard is the fi rst part of 5N/T 1538 , Corresponding to 150 IT5 11133-1 Microbiology of food and animal feeding stuffs-Guidelines on preparation and production of culture media-General guidelines on quality assurance for the preparation of cu

24、lture media in the laboratory (English ver sion , 2000). The consistencdegree of this standard and ISO IT5 11133-1 is modified, the main dif ferent showing bellows: -Modify some editing pattern according to the customs of Chinese; 一Changesome of the expression of International standard to National s

25、tandard of China; -Modfy Foreword , Introduction of original standard; 一Deletethe quotation standard number in the Chapter 3 of the original standard; -5ubstitute 1508402 for 150 19000 and Add GB/T 6682 in normative references; 一Replacespecial international standard with special standard; -Add the s

26、pecial degree of temperature in 4.2.4, e. g. 40C ,20oC ; -Add description of weighing dehydrated medium in 4.3.3; -Add description of sterilization of different media in 4.3.7.1 ; 一Modifythe assembly condition of the filter in 4. 3. 7. 3; -Add note of disposal of media in 4. 5; 一Addtotal plate count

27、ing in 5.2.5; 一Deletenote 1) of the original standard. Annex A , Annex B and Annex C are all informative. This standards was proposed and administrated by National Regulatory Commission for Certification and Accreditation. This standards was drafted by 5hanxi Entry-Exit Inspection and Ouarantine of

28、the Peoples Republic of China and China Inspection and Ouarantine Academy of 5cience. The main drafters of this standard are Li weihua , Zhao guiming , Zhang j ianjun , Li an huifeng , Liu pei and Gong hongxia. This standard is a professional standard of entry-exit inspection and quarantine promulga

29、ted for the first time. 13 SN/T 1538. 1-2005 Introduction The main 0同ectivesare to maintain , resuscitate, grow, detect and/or enumerate a wide varietof microorganisms in the microbiological laboratory, the requirements for media are specific to both the sample and/or organisms to be detected. The p

30、rocedure of microbiology tests depend on culture media being consistent of standard and specifthe minimum requirements , these can provide accu rate and reliable result. SN/T 1538 Guidelines on preparation and production of culture media are an essential part of internal quality control procedures o

31、f microbiology laboratory. It can monitoring the media effectively, and provide the result reliable and effective. This standard is the first part of these series standard , it standard the terms and definitions of media; regulate the general guideline of the storage, preparation ,using and disposal

32、 of media; pro pose the quality control of finished product. Part 2 of these standard will draft whereafter. 14 SN/T 1538. 1-2005 Guidelines on preparation and production of culture media-Part 1 : General guidelines on quality assurance for the preparation of culture media in the laboratory 1 Scope

33、This standard provides the general terminologrelated to quality assurance of the preparation of cul ture media and specifies the minimum requirements to be used for the microbiological analysis of products intended for human consumption or animal feeding. These requirements are applicable to three c

34、ategories of culture media used in laboratories that pre pare and/or use culture media for performing microbiological analyses: 一commerciallymanufactured ready-to-use media; -media prepared form commercially available dehydrated formulations (either complete e. g. plate count agar or basal media to

35、which supplements are added e. g. Baird-Parker agar); 一mediaprepared from its individual components. 2 Normative references This standard incorporates bdated or undated reference , provisions from other publications. These normative references are cited at the appropriate places in the text and the

36、publications are listed hereafter. For dated references , subsequent amendments to or revisions of any of these publications applto this standard only when incorporated in it by amendment or revision. For undated references the latest edition of the publication referred to applies. GB/T 19000 Qualit

37、y management systems-Fundamentals and vocabulary(idt ISO 9000:2000) GB/T 6682 Specification and test method of water in analysis laboratory( eqv ISO 3696: 1987) EN 1659 Invitro diagnostic systems-Culture media for microbiology-Terms and definitions EN 12322 Invitro diagnostic medical devices 一Cultur

38、emedia for microbiology -Performance crite ria for culture media 3 Terminology 3.1 General This clause gives the general definitions related to quality assurance and provides different types of terminology related to culture media and to control cultures. 3.2 Terminology of quality assurance 15 SN/T

39、 1538. 1-2005 3.2.1 Quality assurance AII the planned and systematic activities implemented within the qualitsystem and demonstrated as needed. to provide adequate confidence that an entity will fulfill the requirements for quality. 3.2.2 Qualitcontrol Operational techniques and activities that are

40、used to fulfill the requirements for quality. 3.2.3 Internal qualitcontrol A continuous control program of the laboratory s work prepared bor for them. and based on con trol analsis together with follow-up and if necessar. corrective actions. 3.2.4 Batch of culture media; lot of culture media Fulltr

41、aceable unit of a medium referring to a defined amount of bulk. semi-finished product or end product , which is consistent in type and quality and which has passed the requirements of produc tionCin-process control)and quality assurance testing , and which has been produced within one de fined produ

42、ction period , having been assigned the same lot number. 3.2.5 Performance of culture media The response of a culture medium to challenge btest organisms under defined conditions. 3.3 Terminologof culture media 3.3.1 Culture medium Formulation of substances , in liquid. semi-solid or in solid form,

43、which contain natural and/or syn thetic constituents intended to support the multiplication, or to preserve the viability. of microor ganlsms. When used in connection with compound words , this term is often shortened into medium (e. g. enrichment medi. um). 3.3.2 Culture media classified bcompositi

44、on 3.3.2.1 Chemically defined culture medium Culture medium consisting only of chemically defined constituentsC i. e. of known molecular structure and degree of purity). 16 SN/T 1538. 1-2005 3.3.2.2 Chemically inmplete culture medium Culture medium consisting entirely or partly of natural materials,

45、 processed or otherwise, the chemi cal composition of which is not completely defined. NOTE For the various chemically undefined components used in culture media 150/T口4/5Cghasspecified harmonized designations-see Annex A. 3. 3. 3 Culture media classified by consistency 3.3.3.1 Liquid culture medium consisting of a