【商检行业标准(SN)】 食品中菌落总数的测定 PetrilfilmTM测试片法

中华人民共和国出入境检验检疫行业标准SN/T1897-2007
食品中菌落总数的测定
PetrifilmTM测试片法
Aerobic plate count in foods-PetrifilmTM aerobic count platemethod2007-05-23发布
中华人民共和国
数码防伪
国家质量监督检验检疫总局
全品伙伴网
2007-12-01实施
SN/T1897-2007
本标准主要参考了AOAC990.12《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：中华人民共和国辽宁出人境检验检疫局、中华人民共和国黑龙江出人境检验检疫局、中华人民共和国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。本标准主要起草人：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、王春梅、周振亚、陆苏。本标准系首次发布的出人境检验检疫行业标准。1）PetrifilmTM是由3M公司提供产品的商品名。http：//foodmate.net
1范围
食品中菌落总数的测定
PetrifilmTM测试片法
本标准规定了食品中菌落总数的测定（PetrifilmTM测试片法）。SN/T1897—2007
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。SN0168出口食品平板菌落计数
3术语和定义
下列术语和定义适用于本标准
菌落总数aerobicplatecount
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-formingunits，cfu个细菌在平板计数琼脂培养基上生长形成肉眼可见的菌落。4原理
4.1PetrifilmTM细菌总数测试片（Aerobic countplates)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（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。
4.23MTM快速涂抹棒（QuickSwab）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3MPetrifilmTM测试片一起使用。它包括0.127m（5in）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，0.127m（5in）长的人造纤维涂抹头可涂抹弯曲管道。QuickSwab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。4.33MTMe·Swab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。3Me·Swab涂抹棒可以和大部分3MPetrifilmTM测试片一起使用。它包括人造纤维涂抹头和10mL缓冲蛋白陈水溶液。3MeSwab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。
设备和材料
温箱：36℃土1℃30℃土1℃
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SN/T1897—2007
冰箱：2℃~5℃。
5.3pH计或精密pH试纸。
5.4放大镜或菌落计数器或3MTMPetrifilmTM自动判读仪。6培养基和试剂
6.11mol/L氢氧化钠（NaOH）：称取40gNaOH溶于1000mL蒸馏水中。6.21mol/L盐酸（HCD）移取浓盐酸90ml用蒸馏水稀释至1000mL。6.33MTMPetrifilmTM细菌总数测试片和压板。6.43MTMQuickSwab快速涂抹棒或3MTMe?Swab涂抹棒。7
检验程序
菌落总数的检验程序见图1。Www.bzxZ.net
制备样品匀液
做成几个适当倍数的稀释液
选择2个～3个适宜稀释度
各取1mL分别加入PetrifilmM测试片压板放置中央处，轻压
静置至少1min
计数各测试片菌落数
计算菌落总数结果
菌落总数的检验程序
8操作步骤
8.1检验样品的制备
按SN0168方法进行样品制备。制备1：10的样品匀液后，无菌操作调节该样品匀液的pH为6.6~7.2，酸性样液用1mol/L氢氧化钠（NaOH）调节，碱性样液用1mol/L盐酸（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
按SN0168的程序进行，培养基为PetrifilmTM菌落总数测试片。8.3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2个3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2
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SN/T1897—2007
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，使样液均勾覆盖于圆形的培养面积上，拿起压板，静置至少1min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
表面取样的样品（如拭子）参见附录A。8.5空气取样的样品
空气取样的样品参见附录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，36℃土1℃条件下培养48h土2h（水产品30℃土1℃培养72h士3h），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，可以将测试片放在一15℃条件以下，不超过7d。
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”（toonumerousto 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
计数和记录数据见SN0168。
9结果的表述
菌落计算的规则同SN0168。
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/cm报告。
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SN/T1897—2007
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.1.13MQuickSwab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人管体内，干法取样的无需折断。A. 1. 1. 2
取出涂抹棉签后取样。
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10S。
将样品倒入3MTMPetrifilmM菌落总数测试片。A.1.1.5
3Me·Swab抹棒法
在涂抹棒容器上标记样品信息
打开旋盖，取出涂抹棒。
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。
A1.2.5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LmL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1h，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
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附录B
（资料性附录）
空气取样方法（直接沉降法）
用1mL无菌稀释液水化3MTMPetrifilmTM菌落总数测试片。静置测试片至少1h，使胶体固化（水化好的测试片可以冷藏7d）。在3M固定夹的两端粘上双面胶带。提起上层膜并贴于胶带上固定，将测试片于空气中暴露15min。合上上层膜后置于培养箱内按规定条件培养。http：//
SN/T1897—2007
SN/T 1897—2007
参考文献
[1 AOAC.20o0. Official Method 990.12. Aerobic Plate Count in Foods Rehydratable film(PetrifilmAerobic Count Plate)method,Official Methods of analysis of AOAC International.17th Ed.AOAC International,Gaithersburg,MD.[2JAOAC 2000 Official Method 986.33 Bacterial and Coliform Counts in Milk Dry RehydratableFilm Methods(Petrifilm Aerobic Count Plate and Petrifilm Coliform Count Plate?)Meth-ods.
[3J AOAC Official Method 989.1o Bacterial and Coliform Counts in Dairy Products Dry Rehydratable Film Methods (Petrifilm Aerobic Count Plate? and Petrifilm Coliform CountPlate）Methods.
[4]Aerobic Microorganisms. Enumeration at 3o℃ in Foods by means of PetrifilmTM PlatesNMKLNO.1461993.
[5JUSDA/FSIS MicrobiologyLaboratoryGuidebook 3rd edition 1998Chapter 3Examination ofFresh, Refrigerated and frozen prepared meat, poultry and pasteurized egg products.[6] Health Products And Food Branch (Canada,MFHPB-33).20o1.Enumeration of total aero-bic bacteria in food products and food ingredients using 3MrM PetrifilmTM Aerobic CountPlates.
[7]Association Francaise de Normalisation （AFNOR):Cetrificate No.:3M ol/1-09/89.3MPetrifilm Total Aerobic Count.Tour Europe, 92049 Paris La Defense Cedex.[8] CCFRA Microbiological Methods Manual Standard Plate Count (Total Viable Count)-3MPetrifilmTM Aerobic Count Plate Method 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。[1o] American Public Health Association.1992. Standard Methods For the Examination of DairyProducts,16thed.APHA,Washington,DC.pp.221-222,231-232.[11] American Public Health Association. 1992. Compendium of Methods for the Microbiological Examination of Foods,3rd ed.APHA,Washington,DC.pp.61,80-89.[12]Betts,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 coliCount.Campden Food&DrinkResearch Association,ChippingCampden
Gloucestershire,UK.
[13J U.S.Department of Health and Human Services.1999.Grade\A\Pasteurized milk Ordi-nance:Recommendations of the Public Health Service.Publication No.229.1999 Revision.U.S.Gov't.Print.Off.Washington,DC.
Foreword
SN/T1897—2007
This standard mainly refers toAOAC 990.123M PetrifilmTM)Aerobic Count Plate MethodAnnexAandannexBare informative.This standard was proposed by Certification and Accreditation Adminstrator of the People's RepublicofChina(CNCA),andisunderthejurisdictionofCNCAThe standard was drafted by Liaoning Entry-exit Export Inspection and Quarantine Bureau of PR.China, Heilongjiang Entry-exit Export inspection and Quarantine Bureau of P.R. China, ShenzhengEntry-exit Export Inspection and Quarantine Bureau of P.R.China,Dalian New Era technology devel-opment Itd.,3M China Ltd..
The main drafers of this standard are Lu Xing-an, Cao Jijuan, Li Sulong,Wu Gang,Xie Zhaocong,Qin Cheng,Zheng Qiuyue,Qi Zhenyu,Wang Yuping,Qiu Chi,LiuRan,Yu Chunmei,Zhou ZhenyaandLuSubiao
This standard is a professional standard of entry-exit inspection and quarantine promulgated for thefirst time.
D)Petrifilmisthetrademarkof3Mcompanyhtt
SN/T1897—2007
Aerobicplatecountinfoods
PetrifilmTMaerobiccountplatemethodScope
This standard specifies a testing method of aerobic plate count in foods (PetrifilmTm plate method).This standard is applicable to the aerobic plate count in food and raw materials, as well as the foodcontact container,operationtable,and other surfaceof equipments.2 Normative reference
The following standard contains provisions which,through reference in this text, constitute provi-sions of this standard. For dated referenees, the subsequent amendments (besides the incorrectcontent) or revisions are not applied to this standard. But the person who reach the agreement onthis standard are encouraged to study if using the lastest edtion of these references. For undatedreferences the lastest edition of the publication referred to applies.SN 0168 Plate count for bacterial colonies in food for export3 Terms and definitions
The following terms and defintions are appficable to this standard3.1
aerobic plate count
Aerobic plate count is the total number of bactiera in 1ml(g) sample or one unit area after samplewas treated and then incubated under certain condition such as media component, culture tempera-ture and time, pH and oxygen demands;Based on the assumption that a live organism can reproduceto one countable colony under certain culture condition,the aerobic plate count is used to describethe amount of the bacteria in foods per unit. The result specified by this method includes only themesophilic and aerobic bacteria on the plate count agar media.3.2
colony-forming units,cfu
One bacterium grows on the plate count agar media and forms the visible colony.8
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4Principle
SN/T1897-2007
4,1 3MM Petrifilm M Aerobic count plates:PetrifilmTM plate are a ready-to-use product containingthe standard medium, a cold-water-soluble gelling agent and TTC indicator that can enhance theeffect of colony enumeration.4.2 3MTm Quick Swab:It is a ready-to-use environmental swab system which is applied to the sam-pling program on the surface in food and beverage industry.3M Quick Swab can be used with al the3M PetrifilmTM Plates.It consists of 0.127 m(5in)long Dacron?tipped swab that uses 1.4 mLletheenbrothto facilitatetherecoveryofbacteriaLetheen broth can neutralize the residual saniti-zers on the surface,o.127m(5 in) artifical fibre can swab the curving pipe.Quick Swab canbe usedto the dry and wet sampling mode4.3 3MTm e.Swab:It is a ready-to-use environmental swab system which is applied to the samplingprogram on the surface in food and beverage industry. The 3M e ·Swab is designed to be used withmost 3MM PetrifilmTM plate.It consists of a 10 mm long rayon swab head and contains 10 mL buff-ered peptone water or phosphate buffered saline.The 3M e.Swab has volume scale on sides,and itcan be used to release sample with proper volume onto the Petrifilm M plates.5
Equipmentandmaterials
Incubator:36℃±1℃30℃±1℃Refrigerator:2℃~5℃.
pHmeterorprecisepHpaper.
magnifierand/orcolonycounteror3MMPetrifilmTM platereader.6 Media and agent
6.11mol/LNaOH:Weigh 40 g NaOH and then dissolve it into 1000mLdistilled water.6.21mol/L HCl:Pipe 90 mLHCl,and thendilute it into 1000 mL distilled water.6.33MTMPetrifilmTMAerobicCountPlateandspreader.6.43MTMQuickSwaband3MTMeSwab.0
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SN/T18972007
Flowchart
The procedure of aerobic plate count is indicated in figure 1.Sample
Prepare sample
Prepare serial suitable dilutionSelect 2~3 suitable dilutioninoculate1mLontoPetrifilmplatePut spreader on center of thebottom filmandleaveatleast1minIncubation
Count colonies on plates
Calculate the colony number
Report
Figure 1-The procedure of the aerobic plate countProcedure
Sampling
The sample is prepared in accordance with the provisions referred to in SN o168.After the 1: 10dilution is prepared, the pH of the dilution should be adjusted to 6.6~7.2. 1mol/L NaOH is used toadjust acidic sample, and 1 mol/L Hcl is used to adjust alkaline sample, or do the pH adjustmentaccordingto theproduct specification.Sample dilution
The sample is diluted in accordance with theprovisions referred to in SN0168,and3MrM PetrifilmmPlateisused insteadof agar.
8.3Inoculation
According to food hygiene standard or the contamination level of sample,2~3 suitable dilutions areselected. Place PetrifilmTN plate on flat surface. Lift top film and inoculate 1mL test suspension ontocenter of film base by pipette or micropipette. Carefully place top film down on inoculum, Distribute10
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