大肠杆菌的检测：大肠菌群测定的操作细则

大肠杆菌的检测：大肠菌群测定的操作细则大肠菌群系指一群能发酵乳糖、产酸产气、需氧和兼性厌氧的革兰氏阴性无芽胞杆菌。该菌主要来于人畜粪便,故以此作为粪便污染指标来评价食品的卫生质量推断食品中有否污染肠道致病菌的可能。食品中大肠菌群数系以100mL(g)检样内大肠菌群最可能数(MPN)表示。1设备和材料1.1温箱：36±1°C。1.2冰箱:0〜4°C。1.3恒温水浴：44.5±0.5°C。1.4天平。1.5显微镜。1.6均质器或乳钵。1.7平皿:直径为90mm。1.8试管。1.9吸管。1.10广口瓶或三角烧瓶:容量为500mL。1.11玻璃珠:直径约5mm。1.12载玻片。1.13酒精灯。1.14试管架。2培养基和试剂乳糖胆盐发酵管:按GB4789.28中4.9规定。伊红美蓝琼脂平板:按GB4789.28中4.25规定。乳糖发酵管:按GB4789.28中4.10规定。EC肉汤:按GB4789.28中4.11规定。磷酸盐缓冲稀释液:按GB4789.28中3.22规定。生理盐水。革兰氏染色液:按GB4789.28中2.2规定。检样稀释2.42.5乳糖胆盐发酵管:按GB4789.28中4.9规定。伊红美蓝琼脂平板:按GB4789.28中4.25规定。乳糖发酵管:按GB4789.28中4.10规定。EC肉汤:按GB4789.28中4.11规定。磷酸盐缓冲稀释液:按GB4789.28中3.22规定。生理盐水。革兰氏染色液:按GB4789.28中2.2规定。检样稀释3.1.3另取1mL灭菌吸管，按上条操作依次做10倍递增稀释液，每递增稀释一次，换用1支1mL灭菌吸管。3.1.4根据食品卫生标准要求或对检样污染情况的估计，选择三个稀释度，每个稀释度，接种3管。3.2乳糖发酵试验将待检样品接种于乳糖胆盐发酵管内，接种量在1mL以上者，用双料乳糖胆盐发酵管,1mL及1mL以下者，用单料乳糖胆盐发酵管。每一稀释度接种3管，置36±1C温箱内，培养24±2h,如所有乳糖胆盐发酵管都不产气，则可报告为大肠菌群阴性，如有产气者，则按下列程序进行。3.3分离培养将产气的发酵管分别转种在伊红美蓝琼脂平板上，置36±1C温箱内，培养18-24h，然后取出，观察菌落形态，并做革兰氏染色和证实试验。3.4证实试验在上述平板上，挑取可疑大肠菌群菌落1-2个进行革兰氏染色,同时接种乳糖发酵管，置36±1C温箱内培养24±2h，观察产气情况。凡乳糖管产气、革兰氏染色为阴性的无芽胞杆菌，即可报告为大肠菌群阳性。3.5报告根据证实为大肠菌群阳性的管数，查MPN检索表，报告每100mL(g)大肠菌群的MPN值。4粪大肠菌群(faecalcoliform)4.1用接种环将所有产气的乳糖胆盐发酵管培养物（见3.2条）转种于EC肉汤管内，置44.5±0.2°C水浴箱内（水浴箱内的水面应高于EC肉汤液面），培养24±2h，经培养后,如所有EC肉汤管均不产气，则可报告为阴性;如有产气者，则将所有产气的EC肉汤管分别转种于伊红美蓝琼脂平板上，置培养18-24h,凡平板上有典型菌落者,则证实为粪大肠菌群阳性。4.2结果报告根据证实为粪大肠菌群的阳性管数，查MPN检索表，报告每100mL（g）粪大肠菌群的MPN值乳酸菌的检测：一概述乳酸菌是指一群能分解葡萄糖或乳糖产生乳酸，需氧和兼性厌氧，多数无动力，过氧化氢酶阴性，革兰氏阳性的无芽胞杆菌和球菌。这类细菌在自然界分布广泛，可栖居在人和各种动物的口腔、肠道等器官内，在土壤、食品、饲料、水及一些临床标本中都有乳酸菌的存在。乳酸菌在工业、农业和医药等与人类生活密切相关的领域应用价值很高，相当多的乳酸菌对人、畜的健康起着有益的作用，但个别菌种能对人畜致病，乳酸菌主要包括23个属的细菌。二样本米集检样主要为含乳酸菌活菌的饮料和微生态制剂，样本应放入冰箱保存，心快检验。三检验方法（中华人民共和国国家标准乳酸菌饮料中乳酸菌的微生物学检验GB/T16347-1996）乳酸菌菌总数的测定：乳酸菌菌落总数是指标样在一定条件下培养后，所得1ml检样中所含乳酸菌菌落的总数。（一）检验程序乳酸菌菌落总数检验程序如下：（二）培养基和试剂改良TJA培养基（改良番茄汁琼脂培养基）；改良MC培养基（modifiedChalmers培养基）；0.1%亚甲蓝牛乳培养基；6.5%氯化钠肉汤参照；pH9.6葡萄糖肉汤；40%胆汁肉汤；淀粉水解培养基；精氨酸水解培养基；乳酸杆菌糖发酵管；七叶苷培养基；革兰氏染色液；3%过氧化氢溶液；蛋白胨水、靛基质试剂；明胶培养基；硝酸盐培养基、硝酸盐试剂；生理盐水：定量分装于三角瓶和试管内灭菌。（三）操作步骤1.以无菌操作将经过充分摇匀的检样25ml（或25g）放入含有225ml灭菌生理盐水的来菌广口瓶内做成1：10的均匀稀释液。用1ml灭菌吸管吸取1：10稀释液1m，沿管壁徐徐注入含有9ml灭菌生理盐水的试管内（注意吸管尖端不要触及管内稀释液）。另取1ml灭菌吸管，按上述操作顺序，作10倍增稀释液，如此每递增一次，即换用1支1ml灭菌吸管。选择2〜3个以上适宜稀释度，分别在作10倍递增稀释的同时，即以吸取该稀释度的吸管移1ml稀释液于灭菌平皿内，每个稀释度作两个平皿。稀释液移入平皿后，应及时将冷至50C的乳酸菌计数培养基（改良TJA或改良MC）注入平皿约15ml，并转动平皿使混合均匀。同时将乳酸菌计数培养基倾入加有1ml稀释液检样用的灭菌生理盐水的灭菌平皿内作空白对照，以上整个操作自培养物加入培养皿开始至接种结束须在20min内完成。待琼脂凝固后，翻转平板，置36C±1C温箱内培养72h±3h取出，观察乳酸菌菌特征，选取菌落数在30〜300之间的平板进行计数。计算后，随机挑取5个菌落数进行革兰氏染色，显微镜检查并做过氧氢酶试验。革兰氏阳性，过氧化氢酶阴性，无芽胞的球菌或杆菌可定为乳酸菌。根据证实为乳酸菌菌落计算出皿内的乳酸菌数，然后乘其稀释倍数即得每亳升样品中乳酸菌数。例如，检样10-4的稀释液在改良TJA琼脂平板上，生成的可疑菌落为35个，取5个鉴定，证实的乳酸菌的4个，则1ml检样中乳酸菌数为：35X4/5X104=2.8X105乳酸菌在改良TJA和改良MC培养基上菌落生长形态特征。乳酸菌的鉴定对上述分离到的乳酸菌需进行菌种鉴定时，则作以下试验。菌种制备自平板上挑取菌落，接种于改良TJA或改良MC琼脂斜面，于36^±1°C，24〜48h培养，刮取菌苔，分别进行下列试验。乳酸杆菌鉴定试验极少见还原硝酸盐，不液化明胶，不产生靛基质和硫化氢。常见乳杆菌属内种的碳水化合物反应。产酸乳的链球菌的鉴别试验。DetectionofEscherichiacoli:DeterminationofcoliformbacteriaintheoperatingrulesColiformgroupreferstothegroupoffermentlactose,acidgas,aerobicandfacultativelyanaerobicgram-negativenon-sporingbacillus.Thebacteriamainlycomesfromhumanandanimalfeces,thereforeasthefaecalpollutionindicatorstoevaluatethehygienicqualityoffood,foodisnotinferpollutionintestinalpathogensmay.Thenumberofcoliformbacteriainfoodby100mL(g)samplesincoliformsmostprobablenumber(MPN)said.theequipmentandmaterials1.1:36±1Ctemperaturebox.1.2:0~4Crefrigerator.1.3:44.5±0.5Cconstanttemperaturewaterbath.In1.4days.The1.5microscope.1.6homogenizerormortar.1.7Petridish:diameterof90mm.1.8tube.The1.9straw.1.10jarsorErlenmeyerflask:capacityof500mL.1.11glassbeads:diameterofabout5mm.1.12slides.1.13alcohollamp.The1.14testtuberack.culturemediaandreagentsbilesaltlactosefermentationtube:accordingtoGB4789.284.9provisions.Eosinmethyleneblueagarplate:2.2inGB475provisions.lactosefermentationtube:accordingtoGB4789.284.10provisions.ECbroth:accordingtoGB4789.284.11provisions.phosphatebuffersolutionatGB4789.28:3.22set.saline.Gramstainingsolution:accordingtoGB4789.282.2provisions.sampledilutiontoasepticsample25mL(orG)onthe225mLofsterilesalineorotherdilutionliquidsterilizationglassbottles(bottlestotheappropriatenumberofglassbeads)orsterilizationmortar,afterfullshakingorgrindingtomake1:10uniformdilution.Solidsamplewithhomogenizer,8000-10000r/min1min1:10speedofprocessing,madeofuniformdilution.1mLsterilizingstrawtodraw1:10dilutionof1mL,injectedwith9mLofsterilesalineorotherdilutionliquidinsidethetube,shakingthetubemixing,madeof1:100dilution.another1mLsterilizationstraw,accordingtotheoperationinturndo10timestheincrementaldilution,eachincrementaldilutiononce,for11mLsterilizingstraw.accordingtothestandardsoffoodhygienerequirementsortosamplecontaminationestimation,choiceofthreedilutions,eachdilution,inoculatedwith3tubes.lactosefermentationtestThesampletobedetectedwithbilesaltlactosefermentationtube,inoculationquantityisin1mLabove,usingdoublebilesaltlactosefermentationtube,1mLand1mLbelow,withlactosebilesaltfermentationtube.Eachdilutioninoculation3,is36±1°Ctemperatureis24±2h,culture,suchasallbilesaltlactosefermentationtubewithoutgasproduction,canbereportedascoliformsnegative,suchasgasproduction,accordingtothefollowingprocedures.isolatedcultureThegasproductionofthefermentationpipearerespectivelyusedintheeosinmethyleneblueagarplate,is36±1Ctemperaturebox,18-24hculture,thenremoved,colonymorphologieswereobserved,andGoftestIntheplate,weresuspiciousofcoliformscolony1-2forGramstaining,simultaneousinoculationoflactosefermentationtube,is36±1Ctemperatureinsidecultureof24±2h,observationofgases.Wherelactosetubegasproduction,gramnegativebacilluscanreportno,ascoliforms.ReportAccordingtoconfirmedascoliformstubenumber,checktheMPNretrievaltable,each100mL(g)MPNvalueofcoliformbacteria.4fecalcoliform(faecalcoliform)inoculationloopbyallthegasproducingbilesaltlactosefermentationtubecultures(seeclause3.2)transferredtoECbrothtubes,is44.5+0.2Cwaterbathtank(waterbathtankwaterlevelshouldbehigherthantheECbrothculture),24±2h,aftercultivation,suchasallECbrothtubesarenotgasproduction,canbereportedasnegative;suchasgas,thenallthegasECbrothtubesarerespectivelyswitchedtotheIranianredmethyleneblueagarplate,thecultureof18-24h,wheretheplatehastypicalcolony,isconfirmedforfecalcoliformpositive.findingsreportAccordingtotheconfirmedfecalcoliformpositivenumber,checktheMPNretrievaltable,each100mL(g)MPNvalueoffecalcoliformsDetectionoflacticacidbacteria:AnoverviewLacticacidbacteriaareagroupofdecompositionofglucoseorlactosetoproducelacticacid,aerobicandfacultativeanaerobic,mostunpowered,catalasenegative,grampositivebacillusandcoccuswithout.Thisclassofbacteriawidelydistributedinnature,canliveinthehumanandanimalgutandotherorgans,themouth,inthesoil,food,feed,waterandsomeclinicalspecimensarelacticacidbacteriainthepresenceof.Lacticacidbacteriainindustry,agricultureandmedicineandhumanlifeiscloselyrelatedtothefieldofapplicationofvalueisveryhigh,quiteanumberoflacticacidbacteriaonhuman,animalhealthplaysabeneficialrole,butindividualspeciestohumanandanimalpathogenic,lacticacidbacteriamainlyincludes23generaofbacteria.TwosamplecollectionThesampleismainlycontainslivelacticacidbacteriabeverageandpreparation,samplesshouldbestoredinthefridge,heartfastinspection.Threetestmethods(thepeople'sRepublicofChinanationalstandardsoflacticacidbacteriabeverageoflacticacidbacteriamicrobiologicalexaminationoftheGB/T16347-1996)Lacticacidbacteria:Determinationofthetotalnumberoflacticacidbacteriacolonyistheindexsampleculturedundercertainconditions,the1mlsamplecontainedinthetotalnumberoflacticacidbacteria.(a)inspectionprocedureLacticacidbacteriacolonycountdeterminationprocedureisasfollows:(two)culturemediaandreagentsImprovedTJAmedium(modifiedtomatojuiceagar);improvedMCmedium(modifiedChalmersmedium);0.1%methylenebluemilkmedium;SodiumChlorideBroth6.5%reference;pH9.6glucosebroth;40%BileBroth;starchhydrolysatemedium;argininehydrolysismedium;lactobacillusfermentationtubesevenleafglucoside;culturemedium;Gramstainingsolution;3%hydrogenperoxidesolution;peptonewater,indolereagent;culturemedium;nitratemedium,nitratereagent;saline:quantitativepackagingintriangularbottleandtubesterilization.(three)stepswithsterileoperationwillthoroughlyshakensample25ml(or25g)intoa225mlofsterilesalineinthejarmadeof1:10uniformdilution.with1mlsterilizingstrawtodraw1:10dilutionof1m,alongthepipewallwasinjectedwith9mlofsterilesalineinvitro(Note:donottouchthetipofstrawtubedilution).another1mlsterilizationstraw,accordingtotheorderofoperations,10doublingdilution,soeachincrementaltime,namelythechangewith11mlsterilizingstraw.select2to3ormoreappropriatedilution,respectivelyin10timesdilutedwhileincreasing,namelytodrawthedilutionofthestrawshift1mldilutioninsterilePetridish,eachdilutionoftwoPetridish.dilutionliquidintothePetridish,willbetimelycoolingto50°Cmediumforlacticacidbacteriacounts(modifiedTJAormodifiedMC)intoaPetridishofabout15ml,androtatingtheflatdishmakeuniformmixing.Atthesametime,mediumforlacticacidbacteriacountspourwith1mldilutionofsampleswithsterilesalinesterilePetridishfortheblankcontrol,thewholeoperationfromabovecultureintoPetridishestoinoculationendshallbecompletedwithin20min.agarsolidified,turningplate,is36±1C,thetemperatureinsidecultured72h±3Hremoved,observationoflacticacidbacteriacharacteristics,selectthecolonynumberin30~300betweentheplatecount.Aftercalculation,randomlyselect