大腸桿菌的檢測：大腸菌群測定的操作細則

大腸桿菌的檢測：大腸菌群測定的操作細則大腸菌群系指一群能發(fā)酵乳糖、產(chǎn)酸產(chǎn)氣、需氧和兼性厭氧的革蘭氏陰性無芽胞桿菌。該菌主要來于人畜糞便,故以此作為糞便污染指標來評價食品的衛(wèi)生質(zhì)量推斷食品中有否污染腸道致病菌的可能。食品中大腸菌群數(shù)系以100mL(g)檢樣內(nèi)大腸菌群最可能數(shù)(MPN)表示。1設(shè)備和材料1.1溫箱：36±1°C。1.2冰箱:0?4°C。1.3恒溫水浴：44.5±0.5°C。1.4天平。1.5顯微鏡。1.6均質(zhì)器或乳缽。1.7平皿:直徑為90mm。1.8試管。1.9吸管。1.10廣口瓶或三角燒瓶:容量為500mL。1.11玻璃珠:直徑約5mm。1.12載玻片。1.13酒精燈。1.14試管架。2培養(yǎng)基和試劑乳糖膽鹽發(fā)酵管:按GB4789.28中4.9規(guī)定。伊紅美藍瓊脂平板:按GB4789.28中4.25規(guī)定。乳糖發(fā)酵管:按GB4789.28中4.10規(guī)定。EC肉湯:按GB4789.28中4.11規(guī)定。磷酸鹽緩沖稀釋液:按GB4789.28中3.22規(guī)定。生理鹽水。革蘭氏染色液:按GB4789.28中2.2規(guī)定。檢樣稀釋2.42.5乳糖膽鹽發(fā)酵管:按GB4789.28中4.9規(guī)定。伊紅美藍瓊脂平板:按GB4789.28中4.25規(guī)定。乳糖發(fā)酵管:按GB4789.28中4.10規(guī)定。EC肉湯:按GB4789.28中4.11規(guī)定。磷酸鹽緩沖稀釋液:按GB4789.28中3.22規(guī)定。生理鹽水。革蘭氏染色液:按GB4789.28中2.2規(guī)定。檢樣稀釋3.1.3另取1mL滅菌吸管，按上條操作依次做10倍遞增稀釋液，每遞增稀釋一次，換用1支1mL滅菌吸管。3.1.4根據(jù)食品衛(wèi)生標準要求或?qū)z樣污染情況的估計，選擇三個稀釋度，每個稀釋度，接種3管。3.2乳糖發(fā)酵試驗將待檢樣品接種于乳糖膽鹽發(fā)酵管內(nèi)，接種量在1mL以上者，用雙料乳糖膽鹽發(fā)酵管,1mL及1mL以下者，用單料乳糖膽鹽發(fā)酵管。每一稀釋度接種3管，置36±1C溫箱內(nèi)，培養(yǎng)24±2h,如所有乳糖膽鹽發(fā)酵管都不產(chǎn)氣，則可報告為大腸菌群陰性，如有產(chǎn)氣者，則按下列程序進行。3.3分離培養(yǎng)將產(chǎn)氣的發(fā)酵管分別轉(zhuǎn)種在伊紅美藍瓊脂平板上，置36±1C溫箱內(nèi)，培養(yǎng)18-24h，然后取出，觀察菌落形態(tài)，并做革蘭氏染色和證實試驗。3.4證實試驗在上述平板上，挑取可疑大腸菌群菌落1-2個進行革蘭氏染色,同時接種乳糖發(fā)酵管，置36±1C溫箱內(nèi)培養(yǎng)24±2h，觀察產(chǎn)氣情況。凡乳糖管產(chǎn)氣、革蘭氏染色為陰性的無芽胞桿菌，即可報告為大腸菌群陽性。3.5報告根據(jù)證實為大腸菌群陽性的管數(shù)，查MPN檢索表，報告每100mL(g)大腸菌群的MPN值。4糞大腸菌群(faecalcoliform)4.1用接種環(huán)將所有產(chǎn)氣的乳糖膽鹽發(fā)酵管培養(yǎng)物（見3.2條）轉(zhuǎn)種于EC肉湯管內(nèi)，置44.5±0.2°C水浴箱內(nèi)（水浴箱內(nèi)的水面應(yīng)高于EC肉湯液面），培養(yǎng)24±2h，經(jīng)培養(yǎng)后,如所有EC肉湯管均不產(chǎn)氣，則可報告為陰性;如有產(chǎn)氣者，則將所有產(chǎn)氣的EC肉湯管分別轉(zhuǎn)種于伊紅美藍瓊脂平板上，置培養(yǎng)18-24h,凡平板上有典型菌落者,則證實為糞大腸菌群陽性。4.2結(jié)果報告根據(jù)證實為糞大腸菌群的陽性管數(shù)，查MPN檢索表，報告每100mL（g）糞大腸菌群的MPN值乳酸菌的檢測：一概述乳酸菌是指一群能分解葡萄糖或乳糖產(chǎn)生乳酸，需氧和兼性厭氧，多數(shù)無動力，過氧化氫酶陰性，革蘭氏陽性的無芽胞桿菌和球菌。這類細菌在自然界分布廣泛，可棲居在人和各種動物的口腔、腸道等器官內(nèi)，在土壤、食品、飼料、水及一些臨床標本中都有乳酸菌的存在。乳酸菌在工業(yè)、農(nóng)業(yè)和醫(yī)藥等與人類生活密切相關(guān)的領(lǐng)域應(yīng)用價值很高，相當多的乳酸菌對人、畜的健康起著有益的作用，但個別菌種能對人畜致病，乳酸菌主要包括23個屬的細菌。二樣本米集檢樣主要為含乳酸菌活菌的飲料和微生態(tài)制劑，樣本應(yīng)放入冰箱保存，心快檢驗。三檢驗方法（中華人民共和國國家標準乳酸菌飲料中乳酸菌的微生物學檢驗GB/T16347-1996）乳酸菌菌總數(shù)的測定：乳酸菌菌落總數(shù)是指標樣在一定條件下培養(yǎng)后，所得1ml檢樣中所含乳酸菌菌落的總數(shù)。（一）檢驗程序乳酸菌菌落總數(shù)檢驗程序如下：（二）培養(yǎng)基和試劑改良TJA培養(yǎng)基（改良番茄汁瓊脂培養(yǎng)基）；改良MC培養(yǎng)基（modifiedChalmers培養(yǎng)基）；0.1%亞甲藍牛乳培養(yǎng)基；6.5%氯化鈉肉湯參照；pH9.6葡萄糖肉湯；40%膽汁肉湯；淀粉水解培養(yǎng)基；精氨酸水解培養(yǎng)基；乳酸桿菌糖發(fā)酵管；七葉苷培養(yǎng)基；革蘭氏染色液；3%過氧化氫溶液；蛋白胨水、靛基質(zhì)試劑；明膠培養(yǎng)基；硝酸鹽培養(yǎng)基、硝酸鹽試劑；生理鹽水：定量分裝于三角瓶和試管內(nèi)滅菌。（三）操作步驟1.以無菌操作將經(jīng)過充分搖勻的檢樣25ml（或25g）放入含有225ml滅菌生理鹽水的來菌廣口瓶內(nèi)做成1：10的均勻稀釋液。用1ml滅菌吸管吸取1：10稀釋液1m，沿管壁徐徐注入含有9ml滅菌生理鹽水的試管內(nèi)（注意吸管尖端不要觸及管內(nèi)稀釋液）。另取1ml滅菌吸管，按上述操作順序，作10倍增稀釋液，如此每遞增一次，即換用1支1ml滅菌吸管。選擇2?3個以上適宜稀釋度，分別在作10倍遞增稀釋的同時，即以吸取該稀釋度的吸管移1ml稀釋液于滅菌平皿內(nèi)，每個稀釋度作兩個平皿。稀釋液移入平皿后，應(yīng)及時將冷至50C的乳酸菌計數(shù)培養(yǎng)基（改良TJA或改良MC）注入平皿約15ml，并轉(zhuǎn)動平皿使混合均勻。同時將乳酸菌計數(shù)培養(yǎng)基傾入加有1ml稀釋液檢樣用的滅菌生理鹽水的滅菌平皿內(nèi)作空白對照，以上整個操作自培養(yǎng)物加入培養(yǎng)皿開始至接種結(jié)束須在20min內(nèi)完成。待瓊脂凝固后，翻轉(zhuǎn)平板，置36C±1C溫箱內(nèi)培養(yǎng)72h±3h取出，觀察乳酸菌菌特征，選取菌落數(shù)在30?300之間的平板進行計數(shù)。計算后，隨機挑取5個菌落數(shù)進行革蘭氏染色，顯微鏡檢查并做過氧氫酶試驗。革蘭氏陽性，過氧化氫酶陰性，無芽胞的球菌或桿菌可定為乳酸菌。根據(jù)證實為乳酸菌菌落計算出皿內(nèi)的乳酸菌數(shù)，然后乘其稀釋倍數(shù)即得每亳升樣品中乳酸菌數(shù)。例如，檢樣10-4的稀釋液在改良TJA瓊脂平板上，生成的可疑菌落為35個，取5個鑒定，證實的乳酸菌的4個，則1ml檢樣中乳酸菌數(shù)為：35X4/5X104=2.8X105乳酸菌在改良TJA和改良MC培養(yǎng)基上菌落生長形態(tài)特征。乳酸菌的鑒定對上述分離到的乳酸菌需進行菌種鑒定時，則作以下試驗。菌種制備自平板上挑取菌落，接種于改良TJA或改良MC瓊脂斜面，于36^±1°C，24?48h培養(yǎng)，刮取菌苔，分別進行下列試驗。乳酸桿菌鑒定試驗極少見還原硝酸鹽，不液化明膠，不產(chǎn)生靛基質(zhì)和硫化氫。常見乳桿菌屬內(nèi)種的碳水化合物反應(yīng)。產(chǎn)酸乳的鏈球菌的鑒別試驗。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