課件-growth微生物生長(zhǎng)測(cè)定計(jì)數(shù)

Lecture5-2MicrobialGrowthCHAPTER5BROCKBIOLOGYOFZhao ,ChenSchoolofLifeScienceandBiotechnologyShanghaiJiaoTongUniversity III.MeasuringMicrobial微生物生長(zhǎng)的測(cè)定（計(jì)數(shù)GrowthHowtocountcellnumbersinaDirectcountofmicrobialgrowth:TotalcellcountCountingchambersPlatecountingIndirectcountofmicrobialgrowth:Turbidimetric

ShanghaiJiaoTongDirectmeasurementsofmicrobialgrowth:totalandviablecountsTotalandviablecountingmethodseachhavetheirstrengthsandweaknesses.總菌計(jì)數(shù)與活菌各有利弊。

ShanghaiJiaoTongDirectMeasurements1:TotalcellcountsDirectmicroscopiccount:Thenumberofcellsinapopulationcanbemeasuredbycountingasampleunderthemicroscope.直接顯微,Withliquidsamples,specialcountingmustbeused.液體樣品需使用計(jì)數(shù)室/血球計(jì)數(shù)板

ShanghaiJiaoTongFigureRidgesthatsupportcoverslip

Tocalculatenumberpermilliliterofsample:12cells25largesquares50Sampleaddedhere.Caremustbetakennottoallowoverflow;spacebetweencoverslipandslideis0.02mm(1mm.Wholegridhas25large

cellsarecountedinlarge

Number/mm2(3Number/mm3(1.5 Number/cm3(ml)(1.5squares,atotalareaof1mmatotalvolumeof0.02mm3.

square(16smallsquares):12cells.(Inpractice,severallargesquaresarecountedthenumbersDirectmicroscopiccountingprocedureusingthePetroff-HaussercountingchamberLimitationsofmicroscopicDeadcellsandlivingcellsareallcountedSmallcellsaredifficulttocountNeedphasecontrastmicroscopeNeedhighcellconcentration,106cells/ml(why?)PrecisionisdifficulttoachieveMotilecellsmustbeimmobilizedbeforecounting

ShanghaiJiaoTong DirectMeasurements2:ViableCellCount活菌記數(shù)

ShanghaiJiaoTongFigure

Twomethodsofaviablecount(platecount/colonycount):spread-platemethodandpour-platemethodSampleispipettedontosurfaceofagarplate(0.1mlorless)Pour-plate

Sampleisspreadevenlyoversurfaceofagarusingsterileglass

Sampleispipetted

Sterilemediumisaddedandmixedwellwithinoculum

DilutingcellsuspensionsbeforeWiththeplatemethods,itisimportantthatthenumberofcoloniesdeveloontheplatesnotbetoolarge.Toobtaintheappropriatecolonynumber,thesampletobecountedmustalmostalways

ShanghaiJiaoTongDilutingcellsuspensionsbefore30-300colonies/platewillbe30-300colonies/platewillbeTheQuebeccolony TheAutomatedConcentratecellsuspensionbeforeincubationThemembranefiltrationprocedure.濃縮細(xì)胞懸液Membranewithdifferentporesizesareusedtotrapdifferentmicroorganisms.Incubationtimesformembranesalsovarywiththemediumandmicroorganism.Limitationsofplatingcounting平 Singlecolonymayarisefrommorethanonecell(cellcfu/mlcolony-formingunit/ml菌落形成單位InoculumSuitabilityofIncubationLengthofSizeof

ShanghaiJiaoTongExample:Membrane-filteredsamplesgrownonavarietyofStandardnutrientmediaforatotalbacterialFecalcoliformmediumfordetectingfecalcoliformsthatformbluecolonies.m-EndoagarfordetectingE.coliandothercoliformsthatproducecolonieswithagreensheen.Wortagarforthecultureofyeastsand

ShanghaiJiaoTongIndirectMeasurements:Turbidity濁度Principle:acellsuspensionlookscloudy(turbid)totheeyebecausecellsscatterlightpassingthroughthesuspension.Themorecellspresent,themorelightscatteredandhencethemoreturbidthesuspension.TurbidityMeasurementsofturbidityaremadeMeasurementsofturbidityaremadeinaspectrophotometerorLimitationsofturbiditycountingDeadcellsandlivingcellsarenotParticlesincultureNotgoodathigh

ShanghaiJiaoTongContinuousculture:thechemostat連續(xù)培養(yǎng):恒化器CanwemaintainapopulationatexponentialgrowthGrowthBatchculture-closedsystem批量培FixedvolumeofcultureGrowthrateandgrowthyieldarecontrolledsimultaneously,notmuchyoucando, Continuousculture-opensystem,連續(xù)培FlowsystemofconstantGrowthconditionsremainconstantContinuousadditionofmediumandcontinuousremovalofoverflowCellnumberandnutrientstatusremainGrowthrateandgrowthyieldcanbemanipulatedContinuousculture-thechemostatInInsuchaThepopulationdensityiscontrolledbytheconcentrationoflimitingnutrientinthereservoir;ThegrowthrateiscontrolledbytheflowBothparameterscanbesetbytheexperimenterRelationshipbetweennutrientconcentration,growthrate,andgrowthyieldinabatchculture.Atlownutrientconcentrationsbothgrowthrateandgrowthyieldareaffected.批量培養(yǎng)中營(yíng)養(yǎng)物濃度,生長(zhǎng)速度,與生長(zhǎng)量之間的關(guān)系

GrowthrateandgrowthyieldcanbecontrolledindependentlyinachemostatCellnumberandnutrientstatusremainGrowthrateFlowrateofthefreshmediumintothesystem-dilutionGrowthyieldcontrol(populationConcentrationofalimitingnutrientfactor,carbonsourceornitrogensourceMaintainingapopulationatitsexponentialgrowthphaseforalongtimeShanghaiJiaoTongSteady-staterelationshipsintheThedilutionrateisdeterminedfromtheflowrateandthevolumeoftheculturevessel.稀釋率由流速及培養(yǎng)器體積決定.Athighdilutionrates,growthcannotbalancedilutionandthepopulationwashesSteady-staterelationshipsintheAlthoughthepopulationdensityremainsconstantduringsteadystate,thegrowthrate(doublingtime)canvaryoverawiderange.Thus,theexperimentercanobtainpopulationswithwidelyvaryinggrowthrateswithoutaffectingpopulationdensity.ShanghaiShanghaiJiaoTong Whichgrowthphasehasthelargestincreaseincellnumbersperunitoftime?deathexponentiallagstationary Crypticgrowthoccursduringwhichgrowthphase?deathexponentiallagstationary Whendobatchculturesenterintostationaryphase?whenthecultureistransferredtofreshwhenthenutrientconcentrationinthemediaisdiminishedwhenthetemperatureofthecultureallofthe