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Benefits
QualitativeandQuantitative Analysis ofFoodMicrobiology
FitriyonoAyustaningwarnoS.TP,M.Si
DepartementofNutritionScience FacultyofMedicine DiponegoroUniversity fansaviola@yahoo.com 08156629709

Knowingfoodquality Calculatingfoodpreservation Measuringcellgrowthduringfermentation cellmass

Method
Cellquantitycalculation Cellmasscalculation: direct:ifmediumdoesnotinterferewiththe measurement indirect:observingsubstrate,metabolite

Cellnumbercalculation
4Basicmethodsusedfortotalnumbers are 1. StandardPlateCounts(SPC)forviablecells 2. Themostprobablenumbers(MPN)forviable cells(statisticaldetermination) 3. Dyereductiontechniquesforviablecellsthat possesreducingcapacities 4. DirectMicroscopicCounts(DMC)forboth viableandnonviablecells

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TotalPlateCounts
PlateCounts:Performserialdilutionsofa sample

PlateCount
Inoculate Petriplates fromserial dilutions

Figure 6.15, top portion

Figure 6.16

Afterincubation,countcoloniesonplatesthat have25250colonies(CFUs)

PlateCount

Cell Viability Measurement: Spread Plate

Figure 6.15

Optimal number to count is 30-300 colonies

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BacteriologicalAnalyticalManual
Normalplates(25250).Selectspreaderfree plate(s).Countallcolonyformingunits(CFU), includingthoseofpinpointsize,onselected plate(s).Recorddilution(s)usedandtotal numberofcoloniescounted. http://www.fda.gov/Food/ScienceResearch/La boratoryMethods/BacteriologicalAnalyticalMa nualBAM/default.htm Plateswithmorethan250colonies.When numberofCFUperplateexceeds250,forall dilutions,recordthecountsastoonumerous tocount(TNTC)forallbuttheplateclosestto 250,andcountCFUinthoseportionsofplate thatarerepresentativeofcolonydistribution. Seeref.2fordetailedguidelines.Mark calculatedAPCwithEAPCtodenotethatit wasestimatedfromcountsoutside25250per platerange(see D3).

PlateswithnoCFU
Whenplatesfromalldilutionshaveno colonies,reportAPCaslessthan1timesthe correspondinglowestdilutionused.Mark calculatedAPCwithasterisktodenotethatit wasestimatedfromcountsoutsidethe25250 perplaterange.Whenplate(s)fromasample areknowntobecontaminatedorotherwise unsatisfactory,recordtheresult(s)as laboratoryaccident(LA).
Plateswith25250CFU. Formula:N=C/[(1*n1)+(0.1*n2)]*(d)a. CalculatetheAPCasfollows:

N=NumberofcoloniespermlorgofproductC= Sumofallcoloniesonallplatescountedn1 =Number ofplatesinfirstdilutioncountedn2 =Numberof platesinseconddilutioncounted d=Dilutionfromwhichthefirstcountswere obtained

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fewerthan25CFU
1:100 232,244 33,28 1:1000

Whenplatesfrombothdilutionsyieldfewer than25CFUeach,recordactualplatecount butrecordthecountaslessthan25x1/d whendisthedilutionfactorforthedilution fromwhichthefirstcountswereobtained

1:100 18 1:1000 2 EAPC/ml(g) <2,500

N= 24,000

morethan250CFU
Whenplatesfromboth2dilutionsyieldmore than250CFUeach(butfewerthan100/cm2), estimatetheaerobiccountsfromtheplates (EAPC)nearest250andmultiplybythe dilution.
1:100 TNTC 1:1000 640 EAPC/ml(g) 640,000

4.Allplateswithspreadersand/orlaboratory accident.ReportrespectivelyasSpreader (SPR),orLaboratoryAccident(LA).

TNTC, too numerous to count. EAPC, estimated aerobic plate count.

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AlternativePlatingTechniques
SpiralPlating UsingSpiralplater Continuouslydistributestheliquid inoculumonthesurfaceofarotating agarplate Resultsindepositingthesamplein anArchimedesspiral Afterinoculationcolonieswillgrow intensivelynearthecenterofthe platewhilefewergrowtowardthe edge ApprovedbyAssociationofOfficial AnalyticalChemists(AOAC)

SpiralPlating

MembraneFilters
Membraneswithaporesizewillretain bacteria(generally0.45m) Thenthemembraneisplacedonanagarplate Collectedmicroorganismsareviewedand counted Suitableforsamplesthatcontainlownumbers ofbacteria ExampleofmembranefiltersisHydrophobic GridMembraneFilter(HGMF)

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Filtration

DirectMeasurementsofMicrobial Growth

Figure 6.17a, b

MembraneFilter
Advantage
Morethan100mlsamplescanbetested. Effectiveandacceptabletechnique. Usedto monitordrinkingwateringovernment laboratories. oneofafewmethodsthatwillallowtheisolation andenumerationofmicroorganisms.

HGMF
AdvancedbySharpeandMichaud Widelyusedtoenumerate microorganismsfromavarietyof foodproducts 1,600waxgridsonasingle membranefilter(hydrophobic walltopreventthespreadof colony) Candetectasfewas10cells/g Canbeusedtoenumerateallcfus includingindicatororganisms ApprovedbyAOACfortotal coliforms,fecalcoliforms,and salmonellae

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TheMostProbable Number
-Presumptive test
Determine by gas produced

TheMostProbableNumber

-Confirmed test
Streak on EMB agar plate

-Completed test
Tranferring to lactose broth and NA slant

TheMostProbableNumber
Advantages
Itisrelativelysimple Resultsfromone laboratoryaremorelikely thanSPCresultstoagree withthosefromanother laboratory Specificgroupsofmicrobes canbedeterminedbyuse ofappropriateselective anddifferentialmedia Itisthemethodofchoice fordeterminingfecal coliformdensities

DyeReduction
Estimatethenumberofviableorganismsinsuitableproducts Methyleneblueandresazurin Addpreparedsupernatantsoffoodtostandardsolution Methyleneblue: blue white Resazurin: blue pinkorwhite Timeusedisinverselyproportionaltothenumberof organismsinthesample Alonghistoryofuseinthedairyindustry,especiallyto measuremicrobialqualityfromrawmilk

Disadvantages
Largeamountofglassware isrequired Thelackofopportunityto observethecolonial morphologyofthe organisms Itslackofprecision

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Dye Reduction
Advantages
1. Simple,rapid,and inexpensive 2. Onlyviablecells activelyreducethe dyes

DirectMicroscopicCount
Smearsoffoodspecimensorculturesontoa microscopeslide Stainwithanappropriatedyes Viewandcountcellswiththeaidof microscope Widelyusedindairyindustry

Disadvantages
1. Notallorganisms reducethedyesequally 2. Notapplicabletofood specimensthatcontain reductiveenzymes

DirectMicroscopicCount
Advantages
1. Itisrapidandsimple 2. Cellmorphologycanbe determined 3. Fluorescentprobesfor improvedefficiency

Disadvantages
1. Resultsdependson eachanalyst 2. Bothviableand nonviablearecounted 3. Foodparticlesarenot alwaysdistinguishable frommicroorganisms 4. Somecellmaynottake thestainwell

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DirectMeasurementsofMicrobial Growth
DirectMicroscopicCount

IndirectMeasurementsofMicrobial Growth
Turbidity MetabolicActivity Dryweight

EstimatingBacterialNumbersby IndirectMethods
Turbidity

Optical Density Measurement

600nm to measure Pichia growth

Figure 620

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EstimatingBacterialNumbersbyIndirect Methods
MetabolicActivity measureproductasafunctionoftime Example:Bacterialcultureproducesacidasa metabolicproduct,thereforeyoucanmonitor growthbymonitoringchangeinpHovertime. Dryweightfilterallcells,dryindesiccator,and weighonscale

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