CountingMicrobes

Thursday,February20,2014
11:41AM

Methodsofcellquantification
1. Countingmicroorganisms:determinationofnumberofcellsorconcentrationofcellsina
sampleplaysnumerousandimportantrolesinmicrobiologicalcharacterizationand
experimentation.
A. Directcounts(Actualcountingofmicroorganisms):Amethodbywhichareasonablystatistically
precisemeasurementofcellquantificationisachieved.
1. Thebasisofadirectcountistheactualcountingofeveryorganism(oreverylivingorganism
oftenlimitedtoasingletypeofmicroorganism)presentinasubsampleofapopulation.
2. Types:
a. Viablecounts:onlyviable(alive)cellsarecounted.
i. Techniques:
platecounts(Coloniesfoundedbycells):Coloniesgrowninpetridishesby
variousmethods(notincludingstreaking)maybeusedtodeterminethe
countofviablemicroorganisms
assumesthateverycolonyisfoundedbyasinglecell.
Thatcellmusthavebeenaliveinordertogrowandformacolony.
Problems:
requirelengthyincubation.Cellclumping(2/moreviablecells
stucktogether.Countedassinglecell)=>undercountofviable
cells
toomany/fewcoloniesonaplate;preventionofcrowding
oftenrequiresserialdilution*
toofewcellsrequiresconcentrating,e.g.,bycentrifugationor
filtration**
originalculturemustbeconcentratedpriorto
determiningitsplatecount.
Filtration,whichsievesmicroorganismsoutofthe
medium,isonemethodofconcentrating
microorganisms.
*nomorethan30to300coloniesperplate.**usesa
minimum#whichis10foldsmallerthanthemaximum#,but
greaterthan30.
Note:noneofthesecaveatsareuniquetoplatecounts.
Types:
pourplating:bactinemptypetridish,thenpouragarontop
=>smallcolonies.Badforobligateaerobes.
spreadplating
mostprobablenumbermethod(Noplate):approximateviablecountby
dilutingculturesthengrowingthedilutionculturesinbrothtubes.
Atthedilutionatwhichbrothbecomesturbidornotwithsimilar
likelihood,culturehasbeendilutedtothepointthatthebroth
tubeswereinoculatedwithontheorderofonlyasingle
microorganism(turbid)orfewer(notturbid).
Theconcentrationofthecultureisthentakentobeequaltothe
amountofdilutionnecessarytohavereachedthispoint.
Advantageouswherebrothisadvantageous:
Forexample,manyorganismsarenotgoodatforming
colonies,suchashighlymotileorganisms.
"Whensamplescontaintoofeworganismstogivereliable
measuresofpopulationsizebythestandardplatecount
method,asinfoodandwatersanitationstudies,orwhen
organismswillnotgrowonagar,themostprobablenumber
methodisused."(p.143,Black,1996)
b. Totalcounts:allcellsarecounted,whetherdeadoralive.Generally,requires
microscopes.
i. Technique:
directmicroscopiccount:determinationofthe#ofmicroorganismsfound
withinademarcatedregionofaslideknowntoholdacertainvolumeof
culture.
veryrapid
Problems:
requireshighcellconcentrations(e.g.,10
7
/ml)
potentiallycountingdeadandlivingcellswithequal
probability.
B. estimationsofcellnumber:usevariouscorrelatesofcellnumberratherthandirectcounting
1. oftenpreferableeitherforconvenienceorbecausedirectcountingisdifficultoreven
impossibleinmanysituations(forexample,whenquantifyingfilamentousorganisms).
2. Estimatesofcellnumberincludedeterminationsof:
a. Turbidity:cloudinessofaculture;causedbytheindividualcellsscatteringlight
i. Degreeofturbidityisadirectcorrelateofcellmass.
ii. Asestimatorofcellnumber:1stdetermineaveragecellmassofindividualcells
inaculture
iii. Thisstandardizationusuallyworksbestforculturesgrowinginexponential
phase.
b. metabolicactivity:outputorinputofaculturemaybeusedtoestimateviablecount.
Thismethodhasthesamecaveatsasturbiditymethods.
i. Examplesinclude:"Therateatwhichmetabolicproductssuchasgasesand/or
acidsareformedbyculturereflectsthemassofbacteriapresent...Therateat
whichasubstratesuchasglucoseoroxygenisusedupalsorefelctscellmass."
(p.144,Black,1996)
c. drymass:Estimationofcellcount
Determinationsofdrymasshasalloftheproblemsofturbidityand
metabolicactivitywiththeaddedproblemofhavingtodrycultures
beforeemployingit.
"Tocalculatethedryweightofcells,theymustbeseparatedfromthe
mediumbysomephysicalmeanssuchasfiltrationorcentrifugation.The
cellsarethendried,andtheresultingmassisweighed."(p.144,Black,
1996)
i. Forfilamentousorganismsthismethodworkssufficientlywellcomparedtothe
othermethodslistedthatdrymassdeterminationisemployed.