Chapter10

CulturalMethods
IanL.PepperandCharlesP.Gerba
1.

StudentAperformsasoilextractionwithpeptoneastheextractingsolution.Adilutionandplating
techniqueisperformedusingthespreadplatemethod.Thefollowingresultsareobtained:
a.150coloniesarecountedonanR2Aplateatthe105dilution
b.30coloniesarecountedonanutrientagarplateat104dilution
CalculatethenumberofviablebacteriapergramofsoilforR2Aandnutrientagar.Why
arethecountsfornutrientagarlowerthanfortheR2Amedia?
NumberofviablebacteriapergramofsoilfromR2A:.
Assuming0.1mlofsoilextractisspreadplated.
N=1501/106=1.5108CFU/gsoil
Numberofviablebacteriapergramofsoilfromnutrientagar.
N=301/105=3106CFU/gsoil.
Countsfromnutrientagararelowersinceitisanutrientrichmediumwhichdoesnotallowforthe
growthofoligotrophicbacteriafoundinsoil.R2Aisanutrientpoormediumthatdoesallowfor
oligotrophicgrowth.

2.

StudentBperformsasoilextractionwithpeptoneastheextractingsolution.Adilutionandplating
techniqueisperformedusingthepourplatemethod.Thenumberofbacteriapergramof
soiliscalculatedtobe8108.Whatdilutionplatecontained80colonies?
Sincethepourplatemethodisused,assume1mlofsoilextractisplated.

Number

of CFU

/ g soil

for80colonies

80

Dilution

8 10

dilution

80
8 10

10

# colonies
dilution

8 10

3.

StudentCuseddirectmicroscopicmethodstocountbacteriainthesamesoilasstudentA
(seeproblem1).HowwouldstudentCsnumberscomparetostudentAsnumbers
andwhy?
ThenumberforstudentC(directcounts)wouldexceedthenumberforstudentA
(culturablecount)sincethedirectcountalsomeasuresviablebutnonculturable,viable
butdifficulttoculture,andalsononviablebacteria.

4.

StudentCalsoextractedthesamesoilinpeptonebutlettheextractionsitovernightat
roomtemperaturebeforedilutingandplating.Howwouldthesenumberscompare
tostudentAsnumbersandwhy?
ThenumberforstudentCwouldmostlikelyexceedstudentAsnumbersduetopotential
growthofbacteriawithinthepeptoneextractant.

5.

Identifythemajorerrorsassociatedwithdilutionandplatingtechniques.
Therearemanymajorerrors,including:
a.Only110%ofsoilbacteriaareculturable.
b.Noanaerobesarecultured.
c.Theassumptionof1colonyarisingfrom1cellisusuallyinvalidduetocell
clumping,resultinginanunderestimationofCFUs.
d.Sedimentationofsoilduringpreparationofdilutionscanleadtoan
underestimationofCFUs.

6.

Whatisthedifferencebetweenaprimarycelllineandacontinuouscellline?Whichcell
linedoyouthinkisbetterforisolatingvirusesfromtheenvironment?Why?
Inprimarycellculturethecellscomedirectlyfromtheanimalandonlycanbepasseda
limitednumberoftimes.Incontrast,continuouscellslinescanbepassed

indefinably.