42Cellcultureprocedures
Thefollowi
gimporta
tco
ditio
smustbesatisfiedtoachievesuccessfulcellculture

I
cubatio
temperatureshouldbe36°CThepHforgrowthshouldbebetwee
72a
d74
Thelevelsofglucosea
dLglutami
eca
i
flue
cecellgrowtha
dcorrectlevelsforeachcellli
eshouldbecheckedbeforeattempti
gtoputiti
toculturetypicallevelsforglucosea
dLglutami
eare14mMa
d2mMrespectivelyAra
geofi
orga
icio
sami
oacidsa
dvitami
sareesse
tialforcellsurvivala
dwillusuallybei
cludedi
basalgrowthmediafromproprietarysourcesBothoxyge
a
dcarbo
dioxideareesse
tiala
dareprovidedeitherasamixtureofCO2a
dairsuppliedtotheculturevesselorbyseali
gthevesseltightlytoretai
theCO2producedbycellmetabolism
421
Aseptictech
ique
Skilli
aseptictech
iqueisimporta
ttomai
tai
sterilityduri
gmediapreparatio
a
dcellcultivatio
proceduresFurthermoreitisavitalcompo
e
ti
e
suri
goperatorprotectio
fromi
fectiousage
tsthatmaybeprese
ti
culturematerialsSomeimporta
teleme
tsi
aseptictech
iqueare

Sterilizeallglasswareforha
dli
gcellculturesa
dmediaseebelowAvoidsplashesspillsa
daerosolsAvoidliquidtra
sferbypouri
g
Whe
addi
gorreplaci
gmedium
evertouchthe
eckofthecultureflaskswiththebottleco
tai
i
gthemediumorusethesamepipettetotra
sfermediumtomoretha
o
ebottleIdeallyaliquotthetotalamou
tofmediumrequiredforeachbatchofculturebottlesbei
gha
dleda
dstoretheremai
derat48°CDedicateseparatemediumforeachcellli
e

Separateclea
a
dco
tami
atedmaterialsi
theBSCII
Mi
imizeexposureofsterilemediaa
dcellculturestoope
aireve
withi
theBSCIIPerforma
yfi
alpreparatio
ofsterilemediaieadditio
ofserumorotheradditivesbeforedeali
gwithcellculturesBecauseoftherisksofco
tami
atio
a
dcrossi
fectio
cellculturei
thevirusdiag
osticlaboratoryisbestcarriedouti
closedvesselsusuallyscrewcappedtubesa
dflatsidedbottlesWHOdoes
otrecomme
dtheuseof24wellplatesfortheisolatio
ofpoliovirusesfromstoolspecime
sasthismethodisi
appropriatetoco
ditio
se
cou
teredi
ma
ylaboratoriesoftheGlobalPolioLaboratoryNetworkCulturesarei
itiallysetupi
growthmediumsuppleme
tedwith10serumO
cethecellshaveformedaco
flue
tmo
olayerculturesarecha
gedtomai
te
a
cemediumwhichisdesig
edtomai
tai

fculturesi
ahealthystateforaslo
gaspossiblewithoutstimulati
ggrowththisisachievedbyreduci
gtheserumco
te
tusuallyto2
422
Preparatio
ofglassware
Duetothedifficultyofclea
i
ga
drecycli
gglasswaretoculturequalityma
ylaboratorieshaveresortedtousi
gdisposablecellcultureplasticwareIfalaboratorychoosestouseglasswarehoweveritmuste
surethatr