Roles Ior Aseptic TrunsIer Techniqoes

BeIore Work
1. Remove uII unnecessury ILems Irom LIe work
ureu.
z. Keep IuIr under conLroI.
¸. PuL on Iub couL.
q. WusI Iunds.
¸. GIove up.
6. DIsInIecL work ureu.

Ðoring Work
O $LerIIIze InsLrumenLs beIore LoucIIng cuILure und
uILer.
O Do noL Iuy cuILure Lubes on work ureu.
O Work neur IIume Lo uIIow rIsIng uIr currenLs Lo
keep mIcrobes & dusL uwuy.
O Keep PeLrI dIsI IIds us burrIer beLween mouLI und
nose or IoId enLIre dIsI mInus IId upsIde down
Ior InocuIuLIon.
O Work quIckIy und uvoId excess LuIkIng wIIIe
cuILures ure open.
AIter work
O $LerIIIze uII InsLrumenLs uguIn.
O PuL gIoves und peLrI dIsIes In bIoIuzurd bugs
wIen IInIsIed.
O WusI Iunds LIorougIIy.
O DIsInIecL work ureu.
O PuL used Lubes In rucks Lo be uuLocIuved.
O n generuI, keep LIe work ureu us cIeun us
possIbIe. AvoId producIng excess uerosoIs by
LuIkIng, IuugIIng, eLc. wIIIe cuILures ure open.
O Keep cuILures cIosed us mucI us possIbIe,
workIng quIckIy und uccuruLeIy Is essenLIuI.
O Keep uII ILems uwuy Irom mouLI, uvoId Iood or
drInk In IuboruLory. AIwuys wusI Iunds und work
ureu beIore IeuvIng.
O euve uny cIoLIIng cover used In LIe Iub Lo uvoId
LukIng uny exLru mIcrobes ouL or brIngIng uny In.

Aseptic TrunsIer
AsepLIc LecInIques ure prucLIced becuuse LIey ure
procedures LIuL:
ProLecL LIe cuILure.
ProLecL LIe worker
ProLecL LIe envIronmenL
MuInLuInIng purILy oI cuILure Is essenLIuI In
mIcrobIoIogy II LIe bIoIogIsL Is Lo be ubIe Lo IdenLIIy
bucLerIu, LesL Ior unLIbIoLIc sensILIvILy, or muInLuIn
sLock cuILures. OILen In nuLure u pure cuILure Is
ImpossIbIe Lo come by becuuse specIes IIve
LogeLIer. TIe scIenLIsL Is IeIL workIng wILI mIxed
cuILures. Pure cuILures cun be derIved Irom mIxed
cuILures LIrougI IsoIuLIon oI cuILures und LIIs uIso
requIres LIuL sLerIIe (usepLIc) LecInIques Lo be used.

NormuIIy LrunsIerence Is done Irom
coIonIes. A coIony consIsLs oI usuuIIy severuI
mIIIIon ceIIs LIuL ure ussumed Lo be LIe descendunLs
Irom one ceII.
nocuIuLIons Irom one medIu Lo unoLIer,
LIereIore, ure usuuIIy done by removuI oI u Iew
mIIIIon ceIIs Irom one coIony InLo u new
envIronmenL. TIIs musL be done wILI LIe InLegrILy
oI uII coIonIes remuInIng InLucL. TIrougI LIe use oI
sLerIIe LecInIques, LIIs cun be uccompIIsIed
successIuIIy.
TIere ure u number oI LooIs LIuL ure used
Ior InocuIuLIon procedures. nocuIuLIng Ioops ure
used wIen LrunsIerrIng members oI u broLI cuILure
Lo unoLIer broLI, pIuLed medIu or un ugur sIunL.
nocuIuLIng needIes ure used wIen InocuIuLIng u
broLI cuILure Irom u coIony on pIuLed medIu or
wIen mukIng u sLub In un ugur deep or ugur sIunL
Irom broLI or soIId medIu. ¡orceps ure used Lo
pIuce sLerIIe dIsks conLuInIng some LesLIng ugenL In
u broLI cuILure or on u soIId medIu cuILure. PIpeLs
ure used wIen LrunsIerrIng IIquIds InLo oLIer
IIquIds or onLo soIId medIu.
¡IumIng Is used Lo IncIneruLe uny mIcrobes
IeIL on Ioops und needIes. AIcoIoI IIumIng Is used Lo
sLerIIIze Iorceps. WIen IIumIng InocuIuLIng Ioops
und needIes, cure sIouId be Luken uvoId burnIng LIe
pIusLIc IundIe uL LIe end oI eucI. TIe meLuI oI LIe
Ioop or needIe sIouId gIow red IoL und LIen be
uIIowed Lo cooI beIore dIppIng IL InLo uny cuILures -
II LIe meLuI Is Loo IoL IL wIII kIII LIe orgunIsms LIuL
ure Lo be used Ior InocuIuLIon. AIcoIoI IIumIng Ior
LIe Iorceps Is done by dIppIng LIe Iorceps InLo u
smuII umounL oI uIcoIoI und LIen burnIng LIe
uIcoIoI oII. TIe Iorceps sIouId be dIpped und
burned LIree LImes. Cure sIouId be Luken Lo uvoId
uIcoIoI runnIng up Lowurd LIe Iund.
TIe IIume wIII IoIIow LIe uIcoIoI und burns
wIII resuIL. PIpeLs normuIIy used In Iub ure
prepuckuged, sLerIIe, dIsposubIe pIpeLs.
$omeLImes gIuss pIpeLs ure used und LIese
ure sLored In cuns. TIe gIuss pIpeLs ure dIscurded
InLo u pIpeL jur IIIIed wILI dIsInIecLunL. DIsposubIe
pIpeLs ure deposILed In bIoIuzurd bugs. L Is
ImporLunL LIuL pIpeLLors ure uIwuys used und
pIpeLLIng by mouLI Is proIIbILed.

Procedore 1 - Sterile Pipetting with
Pipettors
1. ObLuIn one Lube oI sLerIIe T$ und one Lube oI
sLerIIe wuLer. PIuce In u Lube ruck.
z. ubeI LIe T$ Lube 'o.¸ m HzO` LIen wrILe your
InILIuIs & LIe duLe.
¸. oosen LIe IIds on LIe z Lubes.
q. Open u prepuckuged, sLerIIIzed 1 m pIpeL und
uLLucI IL Lo LIe bIue pIpeLLor.
¸. Remove LIe IId Irom LIe wuLer Lube, IIume LIe IIp
oI LIe Lube, InserL LIe 1 m pIpeL und wILIdruw o.¸
m oI wuLer. Re-IIume LIe IIp und recup.
6. Remove LIe IId Irom LIe T$ Lube, IIume LIe IIp
oI LIe Lube, deposIL o.¸ m oI wuLer InLo LIe T$,
re-IIume LIe IIp und recup.
;. ReLurn LIe pIpeL Lo LIe puckuge und dIscurd In
LIe bIoIuzurd bug. PIuce LIe Lube wILI LIe wuLer In
LIe ruck desIgnuLed Ior uuLocIuvIng.
8. ncubuLe LIe T$ Lube uL ¸¸°C Ior q8 Iours unLII
nexL Iub sessIon
q. ReLurn LIe sLock cuILure Lo LIe IronL desk.

Procedore ± - Sterile TrunsIer oI Bucteriu
Irom Broth to TSA Slunt
1. ObLuIn u Lube oI T$ conLuInIng E. coII or $.
epIdermIdIs und 1 T$A sIunL. PIuce uII Lubes In Lube
ruck, keepIng LIe ruck neur u IIL unsen burner.
z. ubeI LIe sLerIIe T$A sIunL wILI LIe upproprIuLe
orgunIsm nume, procedure #, your InILIuIs & LIe
duLe.
¸. ¡Iume LIe mouLI oI LIe T$ Lube und LIe sLerIIe
sIunL. Use LIe InocuIuLIng Ioop Lo remove 1 Ioop IuII
oI bucLerIu Irom LIe sLock T$ Lube und cureIuIIy
sLreuk LIe sLerIIe sIunL Irom boLLom Lo Lop. ONE
$TREAK ON e cureIuI Lo noL gouge LIe ugur.
Re-IIume LIe mouLIs oI boLI Lubes.
q. ¡Iume LIe InocuIuLIng Ioop und pIuce Lube In
IncubuLor uL ¸¸°C Ior q8 Iours.
¸. ReLurn LIe sLock cuILure Lo LIe IronL desk.

Procedore ¿ - Sterile TrunsIer oI Bucteriu
Irom TSA Slunts to Broth with u
Stub[Needle
1. ObLuIn u sIunL InocuIuLed wILI $. epIdermIdIs or
E. coII
z. ObLuIn u sLerIIe Lube oI T$.
¸. ubeI LIe sLerIIe T$ Lube wILI LIe upproprIuLe
orgunIsm nume, procedure #, your InILIuIs & LIe
duLe.
q. ¡Iume LIe InocuIuLIng needIe und use LIe needIe
Lo remove u smuII umounL oI growLI Irom LIe sLock
sIunL. A IIgIL LoucI Is suggesLed, IL does noL Luke u
Iurge umounL oI InocuIum Lo obLuIn copIous
umounLs oI orgunIsms. DeposIL LIe InocuIum In LIe
T$ Lube. E $URE Lo IIume LIe IIp oI LIe T$ Lube
und LIe T$A sIunL beIore und uILer InocuIuLIon.
¸. RoII LIe T$ Lube beLween LIe Iunds Lo
LIorougIIy mIx LIe orgunIsms.
(TIIs Is cuIIed 'subbIng` ouL LIe orgunIsm.
L Is u procedure Lo keep LIe orIgInuI cuILure IresI.)
6. ncubuLe uL ¸¸°C Ior q8 Iours.
;. ReLurn LIe sLock cuILure Lo LIe IronL desk.

Procedore q - Sterile TrunsIer Lsing
Alcohol-Ilumed Iorceps
1. ObLuIn 1 Lube T$, Iorceps, smuII beuker oI
uIcoIoI, und vIuI oI sLerIIe dIsks.
z. ubeI LIe T$ Lube '$LerIIe DIsk`, InILIuI und duLe.
oosen LIe IId oI LIe Lube und pIuce In LIe Lube
ruck.
¸. DIp LIe LIps oI LIe Iorceps InLo LIe uIcoIoI und,
IoIdIng LIe Iorceps
HORONTA, pIuce LIem In LIe IIume. e
cureIuI noL Lo IeL LIe uIcoIoI run Lowurds LIe Iund.
q. Open LIe sLerIIe dIsk Lube und IIume LIe IIp.
Remove 1 sLerIIe dIsk wILI LIe sLerIIIzed Iorceps.
¸. Open T$ Lube, IIume LIe IIp, InserL LIe sLerIIe
dIsk InLo LIe Lube.
6. Re-IIume und re-cup boLI LIe sLerIIe Lube und LIe
T$ Lube.
;. ncubuLe LIe Lube uL ¸¸°C Ior q8 Iours.

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