bakter i ident ifikasyonunda kullanılan standart, b iyok imyasal ve f

bakter i ident ifikasyonunda kullanılan standart, b iyok imyasal ve f

91
Konu 11
BAKTERø øDENTøFøKASYONUNDA KULLANILAN
STANDART, BøYOKøMYASAL VE FøZYOLOJøK
TESTLER
$\GÕQ 0 %DNWHUL LGHQWLILNDV\RQXQGD NXOODQÕODQ VWDQGDUW EL\RNLP\DVDO YH IL]\RORMLN WHVWOHU (G &HQJL] 0ÕVÕUOÕJLO $\GÕQ
-
7ÕSYHGLúKHNLPOL÷LQGHJHQHOYH|]HO0LNUREL\RORML.RQX6D *QHú\D\ÕQHYL$QNDUD
%LUoRN LQIHNVL\RQ KDVWDOÕ÷ÕQGD SDWRMHQ PLNURRUJDQL]PDQÕQ NLPOL÷LQLQ WHVSLW HGLOPHVL
WHGDYLQLQ LON EDVDPDNODUÕQGDQ ELULVLQL ROXúWXUXU %LU PLNURRUJDQL]PDQÕQ NLPOLNOHQdirilmesi
identifikasyon DGÕQÕ DOÕU øGHQWLILNDV\RQ LúOHPOHUL R EDNWHULQLQ DLW ROGX÷X JHQXVXQ YH VSHVLILN
HSLWHWLQLQ EHOLUOHQPHVLQH NDGDU GHYDP HWWLULOLU *HUHNWL÷LQGH WLSOHULQH VHURWLS JHQRWLS YV YH
varyetelerine kadar ilerletilir.
Bir bakterinin taNVRQRPLN FHWYHOGH GR÷UX DGUHVLQL WHVSLW HWPHN DQFDN KDQJL RUWDN DWD\Õ
SD\ODúWÕ÷ÕQD EDNDUDN PPNQ ROXU %LU EDNWHULQLQ VR\ D÷DFÕQÕQ WHVSLW HGLOPHVL R EDNWHULQLQ
DWDVÕQGDQJHWLUGL÷LJHQHWLNPLPDUL\L'1$KRPRORMLVLYHU51$ROLJRQNOHRWLWNDWDOR÷XQXHVDVDOÕU
YH KHUKDQJL ELU VHEHSOH JHoLFL RODUDN HGLQPLú RODELOHFH÷L IHQRWLSLN |]HOOLNOHULQGHQ ED÷ÕPVÕ]GÕU
)DNDW KHU ODERUDWXYDUGD UXWLQ RODUDN \DSÕODPD\DFDN NDGDU ]RU ]DPDQ DODQ YH SDKDOÕ LúOHPOHU
gerektirir. Bu nedenle incelenecek bakterinin fenotipik profilLQH EDNÕODUDN LGHQWLILNDV\RQXQXQ
\DSÕOPDVÕ JQFHOGLU KÕ]OÕ VRQXo YHULU YH JHUHNOLGLU %X DPDoOD LQFHOHQHQ EDNWHUL |UQH÷L
ODERUDWXYDUGD oHúLWOL EL\RNLP\DVDO YH IL]\RORMLN WHVWOHUH WDEL WXWXODUDN KDQJL WHVWOHUH QDVÕO
UHDNVL\RQ YHUGL÷L WHVSLW HGLOLU 7HVW VRQXoODUÕ |QFHGHQ KD]ÕUODQPÕú WDEORODU LOH NDUúÕODúWÕUÕODUDN R
bakterinin genus ismi ve spesifik epiteti bulunur.
øGHQWLILNDV\RQD IHQRWLSLN SURILOLQ HVDV DOÕQPDVÕ PRQRWHWLN EDNWHULOHULQ LGHQWLILNDV\RQX LoLQ
\HWHUOLGLU)DNDWSROLWHWLNEDNWHULOHULoLQDúD÷ÕGDNLVHEHSOHUOHED]HQ\DQÕOWÕFÕRODELOPHNWHGLU
- EDNWHUL\HOWDNVRQRPLQLQNDODEDOÕNROPDVÕ
- ED]ÕWUOHULQYDU\HWHOHULQLQEL\RYDUVHURYDUSDWRYDUIDJRYDUPRUIRYDUEXOXQPDVÕ
- IHQRJUDPÕQKL\HUDUúLN\DSÕVÕ
- LQFHOHQHFHN EDNWHUL |UQH÷LQGH KHUKDQJL ELr zamanda olabilecek fenotipik (ve hatta
JHQRWLSLNDGDSWDV\RQHOGH÷LúLPOHU
- EDNWHULOHULQ NRQMXJDV\RQ YH WUDQVGNVL\RQ \ROX LOH ELUELUOHULQH ED]Õ EL\RNLP\DVDO
GDYUDQÕúODUÕ DNWDUDELOL\RU ROPDVÕ |UQH÷LQ SURWHD] UHD] DPLQRDVLW NXOODQÕPÕ ODNWR] VXNUR] rafinoz,
JDODNWR] NVLOR] YH VLWUDW NXOODQÕPÕ DNWDUÕODELOLU |]HOOLNOHUGLU $\UÕFD VWUHSWRNRNODU YH VWDILORNRNODU DUDVÕQGD
,
- KHUEDNWHULQLQELUGHQID]ODIHQRQXQXQEXOXQDELOHFH÷L
- spontan pleiotrofik mutas\RQODURODELOHFH÷L
- D\QL ODERUDWXYDUGD D\QL EDNWHUL |UQH÷LQH \DSÕODQ KHU WHVWWHQ WDQHVLQLQD\NÕUÕVRQXo
YHUGL÷L
J|]|QQH DOÕQGÕ÷ÕQGD GR÷UX LGHQWLILNDV\RQ \DSPDQÕQ ]RU ROGX÷X GDKD L\L DQODúÕOÕU %X IHQRWLSLN
NDUDNWHU GH÷LúLPOHUL WDKPLQOHULQ YH ELOLQenin çok üzerindedir. Bir bakterinin mevcut fenotipik
NLPOL÷LQL \HQL PL ND]DQGÕ÷ÕQÕ \RNVD DWDVDO NLPOL÷L PL ROGX÷XQX '1$ KRPRORML WHVWOHUL\OH ELOH
DQODPDN]RUGXU%DNWHULOHUDUDVÕQGDDNWDUÕODQELU|]HOOLNLGHQWLILNDV\RQGDUHKEHUDOÕQDQELU|]HOOLN
ise kusXUOX VRQXoODUD ND\QDN WHúNLO HGHELOLU $\UÕFD QXPDUDOÕ ND\QDNWD \D]GÕ÷ÕQD J|UH
LGHQWLILNDV\RQ WHVWOHULQGH QHUHGH GXUXODFD÷ÕQÕ ELOPHN ]RUGXU øQFHOHQHQ KHU EDNWHUL |UQH÷L D\QÕ
NRQD÷ÕQEDúNDIORUDVÕQGDQL]ROHHGLOGL÷LQGHKDWWDD\QÕNRQD÷ÕQD\QÕIORUDVÕQGDQEDúNDELUWDULKWH
\HQLGHQL]ROHHGLOGL÷LQGHELOHIDUNOÕELUIHQRWLSJ|VWHUHELOPHNWHGLU+HUEDNWHULVXúXNHQGLQHKDVYH
DQVLNORSHGLN ROPD\DQ NDUPDúÕN ELU GR÷D\D VDKLSWLU øGHQWLILNDV\RQX \DSÕODQ KHUKDQJL ELU EDNWHUL
VXúX KHUKDQJL ELU ]DPDQGD NHQGL JHQXVXQD |]JO RODQ EL\RNLP\DVDO NXUDOODUÕ LKODO HGHELOHFH÷L
KHPROL]LQYHNRDJOD]\DSÕPÕGDDNWDUÕODELOPHNWHGLU
92
JLELNXUDOLKODOHWPH\LDOÕúNDQOÕNKDOLQHJHWLUHQEDNWHULOHUGHYDUGÕUFranciella novicida gibi).
%WQEXELOJLOHUGR÷UXOWXVXQGDRUWD\DoÕNDQJHUoHNRGXUNLLQFHOHQHFHNPLNURRUJDQL]PD\D
laboratuvDUNRúXOODUÕQÕQHOYHUGL÷LHQoRNVD\ÕGDWHVWL\DSPDNYHWHNELUWHVWVRQXFXQDJHUH÷LQGHQ
ID]ODED÷OÕNDOPDPDNJHUHNLU$\UÕFDPPNQROXUVDEDNWHUL\HOLGHQWLILNDV\RQXELOJLVD\DU\DUGÕPÕ
LOHYHWDNVRPHWULNHVDVODUGR÷UXOWXVXQGD\DSPDNHQGR÷UXVRQXFXYHUHEilir.(Bkz. Ek-4).
)ø=<2/2-ø.7(67/(5
%DNWHULKFUHVLQLQPHWDEROLNIDDOL\HWOHULQHGÕúDUGDQKLoELUPGDKDOH\DSÕOPDGÕ÷ÕYHVDGHFH
J|]OHPHVDVOÕWHVWOHUGLUEDúOÕFDODUÕúXQODUGÕU
Anaerop üreme:
7DQÕP%XWHVWEDNWHULQLQ]RUXQOXDQDHURSPXROGX÷XQXLIDGH eder.
Prensip:%DNWHULNHQGLVLQHHQX\JXQLNLD\UÕEHVL\HULQHHNLOHUHNELULVLDHURSGL÷HULDQDHURS
olarak inkübe edilir.
'H÷HUOHQGLUPH $QDHURS EHVL\HULQGH ROPDVÕQD UD÷PHQ D\QL EDNWHUL NRORQLVLQLQ DHURS
EHVL\HULQGHEXOXQPDPDVÕREDNWHULQLQ]RUXQOXDQDHURSROGX÷XQXJ|VWHULU
Subpasajlara bu testi yapmak güvenli sonuç vermeyebilir. Çünkü birkaç pasajdan sonra
ELUoRN DQDHURS EDNWHUL RNVLMHQ WROHUDQVÕ JHOLúWLUHELOLU YH DHURS RUWDPGD NÕVPHQ UHPH\H
EDúOD\DELOLU
<DQOÕúSR]LWLIVRQXoODUHaemophilus genusunda olabilir. Bu bakteriler aerop ortamda faktör
X (Protoporphirin IX) JHUHNVLQGL÷L KDOGH DQDHURS RUWDPGD EXQD JHUHNVLQLP J|VWHUPH]
Haemophilus¶ODUoXNXODWDDJDUGÕúÕQGDELUEHVL\HULQHHNLOHUHNEXWHVW\DSÕOGÕ\VDDQDHURSUHUDPD
aerop üremeyebilir. BöyleELU\DQOÕúSR]LWLIVRQXFDNDUúÕGLNNDWOLROPDNJHUHNLU
<DQOÕú QHJDWLI VRQXoODU LVH GLVJRQLN DQDHURS JHQXVODUGD ROXU %D]Õ DQDHURSODU -7.inci
günden sonra üreyebilirler (Actinomyces gibi). Bu nedenle besiyerleri 2.inci günde yoklanarak,
7.inci gün sonuna kadar bekletilmelidir.
Gram boyama :
7DQÕP%R\DPDGGHVLQHJHoLUJHQRODQKFUHGXYDUÕQÕQPLPDULVLKDNNÕQGDELOJLYHULUGram
NHOLPHVL|]HOLVLPGLUE\NKDUILOHEDúOD\DUDN\D]ÕOPDOÕGÕU).
Prensip: .ULVWDO YL\ROH KFUH GXYDUÕQGDNL SHSWLGRJOLNDQ NRPSOHNVLQH SHQHWUH ROXU (÷HU
KFUHGH GÕú GXYDU \RN LVH yani incelenen bakteri Gram negatif ise), kristal viyole magnezyum
ribonukleat kristalleri halinde gücre içerisine çöker. Bu çökelti, alkolde çözünmez, dekolorizasyon
LOH JLGHULOHPH] (÷HU KFUHGH ELU GÕúGXYDUÕ YDU LVH yani incelen bakteri Gram pozitif ise), uygulanan
NULVWDO YL\ROH KFUH LoHULVLQGH DONROGH o|]QHELOHQ ELOHúLNOHU KDOLQGH NDOÕU %X ER\DQÕQ EX |]HOOL÷L
VD\HVLQGHEDNWHULPHPEUDQODUÕKDNNÕQGDILNLUVDKLELROXQXU
øúOHP Bkz. Konu-7 Mikrobiyolojide boyama yöntemleri.
Koopler's Gram modifikasyonu (anaerop Gram boyama$QDHURSEDNWHULOHUQDULQGLUYHÕVÕLOH
NROD\FD GHIRUPH RODELOHFHNOHUL LoLQ PDWHU\DOLQ ILNVDV\RQX PHWDQRO \DGD DVHWRQ LOH \DSÕOÕU
Dekolorizasyona direnç gösteremeyen anaerop *UDP SR]LWLI EDNWHULOHULQ *UDP SR]LWLI ER\DQPÕú
RODUDNNDOPDODUÕQÕVD÷ODPDNLoLQNULVWDOYL\ROHLOHPXDPHOHVÕUDVÕQGDSUHSDUDW]HULQH-5 damla
WD]H KD]ÕUODQPÕú OLN VRG\XP ELNDUERQDW GDPODWÕOÕU $\UÕFD VXOX IXNVLQ LoHULVLQH VDIUDQLQ LODYH
HGLOLU'L÷HULúOHPOHU.RQX-¶GHDQODWÕOGÕ÷ÕJLEL\DSÕOÕU
%XWHVWDQDHURELNEDNWHULOHUHX\JXODQGÕ÷ÕQGDúXQODUKDWÕUODQPDOÕGÕU
1. (÷HU LQFHOHQHQ DQDHURS EDNWHUL *UDP QHJDWLI LVH UHQNOHUL JHQHOOLNOH ELU *UDP QHJDWLI
EDNWHUL LoLQ ROPDVÕ EHNOHQHQGHQ GDKD VROJXQ YH GDKD DoÕN SHPEHGLU %X QHGHQOH PLNURVNRS
VDKDVÕQGD LON EDNÕúWD ER\D DUWLIDNWÕ YH\D EHVL\HUL NDOÕQWÕVÕ JLEL J|UQHQ ROXúXPODUÕQ ELU DQDHURS
*UDP QHJDWLI EDNWHUL KFUH NPHVL ROXS ROPDGÕ÷Õ GLNNDWOH LQFHOHQPHOLGLU Bacteroides
pneumosintes böyledir).
2. Anaeroplar dekolorizasyona direnç gösteremezler. Gram pozitif anaeroplar daima Gram
negatif boyanmaya meyil gösterirler, bu nedenle Gram pozitif boyananlar genellikle Gram
SR]LWLIWLUOHUDQFDN*UDPQHJDWLIER\DQDQODUÕQEHONLGH*UDPSR]LWLIRODELOHFH÷LKDWÕUODQPDOÕGÕUBir
DQDHUREXQ *UDP SR]LWLI ROGX÷X Tween- LoHUHQ DJDUGD UHPHVLQLQ DUWPDVÕ EUXFHOOD DJDUGD
LQKLEHROPDVÕYHYDQNRPLVLQGLVNLLOHLQKLEHROPDVÕLOHGR÷UXODQDELOLU
93
3.$\QLPLNURVNRSVDKDVÕQGDD\QLKFUHOHUIDUNOÕUHQNWRQXQGDER\DQDELOLUOHUVDINültürden
DOÕQDQDQDHURSEDNWHULOHULQLQEXJ|UQWOHULELUNRQWDPLQDV\RQXGúQGUHELOLU
4.$\QLKFUHQLQELU\DUÕVÕEDúNDUHQNRODELOLU%XER\DQPDVSRUYH\DPHWDNURPDWLNFLVLP
RODUDN\DQOÕú\RUXPODQDELOLUWD]HPDWHU\DOGHVSRUEXOXQPD\DFD÷ÕKDWÕUODQPDOÕGÕU).
'H÷HUOHQGLUPH Bkz. Konu-7 Mikrobiyolojide boyama yöntemleri.
*UDP ER\DPD LoLQ NOWUOHULQ GDLPD WD]H ROPDVÕ X\JXQGXU HVNLPLú NOWUOHU YH ]RUXQOX
DQHURSODUÕQ *UDP QHJDWLI ER\DQPD\D PH÷LOOL ROGX÷X KDWÕUODQPDOÕGÕU \DQOÕú QHJDWLI VRQXo
verebilirler. Protoplastlar daima Gram pozitiftir.
%R\DVROXV\RQODUÕQÕQLoHULVLQGHNRQWDPLQDV\RQDED÷OՁUHPHROPDPDVÕLoLQLoHULVLQHPO
NORURIRUPNDWÕOÕUYH\D&&O4 konur. Bu madde solusyonu muhtemel bir kontaminasyondan korur.
Pozitif kontrol için stafilokoklar, negatif kontrol için E. coli D\QL ODP ]HULQGH LúOHPH WDEL
tutulabilir.
Hareket muayenesi:
7DQÕP %DNWHUL KFUHVLQLQ KDUHNHW RUJDQHOL IODMHOODGÕU $QFDN EXQXQ GÕúÕQGD GD KDUHNHW
HGHELOPHOHUL PPNQGU %R\DVÕ] WD]H SUHSDUDW [ E\OWPH YH DVÕOÕ GDPOD WHNQL÷L LOH
LQFHOHQGL÷LQGHEDúOÕFDúXWLSKDUHNHWOHUJ|]OHQHELOLU
7LWUHúLP KDUHNHWL (Twitching Motility) %X JHUoHN ELU KDUHNHW úHNOL ROPD\ÕS KFUHQLQ
EXOXQGX÷XPLNURVNRSVDKDVÕQGDQEDúNDELUVDKD\DGR÷UX\RODOPDVÕQÕVD÷ODPD]Streptococci ve
Staphilococci dahil olmak üzere pek çok türde bulunur. Bu bakteriler hareketsiz kabul edilir.
2. Ameboid Hareket (Ameboid Movement) +FUHQLQ KDUHNHW HGHFH÷L \|QH GR÷UX
VLWRSOD]PDVÕQÕ VRO-JHO NÕYDPÕQD JHWLUPH \HWHQH÷L YDUGÕU EX GXUXPGD KFUH JLGHFH÷L \|QGH ELU
pseXRGSRG \DODQFÕ D\DNROXúWXUXUYHVLWRSOD]PDVRONÕYDPÕQDJHOHUHNSVHXRGSRGLoLQHGR÷UX
DNDU'DKDVRQUDVLWRSOD]PDHVNLNÕYDPÕQDG|QHU%|\OHFHKFUH\HUGH÷LúWLUPLúROXU
3. Akarak hareket (Gliding Movement)+FUHNDWÕ\]H\HWHPDVÕQGDL\LDoÕNODQPDPÕúELU
PHNDQL]PDLOHKLoELUVDOÕQÕP\DSPDGDQND\DUúHNLOGH\HUGH÷LúWLUHELOLU%XEDNWHULOHUKDUHNHWVL]
kabul edilir.
4. Sarmal hareket (Oscillatory Movement): Tipik olarak Spirochetales
GH J|UOU D\UÕ
hareket gurubunu kapsar, i) Hücre bir ucundanDVÕODUDNSDQGOJLELVDOÕQÕULL6DDW\|QQGH\DGD
WHUVL \|QGH VSLUDO úHNOLQGH G|QHU Vpiroketler, Cristaspira, Treponema, Borrelia JHQXVODUÕ VDDW
istikameti tersine dönerler. Leptospira'lar saat istikametine dönerler.) iii) Hücre endoflajelleriyle
VÕNÕúÕUVRQUDGDERúDOPÕú\D\JLEL|QHGR÷UXIÕUODUBu bakteriler hareketli kabul edilir.
5. Flagellar hareket (Flagellum Mediated Locomotion): Hücre, peritriköz yada monotriköz
IODMHOOHUL\OH DNWLI úHNLOGH KDUHNHW HGHELOLU $NVL EHOLUWLOPHPLúVH KDUHNHWOLOLN EX DQODPGD DOÕQÕU %X
bakteriler hareketli kabul edilir.
6. Dalgalanmalar (Metachronal Waving) 6LO\D HVDVÕQD GD\DQGÕ÷Õ GúQOPHNWHGLU
+FUHQLQ GDOJDODQPD IUHNDQVÕ JHQHOOLNOH +] VDQL\HGH GHID FLYDUÕQGDGÕU %X EDNWHULOHU
hareketsiz kabul edilir.
Prensip: 0LNURVNRS LOH EDNWHUL KFUHVLQLQ KDUHNHWOLOL÷LQLQ J|] LOH WHVSLWLQGHQ LEDUHWWLU Bu
WHVWLQHVDVÕIODMHOODUKDUHNHWLQWHVSLWHGLOPHVLGLU
øúOHP $VÕOÕ GDPOD WHNQL÷L %XQXQ LoLQ ELU NRORQL PDWHU\DOL EXLúLoLQ\DSÕOPÕú|]HOELUODP
üzerinde eziOLU ]HULQH ELU GDPOD VWHULO WX]OX VX NRQXOXU /DP WHUV oHYULOHUHN GDPODQÕQ \]H\
JHULOLPL LOH ODPÕQ \]H\LQGH DVÕOÕ NDOPDVÕ VD÷ODQÕU 0LNURVNRSXQ NDQGDQV|U DúD÷Õ oHNLOLU ODPÕQ
ÕVODN\]NRQGDQV|UHGR÷UXoHYULOLUHN\DYDúFDWDEOD\D\HUOHúWLULOLU[Eyültme ile incelenir.
+DUHNHWWHVSLWLLoLQLQGLUHNW\|QWHPOHUGHYDUGÕU+DUHNHWEHVL\HULEXQXQLoLQX\JXQGXUDJDULoHUHQSHNoRN\DUÕNDWÕEHVL\HULQLQELUXFXQDHNLP\DSÕODUDNLQNEDV\RQXWDNLEHQ\D\ÕOPD
ROXSROPDGÕ÷ÕQDEDNÕOÕU
'H÷HUOHQGLUPH LamÕQ|QFHGHQÕVÕWÕOÕSVR÷XWXOPXúROPDVÕYDUVDKDUHNHWLNDPoÕODU3R]LWLI
kontrol olarak Pseudomonas aeruginosa veya ProteusWUOHULNXOODQÕODELOLU1HJDWLINRQWUROLoLQELU
VWDILORNRNNXOODQÕOÕU
Kapsül muayenesi:
7DQÕP%DNWHULKFUHVLQLQGÕúGXYDUÕQÕ|UWHQJOLNRNDOLNV\DSÕGDNLPXNRLG|UWNDSVOQQ
YDUOÕ÷ÕQÕ DUDPDN LoLQ \DSÕODQ ELU WHVWWLU $QFDN WD]H L]RODWODU LoLQ YDUOÕ÷ÕQÕQ \D GD \RNOX÷XQXQ
94
LGHQWLN GH÷HUL RODELOLU dQN EDNWHULOHU SDVDMODU VÕUDVÕQGD NDSVOOHULQL ND\EHGHUOHU %X WHVW
Pneumococci, Meningococci ve Klebsiella
ODU LoLQ LGHQWLNWLU YH DVOÕQGD YLUODQV IDNW|U RODUDN
GH÷HUOL ELU EXOJXGXU ho WUO NDSVO EXOXQXU 1) Makrokapsül= negatif boyama ile kolayca
izlenir, 2) Mikrokapsül ancak serolojik tekniklerle gösterilir 3) Slim tabaka tam bir kaSVOGH÷LOGLU
daha çok bakterinin adezinleri (dekstran, seluloz, glukan)dir.
Prensip:%XWHVWPDNURNDSVOQER\DPD\|QWHPOHULLOHJ|VWHULOPHVLHVDVÕQDGD\DQÕU
øúOHP%LUNDSVOER\DVÕYH\DEDVLWoHoLQLPUHNNHELLOHER\DQÕU&DQOÕEDNWHUL|UQH÷L
|]JONDSVODQWLNRUXLOHPXDPHOHHGLOHUHNNDSVOúLúPHUHDNVL\RQXDUDQÕU
'H÷HUOHQGLUPH%R\DQDQNDSVO[E\OWPHLOHÕúÕ÷ÕNÕUDQJHQLúSDUODNELUWDEDNDRODUDN
J|UOU 7LSH |]JO VWDQGDUW DQWLNRUODU LOH PXDPHOH HGLOHQ EDNWHULQLQ JHQLúOH\HQ NDSVO úLúHUHN
mikroskopta görülebilir hale gelir.
Selüler morfoloji:
7DQÕP %DNWHUL KFUHVLQLQ PRUIRORMLVL JHQXV VHYL\HVLQGH LGHQWLILNDV\RQD \DUGÕPFÕ ROXU
Prensip:+FUHúHNOLQLQODERUDWXYDUúDUWODUÕQGDWHVSLWLHVDVÕQDGD\DQÕU
øúOHP øQFHOHQHFHN EDNWHUL |UQH÷L WUQH X\JXQ NDWÕ EHVL\HULQGHQ DOÕQPDOÕGÕU 7UQH
X\JXQ ELU ER\DPD PHWRGX LOH ER\DQÕU YH PLNURVNRSWD LQFHOHQLU 'DKD L\LVL FDQOÕ ER\DODUOD
ER\DPDNYH\DPDWHU\DOLGR÷UXGDQPLNURVNRSWDLQFHOHQPHNWLU
'H÷HUOHQGLUPH%N].RQX%DNWHULOHULQVÕQÕIODQGÕUÕOPDODUÕ
0DWHU\DOL DOHYGHQ JHoLUHUHN GH÷LO DVHWRQ \D GD DONROGH EHNOHWHUHN WHVSLW HWPHOL E|\OHFH
EDNWHUL KFUHVLQLQ GHIRUPDV\RQXQD HQJHO ROXQPDOÕGÕU $\UÕFD NXOODQÕODQ WX]OX VX\XQ L]RWRQLN
ROPDVÕúDUWWÕU6ÕYÕEHVL\HULQGHUHWLOHQEDNWHULKFUHVLQLQX]DPD\DPH÷LOOLROGX÷XKDWÕUODQPDOÕGÕU
+FUHGXYDUÕQÕQEXOXQPDGÕ÷ÕEDNWHULOHUGHMycoplasmacaea gibi) labil morfolojiler bulunabilir.
Spor muayenesi:
7DQÕP %D]Õ JHQXVODU Bacillus ve Clostridium JLEL X\JXQ ROPD\DQ \DúDP NRúXOODUÕQGD
spor yapabilirler, böylH ELU |]HOOL÷LQ J|VWHULOPHVL VDGHFH JHQXV YH KDWWD IDPLO\D VHYL\HVLQGH
D\ÕUÕPD\DUGÕPFÕRODELOLU6SRUXQ\DSÕVÕQGDGLSLNROLQLNDVLW(pyridine 2,6-dicarboxylic acid), keratin
ve kitin'e benzer proteinler gibi normalde bakteri hücresinde bulunmayan kompleks \DSÕODU
bulunur.
Prensip: 6SRU JHOLúLPL \DOQÕ]FD IHQD RUWDP NRúXOODUÕQGD PPNQGU %DNWHUL ORJDULWPLN
UHPHQLQVRQXQGDNDUERQD]RWIRVIDWND\QDNODUÕQÕQWNHQPHVLLOHIHQDNRúXOODUDGR÷UX]RUODQÕU
øúOHP6DIEDNWHULNOWUQGHQDOÕQDQoRNPLNWDUGDPDteryal küçük hacimde bir besiyerine
ekilerek 7-10 gün etüvde ve 4- JQ RGD ÕVÕVÕQGD EHNOHWLOLU 6SRUODV\RQX WDKULN DPDFÕ EHVL\HUL
LoHULVLQH EDNWHUL VXúXQD |]JO PLQHUDOOHU LODYH HGLOHELOLU &22 JD]ÕQÕQ RUWDPGDNL NRQVDQWUDV\RQX
D]DOÕUVDClostiridium’lar dDKDNROD\VSRUODQÕUODU'R÷UXGDQ\DGDER\DQDUDNPLNURVNRSWDLQFHOHQLU
'H÷HUOHQGLUPH6SRUODUGH÷LúLNWHUNLSWHNLVSRUER\DODUÕLOHER\DQDELOHFH÷LJLELROJXQODúPÕú
VSRUODU GLUHNW PLNURVNRSL \D GD *UDP ER\DVÕ LOH GH J|]OHQHELOLU +FUH LoHULVLQGH RODELOHFHN
PHWDNURPDWLN FLVLPFLNOHU LOH NDUÕúWÕUPDPDN LoLQ VSRU ER\DODUÕ \DUGÕPÕ LOH WDQÕ NR\PDN GDKD
GR÷UXGXU Erlich-Ziehl-Neelsen ER\DVÕ LOH VSRUODU NÕUPÕ]Õ UHQNWH ER\DQÕUODU 0DODúLW \HúLOL GH
NXOODQÕODELOLU
.OLQLN PDWHU\DOGHQ KD]ÕUODQPÕú WD]H SUHSDUDWWD VSRU aranmaz (yoktur). Logaritmik üreme
ID]ÕQGDNLJHQoNOWUGHQ\DSÕODQSUHSDUDWODUGDGDVSRUDUDQPD]
'H÷LúLNVÕFDNOÕNODUGDUHPH°°C de üreme) :
7DQÕP 7HVWLQ DQODPÕ X\JXQ ROPD\DQ VÕFDNOÕNODUGD EDNWHUL KFUHVLQLQ PHWDEROLN
faaliyetlerin devam ediSHWPH\HFH÷LQLQEHOLUOHQPHVLGLU
Prensip: %D]Õ EDNWHULOHULQ NRQDN GRNX GÕúÕQGD \NVHN YH\D GúN ÕVÕGD PHWDEROLN
IDDOL\HWOHULQHGHYDPHGHELOL\RUROPDVÕRVXúXQNLPOL÷LKDNNÕQGDELOJLYHULFLGLU
øúOHP Türe uygun vasat içerisine inoküle edilen bakteri 22 veya 42 °&
\HD\DUODPÕúHWYGH
YH\DEX]GRODEÕQGD-48 saat inkübe edilir.
'H÷HUOHQGLUPH.RORQLJHOLúLPLSR]LWLIWHVWVRQXFXRODUDN\RUXPODQÕUYH°C için pozitif
kontrol Pseudomonas aeruginosa , 5°C için Listeria monocytogenes NXOODQÕODELOLU
95
YXNDUÕGDDQODWÕODQODUGÕúÕQGDEDNWHULIL]\RORMLVLQLWDQÕPD\D\DUD\DQEDúNDED]ÕX\JXODPDODU
GD YDUGÕU gUQH÷LQ NRORQL PRUIRORMLVL NDo JQGH UHGL÷L NRORQLQLQ IORUHVDQ YHULS YHUPHGL÷L JLEL
%HVL\HULQL NRNODPDN QXPDUDOÕ ND\QDNWD \D]GÕ÷ÕQD J|UH FLGGL VRUXQODUD VHEHS ROPDNWDGÕU
.RNODQPDPDOÕGÕU$QFDNNOWUSOD÷ÕQÕQX]DNWDQGX\XODQELUNRNXVXYDUVDQRWHGLOHELOLU
%ø<2.ø0<$6$/7(67/(5
ùHNHUIHUPHQWDV\RQWHVWOHUL
7DQÕP %LU EDNWHUL KFUHVLQGH KDQJL úHNHUOHUL IHUPHQWH HGHELOHQ PHWDEROL]PDQÕQ
EXOXQGX÷XREDNWHULVXúXLoLQLGHQWLNGH÷HUWDúÕU
Prensip:(VDVLWLEDUÕLOHEXWHVWLQFHOHQHFHNEDNWHULQLQUH\HELOHFH÷LEHVL\HULQLQWHUNLELQH
VDGHFH GHQHQHFHN RODQ NDUERQKLGUDWÕQ LODYH HGLOHUHN IHUPHQWH ROXS ROPDGÕ÷ÕQÕQ L]OHQPHVLQGHQ
ibarettir. Bakteri, inkübasyRQ G|QHPL VRQXQGD EX úHNHUL IHUPHQWH HGLS DVLW ROXúWXUDELOPLú LVH
RUWDPÕQ S+
VÕ VÕQÕU GH÷HULQLQ DOWÕQD GúHFHNWLU S+ LQGLNDW|U YH\D ELU S+-PHWUH \DUGÕPÕ LOH
DVLGLWHWHVSLWL\DSÕOÕU%XUDGDGLNNDWHGLOHFHNQRNWDEHVL\HULLoHULVLQGHGHQHQHFHNúHNHUGHQEDúND
NDUERQKLGUDW EXOXQPDPDVÕ JHUHNWL÷LGLU %|\OHFH EDNWHUL RUWDPGDNL \HJDQH NDUERQKLGUDWÕ
NXOODQPD\D]RUODQÕU%DNWHULQLQYHNDUERQKLGUDWÕQWUQHJ|UHODNWLNVNVLQLNDVHWLNIRUPLNDVLWOHU
ROXúDELOLUOHU+DQJLDVLWOHULQROXúWX÷XQXQELU|QHPL\RNWXU.
øúOHP Saf kültüründen bir öze dolusu koloni materyali, bakteri için uygun olan ve içerisinde
sadece karbonhidrat bulunan besiyerine ekilir. Bakterinin üreyebilmesi için özel besiyeri
JHUHNVLQLPL \RN LVH ELU JHQHO NXOODQÕP VÕYÕ EHVL\HULQH HNLOLU %DNWHULQLQ WUQH X\JXQ VÕFDNOÕNWD
uygun süre inkübe edilir.
ùHNHUIHUPDQWDV\RQED]DOPHGLXP
Pepton
5g
Brom-Timol Mavisi
0.001 g
Distile Su
1 lt
DHQHQHFHNúHNHU
1g
oz, mellibiyoz, rafinoz, ramnoz,
'HQHQHFHN úHNHU $UDELQR] GHNVWUR] JOXNR] NVLOR] VXNUR] ODNWR] PDOWR] PDQQ
sellobiyoz, trehaloz... olabilir)
%X EHVL\HUL LoHULVLQH úHNHU LODYHVL \DSÕOGÕNWDQ VRQUD 'XUKDP WS WHUV oHYULOHUHN KDYDVÕ
DOÕQPDOÕYHWLQGDOL]DV\RQLOHVWHULOL]HHGLOPHOLGLU
$QDHURSODUD \DSÕODFDN WHVWOHUGH LVH 3<-Broth EHVL\HUL NXOODQÕOÕU HNLPGHQ |QFH ND\QDWÕOÕS
VR÷XWXOPDOÕGÕU*HUHNLUVHLoHULVLQHDQDHURSEDNWHULQLQLKWL\DoODUÕYLW.1 , sistein, lizin veya hemin)
LODYHHGLOLU(NLPLWDNLEHQ\]H\LPOVWHULOPLQHUDO\D÷ÕLOHNDSDWÕOPDOÕGÕU
'H÷HUOHQGLUPH øQNEDV\RQ G|QHPL VRQXQGD ROXúDQ VDUÕ UHQN RUWDPGDNL úHNHULQ
IHUPHQWDV\RQX VRQXFXQGD DVLW ROXúWX÷XQX YH SR]LWLI WHVW VRQXFXQX LúDUHW HGHU .RQWURO RODUDN
HNLP\DSÕOPDPÕúELUWSEHVL\HULLOHELUOLNWHLQNEHHGLOPHOLGLU
Brom-WLPRO PDYLVL S+ GH DoÕN \HúLOGLU S+≤5
GH VDUÕ UHQN DOÕU DQFDN S+≥7.5'de ise
NR\X\HúLO\DGDPDYLUHQNDOÕU.DUDUYHULOHPH\HQGXUXPODUGDELUS+-strip ya da pH-PHWUHLOHS+
ÕQ
XQ DOWÕQGD ROGX÷XQX GR÷UXODPDN JHUHNLU - DUDVÕ S+ GHUHFHOHUL ³]D\ÕI DVLW³ úHNOLQGH
\RUXPODQPDOÕGÕU Alcaligenes JLEL ED]Õ EDNWHULOHU YH ED]Õ DQDHURSODU RUWDPGDNL SHSWRQGDQ
DPRQ\DN ROXúWXUDUDN DVLW YDVDWÕ WDPSRQOD\DUDN S+ WHVSLWLQL PDVNHOH\HELOLUOHU %X QHGHQOH X]XQ
LQNEDV\RQGDQ NDoÕQPDOÕGÕU D\UÕFD LQNEDV\RQ ER\XQFD WSOHU EHOLUOL DUDOÕNODUOD \RNODQPDOÕGÕU
+HUKDQJLELUDQGDS+
QÕQ¶XQDOWÕQDGúPHVLYHEHVL\HULQLQVDUÕUHQNDOPDVÕSR]LWLIVRQXoLoLQ
\HWHUOLGLU S+ QÕQ \HQLGHQ \NVHOPHVL YH \HQLGHQ DoÕN \HúLO UHQN ROXúXPX WHVW VRQXFXQX QHJDWLI
yapmaz.
%LOKDVVDDQDHURSRODQED]ÕEDNWHULOHULQNEDV\RQG|QHPLER\XQFDúHNHULGH÷LODPDUHQN
LQGLNDW|UQ GHJUDGH HGHELOPHNWHGLU 2UWDPGD DVLW EXOXQPDGÕ÷Õ KDOGH UHQN LQGLNDW|U VDUÕ UHQN
DODELOLU <DQOÕú SR]LWLI VRQXo YHUHELOLU %HVL\HULQH GDPOD EURPWLPRO PDYLVL GDPODWDUDN S+
ÕQ
JHUoHNWHQDVLWROXSROPDGÕ÷ÕQÕGR÷UXODPDNJHUHNHELOLU
'XUKDPWSOHULLoHULVLQGHROXúPXúJD]NDEDUFÕNODUÕIHUPHQWDV\RQVRQXFXROXúDQNDWDEROLN
gaz ürünlerin (CO2, H2, H26EXOXQGX÷XQXNDOLWDWLIRODUDNJ|VWHULU
96
$QDHURSODU LoLQ JD] \DSÕPÕQÕQ WHVSLWL DPDFÕ LOH GXUKHLP WSOHUL NXOODQÕOmaz, GLC (Gas/LTXLG&KURPRWRJUDSK\\|QWHPLNXOODQÕOÕU
Aminoasit (Arginin-Lysin-Ornithin) dekarboksilasyonu:
7DQÕP 'HNDUERNVLOD]
ODU DPLQRDVLWOHULQ NDUERNVLO NÕVPÕ LOH UHDNVL\RQD JLUHELOHQ ELU JUXS
HQ]LP VXEVWUDWODUÕGÕU &22 DoÕ÷D oÕNDUDQ EX UHDNVL\RQdekarboksilasyon olarak isimlendirilir. Her
ELU GHNDUERNVLODV\RQ HQ]LPL \DOQÕ]FD ELU DPLQRDVLW LoLQ VSHVLILNWLU Arginin, Lysin ve Ornithin
GHNDUERNVLODV\RQX D\UÕ WHVWWLU YH EX DPLQRDVLWOHULQ ELU EDNWHUL |UQH÷L WDUDIÕQGDQ NXOODQÕOPDVÕ
JHQXVED]ÕQGDLGHQWiktir.
Prensip: 'HNDUERNVLODV\RQ WHVWOHUL LoLQ 0RHOOHU GHFDUER[\ODVH EX\\RQ NXOODQÕOÕU (÷HU
LQFHOHQHQEDNWHUL|UQH÷LEHVL\HULQHNRQXODQDPLQRDVLWLNXOODQÕ\RUVDDONDOLDPLQOHURUWD\DoÕNDUYH
pH yükselir. Arginin, önce citrullin’e ve sonra agmatin 'e hidrROL]H ROXU %X ELU GLKLGUROL] ROD\ÕGÕU
Lysine, cadaverin'e; ornithin, putrescine'e deamine olur. Hepsi alkalin aminlerdir. pH indikatörü
EHVL\HULQLQDONDOLROGX÷XQXJ|VWHUL\RUVDROXPOXVRQXoDQODPÕQDJHOLU
øúOHP Moeller decarboxylase buyyon içerisine denenecek amino asitlerden bir tanesi ilave
HGLOLU YH LQFHOHQHFHN EDNWHUL HNLOLU $PLQR DVLW LODYHVL \DSÕOPDPÕú ELU EHVL\HUL NRQWURO LoLQ
NXOODQÕOPDOÕGÕU øQNEDV\RQXQ LON VDDWOHULQGH EHVL\HUL VDUÕ UHQN DODELOLU Bunun sebebi ortamda
bulunan glukozun fermeQWDV\RQXGXU (÷HU LQNEDV\RQ VRQXQGD DPLQR DVLW GHNDUERNVLOH ROXUVD
RUWD\DoÕNDQDONDOLQDPLQOHUS+
ÕQLQ]HULQHoÕNDUÕUS+LQGLNDW|UPDYL-\HúLOUHQNDOÕUVDVRQXo
pozitiftir.
Moeller decarboxylase broth:
Pepton
5 g
Beef extract
5 g
Bromcresol purple
0.01 g
Cresol red
0.005 g
Glucose
0.5 g
Pyridoxal
0.005 g
Amino asit
10 g (L-arginin, L-lysine veya L-ornithin)
Distile su
1 lt
S+ D\DUODQÕU
(Aminoasitlerin D-IRUPXNXOODQÕODFDNVDNDWÕPLNWDUGDNXOODQÕOPDOÕGÕU
'H÷HUOHQGLUme: øON VDDWOHUGH ROXúPDVÕ EHNOHQHQ VDUÕ UHQN GH÷HUOHQGLULOPH] øQNEDV\RQ
VRQXQGD EHVL\HUL UHQJLQLQ NR\X \HúLO ROPDVÕ SR]LWLI VDUÕ RODUDN NDOPDVÕ QHJDWLI VRQXFX J|VWHULU
+LoELU VDUDUPD ROPDGDQ RUMLQDO UHQJLQGH NDOÕS NDOPDGÕ÷Õ HNLP \DSÕOPDPÕú NRQWURO WSü ile
NDUúÕODúWÕUÕODUDN\DQOÕúSR]LWLIVRQXoODUGDQNDoÕQPDNJHUHNLU
Negatif kontrol olarak Enterobacter aerogenes, pozitif kontrol olarak ise Enterobacter
cloacaeNXOODQÕODELOLU
DNAz testi:
7DQÕP %D]Õ EDNWHULOHU deoksiribonuclease enzimi yaparlar. Bu test bakteride böyle bir
içerisinden
HQ]LPDWLN DNWLYLWHQLQ PHYFXGL\HWLQLQ DUDúWÕUÕOPDVÕGÕU %X WHVW Staphylococci
S.aureus
XD\ÕUDELOPHNLoLQSOD]PDNRDJOD]WHVWLQGHQGDKDNÕ\PHWOLGLU
Prensip:%XUDGDNXOODQÕODQEHVL\HULQGH'1$YDUGÕU%DNWHULQLQ'1$]Dktivitesi varsa koloni
oHYUHVLQGH EXOXQDQ '1$ SDUoDODQDFDNWÕU øQNEDV\RQX WDNLEHQ X\JXODQDQ D\ÕUDo ELU '1$
ER\DVÕGÕU.RORQLHWUDIÕQGD'1$EXOXQXSEXOXQPDGÕ÷ÕRUWD\DoÕNDUÕU
øúOHPøQFHOHQHFHNEDNWHULQLQVDINOWUQGHQELUNRORQL'1$]EHVL\HULQHHNLOLU, uygun süre
LQNEH HGLOLUøQNEDV\RQXWDNLEHQROXúDQNRORQLOHU]HULQHGDPODD\ÕUDoX\JXODQÕUùHIIDI]RQ
DUDQÕU
Deoksiribonüklaz Agar:
Desoxyribonucleic acid
2g
Phytone
5g
Sodium chloride
5g
Trypticase
15 g
Agar
15 g
Distile su
1 lt
pH=D\DUODQÕU
97
$\ÕUDo
1N HCl veya %0.1 Toluidin Mavisi
'H÷HUOHQGLUPHøQFHOHQHQEDNWHUL'1$]SR]LWLILVHNRORQLHWUDIÕQGDEHUUDNYHúHIIDIELU]RQ
J|UOU 7ROXLGLQ PDYLVL NXOODQÕOPÕú LVH EX VDKD SDUODN SHPEH UHQN DODFDNWÕU %X J|UQW WHVWLQ
sonucunun SR]LWLI ROGX÷X DQODPÕQD JHOLU '1$] DNWLYLWHVLQL WDKULN HGHELOPHN LoLQ EHVL\HULQH&D++
NDWÕOPDVÕ|QHULOPLúWLU$QFDNEHVL\HULQH7U\SWLFDVH6R\D$JDUNDWÕOÕUVDEXQDJHUHNNDOPD]3R]LWLI
kontrol S. aureus’tur.
Eskulin hidrolizi :
7DQÕP Eskulin (aesculin) bir glikozittir. 6,7-dihydroxycoumarin’in 6-ß-D-glucosylGHULYDWÕGÕU
(VNXOLQLQ KLGUROL]OHQPHVL YH IHUPHQWDV\RQX IDUNOÕ LNL NLP\DVDO UHDNVL\RQGXU (VNXOLQ
IHUPHQWDV\RQXQXQ PLNUREL\RORMLN |QHPL \RNWXU DPD HVNXOLQLQ KLGUROL]OHQPHVL WHVWL ELOKDVVD ED]Õ
anaeroplar ve streptokoklar için identiktir.
Prensip: Eskulin, hidrolize olunca 6-7-dihydroxycoumarinDoÕ÷DoÕNDUEXPDGGHRUWDPGD
EXOXQDQ IHUULN VLWUDW LOH UHDNVL\RQD JLUHU )HUULN VLWUDW XOWUD YL\ROH ÕúÕNWD IORUHVDQV YHUGL÷L KDOGH
reaksiyona girdikten sonra floresans vermez hale gelir.
øúOHPøQFHOHQHFHNEDNWHULQLQVDINOWUQGHQELU|]HGROXVXPDWHU\DOPOHVNXOLQEX\\RQD
HNLOLU (NLOPHPLú NRQWURO EX\\RQ LOH ELUOLNWH WUH X\JXQ VUH ER\XQFD LQNEH HGLOLU YH KHUJQ
XOWUDYL\ROHÕúÕNDOWÕQGD\RNODQÕU
Eskulin Buyyon:
Aesculin
1g
Ferric citrate
0.5 g
Peptonlu su
1 lt
'H÷HUOHQGLUPH (NLP \DSÕOPDPÕú EX\\RQ HNLP \DSÕOPÕú EX\\RQ LOH \DQ\DQD JHWLULOHUHN
XOWUDYL\ROH ODPED\D \DNÕQODúWÕUÕOÕU (NLP \DSÕOPDPÕú EX\\RQ IORUHVDQV YHULUNHQ HNLP \DSÕOPÕú
buyyon ÕúÕPÕ\RULVHSR]LWLIWHVWVRQXFXRODUDN\RUXPODQÕU
Buyyondaki siyah kahverengi renk H26YHGHPLUVOIUOHULQROXúXPXQDED÷OÕROXSHVNXOLQLQ
KLGUROL]OHQGL÷LDQODPÕQÕWDúÕPD]
$QDHURSODU LoLQ HVNXOLQ KLGUROL]L EDNÕODFDNVD DQDHURS DWPRVIHUGHQ oÕNDUÕOGÕNWDn sonra ilk
15-GDNLNDLoHULVLQGHEDNÕOPDOÕGÕU$NVLKDOGH\DQOÕúSR]LWLIVRQXoYHULUOHU
Fenil alanin deaminasyonu:
7DQÕP Phenylalanine IRVIRIHQROSLUXYDWWDQ VHQWH]OHQHQ IHQLO SLUYDW¶ÕQ DPLQOHQPLú ELU
GHULYDV\RQXGXU$URPDWLNELUDPLQRDVLWWLU%D]Õbakteriler bundan fenil pirüvik asit elde edebilirler.
Prensip: %X WHVW LoHULVLQGH IHQLO DODQLQ EXOXQDQ ELU RUWDPGD IHQLO SLUYLN DVLW ROXúXS
ROXúPDGÕ÷ÕQÕQD\ÕUDoLODYHVLLOHWHVSLWHGLOPHVLHVDVÕQDGD\DQÕU
øúOHP øQFHOHQHFHN EDNWHUL WSWH KD]ÕUODQPÕú 3KHQ\ODODQLQH DJDU¶DHNLOLSWUHX\JXQVUH
LQNEHHGLOLUhUHPH\LWDNLEHQNRORQLOHU]HULQHGDPODD\ÕUDoGDPODWÕODUDN\HúLOUHQNDUDQÕU
Phenylalanine agar
2g
D- veya L-Phenylalanine
Yeast extract
3g
Sodium chloride
5g
Sodium phosphate
1g
HCI
2.5 ml
Agar
12 g
Distile su
1 lt
$\ÕUDo
Ferric chloride
12 g
Distile su
100 ml
S+ D\DUODQÕU
98
'H÷HUOHQGLUPH+HPHQROXúDQNR\X\HúLOUHQNRUWDPGDIHQLOSLUYLNDVLWEXOXQGX÷XQX\DQL
pozitif test sonucunu gösterir. %HVL\HULLoHULVLQHNDUERQKLGUDWWDQ]HQJLQELUNDWNÕNXOODQÕOPDPDOÕGÕU
Negatif kontrol için E. coli, pozitif kontrol için herhangi bir Providencia, Proteus veya Morganella
VXúXNXOODQÕOÕU
7LUR]LQVD\GDPODúWÕUPD
7DQÕP Tirozin, fosfofenolpiruvat’tan sentH]OHQHQ KLGURNVLIHQLOSLUXYDW¶ÕQ DPLQOHQPLú ELU
derivasyonudur. Aromatik bir amino asittir. Bilhassa Bacillus, Nocardia, Pseudomonas,
Acetobacter ve eugonic Mycobacteria
ODUEXDPLQRDVLWWHQPHODQLQROXúWXUDELOLU
Prensip: Tirozin’in deamiasyonu tyrosinase HQ]LPL LOH ROXU YH EX WHVW RUWD\D oÕNDQ VL\DK
UHQNOLPHODQLQ
LQWHVSLWHGLOPHVLQHGD\DQÕU
øúOHP Tirozin agar içine veya et infuzyon buyyon içerisine 0.1% tyrosine ilave edilerek
HNLP\DSÕOÕU7UHX\JXQVUHNDGDULQNEHHGLOHUHNKHUJQVL\DKUHQNDUDQÕU
Tyrosine agar (Gordon&Smith, 1955):
Pepton
5g
6Õ÷ÕUHW|]HWL
3g
Agar
15 g
5g
L-Tyrosine
Distile su
1 lt
Tyrosine DJDU
D HNLP \DSÕOPÕúVD VL\DK NRORQL ROXúXPX \DQÕQGD NRORQL oHYUHVLQGH YH
DOWÕQGDDJDULoHULVLQGHVD\GDPODúPDJ|Ulmesi beklenir.
'H÷HUOHQGLUPH Koloni çevresinde hem siyah pigmentasyon ve hem de agarda
VD\GDPODúPD SR]LWLI WHVW VRQXFX RODUDN \RUXPODQÕU øQNEDV\RQ ÕVÕVÕ PPNQ ROGX÷X NDGDU
GúN(30 °&FLYDUÕQGDWXWXOPDOÕGÕU%HVL\HULQHNDUERQKLGUDWLODYHHGLOPHPHOLdir.
øQGROWHVWL7ULSWRIDQ¶GDQLQGRO\DSÕPÕ
7DQÕPøQGROELUbenzyl pyrroleROXSIRVIRIHQROSLUXYDWWDQVHQWH]OHQHQLQGROJOLVHUROIRVIDW
ÕQ
DOGHKLWHED÷ODQPDVÕ\ODRUWD\DoÕNDU$URPDWLNELUDPLQRDVLWWLUTyptophanase enzimine sahip olan
bakteriler triptoIDQÕ|QFHGHDPLQHVRQUDKLGUROL]HHGHUHNLQGROROXúWXUXUODU
Prensip: øQGRO p-dimethylaminobenzaldehyde (Kovac's ve Ehrlich
V D\ÕUDFÕ QÕQ DOGHKLW
N|NLOHUHDNVL\RQDJLUPH\HLVWHNOLGLU%XUHDNVL\RQJHUoHNOHúLUVHEHVL\HULNÕUPÕ]ÕUHQNDOÕU
øúOHP Bakteri örnekleri triptofan'dan zengin bir besiyerinde inkübe edilmelidir. Bu amaç ile,
6XOILGH øQGROH 0RWLOLW\ 6,0 0RWLOLW\ øQGROH 2UQLWKLQH 0,2 YH\D øQGRO 1LWUDW EHVL\HUOHUL
NXOODQÕODELOLUøQGROGHQH\LQGHúXEHVL\HULGHNXOODQÕOÕU
Tryptophan buyyon (1% tryptophan) besiyeri:
Pepton veya pancreatic casein digest
2g
Sodium chloride
0,5g
Distile su
100ml
.RYDF
VD\ÕUDFÕ
Saf amyl veya isoamyl alkol
150ml
10g
p-Dimethylaminobenzaldehyde
HCI
50ml
(KUOLFK
VD\ÕUDFÕ
2g
p-Dimethylaminobenzaldehyde
Etil alkol
190ml
HCl
40ml
7ULSWRIDQLoHUHQEHVL\HULQGHEDNWHUL|UQH÷LQLQ-VDDWOÕNNOWU\DSÕOÕUKovac'sD\ÕUDFÕ
NXOODQÕODFDNVD - GDPOD D\ÕUDo WSQ Lo GXYDUÕQGDQ \DYDúFD DNÕWÕOÕU (÷HU Ehrlich's D\ÕUDFÕ
kullDQÕOÕ\RULVH|QFHPONVLOHQxyleneNRQXODUDNEHVL\HULPXKWHYL\DWÕQÕQWSQGLELQHo|NPHVL
EHNOHQLUYHWSVDUVÕOPDGDQEhrlich'sD\ÕUDFÕNRQXU5HQNGH÷LúLPLJ|]OHQLU
99
'H÷HUOHQGLUPH %LUNDo VDQL\H LoHULVLQGH D\ÕUDo LOH EX\\RQDUDVÕQGDYH\DD\ÕUDoLOH ksilen
WDEDNDVÕDUDVÕQGDROXúDQSDUODNNÕUPÕ]ÕUHQNSR]LWLIVRQXFXELOGLULU%HVL\HULQHNDUERQKLGUDWLODYH
edilmemelidir.
+Õ]OÕ WDQÕ DPDFÕ LOH ILOWUH ND÷ÕGÕQD p-dimethylaminocinnamaldehyde emdirilerek indol
\DSÕPÕDUDQDELOLU%XPDGGHNDUVLQRMHQGLUGLNNDWOLoDOÕúPDNJHUHNLU
Pozitif kontrol için E.coli, negatif kontrol için Klebsiella, Enterobacter, Hafnia veya Serratia
JUXEXEDNWHULOHUNXOODQÕODELOLU
Hidrojen sülfit (H26\DSÕPÕ
7DQÕP %D]Õ EDNWHULOHU LoHULVLQGH NNUW EXOXQDQ JLEL DPLQRDVLW \DGD EDúND VOIDWOÕ
ELOHúLNOHULNXOODQDUDNKLGURMHQVOILW+26ROXúWXUDELOLUOHU
Prensip: øQFHOHQHFHN EDNWHUL NDUERQKLGUDWWDQ IDNLU NNUWO DPLQRDVLWWHQ ]HQJLQ ELU
EHVL\HULQGH UHWLOHUHN DPLQRDVLWOHUL NXOODQPD\D ]RUODQÕU +26 ROXúX\RUVD GHPLUOL YH\D NXUúunlu
indikatörler ile tespit edilir.
øúOHP øQFHOHQHFHN EDNWHULGHQ ELU NRORQL DPDFD HQ X\JXQ RODUDN 'HPLU-Klorürlü-Jelatin
%HVL\HULQHHNLOLUøQNEDV\RQXWDNLEHQROXúDQGHPLUVOIU)H6VHEHEL\OHVL\DKNRORQLROXúXPX
DUDQÕU
'H÷HUOHQGLUPH øQFHOHQHQ EDNWHUL GDKD NROD\ UH\HELOHFH÷L EDúND ELU NDWÕ EHVL\HULQH
HNLOHFHNVH EX EHVL\HUL WS LoHULVLQH G|NOPHOL YH NDUERQKLGUDWWDQ IDNLU ROPDOÕGÕU øQNEDV\RQGDQ
|QFHNXUúXQDVHWDWHPGLULOPLúVWHULOV]JHoND÷ÕGÕEHVL\HULQHGH÷PH\HFHNúHNLOGHWSQD÷]ÕQD
VÕNÕúWÕUÕODUDN \HUOHúWLULOLU +26 YRODWLOGLU øQNEDV\RQX WDNLEHQ ROXúDQ +26 ND÷ÕW ]HULQGH VL\DK
UHQNOL NXUúXQ VOIU 3E6 ROXúWXUXU ø\L RNVLMHQOHQGLULOPLú RUWDPGD +26 ROXúPD\DFD÷Õ
KDWÕUODQPDOÕGÕU%XWHVWDQDHURSODUD\DSÕOGÕ÷ÕQGDDQDHURELNDWPRVIHUER]XOGXNWDQVRQUDLON\DUÕP
VDDWLoHULVLQGHNRORQLHWUDIÕQGDVL\DKELUUHQNOHúPHSR]LWLIVRQXoDQODPÕQDJHOLU
Pozitif kontrol olarak E.coli, B.subtilisNXOODQÕODELOLU
Hemoliz testi:
7DQÕP 7HVWLQ DQODPÕ EDNWHULQLQ KHPROLWLN DNWLYLWHVLQLQ YDUOÕ÷ÕQÕQ WHVSLWLGLU Bu test, aksi
EHOLUWLOPHPLúVHNR\XQNDQÕQÕHVDVDOÕU$QDHURSODULoLQDWYH\DWDYúDQNDQÕNXOODQÕOPDVÕLVWHQHELOLU
.DQOÕ DJDUGD UH\HQ EDNWHUL NRORQLOHULQLQ oHYUHVLQGH HULWURVLWOHULQ SDUoDODQDUDN ELU ]RQ ROXúPDVÕ
RODUDNWDQÕPODQÕU
Prensip: EritrositleULQ SDUoDODQPDVÕ haemolysin HQ]LPL LOH ROXU %X WHVW HVDV LWLEDUÕ LOH
EDNWHULQLQEXHQ]LPL\DSÕS\DSPDGÕ÷ÕQÕQWHVSLWLGLU
øúOHP%LU|]HGROXVXEDNWHULPOONR\XQNDQÕLoHUHQNDQOÕDJDUDHNLOHUHNWUQHX\JXQ
VÕFDNOÕNWDYHVUHLQNEHHGLOLU.RORQLoHYUHVLQGHHULWURVLWOHULQOL]LVROGX÷XELU]RQROXúXPXDUDQÕU
'H÷HUOHQGLUPH Koloninin çevresi mikroskopta 10x büyültme ile daima kontrol edilmeli ve
DJDU GHULQOLNOHUL GDKLO ROPDN ]HUH HULWURVLWOHULQ EXOXQPDGÕ÷Õ ELU ]RQXQ YDUOÕ÷Õ DUDQPDOÕGÕU ‰
Hemoliz). .RORQL oHYUHVLQGH NÕVPHQ SDUoDODQPÕú HULWURVLWOHULQ PHYFXGL\HWLQGH GHPLUOL ELOHúLNOHULQ
ROXúWXUGX÷X \HúLO UHQNOL ELU ]RQ J|]OHQHFHNWLU . +HPROL] .RORQL çevresinde eritrositlerin
SDUoDODQPDGÕ÷ÕGXUXPLVHQHJDWLIWHVWVRQXFXRODUDN\RUXPODQÕUKHPROL].
+HPROL]LQ
OHU 2 YH 6 ROPDN ]HUH LNL IDUNOÕ úHNLOGH EXOXQDELOLUOHU 6 6XUIDFH WLSLQGHNL
KHPROL]LQOHU DJDU \]H\LQGH 2 WLSLQGHNL KHPROL]LQOHU LVH DJDUÕQ GHULQOLNOHULQGH KHPROL] \DSDUODU
2NVLMHQVL]RUWDPGDKHPROL]]RQXJHQLúOHU
Pozitif kontrol için B. cereus veya A grubu streptokok, negatif kontrol için S. epidermidis
NXOODQÕODELOLU
Jelatinaz testi:
7DQÕP -HODWLQ NROODMHQLQ ND\QDWÕOPDVÕ LOH RUWD\D oÕNDQ ELU SURWHLQGLU 6ÕFDN VXGD
EHNOHWLOGL÷LQGHMHONÕYDPÕQDJHOLU28-35 °&DUDVÕQGDVRONÕYDPÕQGDGÕU%LOKDVVDClostridium türleri
EDúWDROPDN]HUHED]ÕEDNWHULOHUMHODWLQD]ED]HQNROODMHQD]HQ]LPOHULLOHMHODWLQLSDUoDODU
Prensip:-HODWLQOLEHVL\HULQGHUH\HQEDNWHULQLQMHODWLQD]HQ]LPLYDUVDNRORQLVLHWUDIÕQGDVRO
NÕYDPÕQGDELU]RQJHOLúPHVLHVDVÕQDGD\DQÕU
øúOHPøQFHOHQHFHNEDNWHULL÷QH|]HLOHDJDU\]H\LQHEDWÕUÕODUDNHNLP\DSÕOPDOÕGÕUQF
VDDWWHQ LWLEDUHQ KHU JQ \RNODQPDOÕGÕU .RORQLQLQ HWUDIÕQGD MHODWLQLQ GHNRPSR]H RODUDN VRO
NÕYDPÕQDJHOPHVLLQFHOHQLU
100
Jelatin Agar:
Pepton
6Õ÷ÕUHW|]HWL
Agar
Gelatin
Distile Su
5g
3g
15 g
4g
1 lt
S+ D\DUODQÕU
'H÷HUOHQGLUPH Jelatinde böyle bir sulanma pozitif test sonucunu gösterir. Besiyerinin her
WDUDIÕQGD VRO NÕYDPÕQGD o|]OPH ROPXúVD - VDDW EX]GRODEÕQGD EÕUDNÕOarak tekrar kontrol
HGLOPHOLGLU %X GXUXPGD EHVL\HULQGHNL MHODWLQ \HQLGHQ MHO NÕYDPÕQD JHoHU DPD NRORQL HWUDIÕQGD
GHNRPSR]HMHODWLQYDUVDKDODVRONÕYDPÕQGDNDODFDNWÕU
%HVL\HULQH NDUERQKLGUDW NRQXOPDPDOÕGÕU %DNWHUL NROD\ HQHUML WHPLQ HGHELOHFH÷L
karbonKLGUDWODUÕ EXOXUVD MHODWLQL VLQGLUPH\H JHUHNVLQLP GX\PD\DELOLU %X GXUXPGD \DQOÕú QHJDWLI
VRQXoODUDOÕQDELOLU%HVL\HULQH&D++LODYHHGLOPHVLYHUHPHÕVÕVÕQÕQELUD]DOWÕQGDLQNEHHGLOPHVL
EDNWHULQLQ YDUVD MHODWLQD] DNWLYLWHVLQL NDPoÕODU .ROODMHQD] DNWivitesi fazla olan proteolitik türler
MHODWLQLGHSDUoDOD\DUDN\DQOÕúSR]LWLIVRQXoYHUHELOLU
$QDHURSODUEXYH\DEDúNDEHVL\HULQGHWHVWHGLOHELOLU%LUDOWHUQDWLIPHWRWRODUDNX\JXQVUH
inkübe edildikten sonra, koloniler üzerine mercuric chloride (Hg2Cl2GDPODWÕOÕU%XPDGGHVHUEHVW
MHODWLQ WDUDIÕQGDQ ED÷ODQDUDN UHQN GH÷LúWLUHFHNWLU 'ROD\ÕVÕ LOH NRORQL HWUDIÕQGD UHQNVL] ELU ]RQ
J|UOPHVLSR]LWLIMHODWLQD]DNWLYLWHVLQLLúDUHWHGHU
Katalaz testi:
7DQÕP Katalaz (catalase EDNWHULOHULQ UHWWL÷L ELU HQ]LPGLU $VOÕQGD ELU KHPRSURWHLQGLU
6SHURNVLW GLVPXWD] YH LQGLUJHQPLú IODYRSURWHLQOHULQ RNVLWOHQPHVL VÕUDVÕQGD EDNWHUL KFUHVL
LoHULVLQGHDoÕ÷DoÕNDQWRNVLNKLGURMHQSHURNVLWLVXYHRNVLMHQHNDWDOL]HGHU.DWDOD]VWUHSWRNRNODU
hariç pekçok aerobik ve fakülWDWLI PLNURRUJDQL]PD WDUDIÕQGDQ UHWLOLU %X QHGHQOH NDWDOD] WHVWL
VÕNOÕNODMicrococcaceae ve StreptococcaceaeD\ÕUÕPÕQGDNXOODQÕOÕU
Prensip: øQFHOHQHFHNEDNWHULQLQNDWDOD]UHWLPLQLWHVSLWHWPHNDPDFÕLOHEDNWHULNRORQLVLQH
H2O2 GDPODWÕOÕU %DNWHULQLQ NDWDOD] HQ]LPL YDUVD NDEDUFÕNODU úHNOLQGH 22 JD]Õ oÕNÕúÕ WHVSLW HGLOLU
5HDNVL\RQú|\OHGLU2H2O2 F!+2O + O2.
øúOHP incelenecek bakteri kolonisi kürdan kullanarak lam üzerinde ezilir. Üzerine 1 damla
KLGURMHQSHURNVLWGDPODWÕOÕU*D]NDEDUFÕNODUÕDUDQÕU
'H÷HUOHQGLUPH +Õ]OD EDúOD\DQ YH NÕVD VUH LoHULVLQGH ND\EROPD\DQ NDEDUFÕNODU SR]LWLI
UHDNVL\RQ DQODPÕQD JHOLU %D]Õ EDNWHULOHU NDWDOD]GDQ EDúND 1RQ-hem katalaz) enzimler ile
hidrojenperoksiti dekompoze edebilir. Bu nedenle 20-30 saniye sonra oluúDQYH\DELUNDoNoN
NDEDUFÕN úHNOLQGH ROXúDUDN KHPHQ ND\ERODQ JD] NDEDUFÕNODUÕ SR]LWLI WHVW VRQXFX RODUDN
yorumlanmaz.
.DQOÕ EHVL\HULQGHQ DOÕQDQ NRORQLOHU \DQOÕú SR]LWLI VRQXo YHUHELOLU oQN HULWURVLWOHULQ
içerisinde de katalaz enzimi mevcuttur ve özH LOH NRORQL PDWHU\DOL DOÕQÕUNHQ EHVL\HULQH WHPDV
HGHUHN\DQOÕúOÕNODRUDGDQHULWURVLWOHULDOPDNPPNQGU
$QDHURSEDNWHULOHULoLQWHVW|QFHVLPDWHU\DO\DUÕPVDDWNDGDUKDYDLOHWHPDVWDEÕUDNÕOPDOÕ
YHNXOODQÕODQSHURNVLWVROV\RQXOLNROPDOÕGÕU
H2O2 VWRN VROXV\RQ ÕúÕN DOPD\DQ NDKYHUHQNOL ELU úLúH LoHULVLQGH YH EX]GRODEÕQGD
VDNODQPDOÕGÕU $NVL KDOGH LQDNWLYH RODUDN \DQOÕú QHJDWLI VRQXo YHUHELOLU 3R]LWLI NRQWURO RODUDN
Staphylococcus aureus, negatif kontrol olarak herhangi bir Streptococcus NXOODQÕODEilir. Burada
DQODWÕODQNDWDOD]WHVWVWDQGDUWODUÕYH\|QWHPOHULMycobacterium
ODULoLQJHoHUOLGH÷LOGLU
Potasyum siyanit (KCN)’te üreme:
7DQÕP Potasyum siyanit solunum fermentlerini (cytochrom, porphyrin) bloke eder. Bir
bakterinin KCN içeren bir besiyeULQGH UH\HELOPHVL KFUHQLQ QRQIHUPHQWDWLI \RO GÕúÕQGD ELU
VROXQXP PHNDQL]PDVÕQÕQ EXOXQGX÷XQX J|VWHULU YH ELOKDVVD Arizona ve Salmonella JHQXVODUÕQÕQ
CitrobacterJHQXVXQGDQD\ÕUÕPÕQGD|QHPWDúÕU
Prensip: øQFHOHQHFHN EDNWHUL |UQH÷L .&1 LoHUHQ EHVL\HULQH ekilerek inkübe edilir.
hUH\HELOL\RUVDREDNWHULQLQIHUPHQWDWLIVROXQXPPHNDQL]PDVÕYDUGHPHNWLU
101
øúOHP $úD÷ÕGDNL ED]DO EX\\RQD .&1 LODYH HGLOPHGHQ ELU WSH D\UÕOÕU YH NRQWURO EHVL\HUL
RODUDN NXOODQÕOÕU .&1 LODYH HGLOPLú EX\\RQ LOH ELUOLNWH HNLP \DSÕOarak türüne uygun süre kadar
inkübe edilir. +HUJQ\RNODQÕU
KCN Buyyon (Moeller, 1954):
Pepton
10 g
Sodium Chloride NaCl
5g
Potassium Phosphate, KH2PO4
0.2 g
Disodium Phosphate, Na2HPO4 .2H2O
5.64 g
Distile su
1 lt
Potassium Cyanide, KCN %5
15 ml
'H÷HUOHQGLUPH KCN içeren ve içermeyen besiyerinin herikisinde de üreme tespit edilirse
WHVW VRQXFX SR]LWLI RODUDN \RUXPODQÕU .&1
OL EX\\RQGD UHPH \RN LNHQ NRQWURO EX\\RQGD UHPH
YDUVDQHJDWLIWHVWVRQXFXRODUDN\RUXPODQÕU. Her ikisinde de üreme yok ise o bakterinin üremesi
LoLQ PXWODN JHUHNVLQGL÷L |]HO EHVL\HULQH VRQ NRQVDQWUDV\RQX RODFDN úHNLOGH .&1
NDWÕODUDN WHVW WHNUDUODQPDOÕGÕU 7HVW NDOLWDWLIWLU UHPHQLQ D] \DGD ID]OD ROPDVÕ WHVW VRQXFXQX
etkilemez.
Pozitif kontrol olarak Citrobacter, Providencia, Proteus, Klebsiella, Hafnia, Serratia,
Enterobacter NXOODQÕODELOLU 1HJDWLI NRQWURO RODUDN Shigella, Salmonella, Arizona veya E.coli
NXOODQÕODELOLU
.&1 WRNVLN ROXS NLOLW DOWÕQGD EXOXQPDOÕGÕU YH EXKDUODúPDVÕQÕQ HQgellenmesi için lastik
NDSDNOÕ WSOHUGH VDNODQPDOÕGÕU .&1 LoHUHQ EHVL\HUOHUL NXOODQÕOGÕNWDQ VRQUD GR÷UXGDQ RWRNODYD
NRQXOPD] /DERUDWXDU JYHQOL÷LQL VD÷ODPDN LoLQ DWÕODFDN EHVL\HUOHUL LoHULVLQH |QFH .2+
GHQ
HQD]PONRQXOPDOÕYHWSHELUSDUoDNULVtalize FeSO4DWÕODUDNVL\DQUQWDPDPHQED÷ODQPDVÕ
VD÷ODQPDOÕGÕU
Koagülaz (Coagulase) test:
7DQÕP.RDJOD]¶ODUVLWUDWOÕYH\DRN]DODWOÕSOD]PD\ÕNRDJOHHGHELOHQELUELULQGHQoRNIDUNOÕ
LNLWDQHEDNWHULHQ]LPLGLU2UWDPDVDOÕQDQserbest koagülaz: KültUILOWUDWODUÕQDJHoHUWSWHVWL
JHUHNWLULU+FUHGXYDUÕQDED÷OÕNRDJOD]FOXPSLQJIDNW|U.OWUILOWUDWODUÕQGDEXOXQPD]/DP
WHVWL\HWHUOLGLU.RDJOD]ODUÕQWHVSLWLELOKDVVDStaphylococcus aureus'un ve Yersinia pestis’in kendi
JHQXVXQXQGL÷HU\HOHULQGHQD\ÕUÕPÕQD\DUGÕPFÕROXU
Prensip:øQFHOHQHFHNEDNWHULODPGDYHWSWHWDYúDQSOD]PDVÕLOHPXDPHOHHGLOLSSOD]PDGD
NRDJODV\RQDUDQÕU
øúOHP YH GH÷HUOHQGLUPH 'HQH\GH VLWUDWOÕ YH\D RN]DODWOÕ WDYúDQ YH\D LQVDQ SOD]PDVÕ
NXOODQÕOÕU3OD]PDVLWUDWOÕLVHVLWUDWÕNXOODQDQEDNWHULOHUSOD]PDLoHULVLQGHEXOXQDQVLWUDWÕNXOODQDUDN
NRDJODV\RQ \DSDELOLU YH \DQOÕú SR]LWLI VRQXo YHUHELOLUOHU %X VHEHSOH SR]LWLI NRQWURO RODUDN
Staphylococcus aureus, negatif kontrol olarak Staphylococcus epidermidis NXOODQÕOPDOÕGÕU YH\D
DQWLNRDJXODQRODUDNVLWUDW\HULQHRN]DODWNXOODQÕOPDOÕGÕU
a) %D÷OÕ NRDJOD]
ÕQ WHVELWL /DP 7HVW øQFHOHQHFHN EDNWHUL NRORQLVL ODP ]HULQGH LNL D\UÕ
\HUGHH]LOLUELUWDQHVL]HULQHGDPODL]RWRQLNWX]OXVXGL÷HULQHSOD]PDLODYHHGLOLU0XNayeseli
RODUDNJ|]YHPLNURVNRSODDJOWLQDV\RQDUDQÕU
VDQL\H LoHULVLQGH NRDJODV\RQ ROXúXPX SR]LWLI UHDNVL\RQX J|VWHULU GDNLND LoHULVLQGH
ROXúPDPÕú LVH WHVW VRQXFX QHJDWLIWLU 3R]LWLI WHVW VRQXoODUÕ ODP ]HULQGH SOD]PD LKWLYD HWPH\HQ
koloni ile daimD NDUúÕODúWÕUÕOPDOÕGÕU +HU LNLVLQLQ GH SR]LWLI ROGX÷X GXUXPODUGD ELU GH÷HUOHQGLUPH
\DSPDNWDQNDoÕQÕOPDOÕGÕUoQNL]RWRQLNROVDELOHWX]OXVXLOHWHPDVHGHQED]ÕEDNWHULOHUVSRQWDQ
aglütine olabilir.
b) 6HUEHVW NRDJOD]ÕQ WHVELWL 7S 7HVW øQFHOHQHFHk bakterinin 18- VDDWOÕN EX\\RQ
NOWUQGHQ PO WD]H SOD]PD ]HULQH HNOHQLS VDDW DUDOÕNODUOD NRDJODV\RQ DUDQÕU EXOXQDPD]
LVH VDDW RGD ÕVÕVÕQGD EÕUDNÕODUDN WHNUDU RNXQPDOÕGÕU 7S LQFHOHQLUNHQ KDILIoH H÷LOPHOL DQFDN
oDONDODQPDPDOÕGÕU3OD]PDGDNRDgülasyon görülmesi pozitif sonuçtur.
102
7KLRPHUVDO NXOODQDQ ELUH\OHULQ SOD]PDVÕ NXOODQÕOGÕ÷ÕQGD NRDJODV\RQX HQJHOOHU KHSDULQ
engellemez. 3OD]PD LoHULVLQH PO NDOVL\XP NORULW HNOHQPHVL YH\D úHNHU KDVWDODUÕQÕQ
SOD]PDVÕQÕQNXOODQÕOPDVÕEXWHVWLGDKDGX\DUOÕYHJYHQOL\DSDU
<RNODPDODUVÕUDVÕQGDELUGHIDNRDJODV\RQJ|UOPHVLLOHUOH\HQ\RNODPDODUGDNRDJODV\RQ
ND\EROVD ELOH SR]LWLI VRQXo DQODPÕQD JHOLU dQN LQNEDV\RQ VÕUDVÕQGD ROXúDELOHFHN SÕKWÕ D\QL
EDNWHULQLQ ROXúWXUDELOHFH÷L ILEULQROL]LQ HQ]LPi ile tekrar yok edilebilir. Bu sebeple daha uzun
LQNEDV\RQGDQNDoÕQÕOPDOÕGÕU
1HJDWLI ODP WHVW J|VWHUHQ EWQ VXúODU WS WHVWLQH DOÕQPDOÕGÕU dQN ED÷OÕ NRDJOD]Õ
ROPD\DQ EDNWHULOHULQ VHUEHVW NRDJOD]ODUÕ EXOXQDELOLU $PD ELU EDNWHULQLQ VHUEHVW NRDJOD]ODUÕ
YDUVDJHQHOOLNOHED÷OÕNRDJOD]ODUÕGDYDUGÕU
Lipaz ve Lesitinaz testi:
7DQÕP /LSROLWLN EDNWHULOHU OLSD] HQ]LPOHUL LOH \D÷ODUÕ SDUoDOD\DELOLUOHU %D]Õ EDNWHULQLQ
identifikasyonunda önemli bir kriterdir.
Prensip: <D÷ODU OLSROL] \D GD KLGUROL] LOH JOLVHULQ YH \D÷ DVLWOHULQH SDUoDODQÕU 'HQH\LQ
SUHQVLELEHVL\HULQGHEXPDGGHOHULQS+LQGLNDW|ULOHWHVSLWHGLOPHVLQHGD\DQÕU
øúOHP%DNWHULQLQVDINOWUQGHQOLSD]EHVL\HULQHHNLP\DSÕODUDNWUQHX\JXQVUHLQNEH
edilir.
Lipaz Besiyeri (Davis & Ewing, 1964):
Pepton
10 g
Yeast Ectract.
3g
Sodium Chloride, NaCl
5g
Agar
20 g
Victoria Blue, 1:1500 aq.sol.,
100 ml
50 ml
0ÕVÕU\D÷ÕYY
Distile su
900 ml
'H÷HUOHQGLUPH Koloni HWUDIÕQGD ROXúDQ PDYL UHQN \D÷ÕQ SDUoDODQGÕ÷ÕQÕ GROD\ÕVÕ LOH R
EDNWHULQLQ OLSD]ODUÕ ROGX÷XQX YH SR]LWLI WHVW VRQXFXQX LúDUHW HGHU <DQGDQ EH\D] ÕúÕN LOH
D\GÕQODWÕOGÕ÷ÕQGDNRORQLELULQFLWDQHVLJLELSDUOÕ\RULVHOLSD]DNWLYLWHVLYDUGÕU5HQNROXúPDPDVÕYH\D
NÕUPÕ]ÕUHQNROXúPDVÕQHJDWLIWHVWVRQXFXRODUDNGH÷HUOHQGLULOPHOLGLU
$JDULoHULVLQGHYHNRORQLoHYUHVLQGHSUHVLSLWDWODUÕQEXOXQPDVÕEDNWHULQLQOHVLWLQD]DNWLYLWHVLQL
J|VWHULU .RORQL ]HULQH GDPOD VX NRQXODUDN EDNÕOGÕ÷ÕQGD NRORQL HWUDIÕQGD SUHVLSLWDW LKWLYD
HWPH\HQSDUODNJHQLúELU]RQYDULVHEXOHVLWLQD]SR]LWLIDQODPÕQDJHOLU
Malonat fermentasyonu :
7DQÕP %D]Õ EDNWHULOHU Vodyum malonat
Õ NXOODQPD YH IHUPHQWOHPH |]HOOL÷LQH VDKLSWLU
Salmonella - Arizona ve Klebsiella -Aerobacter –SerratiaD\ÕUÕPÕQGDID\GDOÕGÕU
Prensip: 0DORQDW¶ÕQ \ÕNÕP rünleri baziktir. Bu test bakteri üreyen ve içerisinde malonat
EXOXQDQEHVL\HULQLQS+VÕQÕQWHVSLWLHVDVÕQDGD\DQÕU
øúOHP øQFHOHQHFHN EDNWHUL WUQH X\JXQ VUH PDORQDW EX\\RQGD LQNEH HGLOLU S+
LQGLNDW|ULOHDONDOLGH÷LúLPDUDQÕU
Modifiye Malonat Buyyon:
Ammonium sulphate
2g
Dipotassium phosphate, K2HPO4
0.6 g
Potassium phosphate, KH2PO4
0.4 g
Sodium chloride
2g
Sodium malonate
3g
Brom-timol mavisi, %5 alk.sol
5 ml
Distile su
1 lt
'H÷HUOHQGLUPH0DYLYHNR\X\HúLOUHQNOHúPHS+
ÕQ\NVHOGL÷LQL\DQLSR]LWLIWHVWVRQXFXQX
J|VWHULU5HQJLQGH÷LúPHPHVL\DGDVDUÕ\DG|QúPHVLQHJDWLIWHVWVRQXFXQXJ|VWHULU'DLPDHNLP
\DSÕOPDPÕú ELU EX\\RQ LOH ELUOLNWH LQNEH HGLOPHOLGLU Besiyerine karbonhidrat konulmaz. Çünkü
103
karbonhidrat fermHQWOHQHUHNDVLWROXúWXUXUYHYDUVDDONDOLPDGGHOHULWDPSRQOD\DUDN\DQOÕúQHJDWLI
sonuçlara sebebiyet verebilir. Serratia¶ODUQHJDWLINRQWURORODUDNNXOODQÕODELOLU
Mukat fermentasyonu:
7DQÕP 0XNDW ED]Õ EDNWHULOHU WDUDIÕQGDQ IHUPHQWOHQLU Shigella , Escherichia, Salmonella,
Arizona ve CitrobacterD\ÕUÕPÕQGD|QHPWDúÕU
Prensip: 0XNDW PXNLN DVLW IHUPHQWH RODELOLU \DSÕGDGÕU YH VRQ UQ NXYYHWOL ELU DVLWWLU %X
WHVWPXNDWEXOXQDQEHVL\HULQGHVRQUQRODQDVLWOHULQROXúWX÷XQXJ|]OHPHSUHQVLELQHGD\DQÕU
øúOHP Bakterinin saf kültürün organik asit besiyerine ekilerek türüne uygun süre inkübe
HGLOLUøQGLNDW|ULOHS+WHVSLWHGLOLU
Organik Asit Besiyeri (Ellis et al.,1957):
Pepton
10 g
Brom-timol mavisi %2
12 ml
Mukat
10 g
Distile su
1 lt
S+ D\DUODQÕU
(Bu besiyeri, D-7DUWDUDW
YH VLWUDW NXOODQÕPÕQÕ WHVSLW HWPHN LoLQ GH NXOODQÕODELOLU
Bu durumda, besiyerine
NXOODQÕODQPXNDW\HULQHWDUWDUDWYH\DVLWUDWNRQXU
'H÷HUOHQGLUPH .DUERQKLGUDW IHUPHQWDV\RQ WHVWOHULQGH ROGX÷X JLEL S+ LQGLNDW|rünün koyu
\HúLOGHQVDUÕUHQJHGR÷UXG|QúPHVLS+
ÕQDOWÕQDGúHUHNDVLWROXúWX÷XDQODPÕQDJHOLUSR]LWLI
VRQXFXLúDUHWHGHU%XEHVL\HULQHNDUERQKLGUDWLODYHVLQGHQNDoÕQPDNJHUHNLU
%D]Õ ND\QDNODU VRQXFXQ GR÷UXODQPDVÕ DPDFÕ LOH KHP NRQWURO WSQH YH hem de ekim
\DSÕOPÕúWSQHQ|WUDONXUúXQDVHWDWHUL\L÷LQLQPOLODYHVLQLJHUHNOLJ|UPHNWHGLUOHU
Pozitif kontrol olarak E.coli, negatif kontrol olarak da herhangi bir Shigella veya
Edwardsiella\HVLNXOODQÕODELOLU
0HWLONÕUPÕ]ÕVÕWHVWL
7DQÕP Karbonhidratlardan ileri seviyede (pH”DVLWROXúXPXQXWHVELWLoLQNXOODQÕOÕU%X
GXUXPDQFDNNDUÕúÕNDVLWIHUPHQWDV\RQ\ROXQXNXOODQDQEDNWHULOHUGHPPNQGU
Prensip: +HU EDNWHULQLQ UDKDWoD IHUPHQWH HGHELOHFH÷L ELU úHNHU PHVHOD JOXNR] EXOXQDQ
besiyerinGH UHWLOHQ EDNWHULQLQ S+ µÕ ¶Q DOWÕQD GúUHFHN NDGDU NXYYHWOL DVLW \DSÕS
\DSPDGÕNODUÕQÕQS+LQGLNDW|UYH\DS+-PHWUHLOHDUDQPDVÕHVDVÕQDGD\DQÕU
øúOHP Bu test için Voges-3URVNDXHU0593EX\\RQNXOODQÕOÕU(QD]VDDWLQNEHHGLOLU
sürenin uzXQROPDVÕELOKDVVDJHUHNOLGLUVDDWGROPDGDQRNXPD\DSÕOPD]7SHGDPODPHWLO
UHGD\ÕUDFÕEHVL\HULQLQKHUPLOLOLWUHVLQHGDPODD\ÕUDoilave edilir ve kÕUPÕ]ÕUHQNDUDQÕU
Methyl-Red/Voges-Proskauer (MR/VP) buyyon
MR/VP broth Polypepton
7g
Glucose
5g
Dipotassium phosphate
5g
Distile su
1 lt
S+ D\DUODQÕU
0HWLOUHGD\ÕUDFÕ
Methyl red
0.1 g
Ethyl alcohol 95%
300 ml
Distile su
200 ml
'H÷HUOHQGLUPH%HVL\HUL\]H\LQGHWDPDPÕQGDROPD\DELOLUNÕUPÕ]ÕELUUHQNROXúXPXSR]LWLI
VRQXoWXUWXUXQFX\DGR÷UXJLGHQDUDUHQNOHUSR]LWLIGH÷LOGLU'DKDL\LVLELUS+-metre ile besiyeri pH
VÕQÕQVÕQÕUÕQÕQDOWÕQGDROXSROPDGÕ÷ÕQÕQRNXPDNWÕU
3HNoRN EDNWHUL EDVLW ELU úHNHU RODQ JOXNR]X IHUPHQWH HGHELOLU GL\H GúQOHUHN Eu
EHVL\HULQH NDUERQKLGUDW RODUDN JOXNR] VHoLOPLú YH \HWHUOL J|UOPúWU +DOEXNL Gardnerella
vaginalis ve Eubacterium WUOHUL JLEL ED]Õ EDNWHULOHU JOXNR]X NXOODQDPDGÕ÷Õ KDOGH EDúND
NDUERQKLGUDWODUGDQ oRN NXYYHWOL SK DVLW ROXúWXUDELOLU %HONLGH EX WHVW LoLQ NXOODQÕODQ 0593
104
EX\\RQ NDUERQKLGUDW EDNÕPÕQGDQ GDKD ]HQJLQ KDOH JHWLULOPHOLGLU %|\OHFH SRWDQVL\HO \DQOÕú
QHJDWLIVRQXoODUHQJHOOHQPLúRODFDNWÕU
3R]LWLI NRQWURO DPDFÕ LOH E.coli QHJDWLI NRQWURO DPDFÕ LOH Enterobacter aerogenes veya
Klebsiella pneumoniaNXOODQÕODELOLU
Bu test bu safhada Voges-Proskauer testi ile devam ettirilebilir:
Voges-Proskauer testi:
%XWHVWLX\JXODPDNLoLQ\XNDUGDDQODWÕODQ0HWLONÕUPÕ]ÕWHVWLQLQ\DSÕOPÕúROPDVÕJHUHNLU
7DQÕP %D]Õ EDNWHULOHU NDUERQKLGUDWÕ EXWDQHGLRl fermentasyon yolu ile fermente ederler.
Sonuçta, acetyl-methyl carbinol, veya onun kursörleri acetoin ve 2,3,butanediol ROXúXU %D]Õ
enterobakteriler (Klebsiella, Enterobacter, Hafnia, Serratia ) için identiktir.
Prensip: øQFHOHQHQEDNWHULEXWDQHGLROIHUPHQWDV\RQ\ROXQXNXOODQGÕ\VDRUWDPGDEXOXQDQ
acetoin ve 2,3,butanediol D\ÕUDoODUÕQ LODYHVL\OH GLDVHWLOHRNVLWOHQLUYH.-naphtolLOHELUOHúLSNÕUPÕ]Õ
UHQNYHULU7HVWLQDPDFÕEXNÕUPÕ]ÕUHQJLQDUDQPDVÕGÕU
øúOHP 0HWLO UHG WHVWLQGH DQODWÕOGÕ÷Õ JLEL UHWLOHQ EDNWHUL |UQH÷L ]HULQH EHVL\HULQLQ KHU
PLOLOLWUHVLQH PO D\ÕUDo- YH PO D\ÕUDo- LODYH HGLOLU 7S NXYYHWOH oDONDODQÕS \DWÕN
SR]LV\RQGD \DUÕP VDDW EHNOHQLU .ÕUPÕ]Õ UHQN DUDQÕU Bu yöntem Barritt metodudur. O'meara
metodunda ise .-naphtol yerine creatineNXOODQÕOÕU
$\ÕUDo-1
.-Naphthol (5%)
5g
Ethyl alcohol
100 ml
$\ÕUDo-2
Potassium hydroxide (40%)
40 g
Distile su
100 ml
'H÷HUOHQGLUPH $\ÕUDo LODYHVLQGHQ - GDNLND VRQUD ROXúDQ NÕUPÕ]Õ UHQN SR]LWLI VRQXo
DQODPÕQD JHOLU 'H÷HUOHQGLUPH VDDWWHQ VRQUD \DSÕOPDPDOÕGÕU oQN QHJDWLI VRQXoODQDQ
WHVWOHUGHELOH\DNODúÕNELUVDDWVRQUDEDNÕUUHQJLROXúXU
Nitrat redüksiyonu:
7DQÕP %DNWHULQLQ QLWUDWGDQ RNVLMHQ NRSDUDELOGL÷LQLQ DUDúWÕUÕOPDVÕGÕU 1LWUDWODUÕ QLWULWH
indirgeyen nitrate reductase enzimleri Pantoa agglomerans,ED]ÕSerratia ve Yersinia türleri hariç
bütün EnterobacteriaceaeIDPLO\DVÕQGDYHDQDHURSODUGDQLWUDWUHGNWD]ODUJHQHOOLNOHEXOXQXU%X
test Haemophilus, Neisseria ve Branhamella genusuna ait üyelerin de birbirlerindeQD\ÕUÕPODUÕQÕQ
\DSÕODELOPHVLLoLQID\GDOÕELUWHVWWLU
Prensip:5HDNVL\RQXQNLP\DVDOSURILOLú|\OHGLUNO3- + 2e- + 2H FF!122 + H20
7HVWLQ SUHQVLEL LQFHOHQHQ EDNWHULQLQ UHWLOGL÷L QLWUDWOÕ RUWDPGD QLWULW DUDQPDVÕGÕU %DNWHUL NRORQLVL
üzerine
veya
buyyonD
D\ÕUDoODU
GDPODWÕOGÕ÷ÕQGD
RUWDPGD
QLWULW
YDUVD
p-sulfobenzene-azo-.-naphthylamineROXúXU%XPDGGHNÕUPÕ]ÕUHQNOLELUGLD]RQLXPER\DVÕGÕU
øúOHP%XWHVWLoLQEDNWHUL|UQH÷LDúD÷ÕGDNLEHVL\HULQGHWUQHX\JXQVUHLQNEHHGLOLU+HU
LNLD\ÕUDoVÕUDVÕ\ODPOGDPODWÕOÕU5HQNGH÷LúLPLJ|]OHQLU
Nitrate Broth or Nitrate Agar:
Beef extract
3g
Pepton
5g
Potassium nitrate (KNO3)
1g
Agar (nitrit içermeyen)
12 g
Distile su
1 lt.
$\ÕUDo$
Sulfanilic acid
8g
Acetic acid 5N,30%
1 lt
$\ÕUDo%
.-Naphthylamine
5g
Acetic acid (5 N),30%
1 lt
105
'H÷HUOHQGLUPH VDQL\H LoHULVLQGH NÕUPÕ]Õ UHQN ROXúPDVÕ SR]LWLI VRQXo DQODPÕQD JHOLU
%HVL\HULQLQUHQJLQGHELUGH÷LúLPROPDPDVÕúXVRQXoODUGDQELULVLQLLIDGHHGHU%XEDNWHULQLWUDWÕ
nLWULWH UHGNOHPHPLúWLU EX KDOGH WHVWLQ VRQXFX JHUoHNWHQ QHJDWLIWLU %X EDNWHUL QLWUDWÕ QLWULWH
UHGNOHPLú DPD QLWULW VUDWOH EDúND NRPSRQHQWOHUH ED÷ODQDUDN D\ÕUDoODUGDQ JL]OHQPLúWLU 7HVWLQ
sonucu pozitiftir ama negatif görünmektedir. (gUQH÷LQQLWUDWLNLGHIDUHGNOHQLUVHVRQXoWDQLWULWGH÷LO1+3, N2,
NO, N2O veya hydroxylamine úHNOLQH G|QúU $\ÕUDoODU EXQODUÕ WHVSLW HGHPH]). Bunu anlamak için, negatif
VRQXo YHUHQ EHVL \HUL LoHULVLQH NoN ELU PHWDOLN oLQNR SDUoDVÕ DWÕODUDN QLWUDWODU PDQXHO RODUDN
nLWULWH LQGLUJHQPHOL YH NÕUPÕ]Õ UHQN ROXúXPX DUDQPDOÕGÕU %X GR÷UXODPD LúOHPLQLQ VRQXFXQGD
NÕUPÕ]Õ UHQN J|UOPHVL RUWDPGD LQGLUJHQPHPLú QLWUDW EXOXQGX÷XQX YH EDNWHULQLQ QLWUDW
UHGNWD]ODUÕQÕQEXOXQPDGÕ÷ÕQÕ\DQLWHVWLQVRQXFXQQHJDWLIROGX÷XQXJ|VWHULU
KonWURO DPDFÕ LOH QHJDWLI WHVWOHU WHNUDUODQDELOLU YH\D QHJDWLI NRQWURO RODUDN Acinetobacter
baumannii, pozitif kontrol olarak ise Escherichia coliNXOODQÕODELOLQLU
Bu test Mycobacterium¶ODU LoLQ \DSÕODFDNVD EHVL\HUL PRGLIL\H HGLOPHOL ODNWLN DVLW LODYH
edilmHOLYHLQNEDV\RQVUHVLWUHX\JXQRODUDNX]DWÕOPDOÕGÕU
Oksidaz testi:
7DQÕP Cytochrome C oxydaseHQ]LPLQLQYDUOÕ÷ÕQÕDUD\DQELUWHVWWLU%XHQ]LPGHPLULoHUHQ
ELUKHPRSURWHLQGLUQRQIHUPHQWDWLIDHURELNVROXQXPDNDWÕOÕUPseudomonas, Neisseria, Vibrio ve
Pasteurella’larda bulunur zorunlu anaeroplarda genellikle bulunmaz.
Prensip:øQFHOHQHQEDNWHULGHEXHQ]LPEXOXQX\RUVDD\ÕUDoÕLQGRIHQROPDYLVL¶QHRNVLWOHUYH
PDYLUHQNDoÕ÷DoÕNDU
øúOHP %1 lik Kovac
V RNVLGD] D\ÕUDFÕ Tetramethyl-p-phenylenediamine dihydrochloride)
veya Gordon - McLeod
V RNVLGD] D\ÕUDFÕ Dimethyl-p-phenylenediamine dihydrochloride) veya
WLFDUL RNVLGD] GLVN YH D\ÕUDoODUÕ NXOODQÕODELOLU Direkt Plak Teknik: 2- GDPOD D\ÕUDo GR÷UXGDQ
EDNWHUL NRORQLVL ]HULQH GDPODWÕOÕU øQGLUHNW 6WULS Teknik: $\ÕUDoODUGDQ ELULVL ELU V]JHo ND÷ÕGÕQD
emdirilir ve üzerine bakteri kolonisi tatbik edilir. Önerilen metot budur.
'H÷HUOHQGLUPHVDQL\HLoHULVLQGHEDNWHULNRORQLVLQLQWHPDVHWWL÷LV]JHoND÷ÕGÕQÕQYH\D
]HULQH D\ÕUDo GDPODWÕODQ NRORQLQLQ NR\X PDYL UHQN DOPDVÕ SR]LWLI VRQXoWXU VDQL\H LoHULVLQGH
ROXúDQ UHQNOHúPH GXUXPXQGD WHVW WHNUDUODQPDOÕGÕU GDKD JHo ROXúDELOHFHN UHQNOHúPHOHU
GH÷HUOHQGLULOPH] 1LNHO NURP YH SDVODQPD] oHOLNGHQ \DSÕOPÕú |]HOHU \HULQH D÷DoWDQ \DSÕOPÕú
NUGDQODU NXOODQÕOPDOÕGÕU dQN PHWDO \]H\LQGH ROXúDQ RNVLWOHU WHVWLQ VRQXFXQX \DQOÕú SR]LWLI
yapar. Negatif kontrol olarak E. coli , pozitif kontrol için ise P. aeruginosaNXOODQÕODELOLQLU
ß-D-galaktozidaz (ONPG) testi:
7DQÕP Bakteri hücresi laktozu kullanabilmek için önce: 1) Laktozu bir permease enzimi ile
KFUH LoHULVLQH DOÕU GDKD VRQUD KFUH LoHULVLQH DOGÕ÷Õ ODNWR]X LQWUDVHOOHU ELU HQ]LP RODQ ßgalactosidase ile hidrolize eder. Bu test, ß-JDODNWR]LGD]
ÕQ YDUOÕ÷ÕQÕ \DQL ODNWR]D SRWDQVL\HO HWNLOL
bakterileri ortayDoÕNDUWÕU%XWHVWVRQXFXSR]LWLIRODQELUEDNWHULNDNWR]XIHUPHQWHHWPHN]RUXQGD
GH÷LOGLU
Prensip:(÷HUEDNWHULGH‰-galaktozidaz enzimi varsa ortamda bulunan O-Nitrophenyl-ß-D
Galactopyranoside 213* SDUoDODQDFDNWÕU6RQXoWD JDODNWR] YHO-NitrophenylDoÕ÷DoÕNDFDNWÕU
Bunlardan O-NitrophenylVDUÕUHQNOLGLU
øúOHP øQFHOHQHFHN EDNWHULQLQ VDI NOWUQGHQ ELU NRORQL 213* EX\\RQD HNLOHUHN WUQH
X\JXQVUHLQNEHHGLOLU6DUÕUHQNDUDQÕU
ONPG Buyyon:
Peptonlu su
750 ml
ONPG stok solusyonu
250 ml
ONPG Stok Solusyonu:
ONPG
0.01M Na2HPO4
6g
1 lt
'H÷HUOHQGLUPH øQNEDV\RQX WDNLEHQ ROXúDQ VDUÕ UHQN SR]LWLI WHVW VRQXFXQX YHULU 213*
VWRNVROXV\RQXEX]GRODEÕQGDYHNDUDQOÕNWDVDNODQPDOÕGÕU
106
Safraya tolerans:
7DQÕP %D]Õ EDNWHULOHU EDUVD÷ÕQ GR÷DO NRQDNoÕVÕ ROXS ELU NDUDFL÷HU VDOJÕVÕ RODQ VDIUDQÕQ
NRQVDQWUDV\RQODUÕQD ELOH WROHUDQV J|VWHULUOHU %X WHVW R EDNWHULQLQ VDIUDGD \DQL EDUVDNWD
\DúD\ÕS\DúD\DPDGÕ÷ÕQÕWHVSLWHGHUB. fragilis
LQGL÷HU*UDPQHJDWLIDQDHURSODUGDQD\ÕUÕPÕQGDYH
ED]Õ HQWHUREDNWHULOHU YH ' JUXEX VWUHSWRNRNODUÕQ D\ÕUÕPÕQGD DQDKWDU WHVWWLU %X WHVW
PneumococcusJHQXVXQDX\JXODQDQVDIUDGDOL]LVWHVWLQGHQED÷ÕPVÕ]GÕU
Prensip: øoHULVLQGH VDIUD EXOXQDQ ELU EHVL\HULQGH UHPH ROXS ROPDGÕ÷ÕQD
EDNÕOPDVÕQGDQLEDUHWWLU
øúOHP: øQFHOHQHFHN EDNWHULQLQ VDI NRORQLVL VDIUDOÕ DJDU LoHULVLQH HNLOLU WUQH X\JXQ
VÕFDNOÕNWDX\JXQVUHLQNEHHGLOLUUHPHDUDQÕU
6DIUDOÕ$JDU
40 g
.XUXWXOPXú|N]VDIUDVÕ
At serumu
50 ml
Et infuzyon agar(veya türe uygun besiyeri)
1 lt
De÷HUOHQGLUPHøQNEDV\RQXWDNLEHQVDGHFHUHPHDUDQÕU.RORQLPRUIRORMLVLNRORQLUHQJL
YH\DPLNURVNRSLEDNÕOPD]%DNWHULNRORQLVLQLQPHYFXGL\HWLSR]LWLIWHVWVRQXFXLoLQ\HWHUOLGLU
7LFDUHWWHQ WHPLQ HGLOHELOHQ VDIUD GLVNOHUL GH YDUGÕU DQWLEL\RWLN GLVNOHUL JLEL HNLP \DSÕOPÕú
DJDU\]H\LQHEÕUDNÕOÕS]RQ|OoP\DSÕODELOLU'LVNNXOODQÕPÕDQDHURSODULoLQWHUFLKHGLOLU
Safrada lizis :
7DQÕP 3Q|PRNRNODUÕ GL÷HU VWUHSWRNRNODUGDQ D\ÕUPDN LoLQ \DSÕODQ ELU WHVWWLU 3Q|PRNRNODU
enterik ekolojiye (safraya) fazla GX\DUOÕGÕUODU2UWDPGDVDIUDPHYFXGL\HWLQGHSQ|PRNRNRWROL]LQOHUL
(alanin-muramyl-amidaseDNWLYHROXUYHEDNWHULKFUHVLSDUoDODQÕU%XWHVWVDIUD\DWROHUDQVWHVWL
GH÷LOGLUYHEXWHVWLoLQVDIUDGDHULPHLIDGHVLPDNXOELUWHULPGH÷LOGLU
Prensip: Bakteri kOWU]HULQHVDIUDHNOHQLUEX\\RQXQRSWLNJHoLUJHQOL÷LQHEDNÕODUDNOL]LV
DUDQÕU 2WROL]LVH GLUHQHQ SQ|PRNRNODUÕQ \DQOÕú QHJDWLI VRQXoODUÕQÕ HQJHOOHPHN LoLQ YH EDNWHULQLQ
otolizinlerini aktive etmek için buyyona sodium deoxylate eklenebilir.
øúOHP PnömoNRN ROGX÷XQGDQ úSKHOHQLOHQ VWUHSWRNRN |UQH÷L LNL D\UÕ EX\\RQGD EROFD
UHWLOLS S+ VÕ - \D D\DUODQÕU OXN sodium deoxylate solüsyonu eklenir. Bir tanesinin
üzerine %9- OLN |N] VDIUDVÕ YH\D RQXQ \HULQH RQD HúGH÷HU RODUDN VDIUD DVLWL RODQ sodium
taurocholate HNOHQLU GDNLND HWYGH EÕUDNÕOÕU +HU LNL WS RSWLN RNX\XFXGD YH\D J|] LOH
EDNÕODUDN PXND\HVH HGLOLU NRQWURO WSQH NÕ\DVOD NOWU VÕYÕVÕQÕQ UHQJLQLQ DoÕOÕS DoÕOPDGÕ÷Õ
\RNODQÕU
'H÷HUOHQGLUPH Safra ilave edilen buyyonda, kontrol tüpünH NÕ\DVOD UHQN DoÕOPDVÕ SR]LWLI
test sonucudur. 1HJDWLINRQWURORODUDNHQWHURNRNNXOODQÕODELOLU
6LWUDWNXOODQÕPÕ :
7DQÕP Sodium citrate bir sitrik asit tuzudur ve trikarboksilik asit siklusu (Tricarboxylic Acid
Cycle, TCA, Krebs' cycle) içerisinde bir mHWDEROLWRODUDN\HUDOÕU%D]ÕEDNWHULOHUNDUERQKLGUDWODUÕQ
GÕúÕQGDEXPHWDEROLWLGHELUHQHUMLYHNDUERQND\QD÷ÕRODUDNNXOODQDELOLUOHU
Prensip:%XWHVW\DSÕOÕUNHQEDNWHUL|UQH÷LSURWHLQYHNDUERQKLGUDWODUGDQ\RNVXQELURUWDPD
HNLOHUHN VLWUDWÕ NXOODQPD\D ]RUODQÕU (÷HU EDNWHUL VLWUDWÕ NXOODQDELOLUVH ED]LN UQOHU DoÕ÷D oÕNDUÕU
dQN EHVL\HUL LoHULVLQGH VLWUDWD ED÷OÕ RODUDN EXOXQDQ NDW\RQODU VLWUDW D\UÕOÕQFD DPRQ\D÷D
amonyuma (NH4+) ve amonyum hidroksit’e (NH42+ G|QúU 2OXúDQ DONDOL RUWDPGD EXOXQDn
EURPWLPROPDYLVLS+LQGLNDW|URODUDNED]ROXúXPXQXJ|VWHULU
øúOHP Denecek bakterinin saf kültüründen bir koloni Simmon’s Sitrat agar'a (veya Koser’s
VLWUDWDJDUÕQDHNLOLURSWLPDOVUHGHQELUD]GDKDX]XQELUVUHLQNEHHGLOLUhUHPHYHPDYLUHQN
arDQÕU
Simmons Citrate agar:
Ammonium dihydrogen phosphate
1g
Dipotassium phosphate
1g
Sodium chloride
5g
Sodium citrate
2g
107
Magnesium sulfate
Agar
Bromthymol blue
Distile su
0.20 g
15 g
0.08 g
1 lt
'H÷HUOHQGLUPe: VDDWOÕN LQNEDV\RQX WDNLEHQ NRORQL JHOLúPHVL SR]LWLI WHVW VRQXFX LoLQ
\HWHUOLGLU dQN EX EHVL\HULQGH EDNWHULQLQ NXOODQDELOHFH÷L VLWUDWWDQ EDúND ELU NDUERQ ND\QD÷Õ
\RNWXU .RORQL JHOLúL\RUVD VLWUDWÕ NXOODQDELOL\RU GHPHNWLU .RORQLOHU HWUDIÕQGD NR\X mavi renk
ROXúXPXSR]LWLIVRQXFXGHVWHNOHU.RORQLJHOLúPLúDQFDNPDYLUHQNDOPDPÕúEHVL\HUOHULQLQGDKD
X]XQVUHLQNEDV\RQX\DSÕODUDNUHQNROXúXPXDUDQPDVÕQDJHUHN\RNWXU$QFDNEHNOHQLUVHPDYL
UHQNROXúDFDNWÕU
Pozitif kontrol için Enterobacter aerogenes, negatif kontrol için Esherichia coliNXOODQÕODELOLU
Üreaz testi:
7DQÕP Üre (NH2-CO-NH2), karbonik asitin bir diamide türevidir. Bütün diamitler kolayca
amonyak ve CO2
HKLGUROL]HROXUODUGDKDVRQUDDPRQ\XPNDUERQDWDG|QúUOHU%D]ÕEDNWHULOHU
üreaz (ureaseHQ]LPOHULLOHúXUHDNVL\RQXNDWDOL]OHUOHU(NH2-CO-NH2) + 2 H2O + urease F!&22
+ H2O + 2NH3 <F!1+4)2CO3
Prensip: %DNWHUL LoHULVLQGH UH EXOXQDQ EHVL\HULQGH UHWLOHUHNUH¶QLQED]LN\ÕNÕPUQOHUL
pH indikatörü ile izlenir.
øúOHP øQFHOHQHcek bakterinin saf kültüründen bir öze dolusu Stuart's üre buyyona veya
Christensen
VUHDJDU
DHNLP\DSÕODUDNWUQHX\JXQVUHLQNEHHGLOLUS+LQGLNDW|UQGHNLUHQN
GH÷LúLPLJ|]OHQLU
Stuart's Urea Broth
Yeast extract
0.1 g
Monopotassium phosphate
9.1 g
Disodium phosphate
9.5 g
Urea
20 g
Phenol red
0.01 g
S+ D\DUODQÕU
Christensen's Urea Agar
Pepton
1g
Glucose
1g
Sodium chloride
5g
Monopotassium phophate
2g
Urea
20 g
Phenol red
0.012 g
Agar
15 g
S+ D\DUODQÕU
'H÷HUOHQGLUPH %D]Õ PLNURRUJDQL]PDODU (Proteus ve Actinobacillus gibi) 1-2 saat içinde
üreyi hidrolize edebilir, daha az aktif türler ise 2 veya 3 gün gereksinebilir. Her iki besiyeri de alkali
S+
GD NÕUPÕ]Õ UHQN DOÕU %X UHQN GH÷LúLPL SR]LWLI UHDNVL\RQ DQODPÕQD JHOLU %HVL\HUL VDUÕ UHQJLQL
NRUXPXúLVHQHJDWLIUHDNVL\RQRODUDN\RUXPODQÕU
Pozitif kontrol için Proteus'lar, negatif kontrol için Escherichia coli]D\ÕISR]LWLINRQWUROLoLQ
ise KlebsiellaWUOHULNXOODQÕODELOir.
'L÷HUEL\RNLP\DVDOWHVWOHU :
Adanitol (ribitol): 5LER]
XQ LQGLUJHQPH LOH \ÕNÕOPÕú UQGU %D]Õ EDNWHULOHU EXQX SROLKLGULN ELU
DONROHG|QúWUHELOLU
Dulsitol (galactitol) ve sorbitol (glucitol): *OXNR]XQ DOGHKLW JXUXEXQXQ LQGLUJHQPHVL VÕUDVÕQGD
ortayD oÕNDQ SROLKLGULN DONROOHUGLU & JUXEX VWUHSWRNRNODU, E. coli, K. pneumonia ve birçok
Salmonella için identiktir.
Mannitol'e etki: 0DQQR]YH\DIUXNWR]XQ\ÕNÕPUQOHULQGHQROXSSROLKLGULNELUDONROGXUE. coliED]Õ
Salmonella, Mycobacterium ve Bacillus WUOHULWDUDIÕQGDQPHWDEROL]HHGLOLU
108
Eritritol: Eritroz (erythrose LVLPOL úHNHULQ SDUoDODQPDVÕ LOH RUWD\D oÕNDQ ELU DONROGXU .LP\DVDO
\DSÕVÕ&+2OH(CHOH)2CH22+GÕU%D]ÕEDNWHULOHUHULWULWROXPHWDEROL]HHGHELOLUOHU
øQRVLWRO
HHWNL øQRVLWROhexahydroxycyclohexane genel ismi ile bilinir. Bakterilerin fosfolipitlerinde
\HUDOÕU 'RNX] IDUNOÕ VWHUHRNLP\DVDO IRUPX EXOXQXU %DúOÕFDODUÕ úXQODUGÕU : Myo-inositol, scyllitol
DPLQRJOLNR]LW DQWLEL\RWLNOHULQ oHNLUGH÷LQL ROXúWXUXU d-inositol, ve l-inositol vs. BD]Õ EDNWHULOHULQ
beslenme gereksinimidir. Bilhassa K. pneumoniaD\ÕUÕPÕQGDID\GDOÕGÕU
Salisin'e etki: O-Hydroxymethil-Phenyl-ß-D-glucopyranoside %X PDGGHQLQ EDNWHUL WDUDIÕQGDQ
PHWDEROL]HHGLOLSHGLOPHGL÷LELOKDVVDS.pyogenes, K.pneumonia ve S.marcences için identiktir.
Gliserol fermentasyonu: Gliserol bir alkoldür. Alkolik fermentasyonun bir minör ürünüdür. Fakat
alkali ortamda veya ortamda bisülfit mevcudiyetinde çok miktarda dihydroxyacetone phosphate
DoÕ÷D oÕNDU YH 1$'+2 WDUDIÕQGDQ glycerol-3-phosphate¶D G|QúWUOU 'DKD VRQUD EX PDGGH
GHIRVIRULOHHGLOHUHNHQHUMLDoÕ÷DoÕNDUÕOÕUYHJOLVHUROPH\GDQDJHOLU%X\ROXNXOODQDUDNHQHUMLWHPLQ
ED]ÕEDNWHULOHULoLQEXWHVWLGHQWLNWLUBacillus gibi).
1LúDVWD KLGUROL]L Bakterinin türüne uygun, proteinden zengin ama karbonhidrattan fakir bir
EHVL\HULQHQLúDVWDLODYHHGLOLUøQRNODV\RQXWDNLEHQX\JXQVUHLQNEHHGLOLUNolonilerinin üzerine
Gram's iodine solusyonundan 1-GDPODEÕUDNÕOÕU5HQNVL]NDOPDVÕQLúDVWDQÕQKLGUROL]HROGX÷XQX
gösterir.
Kazein hidrolizi: Bakterinin türüne uygun, karbonhidrattan zengin ama proteinden fakir bir
besiyerine inek sütü ilave edilir. Uygun süre inkübe edilir, kD]HLQKLGUROL]HROPXúVDNRORQLoHYUHVL
úHIIDIWÕU
$1$(523/$5,1ø'(17ø)ø.$6<218
Yukardaki testler anaerobik atmosIHU DOWÕQGD DQDHURSODU LoLQ GH \DSÕODELOLU (÷HU WHVWLQ
DQDHURELNEDNWHULOHULoLQ\DSÕOPDVÕELU|]HOOLNDU]HGL\RUVDEXGXUXPWHVWDQODWÕOÕUNHQDoÕNODQPÕúWÕU
.OLQLN |QHPL RODQ ED]Õ DQDHURELN EDNWHULOHULQ NRORQL PRUIRORMLVL SLJPHQW YH IORUHVDQV |]HOOLNOHUi
Tablo 11-µGHYHULOPLúWLU
Tablo 11-1 .OLQLN|QHPLRODQED]ÕDQDHURSEDNWHULOHULQNOWU|]HOOLNOHUL
.OWU|]HOOL÷L
0XKWHPHOEDNWHULoHúLGL
Bacteroides ureolyticus grubu, Ekinella
$JDUÕGHOHQNRORQL
Bacteroides levii, B. macacae, Porphyromonas
Siyah pigmentli koloni
asaccharolytica, P.gingivalis ve P. endodontalis, Prevotella
corporis, P. loeschii, P. denticola, P. melaninogenica, P.
nigrescens veya herhangi bir anaerop Gram negatif çomak.
Prevotella intermedia, Prevotella loeschii
.ÕUPÕ]ÕIORUHVDQV
Fusobacterium türleri
<HúLOLPVLIORUHVDQV
Fusobacterium türleri
%HVL\HULQLQ\HúLOOHQPHVL
Clostridium perfringens
Çift zonlu ß hemoliz
Sahanda yumurta görünümü Fusobacterium necrophorum, Fusobacterium varium
Clostridium türleri
øULYHoHQWLNOLNRORQL
Clostridium septicum
0HG]EDúÕNRORQL
Actinomyces türleri
$]ÕGLúLJ|UQWV
Fusobacterium nucleatum
Benekli koloni
Clostridium septicum, Clostridium sordelli
%X\\RQGDNPHOHúPH
gQ7DQÕ3ODNODUÕ3UHVXPSWRSODWHV3 lü kuadrant plaklar):
109
ùHNLO-1 gQWDQÕSODNODUÕLOHDQDHrop identifikasyon yöntemi. Herbirisi
G|UGHE|OQPú$'YHLoHULVLQGHGH÷LúLNNDWNÕPDGGHOHULEXOXQDQ/'DJDU
(Lomber-Dowel agar) EHVL\HULYHWDQH&'&NDQOÕDJDUDHNLP\DSÕOÕU3ODN1A-'VÕUDVÕLOH/'HVNXOLQDJDU/'\XPXUWDVDUÕVÕDJDU/'VDIUDDgar, sade LD
agar içerir. Plak-2A-'VÕUDVÕLOH/'QLúDVWDDJDU%-LD süt agar, LD DNA agar,
LD glikoz agar içerir. Plak-3A-'VÕUDVÕLOHLD laktoz agar, LD ramnoz agar, LD
jelatin agar, LD mannitol agar içerir.
DJDUGDNXOODQÕOÕU%N]ùHNLO-1.
7DQÕP Lombard ve
'RZHOO WDUDIÕQGDQ JHOLúWLULOPLú
YH &'& WDUDIÕQGDQ NDEXO
J|US VWDQGDUGL]H HGLOPLú ELU
anaerop
identifikasyon
SURVHGUGU %X LúOHP LoLQ
her birisL G|UGH E|OQPú o
tane standart petri kutusu
NXOODQÕOÕUBu plaklar 1 den 3 e
NDGDUQXPDUDODQGÕUÕOÕU+HUELU
SHWUL NXWXVX VD÷ VWWHQ
EDúOD\ÕS VDDW LVWLNDPHWLQH
GR÷UX G|QHFHN úHNLOGH $
harfinden D harfine kadar
isimlendirilir
(1A,1B,1C,1D;
2A,2B,2C,2D; 3A,3B,3C,3D)
ve her birisine LD agar
(Lomber-'RZHODJDUYHIDUNOÕ
NDWNÕ
PDGGHOHUL
LoHUHQ
besiyerleri konulur. Böylece
ELUELULQGHQIDUNOÕ|]HOOLNWHRODQ
WDQHSODNVHULVLROXúWXUXOXU
%XoSHWULNXWXVXQGDQEDúND
DGHW &'& DQDHURS NDQOÕ
Prensip: 7DQÕPODQDQ EX \|QWHPGH DQDHURS LGHQWLILNDV\RQD SUDWLN ELU \DNODúÕP HVDV
DOÕQPÕúWÕU 7HN ELU EDNWHUL |UQH÷LQH WDQH EL\RNLP\DVDO YH IL]\RORMLN WHVWL WHN ELU LQNEDV\RQGD
yapmak mümkündür. Besiyeri modifiye edilebilir. LD agar\HULQH|UQH÷LQ3<DJDUNXOODQÕODELOLUYH\D
EHVL\HUL VD\ÕVÕ DPDFD |]HO úHNLOGH DUWÕUÕODELOLU %|\OHFH DQDHURS LGHQWLILNDV\RQD ELU HVQHNOLN YH
oDEXNOXNJHWLULOPLúWLU
øúOHP øQFHOHQHFHN EDNWHULQLQ VÕYÕ EHVL\HULQGHNL VDDWOÕN VDI NOWU 0F)DUODQG 1R
bXODQÕNOÕNVHYL\HVLQHD\DUODQÕUYHKHUSODNWDNLoH\UH÷HYHWDQHNDQOÕDJDUDHúLWPLNWDUGDHNLP
\DSÕOÕU%LULQFLSOD÷ÕQ'oH\UH÷LQHHNLP\DSÕOGÕNWDQVRQUD(LD agar bulunan sol-üst çeyrek) agar yüzeyine
VWHULOELUNXUXWPDND÷ÕGÕ\HUOHúWLULOLUEXND÷ÕWGDKDVRQUDLQGROWHVWLQGHNXOODQÕODFDNWÕU
&'& NDQOÕ DJDU ]HULQH ,8 SHQLVLOLQ JPO NRQVDQWUDV\RQGD NDQDPLVLQ JPO
NRQVDQWUDV\RQGD ULIDPSLQ HPGLULOPLú GLVNOHU \HUOHúWLULOLU %XQODUÕQ WLFDUL RODQODUÕ GD YDUGÕU %HFWRQ
Dickinson, MD).
øNLQFL&'&DQDHURSNDQOÕDJDU]HULQH636YHQLWUDWHPGLULOPLúGLVNOHU\HUOHúWLULOLU636GLVN
KD]ÕUODPDNLoLQOLN636VROXV\RQXQGDQODOÕQÕUVWDQGDUWDQWLEL\RWLNGX\DUOÕNGLVNLQHóLQFK
HPGLULOLU NXUXWXOXU 7LFDUHWWH *URED[ GLVNOHUL DGÕ LOH ELOLQLU 5RFhe diagnostic, Nutley, NJ). Bunlar
RGDÕVÕVÕQGDD\EX]GRODEÕYHNDUDQOÕNWDGDKDX]XQVUHVWDELOGLU1LWUDWGLVNLKD]ÕUODPDNLoLQJ
KNO3 ve 0.1g Na2MoO4.2H2O (sodium molibdate) PO GLVWLOH VX LoHULVLQGH o|]OU 3RU oDSÕ
PRODQILOWUHNXOODQÕODUDNVWHULOOHQLUNXUXWPDND÷ÕGÕGLVNLQHORODUDNHPGLULOLUYHNXUXWXOXU
øQRNOHHGLOHQSODNODUEDNWHULQLQWUQHX\JXQVUHLQNEHHGLOLU
'H÷HUOHQGLUPH WDQH &'& NDQOÕ DJDUGD KHPROL] SLJPHQW IORUHVDQV *UDP UHDNVL\RQ
hareket ve penisilin, ULIDPSLQNDQDPLVLQGLUHQFLEDNÕOÕU6363HQLVLOLQYHNDQDPLVLQGLVNLLoLQ
PP ULIDPSLQ GLVNL LoLQ PP GHQ GDU ]RQODU GLUHQoOL GDKD JHQLú ]RQODU LVH GX\DUOÕ RODUDN QRW
HGLOLU 1LWUDW GLVNL ]HULQH QLWUDW UHGNVL\RQ WHVWLQGH DQODWÕODQ D\ÕUDoODU GDPODWÕOÕU YH RUDGD
DQODWÕOGÕ÷ÕúHNLOGHGH÷HUOHQGLULOLU$QWLEL\RWLNGX\DUOÕOÕNGLVNOHULQLQ\RUXPX7DEOR-¶GHYHULOPLúWLU
Plak-¶LQ $ oH\UH÷L úX WHVWOHUH KL]PHW HGHU HVNXOLQ KLGUROL]L +26 ROXúXPX YH NDWDOD] 3ODN-1B
oH\UH÷LQGHOLSD]YHOHVLWLQD]DNWLYLWHVLEDNÕOÕU3ODN-&YH'oH\UHNOHULQGHVÕUDVÕLOHVDIUD\DWROHUDQV
110
YH LQGRO EDNÕOÕU 3ODN-¶GH VÕUD LOH QLúDVWDQÕQ KLGUROL]L ND]HLQ KLGUROL]L '1$] DNWLYLWHVL YH JOXNR]
IHUPHQWDV\RQXEDNÕOÕU3ODN-¶WH$%YH'oH\UHNOHULQGHVÕUDVÕLOHODNWR]UDPQR]IHUPentasyonu ve
PDQQLWRO¶XNXOODQPDDUDúWÕUÕOÕU&oH\UH÷LQGHMHODWLQKLGUROL]LLQFHOHQLU%XWHVWOHULQWHNHUWHNHUQDVÕO
\RUXPODQDFD÷Õ EX NRQXQXQ LoHULVLQGH LOJLOL EDúOÕN DOWÕQGD DQODWÕOPÕúWÕU Bakteri identifikasyon
WDEORODUÕ(N-WHYHULOPLúWLU
Tablo 11-2 gQ WDQÕ SODNODUÕQGD NXOODQÕODQ DQWLEL\RWLN GLVNOHULQLQ \RUXPODQPDVÕ {R, dirençli; S,
GX\DUOÕ±GH÷LúNHQVRQXoODUÕLIDGHHGHU`
Denenen antibiyotik
Üretilen anaerop mikroorganizma
Colistin
Kanamisin Vancomycin
(10g)
(1 mg)
(5g)
Bütün Gram pozitifler
R
R
S
Gram negatif koklar
S
S
R
Bacteroides fragilis grubu
R
R
R
Bacteroides ureolyticus grubu
S
S
R
'L÷HUBacteroides’ler
±
R
R
Fusobacterium’lar
S
S
R
Prevotella
±
R
R
Anaerop identifikasyon kitleri:
øGHQWLILNDV\RQGD NXOODQPDN ]HUH ODERUDWXYDUGD KD]ÕUODQDQ EWQ NLP\DVDO PDGGHOHU
EHVL\HUOHULYHD\ÕUDoODUNoNDPSXOOHUHYHúLúHOHUHGROGXUXOXSNXWXLoHULVLQGHNXOODQÕPDKD]ÕUELU
úHNLOGH VDWÕOÕU Bunlara identifikasyon kitleri dHQLU 7LFDUL LGHQWLILNDV\RQ NLWOHULQLQ VD\ÕVÕ Q
]HULQGHGLU DQFDN ]HULQGH HQ oRN oDOÕúPD \DSÕODQ $3,-20A (Analytab,NY) ve Minitek (Becton
Dickinson, MD)’tir. Her ikisi de asakkarolitik anaeroplar ve Gram negatif koklar için yetersiz
görünmektedirler<DSÕODQNDUúÕODúWÕUPDOÕoDOÕúPDODU3UHVXPSWR3ODWHVgQWDQÕ3ODNODUÕ\|QWHPLQLQ
KHU WUO LGHQWLILNDV\RQ NLWLQGHQ GDKD GH÷HUOL ROGX÷XQX J|VWHUPLúWLU %LU oDOÕúPDGD $3,-ZYM
(Analtab, NY), API An-IDENT (Analytab,NY), IDS RapID-ANA II (Innovate Diagnostic, GA), Vitek
Anaerobe Identification ANI (Vitek Systems, MO), MicroScan Rapid Anaerobe Identification
System (American MicroScan CA) ve ATB 32A Anaerobes ID (bioMerieux, France) isimli kitler
NDUúÕODúWÕUÕOPÕú VRQXoWD EX NLWOHULQ LGHQWLILNDV\RQ GR÷UXOXN GH÷erinin ortalama %60- DUDVÕQGD
ROGX÷X EXOXQPXúWXU <D]DUODU EX YH EHQ]HU NLWOHULQ NXOODQÕPGDQ NDOGÕUÕOPDVÕ WHPPHQLVLQGH
EXOXQPXúODUGÕU
5DS,'$1$,,NLWLNXOODQDUDNGDKD|QFHGHQQHROGX÷XELOLQHQDQDHUREXQ
VLQLGR÷UX
olarak identifiye edilebilmiúWLU%LUEDúNDoDOÕúPDGDLVHDQDHURSEDNWHULNXOODQÕOPÕúYHD\QLNLW
LOHDQFDNEDNWHUL|UQH÷LGR÷UXRODUDNLGHQWLIL\HHGLOHELOPLúWLU
%D]ÕDQDHURSLGHQWLILNDV\RQNLWOHUL\R÷XQLQRNXOXP0DF)DUODQG1RNXOODQPDNVXUHWLLOH
VDDWOÕN LQNEDV\RQ dönemi sonunda bakterinin enzimatik (glycosidase, aminopeptidase)
aktivitesine dayanarak identifikasyon yapabilmektedir. 24-VDDWOÕNLQNEDV\RQG|QHPLRODQNLWOHU
LOHEXVDDWOLNNLWOHUDUDVÕQGDNDUúÕODúWÕUPD\DSDQELUoDOÕúPDGDDQDHURSEDNWHULNXOODQÕOPÕúWÕU
Bu bakterilerden 268 tane Bacteroides ve Fusobacterium’un %74'ü, 97 tane anaerobik kokun
L WDQH VSRUVX] *UDP SR]LWLI DQDHURELN oRPD÷ÕQ L WDQH Clostridium‘un %64'ü
GR÷UXLGHQWLIL\HHGLOHELOPLúWLU6RQXoWDDQODúÕOPÕúWÕUNLKHULNLúHNLOGHHOGHHGLOHQVRQXoODUELUELULQH
DQODPOÕúHNLOGH\DNÕQoÕNPDNWDGÕUDPDGR÷UXOXNODUÕWDUWÕúPDOÕGÕU<D]DUODUÕQRUWDNGúQFHVLLODYH
WHVWOHU JHUHNWL÷L YH KÕ]OÕ VRQXo JHUHNVLQLPL GÕúÕQGD KLoELU NLWLQ DQDHURELN LGHQWLILNDV\RQ DPDFÕ LOH
NXOODQÕOPDPDVÕ JHUHNWL÷LGLU $\UÕFD NLWOHUH LQRNOH HGLOHFHN EDNWHUL |UQH÷LQLQ UHWLFL ILUPDODUÕQ
EHOLUOHGL÷L EHVL\HUOHULQGH KD]ÕUODQPDVÕ JHUHNPHNWHGLU gUQH÷LQ LQFHOHQHFHN EDNWHUL |UQH÷L
Schaedler Blood Agar veya Cycloserine-&HIL[LWLQ)UXFWRVH$JDULoHULVLQGHUHWLOPLúVHNLWOHUKDWDOÕ
VRQXoODU YHUPHNWHGLU 'R÷UX LGHQWLILNDV\RQ DPDFÕ\OD UHWLFL ILUPDODU NLWOHULQ LoHULVLQH JHUH÷LQGHQ
ID]ODoHúLWOLOLNWHEHVL\HUOHULLODYHHWPHNWHGLUEXQODULGHQWLILNDV\RQNLWLQLQPDOL\HWLQLDUWÕUPDNWDGÕUYH
UHWLFL ILUPD\D ED÷ÕPOÕ KDOH JHWLUPHNWHGLU %LU EDúND SUREOHP ]DPDQOD ER]XQDELOHFH÷L KDNOÕ
111
gerekçesi ile vit K1, L-F\VWLQH YH \HDVW H[WUDFW NLWOHUH HNOHQHPHPLúWLU 2\VDNL EXQODU ED]Õ
Bacteroides ve PorphyromonasWUOHULQLQED÷ÕPOÕROGX÷XPDGGHOHUGLU
Gaz likit kromotografisi (Gas-Liquid-Chromotography, GLC):
7DQÕP$QDHUREL]PNRQXVXQGDDQODWÕOGÕ÷Ձ]HUHDQDHURSEDNWHULOHULQEmbden-Meyerhof3DUQDV \ROX LOH HOGH HWWLNOHUL KHNVR] \ÕNÕP UQOHUL kendi türlerine özgüldür ve bu kimyasal
maddeleri üredikleri ortama salarlar. GLC ile bakteri iGHQWLILNDV\RQX EDNWHUL\L GH÷LO EDNWHULQLQ
UHGL÷L RUWDPGDNL KHNVR] \ÕNÕP UQOHULQL WHVSLW HWPH HVDVÕQD GD\DQÕU %X UQOHU YRODWLO YH\D
QRQYRODWLO\D÷DVLWOHULGLU*/&LOHLQFHOHQHQEDNWHULQLQJHQXVXWDPRODUDNV|\OHQHELOLUDPDVSHVLILN
epitetini kesin RODUDN V|\OHPHN ]RUGXU gUQH÷LQ ELU EDNWHULQLQ UHGL÷L RUWDPD EWLULN DVLW YHUGL÷L
tespit edilirse, bunun hangi ClostridiumROGX÷XQXV|\OHPHN]RUGXUdQNClostridium’lar içerisinde
WDQHVLUHGL÷LRUWDPDEWLULNDVLWVDODUPropinobacterium genusundaki bütün bakteriler ortama
SURSL\RQLN DVLW VDODU */& EXOJXODUÕ LOH GL÷HU VWDQGDUW EL\RNLP\DVDO YH IL]\RORMLN WHVW VRQXoODUÕ
ELUOHúWLULOGL÷LQGHGDKDGR÷UXELULGHQWLILNDV\RQ\DSÕODELOLU.DEXOJ|UHQWDQH*/&VWDQGDUGÕYDUGÕU
Thermal Conductivity DetectorV7&'+HJD]ÕEDVÕQFÕOELQ2, NRORQÕVÕVՃ&HQMHNW|UÕVÕVÕ
ƒ&GHGHNW|UÕVÕVՃ&YH+\GURJHQ)ODPH,RQL]DWLRQ'HWHFWRUV),'+HJD]ÕEDVÕQFÕ
lb/in2NRORQÕVÕVՃ&HQMHNW|UÕVÕVՃ&GHGHNW|UÕVÕVՃ&+HULNLVLQGHGHWDúÕ\ÕFÕJD]
helyum’dur ve okumalar 1 saniyede 1 mV seviyesindedir. FID, nonvolatil asitlerin tayini için
önerilmez.
A
B
112
C
D
ùHNLO-2 Gaz likit kromotografi yönteminde NDOLEUDV\RQVROV\RQXQXQYHUGL÷LSLNOHU$
muhtelif bakteri örneklerinin 3<*EX\\RQGDƒ&GHYHVDDWOÕNNOWUQQ7KHUPDO
&RQGXFWLYLW\'HWHFWRUFLKD]ÕLOHYHUGL÷LSLNOHU%&'%DNWHULOHUWDUDIÕQGDQKDQJL\D÷DVLWOHULQLQ
EHVL\HULQHVDOÕQGÕ÷ÕWHVSLWHGLOLUYHLGHQWLILNDV\RQ\DSÕOÕU
Pa, propionic acid; Aa, acetic acidø%$ isobutyric acid; Ba, butyric acidø9Disovaleric acid; Va, valeric acidø&D
isocaproic acid; Ca, caproic acid.
Prensip:%DNWHULQLQUHWLOGL÷LVÕYÕEHVL\HULÕVÕWÕOPÕúKHO\XPJD]ÕLoHULVLQGHQJHoLULOLUEXVÕUDGD
ROXúDQ VÕFDNOÕN GH÷LúLPOHUL FLKD] WDUDIÕQGDQ HOHNWULNVHO VLQ\DOOHUH G|QúWUOU DPSOLIL\H HGLOHUHN
ND\ÕWODQÕU %HVL\HULQGHNL KHU \D÷ DVLWL EHOLUOL VUHOHUGH \DQDU YH YROWDM VÕoUDODPDODUÕ SLN \DSDU
.DoÕQFÕGDNLNDEXHOHNWULNVLQ\DOGH÷LúLPLPH\GDQDJHOGL\VHRVÕUDGD\DQPDNWDRODQ\D÷ asitinin
QHROGX÷XQXNDQWLWDWLIRODUDNLIDGHHGHU%XQXQLoLQ|QFHGHQLoHULVLQGHELOLQHQPLNWDUGDELOLQHQ\D÷
DVLWOHUL EXOXQDQ ELU VÕYÕ FLKD]GD RNXQXS ND\ÕW HGLOHUHN FLKD] NDOLEUH HGLOPHOLGLU Volatil asit
kalibrasyon solüsyonu için bir tüpe acetic, formic, propionic, isobutyric, butyric, isovaleric, valeric,
isocaproic, caproicDVLWOHUYHGLVWLOHVXVÕUDVÕ\OD
PO NRQXU +D]ÕU VDWÕODQÕ GD YDUGÕU (Supelco, Inc.). Nonvolatil asit kalibrasyon
solüsyonu için bir tüpe pyruvic, lactic (%85), succinic, phenylacetic DVLWOHU YH GLVWLOH VX VÕUDVÕ\OD
PO NRQXU YH FLKD]D RNXWWXUXOXU ùHNLO-2A’daki gibi voltaj pikleri
HOGHHGLOLU+HUELUSLNKDQJL\D÷DVLWLQLQQHPLNWDUGDEXOXQGX÷XQXDQODWÕU
øúOHP %DNWHULQLQ NOWUQQ \DSÕOGÕ÷Õ RUWDPGD YRODWLO \D÷ DVLWOHUL acetic, propionic,
isobutyric, butyric, isovaleric, valeric, isocaproic ve caproicDVLWOHULQYDUOÕ÷ÕQÕWHVSLWHWPHNLoLQ :
øQFHOHQHFHNEDNWHULPO3<*EX\\RQDHNLOLUDQDHURELNúDUWODUGDX\JXQVUHLQNEHHGLOLUPO
3<*EX\\RQ[PPOLNWHPL]ELUYLGDNDSDNOÕWSHDOÕQÕUS+ YH\DDOWÕQGDROXQFD\DNDGDU
%50 (V/V) H2SO4LODYHHGLOLUEXLúOHPHWHUGHo|]QUO÷DUWÕUDFDNWÕUPOethyl ether ilave edilir,
tEQNDSD÷ÕVÕNÕOÕUYHNXYYHWOHoDONDODQÕUEXVÕUDGDXoXFX\D÷DVLWOHULHWHULoHULVLQGHo|]QHFHNWLU
GDKDD]SDUOD\ÕFÕROGX÷XQGDQHWHU\HULQHMetyl-tert-butylNXOODQÕODELOLU-2000 rpm 15 dakika
santrifüjlenerek eter-kültür emülsiyonu çöktürülür, tüp, -ƒ& GH EHNOHWLOLU EX LúOHP VÕUDVÕQGD
EHVL\HULGRQDUDPDHWHUVÕYÕKDOGHNDODFDNWÕU7SQNDSD÷ÕDoÕODUDNHWHUELUEDúNDWSHDOÕQÕUYH
istenirse üzerine birkaç parça CaCl2 SDUoDVÕ DWÕODUDN YDUVD NDOÕQWÕ VX ED÷ODQÕU */& SDNHWL 6S1220) içerisine 14 l enjekte edilir.
113
Acetic acid
Propionic acid
Isobutyric acid
Butyric acid
Isovaleric acid
Valeric acid
Isocaproic acid
Caproic acid
Lactic acid
Succinic acid
Phenyl acetic acid
1RQYRODWLO \D÷ DVLWOHUL Pyruvic, lactic, fumaric, succinic, hydrocinnamic ve phenylacetic
DVLWOHULQWHVSLWLLoLQPO3<*EX\\RQGDNLVDINOWUGHQWHPL]ELUYLGDNDSDNOÕWSHDOÕQÕUPO
H2SO4 ve 2 ml metanol eklenir, 55 °C lik su banyosunda en az 1 saat ve hatta mümkünse bir gece
EHNOHWLOLU PO GLVWLOH VX YH PO NORURIRUP HNOHQLUEXVDIKDGD\D÷DVLWOHULNORURIRUPLoHULVLQGH
o|]QHFHNWLUVDQWULIMOHQHUHNNORURIRUPHPOVL\RQXQXQWSQGLELQHo|NPHVLVD÷ODQÕU(QMHNW|ULOH
tüpQGLELQGHNLNORURIRUPDOÕQÕUHQMHNW|UL÷QHVLQLQGÕú\]H\LGLVWLOHVXLOH\ÕNDQÕUl emülsiyon
GLC paketine (SP-1000) enjekte edilir.
Tablo 11-3 .OLQLN|QHPLRODQED]ÕDQDHURSODUÕQYHgaz likit kromotografisi ile
LGHQWLILNDV\RQXQXVD÷OD\DQ\D÷DVLWLSURILOLPeptone-Yeast-Extract-Glucose buyyonda, 35
ƒ&GHYHVDDWOÕNNOWUNXOODQÕOPÕú|OoPOHU7KHUPDO&RQGXFWLYLW\'HWHFWRUFLKD]ÕLOH
\DSÕOPÕúWÕU
Bacteroides capillosus
+
±
-
-
-
-
-
-
-
+
Bacteroides distasonis
Bacteroides fragilis
Bacteroides ovatus
Bacteroides splanchnicus
Bacteroides thetaiomicron
Bacteroides uniformis
Bacteroides ureolyticus
Bacteroides vulgatus
Porphyromonas asaccharolytica
Porphyromonas gingivalis
Prevotella bivia
Prevotella disiens
Prevotella intermedia
Fusobacterium gonidiaformans
Fusobacterium mortiferum
Fusobacterium naviforme
Fusobacterium nechroforum
Fusobacterium nucleatum
Fusobacterium varium
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
±
±
+
+
±
±
+
+
+
±
±
+
±
±
+
+
+
±
±
+
±
±
±
+
+
±
±
±
-
+
+
+
+
+
+
+
+
+
±
±
±
±
±
±
+
+
±
±
+
-
±
±
-
-
-
±
+
±
±
±
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
±
±
+
+
+
-
'H÷HUOHQGLUPH Elde edilen pikler kalibrasyon pikleri ile mukayese edilerek incelenen
anaerop bakterinin ortama salGÕ÷ÕKHNVR]\ÕNÕPUQOHULQLQQHROGX÷XQRWHGLOLUYH7DEOR-3 ile
NDUúÕODúWÕUÕOÕU'L÷HUWHVWVRQXoODUÕGDX\XPOX\VDLGHQWLILNDV\RQWDPDPODQÕU
.RQWURO DPDFÕ\OD HNLOPHPLú ELU 3<* EX\\RQD D\QL LúOHPOHU \DSÕODUDN oDOÕúPD\D GDKLO
edilmelidir.
PYG buyyon yerine Chopped-Meat-*OXFRVHEX\\RQNXOODQÕOÕUVDVUSUL]DVHWLNYHODNWLNDVLW
SLNOHUL YHUHELOLU (NLOL ROPDVD ELOH EHNOHWLOPLú EHVL\HUOHUL |QFHGHQ NHVWLULOHPH\HQ SLNOHU YHULU
(NLOPHPLú EX\\RQ SLNOHUL YDUVD HNLOL EX\\RQGDQ HOGH HGLOHQ SLNOHUGHQ EX SLNOHrin büyüklüklerini
114
oÕNDUPDNJHUHNHELOLUgUQH÷LQHNLOPLúEX\\RQGDEXWLULNDVLWSLNLP9HNLOPHPLúEX\\RQGDP9
LVHJHUoHNSLNDVOÕQGDP9¶WXU
KAYNAKLAR:
1. Akan E. Staphylococcus. In$NDQ(HG7ÕEEL0LNUREL\RORMLø]PLU6DUD\.LWDSHYL-19.
2. $\GÕQ 0 *QD\ ø .|NVDO ) 6HULQ 06 7DNVRPHWUL YH EDNWHUL\HO LGHQWLILNDV\RQGD ELOJLVD\DU NXOODQÕPÕ
Mikrobiyol Bült., 1996; 30:281-287.
3. Ballows A, Hausler WJ, Herrman KL, Isenbirg HD, Shadomy HJ(ed.). Manual of Clinic Microbiology, 4.th
ed., American Society for Microbiology, Washington D.C. 1991.
4. Barry AL. Improved 18-hour methyl red test. Appl Microbiol, 1970; 20: 866-870.
5. Elmer WK, Stephen DA, William MJ, Paul CS, Washington CW (eds). Color atlas and textbook of
diagnostic microbiology. 4.th ed, Lippincott JB Company, Philadelphia, 1992.
6. Finegold SM, Martin WJ, Scoot EG. Bailey and Scoott's
Diagnostic Microbiology, 5th ed, St.Louis, CV Mosby, 1978:490-.
7. Lennette EH, Balows A, Hausler WJ Jr, Shadomy EJ(eds). Manual of Clinical Microbiology, 4th ed.
Washington, DC, American Society for Microbiology, 1985.
8. MacFaddin JF. Biochemical Tests for Identification of Medical
Bacteria, 2nd ed, Baltimore, Williams&Wilkins, 1980:78-.
9. Michael JJ, Abbott SL. Bacterial Identification for Publication: When Is Enough Enough? J. Clin.
Microbiol., 2002; 40:1887-1891.
10. Miller JM, Wright JW. Spot indole test: Evaluation of four reagents. J Clin Microbiol, 1982; 15:589-592.
11. Noel RK, John GH. Bergey's Manual of Systematic Bacteriology.
Ed. Barbara Tansill, Vol.1,2,3 1984.
12. Norell SA, Messley KE. Microbiology Lab Manual: Principles ans Applications. New Jersey: PrenticeHall, Inc. 1997: 54-.
13. Sarkonen N, Könönen E, Summanen P, et al. Phenotypic Identification of Actinomyces and Related
Species Isolated from Human Sources. Journal of Clinical Microbiology, 2001; 39(11): 3955-3961.
14. Selvakumar N, Rahman F, Rajasekaran S, et al. Inefficiency of 0.3% Carbol Fuchsin in Ziehl-Neelsen
Staining for Detecting Acid-Fast Bacilli. Journal of Clinical Microbiology, 2002; 40(8):3041-3043.
15. Singleton P, Sainsbury D. Dictionary of Microbiology. NewYork, Johns WileySons, 1978.
16. Timothy B, Taylor MB. Sniffing Bacterial Cultures on Agar Plates: a Useful Tool or a Safety Hazard? J.
Clin. Microbiol. 2002;40:3877-.