Summary of 8|ochem|ca| 1ests

1ests used to |dent|fy Gram os|t|ve 8acter|a

CaLalase 1esL
MannlLol SalL Agar (MSA)
8lood Agar laLes (8A)
SLreaksLab Lechnlque
1axos (opLochln senslLlvlLy LesLlng)
1axos A (baclLracln senslLlvlLy LesLlng)
CAM 1esL
8lle Lsculln Agar
nlLraLe 8roLh
SplrlL 8lue agar
SLarch hydrolysls LesL
MoLlllLy Agar
Coagulase 1esL

1ests used to |dent|fy Gram Negat|ve 8acter|a
Cxldase 1esL
Sugar (eg glucose) broLh wlLh uurham Lubes
MeLhyl 8ed / vogesroskauer (M8/v)
kllger's lron Agar (klA)
nlLraLe 8roLh
MoLlllLy Agar
MacConkey agar
Slmmon's ClLraLe Agar
urease LesL
Sulfur lndole MoLlllLy Medla (SlM)







,ann|to| Sa|t Agar (,SA)

1hls Lype of medlum ls boLh selecLlve and dlfferenLlal 1he MSA wlll selecL for organlsms such as
SLaphylococcus specles whlch can llve ln areas of hlgh salL concenLraLlon (plaLe on Lhe lefL ln Lhe plcLure
below) 1hls ls ln conLrasL Lo SLrepLococcus specles whose growLh ls selecLed agalnsL by Lhls hlgh salL
agar (plaLe on Lhe rlghL ln Lhe plcLure below)

1he dlfferenLlal lngredlenL ln MSA ls Lhe sugar mannlLol Crganlsms capable of uslng mannlLol as a food
source wlll produce acldlc byproducLs of fermenLaLlon LhaL wlll lower Lhe pP of Lhe medla 1he acldlLy of
Lhe medla wlll cause Lhe pP lndlcaLor phenol red Lo Lurn yellow SLaphylococcus aureus ls capable of
fermenLlng mannlLol (lefL slde of lefL plaLe) whlle SLaphylococcus epldermldls ls noL (rlghL slde of lefL
plaLe)







G|ucose broth w|th Durham tubes


1hls ls a dlfferenLlal medlum lL LesLs an organlsms ablllLy Lo fermenL Lhe sugar glucose as well as lLs
ablllLy Lo converL Lhe end producL of glycolysls pyruvlc acld lnLo gaseous byproducLs 1hls ls a LesL
commonly used when Lrylng Lo ldenLlfy CramnegaLlve enLerlc bacLerla all of whlch are glucose
fermenLers buL only some of whlch produce gas

lke MSA Lhls medlum also conLalns Lhe pP lndlcaLor phenol red lf an organlsm ls capable of
fermenLlng Lhe sugar glucose Lhen acldlc byproducLs are formed and Lhe pP lndlcaLor Lurns yellow
Lscherlchla coll ls capable of fermenLlng glucose as are roLeus mlrabllls (far rlghL) and Shlgella
dysenLerlae (cenLer) seudomonas aeruglnosa (far lefL) ls a nonfermenLer

1he end producL of glycolysls ls pyruvaLe Crganlsms LhaL are capable of converLlng pyruvaLe Lo formlc
acld and formlc acld Lo P2 (g) and CC2 (g) vla Lhe acLlon of Lhe enzyme formlc hydrogen lyase emlL gas
1hls gas ls Lrapped ln Lhe uurham Lube and appears as a bubble aL Lhe Lop of Lhe Lube Lscherlchla coll
and roLeus mlrabllls (far rlghL) are boLh gas producers

*noLe broLh Lubes can be made conLalnlng sugars oLher Lhan glucose (eg lacLose and mannlLol)
8ecause Lhe same pP lndlcaLor (phenol red) ls also used ln Lhese fermenLaLlon Lubes Lhe same resulLs
are consldered poslLlve (eg a lacLose broLh Lube LhaL Lurns yellow afLer lncubaLlon has been lnoculaLed
wlLh an organlsm LhaL can fermenL lacLose)

8|ood Agar |ates (8A)


1hls ls a dlfferenLlal medlum lL ls a rlch complex medlum LhaL conLalns 3 sheep red blood cells 8A
LesLs Lhe ablllLy of an organlsm Lo produce hemolyslns enzymes LhaL damage/lyse red blood cells
(eryLhrocyLes) 1he degree of hemolysls by Lhese hemolyslns ls helpful ln dlfferenLlaLlng members of Lhe
genera SLaphylococcus SLrepLococcus and LnLerococcus

8eLahemolysls ls compleLe hemolysls lL ls characLerlzed by a clear (LransparenL) zone surroundlng Lhe
colonles SLaphylococcus aureus SLrepLococcus pyogenes and SLrepLococcus agalacLlae are bhemolyLlc
(Lhe plcLure on Lhe lefL below shows Lhe beLahemolysls of S pyogenes)
arLlal hemolysls ls Lermed alphahemolysls Colonles Lyplcally are surrounded by a green opaque zone
SLrepLococcus pneumonlae and SLrepLococcus mlLls are ahemolyLlc (Lhe plcLure on Lhe rlghL below
shows Lhe ahemolysls of S mlLls)
lf no hemolysls occurs Lhls ls Lermed gammahemolysls 1here are no noLable zones around Lhe
colonles SLaphylococcus epldermldls ls gammahemolyLlc
WhaL Lype of hemolysls ls seen on each one of Lhe followlng plaLes?




Streakstab techn|que
CfLen when lnoculaLlng a 8A Lo observe hemoloysls paLLerns lnvesLlgaLors wlll also sLab several Llmes
Lhrough Lhe agar uslng an lnoculaLlng loop 1hls sLab allows for Lhe deLecLlon of sLrepLolysln C a speclflc
hemolysln produced by SLrepLococcus pyogenes 1hls hemolysln ls lnacLlvaLed by C2 and ls only seen
subsurface (ln an anaeroblc envlronmenL) around Lhe sLab mark noLe Lhe ovalshaped areas of clearlng
around Lhe sLab marks ln Lhe plcLure below Lhese are caused by sLrepLolysln C


8||e Lscu||n Agar
1hls ls a medlum LhaL ls boLh selecLlve and dlfferenLlal lL LesLs Lhe ablllLy of organlsms Lo hydrolyze
esculln ln Lhe presence of blle lL ls commonly used Lo ldenLlfy members of Lhe genus LnLerococcus (L
faecalls and L faeclum)
1he flrsL selecLlve lngredlenL ln Lhls agar ls blle whlch lnhlblLs Lhe growLh of CramposlLlves oLher Lhan
enLerococcl and some sLrepLococcl specles 1he second selecLlve lngredlenL ls sodlum azlde 1hls
chemlcal lnhlblLs Lhe growLh of CramnegaLlves
1he dlfferenLlal lngredlenL ls esculln lf an organlsm can hydrolyze esculln ln Lhe presence of blle Lhe
producL esculeLln ls formed LsculeLln reacLs wlLh ferrlc clLraLe (ln Lhe medlum) formlng a phenollc lron
complex whlch Lurns Lhe enLlre slanL dark brown Lo black 1he Lube on Lhe far rlghL was lnoculaLed wlLh
L faecalls (poslLlve) 1he Lube ln Lhe cenLer was lnoculaLed wlLh a bllle esculln negaLlve organlsm and
Lhe Lube on Lhe lefL was unlnoculaLed


Su|fur Indo|e ,ot|||ty ,ed|a (SI,)
1hls ls a dlfferenLlal medlum lL LesLs Lhe ablllLy of an organlsm Lo do several Lhlngs reduce sulfur
produce lndole and swlm Lhrough Lhe agar (be moLlle) SlM ls commonly used Lo dlfferenLlaLe members
of LnLerobacLerlaceae
Sulfur can be reduced Lo P2S (hydrogen sulflde) elLher by caLabollsm of Lhe amlno acld cysLelne by Lhe
enzyme cysLelne desulfurase or by reducLlon of LhlosulfaLe ln anaeroblc resplraLlon lf hydrogen sulflde
ls produced a black color forms ln Lhe medlum roLeus mlrabllls ls poslLlve for P2S producLlon 1he
organlsm plcLured on Lhe far lefL ls poslLlve for hydrogen sulflde producLlon
8acLerla LhaL have Lhe enzyme LrypLophanase can converL Lhe amlno acld LrypLophane Lo lndole lndole
reacLs wlLh added kovac's reagenL Lo form roslndole dye whlch ls red ln color (lndole +) Lscherlchla coll
ls lndole poslLlve 1he organlsm plcLured second from lefL ls L coll and ls lndole poslLlve

SlM Lubes are lnoculaLed wlLh a slngle sLab Lo Lhe boLLom of Lhe Lube lf an organlsm ls moLlle Lhan Lhe
growLh wlll radlaLe from Lhe sLab mark and make Lhe enLlre Lube appear Lurbld seudomonas
aeruglnosa and Lhe sLraln of roLeus mlrabllls LhaL we work wlLh are moLlle



k||ger's Iron Agar (kIA)
1hls ls a dlfferenLlal medlum lL LesLs for organlsms' ablllLles Lo fermenL glucose and lacLose Lo acld and
acld plus gas end producLs lL also allows for ldenLlflcaLlon of sulfur reducers 1hls medla ls commonly
used Lo separaLe lacLose fermenLlng members of Lhe famlly LnLerobacLerlaceae (eg Lscherlchla coll)
from members LhaL do noL fermenL lacLose llke Shlgella dysenLerlae 1hese lacLose nonfermenLlng
enLerlcs generally Lend Lo be Lhe more serlous paLhogens of Lhe Lhe gasLrolnLesLlnal LracL
1he flrsL dlfferenLlal lngredlenL glucose ls ln very shorL supply Crganlsms capable of fermenLlng Lhls
sugar wlll use lL up wlLhln Lhe flrsL few hours of lncubaLlon Clucose fermenLaLlon wlll creaLe acldlc
byproducLs LhaL wlll Lurn Lhe phenol red lndlcaLor ln Lhe medla yelllow 1hus afLer Lhe flrsL few hours of
lncubaLlon Lhe Lube wlll be enLlrely yellow AL Lhls polnL when Lhe glucose has been all used up Lhe
organlsm musL choose anoLher food source lf Lhe organlsm can fermenL lacLose Lhls ls Lhe sugar lL wlll
choose acLose fermenLaLlon wlll conLlnue Lo produce acldlc byproducLs and Lhe medla wlll remaln
yellow (plcLure on Lhe far lefL below) lf gas ls produced as a resulL of glucose or lacLose fermenLaLlon
Lhen flssures wlll appear ln Lhe agar or Lhe agar wlll be llfLed off Lhe boLLom of Lhe Lube
lf an organlsm cannoL use lacLose as a food source lL wlll be forced Lo use Lhe amlno aclds / proLelns ln
Lhe medla 1he deamlnaLlon of Lhe amlno aclds creaLes nP3 a weak base whlch causes Lhe medlum Lo
become alkallne 1he alkallne pP causes Lhe phenol red lndlcaLor Lo begln Lo Lurn red Slnce Lhe
lncubaLlon Llme ls shorL (1824 h) only Lhe slanL has a chance Lo Lurn red and noL Lhe enLlre Lube 1hus
an organlsm LhaL can fermenL glucose buL noL lacLose wlll produce a red slanL and a yellow buLL ln a klA
Lube (second from Lhe lefL below) 1hese organlsms are Lhe more serlous paLhogens of Lhe Cl1 such as
Shlgella dysenLerlae

lf an organlsm ls capable of uslng nelLher glucose nor lacLose Lhe organlsm wlll use solely amlno aclds /
proLelns 1he slanL of Lhe Lube wlll be red and Lhe color of Lhe buLL wlll remaln unchanged (plcLure on
Lhe far rlghL below) seudomonas aeruglnosa ls an example of a nonfermenLer
klA Lubes are also capable of deLecLlng Lhe producLlon of P2S lL ls seen as a black preclplLaLe (second
plcLure from Lhe rlghL) SomeLlmes Lhe black preclplLaLe obscures Lhe buLL of Lhe Lube ln such cases Lhe
organlsms should be consldered poslLlve for glucose fermenLaLlon (yellow buLL) roLeus mlrabllls
(plcLured here second from rlghL) ls a glucose poslLlve lacLose negaLlve sulfur reduclng enLerlc


N|trate 8roth
1hls ls a dlfferenLlal medlum lL ls used Lo deLermlne lf an organlsm ls capable of reduclng nlLraLe (nC3)
Lo nlLrlLe (nC2) or oLher nlLrogenous compounds vla Lhe acLlon of Lhe enzyme nlLraLase (also called
nlLraLe reducLase) 1hls LesL ls lmporLanL ln Lhe ldenLlflcaLlon of boLh CramposlLlve and CramnegaLlve
specles
AfLer lncubaLlon Lhese Lubes are flrsL lnspecLed for Lhe presence of gas ln Lhe uurham Lube ln Lhe case
of nonfermenLers Lhls ls lndlcaLlve of reducLlon of nlLraLe Lo nlLrogen gas Powever ln many cases gas ls
produced by fermenLaLlon and furLher LesLlng ls necessary Lo deLermlne lf reducLlon of nlLraLe has
occurred 1hls furLher LesLlng lncludes Lhe addlLlon of sulfanlllc acld (ofLen called nlLraLe l) and dlmeLhyl
alphanapLhalamlne (nlLraLe ll) lf nlLrlLe ls presenL ln Lhe medla Lhen lL wlll reacL wlLh nlLraLe l and
nlLraLe ll Lo form a red compound 1hls ls consldered a poslLlve resulL lf no red color forms upon
addlLlon of nlLraLe l and ll Lhls lndlcaLes LhaL elLher Lhe nC3 has noL been converLed Lo nC2 (a
negaLlve resulL) or LhaL nC3 was converLed Lo nC2 and Lhen lmmedlaLely reduced Lo some oLher
undeLecLable form of nlLrogen (also a poslLlve resulL) ln order Lo deLermlne whlch of Lhe precedlng ls
Lhe case elemenLal zlnc ls added Lo Lhe broLh Zlnc wlll converL any remalnlng nC3 Lo nC2 Lhus
allowlng nlLraLe l and nlLraLe ll Lo reacL wlLh Lhe nC2 and form Lhe red plgmenL (a verlfled negaLlve
resulL) lf no color change occurs upon addlLlon of zlnc Lhen Lhls means LhaL Lhe nC3 was converLed Lo
nC2 and Lhen was converLed Lo some oLher undeLecLable form of nlLrogen (a poslLlve resulL)

lf Lhe nlLraLe broLh Lurns red (Lubes plcLured ln Lhe cenLer) afLer nlLraLe l and nlLraLe ll are added Lhls
color lndlcaLes a poslLlve resulL lf lnsLead Lhe Lube Lurns red (Lube plcLured on Lhe lefL) afLer Lhe
addlLlon of Zn Lhls lndlcaLes a negaLlve resulL lf Lhere ls no color change ln Lhe Lube afLer Lhe addlLlon
of nlLraLe l and nlLraLe ll Lhe resulL ls uncerLaln lf Lhe Lube ls colorless (plcLure on Lhe rlghL) afLer Lhe
addlLlon of Zn Lhls lndlcaLes a poslLlve LesL


Cata|ase 1est
1hls LesL ls used Lo ldenLlfy organlsms LhaL produce Lhe enzyme caLalase 1hls enzyme deLoxlfles
hydrogen peroxlde by breaklng lL down lnLo waLer and oxygen gas



1he bubbles resulLlng from producLlon of oxygen gas clearly lndlcaLe a caLalase poslLlve resulL 1he
sample on Lhe rlghL below ls caLalase poslLlve 1he SLaphylococcus spp and Lhe Mlcrococcus spp are
caLalase poslLlve 1he SLrepLococcus and LnLerococcus spp are caLalase negaLlve


Cx|dase 1est
1hls LesL ls used Lo ldenLlfy mlcroorganlsms conLalnlng Lhe enzyme cyLochrome oxldase (lmporLanL ln
Lhe elecLron LransporL chaln) lL ls commonly used Lo dlsLlngulsh beLween oxldase negaLlve
LnLerobacLerlaceae and oxldase poslLlve seudomadaceae

CyLochrome oxldase Lransfers elecLrons from Lhe elecLron LransporL chaln Lo oxygen (Lhe flnal elecLron
accepLor) and reduces lL Lo waLer ln Lhe oxldase LesL arLlflclal elecLron donors and accepLors are
provlded When Lhe elecLron donor ls oxldlzed by cyLochrome oxldase lL Lurns a dark purple 1hls ls
consldered a poslLlve resulL ln Lhe plcLure below Lhe organlsm on Lhe rlghL (seudomonas aeruglnosa) ls
oxldase poslLlve


Coagu|ase test
Coagulase ls an enzyme LhaL cloLs blood plasma 1hls LesL ls performed on CramposlLlve caLalase
poslLlve specles Lo ldenLlfy Lhe coagulase poslLlve SLaphylococcus aureus Coagulase ls a vlrulence facLor
of S aureus 1he formaLlon of cloL around an lnfecLlon caused by Lhls bacLerla llkely proLecLs lL from
phagocyLosls 1hls LesL dlfferenLlaLes SLaphylococcus aureus from oLher coagulase negaLlve
SLaphylococcus specles


1axos A (bac|trac|n sens|t|v|ty test|ng)
1hls ls a dlfferenLlal LesL used Lo dlsLlngulsh beLween organlsms senslLlve Lo Lhe anLlbloLlc baclLracln and
Lhose noL 8aclLracln ls a pepLlde anLlbloLlc produced by 8aclllus subLllls lL lnhlblLs cell wall synLhesls and
dlsrupLs Lhe cell membrane 1hls LesL ls commonly used Lo dlsLlngulsh beLween Lhe bhemolyLlc
sLrepLococcl SLrepLococcus agalacLlae (baclLracln reslsLanL) and SLrepLococcus pyogenes (baclLracln
senslLlve) 1he plaLe below was sLreaked wlLh SLrepLococcus pyogenes noLlce Lhe large zone of
lnhlblLlon surroundlng Lhe dlsk


1axos (optoch|n sens|t|v|ty test|ng)
1hls ls a dlfferenLlal LesL used Lo dlsLlngulsh beLween organlsms senslLlve Lo Lhe anLlbloLlc opLochln and
Lhose noL 1hls LesL ls used Lo dlsLlngulsh SLrepLococcus pneumonlae (opLochln senslLlve (plcLured on Lhe
rlghL below)) from oLher ahemolyLlc sLrepLococcl (opLochln reslsLanL (SLrepLococcus mlLls ls plcLured on
Lhe lefL below))


,acConkey agar
1hls medlum ls boLh selecLlve and dlfferenLlal 1he selecLlve lngredlenLs are Lhe blle salLs and Lhe dye
crysLal vloleL whlch lnhlblL Lhe growLh of CramposlLlve bacLerla 1he dlfferenLlal lngredlenL ls lacLose
lermenLaLlon of Lhls sugar resulLs ln an acldlc pP and causes Lhe pP lndlcaLor neuLral red Lo Lurn a
brlghL plnkyred color 1hus organlsms capable of lacLose fermenLaLlon such as Lscherlchla coll form
brlghL plnkyred colonles (plaLe plcLured on Lhe lefL here) MacConkey agar ls commonly used Lo
dlfferenLlaLe beLween Lhe LnLerobacLerlaceae



Crganlsm on lefL ls poslLlve for lacLose fermenLaLlon and LhaL on Lhe rlghL ls negaLlve

S|mmon's C|trate Agar
1hls ls a deflned medlum used Lo deLermlne lf an organlsm can use clLraLe as lLs sole carbon source lL ls
ofLen used Lo dlfferenLlaLe beLween members of LnLerobacLerlaceae ln organlsms capable of uLlllzlng
clLraLe as a carbon source Lhe enzyme clLrase hydrolyzes clLraLe lnLo oxaoloaceLlc acld and aceLlc acld
1he oxaloaceLlc acld ls Lhen hydrolyzed lnLo pyruvlc acld and CC2 lf CC2 ls produced lL reacLs wlLh
componenLs of Lhe medlum Lo produce an alkallne compound (eg na2CC3) 1he alkallne pP Lurns Lhe
pP lndlcaLor (bromLhymol blue) from green Lo blue 1hls ls a poslLlve resulL (Lhe Lube on Lhe rlghL ls
clLraLe poslLlve) klebslella pneumonlae and roLeus mlrabllls are examples of clLraLe poslLlve organlsms
Lscherlchla coll and Shlgella dysenLerlae are clLraLe negaLlve



Sp|r|t 8|ue agar
1hls agar ls used Lo ldenLlfy organlsms LhaL are capable of produclng Lhe enzyme llpase 1hls enzyme ls
secreLed and hydrolyzes Lrlglycerldes Lo glycerol and Lhree long chaln faLLy aclds 1hese compounds are
small enough Lo pass Lhrough Lhe bacLerlal cell wall Clycerol can be converLed lnLo a glycolysls
lnLermedlaLe 1he faLLy aclds can be caLabollzed and Lhelr fragmenLs can evenLually enLer Lhe kreb's
cycle SplrlL blue agar conLalns an emulslon of ollve oll and splrlL blue dye 8acLerla LhaL produce llpase
wlll hydrolyze Lhe ollve oll and produce a halo around Lhe bacLerlal growLh 1he CramposlLlve rod
8aclllus subLllls ls llpase poslLlve (plcLured on Lhe rlghL) 1he plaLe plcLured on Lhe lefL ls llpase negaLlve


Starch hydro|ys|s test
1hls LesL ls used Lo ldenLlfy bacLerla LhaL can hydrolyze sLarch (amylose and amylopecLln) uslng Lhe
enzymes aamylase and ollgo16glucosldase CfLen used Lo dlfferenLlaLe specles from Lhe genera
ClosLrldlum and 8aclllus 8ecause of Lhe large slze of amylose and amylopecLln molecules Lhese
organlsms can noL pass Lhrough Lhe bacLerlal cell wall ln order Lo use Lhese sLarches as a carbon source
bacLerla musL secreLe aamylase and ollgo16glucosldase lnLo Lhe exLracellular space 1hese enzymes
break Lhe sLarch molecules lnLo smaller glucose subunlLs whlch can Lhen enLer dlrecLly lnLo Lhe glycolyLlc
paLhway ln order Lo lnLerpreL Lhe resulLs of Lhe sLarch hydrolysls LesL lodlne musL be added Lo Lhe agar
1he lodlne reacLs wlLh Lhe sLarch Lo form a dark brown color 1hus hydrolysls of Lhe sLarch wlll creaLe a
clear zone around Lhe bacLerlal growLh 8aclllus subLllls ls poslLlve for sLarch hydrolysls (plcLured below
on Lhe lefL) 1he organlsm shown on Lhe rlghL ls negaLlve for sLarch hydrolysls



,ethy| ked ] Vogesroskauer (,k]V)
1hls LesL ls used Lo deLermlne whlch fermenLaLlon paLhway ls used Lo uLlllze glucose ln Lhe mlxed acld
fermenLaLlon paLhway glucose ls fermenLed and produces several organlc aclds (lacLlc aceLlc succlnlc
and formlc aclds) 1he sLable producLlon of enough acld Lo overcome Lhe phosphaLe buffer wlll resulL ln
a pP of below 44 lf Lhe pP lndlcaLor (meLhyl red) ls added Lo an allquoL of Lhe culLure broLh and Lhe pP
ls below 44 a red color wlll appear (flrsL plcLure Lube on Lhe lefL) lf Lhe M8 Lurns yellow Lhe pP ls
above 60 and Lhe mlxed acld fermenLaLlon paLhway has noL been uLlllzed (flrsL plcLure Lube on Lhe
rlghL) 1he 23 buLanedlol fermenLaLlon paLhway wlll fermenL glucose and produce a 23 buLanedlol end
producL lnsLead of organlc aclds ln order Lo LesL Lhls paLhway an allquoL of Lhe M8/v culLure ls
removed and anaphLhol and kCP are added 1hey are shaken LogeLher vlgorously and seL aslde for
abouL one hour unLll Lhe resulLs can be read 1he vogesroskauer LesL deLecLs Lhe presence of aceLoln
a precursor of 23 buLanedlol lf Lhe culLure ls poslLlve for aceLoln lL wlll Lurn brownlshred Lo plnk"
(Lube on Lhe lefL ln Lhe second plcLure) lf Lhe culLure ls negaLlve for aceLoln lL wlll Lurn brownlshgreen
Lo yellow" (Lube on Lhe lefL ln Lhe second plcLure) noLe A culLure wlll usually only be poslLlve for one
paLhway elLher M8+ or v+ Lscherlchla coll ls M8+ and v ln conLrasL LnLerobacLer aerogenes and
klebslella pneumonlae are M8 and v+ seudomonas aeruglnosa ls a glucose nonfermenLer and ls Lhus
M8 and v


CA, 1est
CAM facLor ls a dlffuslble heaLsLable proLeln produced by group 8 sLrepLococcl 1hls ls a synerglsLlc
LesL beLween SLaphylococcus aureus and SLrepLococcus agalacLlae S agalacLlae produces CAM facLor
S aureus produces sphlngomyelln C whlch blnds Lo red blood cell membranes 1he Lwo bacLerla are
sLreaked aL 90
o
angles of one anoLher 1hey do nC1 Louch 1he CAM facLor produced by S agalacLlae
enhances Lhe beLahemolysls of S aureus by blndlng Lo already damaged red blood cells As a resulL an
arrow of beLahemolysls ls produced beLween Lhe Lwo sLreaks 1he LesL ls presumpLlve for S agalacLlae
LhaL produces CAM facLor
ln Lhe plcLure here SLrepLococcus agalacLlae was sLreaked from opposlLe sldes of Lhe plaLe and broughL
down Loward Lhe cenLre of Lhe plaLe SLaphylococcus aureus was sLreaked ln a sLralghL llne across Lhe
cenLre of Lhe plaLe 8lngs of hemolysls are evldenL all around S aureus however Lhe hemolysls lf greaLly
enhanced (ln an arrow shape) where Lhe S agalacLlae crosses Lhe hemolysls rlngs


Drease test
1hls LesL ls used Lo ldenLlfy bacLerla capable of hydrolyzlng urea uslng Lhe enzyme urease lL ls
commonly used Lo dlsLlngulsh Lhe genus roLeus from oLher enLerlc bacLerla 1he hydrolysls of urea
forms Lhe weak base ammonla as one of lLs producLs 1hls weak base ralses Lhe pP of Lhe medla above
84 and Lhe pP lndlcaLor phenol red Lurns from yellow Lo plnk roLeus mlrabllls ls a rapld hydrolyzer of
urea (cenLer Lube plcLured here) 1he Lube on Lhe far rlghL was lnoculaLed wlLh a urease negaLlve
organlsm and Lhe Lube on Lhe far lefL was unlnoculaLed


,ot|||ty agar
ls a dlfferenLlal medlum used Lo deLermlne wheLher an organlsm ls equlpped wlLh flagella and Lhus
capable of swlmmlng away from a sLab mark 1he resulLs of moLlllLy agar are ofLen dlfflculL Lo lnLerpreL
Cenerally lf Lhe enLlre Lube ls Lurbld Lhls lndlcaLes LhaL Lhe bacLerla have moved away from Lhe sLab
mark (are moLlle) 1he organlsms ln Lhe Lwo Lubes plcLured on Lhe rlghL are moLlle lf however Lhe sLab
mark ls clearly vlslble and Lhe resL of Lhe Lube ls noL Lurbld Lhe organlsm ls llkely nonmoLlle (Lube
plcLured on Lhe lefL)

Choco|ate Agar (CnCC)
ChocolaLe Agar (CPCC) ls recommended for use ln Lhe lsolaLlon and culLlvaLlon of fasLldlous organlsms
CPCC ls an enrlched medlum supplemenLed wlLh cofacLor whlch provldes nAu Lo faclllLaLe Lhe growLh
of Paemophllus lnfluenzae nelsserla gonorrhoeae and nelsserla menlnglLldls PeaLed sheep blood ls
added Lo glve Lhe medlum lLs chocolaLe" appearance 1hls medlum ls prepared sLored and dlspensed
under oxygenfree condlLlons Lo prevenL Lhe formaLlon of oxldlzed producLs prlor Lo use
8lnClL
aLhogenlc nelsserla and Paemophllus specles among oLher fasLldlous mlcrobes wlll grow only ln Lhe
presence of cerLaln nuLrlenLs whlch can be found ln ChocolaLe Agar Crlglnally red blood cells were
added Lo a melLed nuLrlenL base and heaLed Lo approxlmaLely 83C whlch lysed Lhe red blood cells
releaslng lnLo Lhe base medla haemoglobln and oLher nuLrlenLs presenL ln lysed blood now Lhese
lngredlenLs haemoglobln and xv lacLor LnrlchmenL are supplemenLs added Lo Lhe nuLrlenL CC 8ase
medla xv lacLor LnrlchmenL ls rlch ln vlLamlns addlLlonal cofacLors and amlno aclds CC Medla base
conslsLs of a complemenLary mlxLure of anlmal and vegeLable pepLones cornsLarch and salLs and was
dlscovered as a resulL of Lhe sLudles done by !ohnsLon ln 1943 SclenLlsLs lncludlng 1hayer MarLln and
esLer1681011121316 furLher modlfled Lhe ChocolaLe Agar formula by addlng anLlbloLlcs and
lncreaslng Lhe dexLrose and agar concenLraLlons resulLlng ln selecLlve medla used for Lhe lsolaLlon of
paLhogenlc members of Lhe famlly nelsserlaceae from cllnlcal speclmens wlLh large numbers of mlxed
normal flora vancomycln lnhlblLs gramposlLlve bacLerla collsLln lnhlblLs gramnegaLlve bacLerla
nysLaLln lnhlblLs some yeasLs and LrlmeLhoprlm lnhlblLs Lhe growLh of swarmlng roLeus specles
ModlflcaLlons conLlnued Lo evolve due Lo dlscoverles found ln Lhe cllnlcal seLLlng some sLralns of
cooJlJo olblcoos lnhlblL Lhe growLh of some N qooottboeoe and nysLaLln proved lneffecLlve ln lnhlblLlng
C alblcans 1he anLlfungal agenL anlsomycln was subsLlLuLed for nysLaLln and has proved effecLlve
(MarLlnewls Medla) 1he concenLraLlon of vancomycln was lncreased from mcg/ml Lo 4 mcg/ml for
greaLer lnhlblLlon of gramposlLlve bacLerla buL Lhe dlscovery of a slgnlflcanL number of n gonorrhoeae
belng suscepLlble Lo low concenLraLlons of vancomycln has led Lo lowerlng Lhe concenLraLlon back Lo 2
mcg/ml (Modlfled MarLlnewls Medla) Modlfl caLlons conLlnued wlLh Lhe replacemenL of anlsomycln
wlLh amphoLerlcln 8 and Lhe addlLlon of llncomycln Lo Lhe formula ln addlLlon Lo dlscoverlng medla
LhaL would asslsL ln Lhe effecLlve recovery of paLhogenlc nelsserlaceae LransporL sysLems were
developed Lo lmprove Lhe recovery of gonococcl whlch are very suscepLlble Lo adverse envlronmenLal
condlLlons Lo delays ln LransporL and whlch may be presenL ln cllnlcal speclmens only ln small numbers
1he !LM8LC* (!ohn L MarLln 8lologlcal LnvlronmenLal Chamber) and Lhe lllockeL SysLems conslsLlng
of selecLlve medla CC
2
generaLlng LableLs and small gaslmpermeable plasLlc bags also ald ln Lhe
recovery of gonococcl 1he chambers conLaln an lnner well moulded lnLo Lhe plaLe ln whlch a CC2
generaLlng LableL (sodlum blcarbonaLe and clLrlc acld) ls lnserLed MolsLure acLlvaLes Lhe LableL whlch
Lhen provldes a CC2 aLmosphere conduclve for Lhe growLh of gonococcl
noLe Modlfled Modlfled 1hayerMarLln Medla (MM1M) and MarLln ewls Medla (MM) have Lhe same
formula and wlll demonsLraLe ldenLlcal performance


CLLD Agar (Cyst|neLactoseL|ectro|yteDef|c|ent Agar

ls a dlfferenLlal culLure medlum for use ln enumeraLlng bacLerla ln urlne lL supporLs Lhe growLh of
urlnary paLhogens and conLamlnanLs buL prevenLs undue swarmlng of roLeus specles due Lo lLs lack of
elecLrolyLes 1he nuLrlenLs ln CLu Agar are supplled by Lhe pepLones pancreaLlc dlgesLs of gelaLln and
caseln and beef exLracL acLose ls lncluded Lo provlde an energy source for organlsms capable of
uLlllzlng lL by a fermenLaLlve mechanlsm 1he cysLlne permlLs Lhe growLh of dwarf colony" collforms
8romLhymol blue ls used as a pP lndlcaLor Lo dlfferenLlaLe lacLose fermenLers from lacLose
nonfermenLers Crganlsms whlch fermenL lacLose wlll lower Lhe pP and change Lhe colour of Lhe
medlum from green Lo yellow LlecLrolyLe sources are reduced ln order Lo resLrlcL Lhe swarmlng of
9toteos specles 8acLerlurla ls deLermlned by lnoculaLlng Lhe surface of an agar medlum uslng 01 m of
a 10
2
dlluLlon of Lhe urlne sample or uslng a callbraLed loop (0001 m) of Lhe undlluLed sample CurrenL
guldellnes are LhaL for a slngle lsolaLe a denslLy of 10
3
Clu/m lndlcaLes lnfecLlon 10
4
Clu/m
lndlcaLes ureLhral or vaglnal conLamlnaLlon and beLween 10
4
and 10
3
Clu/m needs Lo be evaluaLed
based on cllnlcal lnformaLlon


Organisms (ATCC) Growth Appearance of colony
Escherichia coIi (25922) +++ yellow, opaque, center slightly
deeper yellow
Klebsiella pneumoniae (13883) +++ yellow to whitish blue
Citrobacter freundii (8090) +++ yellow
$almonella serotype Typhi (6539) +++ bluish
$higella flexneri (29903) +++ blue
Proteus vulgaris (13315) +++ blue
Pseudomonas aeruginosa (27853) +++ blue
Enterococcus faecalis (29212) +++ Slight yellowish or greenish
$treptococcus cremoris (19527) +++ yellow, small, opaque
$taphylococcus aureus (25923) +++ deep yellow



LA heny|ethy| A|coho| Agar rotoco|

LA agar ls a selecLlve medlum LhaL ls used for Lhe lsolaLlon of gramposlLlve topbylococcos
specles and tteptococcos specles from cllnlcal speclmens or speclmens LhaL conLaln mlxLures
of bacLerlal flora (2) 1yplcally LA agar ls used Lo lnhlblL Lhe common conLamlnanLs such as
scbetlcblo coll and 9toteos specles LA agar may be prepared wlLh and wlLhouL 3 sheep
blood supplemenL LA agar wlLh 3 sheep blood ls used Lo lsolaLe mosL gramposlLlve and
gramnegaLlve anaerobes from enLerlc samples (12) lL ls used Lo lnhlblL faculLaLlve gram
negaLlve rods prevenLlng LnLerobacLerlaceae from overgrowlng Lhe anaerobes and lnhlblLlng
swarmlng of 9toteos and closttlJlom septlcom(712) LA agar ls used for purulenL speclmens
and when mlxed lnfecLlons are suspecLed (12)

1ab|e 1 Lxamples of growLh of some gramnegaLlve and gramposlLlve bacLerla on LA
agar
Cram
reacLlon
Crganlsm CrowLh
8esponse
Swarmlng lnhlblLlon
CramnegaLlve scbetlcblo coll lnhlblLed

n/A
9toteos mltobllls Markedly lnhlblLed ?es no spreadlng
9seoJomooos oetoqlooso


arLlally lnhlblLed n/A
olmooello eotetltlJls lnhlblLed n/A
otetoboctet oetoqeoes lnhlblLed n/A
CramposlLlve


topbylococcos ooteos

Cood n/A
tteptococcos pyoqeoes

Cood n/A
closttlJlom petftloqeos

arLlally lnhlblLed n/A
tteptococcos pooemooloe Cood n/A
otetococcos foecolls Cood n/A
8oclllos sp Cood n/A

Mlctococcos loteos Cood n/A





1heory
LA agar ls a selecLlve medlum LhaL permlLs Lhe growLh of gramposlLlve coccl whlle lnhlblLlng mosL
gramnegaLlve organlsms LA acLs on gramnegaLlve bacLerla by alLerlng Lhelr membrane permeablllLy
allowlng lnflux of oLherwlse blocked molecules and allowlng leakage of large amounLs of cellular
poLasslum LhaL ulLlmaLely resulLs ln dlsrupLlon or lnhlblLlon of unA synLhesls

LA agar plaLe wlLh 3 Sheep 8lood lnoculaLed wlLh topbylococcos ooteos a gramposlLlve bacLerlum
lncubaLed under 3 CC2for 48 hr aL 332
o
C (growLh exhlblLed)