lecture #4. kb_indonesia 2015

7/21/2019 Lecture #4. KB_Indonesia 2015

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DISK DIFFUSION (KIRBY-BAUER)SUSCEPTIBILITY TESTING



OUTLINE

I. Kirby-Bauer AST ProcedureFlowchart

II. Storage of Disks

III. Inoculu Pre!aration

I". Inoculation of Plates

". A!!lication of Disks to InoculatedAgar Plates

"I.#edia and Incubation $onditions

"II. %eading and Inter!retation'



(



STORAGE OF DISKS

)

$*SI #+'-A,' PA ,/



INOCULUM PREPARATION (1)

Two ethods0 Direct colony and rowth#ethod

• Direct colony sus!ension

$an be used with ost organiss

%ecoended for fastidious organiss

For testing $efo1itin and Sta!h to detectethicillin resistance

Fro an ,2-') hour agar !late0Select (-3 well isolated colonies of the saeor!hological ty!e. Sus!end in abroth4saline. Ad5ust the sus!ension toachie6e a turbidity e7ui6alent to +.33



• rowth #ethod when colony growth is di8cult to

sus!end

for nonfastidious organiss whenfresh 9')-hour: colonies; asre7uired for the direct colony

sus!ension ethod; are nota6ailable 9e1ce!t Sta!h:

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Fro an agar !late0• Select (-3 well isolated colonies of

the sae or!hological ty!e.

•Sus!end in a broth. Incubate at (3='o$ until it achie6es or e1ceeds theturbidity of the +.3 #cFarland

standard• Add broth or saline to the broth

culture to achie6e a turbiditye7ui6alent to a +.3 #cFarlandstandard >




To standardi?e the inoculu; useturbidity standard 9+.3 #cFarland:

• *ate1 !article sus!ension9coercially a6ailable:

• BaS@) 9$*SI #+'; A!!endi1 B:

• Photoetric de6ice

2




INOCULATION OF PLATES

•Di! a sterile cotton swab into thesus!ension

• %otate the swab se6eral ties and !ress&rly on the onside wall of the tube abo6ethe uid le6el

• Streak the swab o6er the entire surface ofthe agar

•%e!eat !rocedure '1 ore; rotating the!late to a!!ro1iately <+o each tie

• Swab the ri of the agar

• *ea6e for (-3 inutes 9no ore than ,3inutes: before a!!lying the disks/



APPLICATION OF DISKS TO

INOCULATED PLATES

• Disks ust be distributed e6enly 9nocloser than ') fro center to center:

• ,3+ !late no ore than ,' disksC

• ,++ !late no ore than < disksCCde!ending on the organis

• ot too close to the edge of the !late

•Do not relocate disk once it has coe intocontact with the agar surface

• Place disks that gi6e sall ?ones to thosethat gi6e larger ?ones to a6oid o6erla!!ing

?ones ,+



• The surface of the !lates should be oist;but no dro!lets of oisture should bea!!arent on the surface of the ediu or

on the Petri !late co6ers when the !latesare inoculated

• If 5ust before use; e1cess surface oistureis !resent on the !lates; !lace the in the

incubator or lainar ow hood at roote!erature with lids a5ar until e1cesssurface oisture is reo6ed bye6a!oration 9usually ,+-(+ inutes:

,,

Media ad I!"#a$i%

C%di$i%&



Media ad I!"#a$i%

C%di$i%& (2)

$hoice of edia and incubation conditionsde!end on the organis being tested0

• #ueller Einton Agar 9#EA:

• #EA with 3 shee! blood• Eaeo!hilus Test #ediu 9ET#:

• $ agar base G , de&ned growth

su!!leent

,'



Media ad I!"#a$i%

C%di$i%& (3)

,(

$*SI #+'-A,'; PAS <,-<(



Media ad I!"#a$i%

C%di$i%& (4)

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Media ad I!"#a$i%

C%di$i%& (')

,3



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$*SI #,++-S'3 PA >'

Media ad I!"#a$i%

C%di$i%& ()



READING AND INTERPRETING

RESULTS

,>

Pro!erly inoculated !late0• $onuent lawn of growth• Hone of inhibition uniforly

circular

Poorly inoculated !late0• Indi6idual colonies are

a!!arent across the !late• Inoculu too light re!eat

test



,2


RESULTS (2)

• #easure the diaeter of the ?ones of co!lete

inhibition; as 5udged by the unaided eye;including the diaeter of the disk

• #easure the ?ones to the nearest whole using a sliding cali!er or ruler which is held on

the back of the in6erted !late

Eold the !late afew inches abo6ea black;nonreecting

backgroundilluinated withreected light



,/

• The ?one arginshould beconsidered the area

showing no ob6ious;6isible growth thatcan be detectedwith the unaidedeye

Ignore faint growth of tiny colonies that can bedetected only with a agnifying lens at the edge ofthe ?one of inhibited growth


RESULTS (3)



• If there are discrete colonies growingwithin a clear ?one of inhibition; thetest should be re!eated with a !ure

culture or sub culture of a singlecolony fro the !riary culture !late

• If discrete colonies continue to grow

within the ?one of inhibition; thecolony-free inner ?one should beeasured.

'+


RESULTS (4)



• Strains of Proteus s!! ay swar intoareas of inhibited growth around certainantiicrobial agents. For Proteus s!!.;

ignore the thin 6eil of swaring growth inan otherwise ob6ious ?one of inhibition

• For trietho!ri and sulfonaides;antagonists in the ediu ay allow

soe slight growth therefore; disregardslight growth 9'+ or less of the lawngrowth:; and easure the ore ob6iousargin to deterine the ?one diaeter

',


RESULTS (')



''


RESULTS ()

If the ?ones ofad5acentantibiotic diskso6erla!; the ?one

diaeter can bedeterined byeasuring theradius of the?one.

• #easure fro the center of the antibiotic disk to a !oint onthe circuference of the ?one where a distinct edge is!resent.

• #ulti!ly this easureent by ' to deterine the diaeter

of the ?one of inhibition



'(


RESULTS ()

hen using bloodsu!!leented edia9as with stre!tococci:;easure the ?ones

fro the u!!ersurface of the agarilluinated withreected light withthe co6er reo6ed

ith heolytic organiss; the ?one of growthinhibition should be easured; not the ?one ofheolysis or inhibition of heolysis.



• Inter!ret the si?es of ?ones ofinhibition by referring to $*SI #,++

Tables 'A through 'I;

• %e!ort the organiss as susce!tible;interediate or resistant to theagents that ha6e been tested

•

Soe agents ay be re!orted onlyas susce!tible or nonsuce!tible;because only susce!tible break!oints

are gi6en; or no or 6ery few resistant')


RESULTS (*)



'3


RESULTS (+)



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RESULTS (1,)



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RESULTS (11)



'2


RESULTS (12)Stre!tococcus s!! L-heolytic rou!



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$*SI #,++-S'3 PA )+


RESULTS (13)



C%./a$i% %0 AST

Re&"$&•

A!!lies to all AST ethods• Before re!orting; re6iew results fro

all the drugs tested on a !atientisolates. $heck that

the AST results consistent with theidenti&cation of the isolate

the results fro indi6idual agentwithin a s!eci&c drug class followthe established hierarchy ofacti6ity rules 9(rd generation

ce!halos!orin ore acti6e than ,st



the isolate is susce!tible to thoseantiicrobial agents for whichresistance ha6e not been docuented

9e.g. "anco and Stre!.: the isolate susce!tible to those

antiicrobial agents for which onlyMsusce!tibleN inter!reti6e criteria e1ist

in $*SI #,++O• A re!eat of the AST 9and aybe ID: ay

be needed

to con&r unusal or inconsistent re!orts(,

C%./a$i% %0 AST

Re&"$& (2)



('

$*SI #,++-S'3 PA ,//C%./a$i% %0 AST

Re&"$& (3)



•

Dangerously isleading results D@@T re!ort as susce!tible ,st and 'nd gen ce!halos!orins;

ce!haycins; and ainoglycosides

against Salonella and Shigella Penicillins; L-lacta4L-lactaase

inhibitor cobinations;

antista!hylococcal ce!hes; andcarba!enes against @1acillin-resistantSta!. S!!.

Ainoglycosides 9e1ce!t high

concentrations:; ce!halos!orins;((

C%./a$i% %0 AST

Re&"$& (4)



()

$*SI #,++-S'3 PA '>

C%./a$i% %0 AST

Re&"$& (')



(3

$*SI #,++-S'3 PA ()

$*SI #,++-S'3 PA '>

C%./a$i% %0 AST

Re&"$& ()



(<

C%./a$i% %0 AST

Re&"$& ()



(>

C%./a$i% %0 AST

Re&"$& (*)



• $on&ration of unusual orinconsistent results ach laboratory ust de6elo! its own

!olicy

• De6elo!ent of resistance andtesting of re!eat isolates

*aboratory guidelines on when to!erfor susce!tibility testing on re!eatisolates should be deterined inconsultation with the edical sta

(2

C%./a$i% %0 AST

Re&"$& (+)



Thank you

(/

lecture #4. kb_indonesia 2015

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