Upload
ridho-wahyutomo
View
212
Download
0
Embed Size (px)
DESCRIPTION
mk
Citation preview
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 1/39
DISK DIFFUSION (KIRBY-BAUER)SUSCEPTIBILITY TESTING
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 2/39
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'
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 3/39
(
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 4/39
STORAGE OF DISKS
)
$*SI #+'-A,' PA ,/
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 5/39
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 $efo1itin and Sta!h to detectethicillin resistance
Fro an ,2-') hour agar !late0Select (-3 well isolated colonies of the saeor!hological ty!e. Sus!end in abroth4saline. Ad5ust the sus!ension toachie6e a turbidity e7ui6alent to +.33
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 6/39
• rowth #ethod when colony growth is di8cult to
sus!end
for nonfastidious organiss whenfresh 9')-hour: colonies; asre7uired for the direct colony
sus!ension ethod; are nota6ailable 9e1ce!t Sta!h:
<
INOCULUM PREPARATION (2)
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 7/39
Fro an agar !late0• Select (-3 well isolated colonies of
the sae or!hological ty!e.
•Sus!end in a broth. Incubate at (3='o$ 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 >
INOCULUM PREPARATION (3)
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 8/39
To standardi?e the inoculu; useturbidity standard 9+.3 #cFarland:
• *ate1 !article sus!ension9coercially a6ailable:
• BaS@) 9$*SI #+'; A!!endi1 B:
• Photoetric de6ice
2
INOCULUM PREPARATION (4)
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 9/39
INOCULATION OF PLATES
•Di! a sterile cotton swab into thesus!ension
• %otate the swab se6eral ties and !ress&rly on the onside wall of the tube abo6ethe uid le6el
• Streak the swab o6er the entire surface ofthe agar
•%e!eat !rocedure '1 ore; rotating the!late to a!!ro1iately <+o each tie
• Swab the ri of the agar
• *ea6e for (-3 inutes 9no ore than ,3inutes: before a!!lying the disks/
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 10/39
APPLICATION OF DISKS TO
INOCULATED PLATES
• Disks ust be distributed e6enly 9nocloser than ') fro center to center:
• ,3+ !late no ore than ,' disksC
• ,++ !late no ore than < disksCCde!ending on the organis
• ot too close to the edge of the !late
•Do not relocate disk once it has coe intocontact with the agar surface
• Place disks that gi6e sall ?ones to thosethat gi6e larger ?ones to a6oid o6erla!!ing
?ones ,+
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 11/39
• The surface of the !lates should be oist;but no dro!lets of oisture should bea!!arent on the surface of the ediu or
on the Petri !late co6ers when the !latesare inoculated
• If 5ust before use; e1cess surface oistureis !resent on the !lates; !lace the in the
incubator or lainar ow hood at roote!erature with lids a5ar until e1cesssurface oisture is reo6ed bye6a!oration 9usually ,+-(+ inutes:
,,
Media ad I!"#a$i%
C%di$i%&
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 12/39
Media ad I!"#a$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
,'
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 13/39
Media ad I!"#a$i%
C%di$i%& (3)
,(
$*SI #+'-A,'; PAS <,-<(
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 14/39
Media ad I!"#a$i%
C%di$i%& (4)
,)
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 15/39
Media ad I!"#a$i%
C%di$i%& (')
,3
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 16/39
,<
$*SI #,++-S'3 PA >'
Media ad I!"#a$i%
C%di$i%& ()
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 17/39
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
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 18/39
,2
READING AND INTERPRETING
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
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 19/39
,/
• 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
READING AND INTERPRETING
RESULTS (3)
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 20/39
• 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.
'+
READING AND INTERPRETING
RESULTS (4)
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 21/39
• 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
',
READING AND INTERPRETING
RESULTS (')
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 22/39
''
READING AND INTERPRETING
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
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 23/39
'(
READING AND INTERPRETING
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.
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 24/39
• 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')
READING AND INTERPRETING
RESULTS (*)
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 25/39
'3
READING AND INTERPRETING
RESULTS (+)
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 26/39
'<
READING AND INTERPRETING
RESULTS (1,)
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 27/39
'>
READING AND INTERPRETING
RESULTS (11)
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 28/39
'2
READING AND INTERPRETING
RESULTS (12)Stre!tococcus s!! L-heolytic rou!
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 29/39
'/
$*SI #,++-S'3 PA )+
READING AND INTERPRETING
RESULTS (13)
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 30/39
C%./a$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
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 31/39
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)
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 32/39
('
$*SI #,++-S'3 PA ,//C%./a$i% %0 AST
Re&"$& (3)
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 33/39
•
Dangerously isleading results D@@T re!ort as susce!tible ,st and 'nd gen ce!halos!orins;
ce!haycins; and ainoglycosides
against Salonella and Shigella Penicillins; L-lacta4L-lactaase
inhibitor cobinations;
antista!hylococcal ce!hes; andcarba!enes against @1acillin-resistantSta!. S!!.
Ainoglycosides 9e1ce!t high
concentrations:; ce!halos!orins;((
C%./a$i% %0 AST
Re&"$& (4)
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 34/39
()
$*SI #,++-S'3 PA '>
C%./a$i% %0 AST
Re&"$& (')
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 35/39
(3
$*SI #,++-S'3 PA ()
$*SI #,++-S'3 PA '>
C%./a$i% %0 AST
Re&"$& ()
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 36/39
(<
C%./a$i% %0 AST
Re&"$& ()
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 37/39
(>
C%./a$i% %0 AST
Re&"$& (*)
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 38/39
• $on&ration of unusual orinconsistent results ach laboratory ust de6elo! its own
!olicy
• De6elo!ent of resistance andtesting of re!eat isolates
*aboratory guidelines on when to!erfor susce!tibility testing on re!eatisolates should be deterined inconsultation with the edical sta
(2
C%./a$i% %0 AST
Re&"$& (+)
7/21/2019 Lecture #4. KB_Indonesia 2015
http://slidepdf.com/reader/full/lecture-4-kbindonesia-2015 39/39
Thank you
(/