Lastprinted9/20/200410:55:00PM

ManualonAntimicrobialSusceptibilityTesting
(UndertheauspicesofIndianAssociationofMedicalMicrobiologists)

Dr.M.K.Lalitha
ProfessorofMicrobiology
DepartmentofMicrobiology
ChristianMedicalCollege
Vellore,TamilNadu

CONTENTS

PAGENo.

1. Introduction

2. Principle

3. FactorsInfluencingAntimicrobialSusceptibilityTesting

4. MethodsofAntimicrobialSusceptibilityTesting

4.1DiskDiffusion

4.2Dilution

14

4.3DilutionAndDiffusion.

20

5. SusceptibilityTestingOfFastidiousBacteria

21

6. ErrorsinInterpretationandreportingResults

28

7. QualityControlinAntimicrobialSusceptibilityTesting

29

8. StandardMethodsfortheDetectionofAntimicrobialResistance.

30

9. ApplicationofComputersinAntimicrobialSusceptibilityTesting

39

10. SelectedBibliography

41

Annexure

43

I. GuidelinesforAntimicrobialSusceptibilityTesting
I. SuggestedDilutionRangesforMICTesting
II. SolventsandDiluentsforAntibiotics

1.Introduction
Resistancetoantimicrobialagents(AMR)hasresultedinmorbidityandmortalityfrom
treatmentfailures andincreasedhealth carecosts.Althoughdefiningtheprecisepublic
healthriskandestimatingtheincreaseincostsisnotasimpleundertaking,thereislittle
doubtthatemergentantibioticresistanceisaseriousglobalproblem.
Appropriate antimicrobial drug use has unquestionable benefit, but physicians and the
publicfrequentlyusetheseagentsinappropriately.Inappropriateuseresultsfromphysicians
providingantimicrobialdrugstotreatviralinfections,usinginadequatecriteriafordiagnosis
ofinfectionsthatpotentiallyhaveabacterialaetiology,unnecessarilyprescribingexpensive,
broadspectrumagents,andnotfollowingestablishedrecommendationsforusingchemo
prophylaxis. The availability of antibiotics over the counter, despite regulations to the
contrary,alsofuelinappropriateusageofantimicrobialdrugsinIndia.Theeasyavailability
ofantimicrobialdrugsleadstotheirincorporationintoherbalor"folk"remedies,whichalso
increasesinappropriateuseoftheseagents.
Widespreadantibioticusageexertsaselectivepressurethatactsasadrivingforceinthe
development of antibiotic resistance. The association between increased rates of
antimicrobialuseandresistancehasbeendocumentedfornosocomialinfectionsaswellas
for resistant community acquired infections. As resistance develops to "firstline"
antibiotics,therapywithnew,broaderspectrum,moreexpensiveantibioticsincreases,butis
followedbydevelopmentofresistancetothenewclassofdrugs.
Resistancefactors,particularlythosecarriedonmobileelements,canspreadrapidlywithin
humanandanimalpopulations.Multidrugresistantpathogenstravelnotonlylocallybut
also globally, with newly introduced pathogens spreading rapidly in susceptible hosts.
Antibioticresistancepatternsmayvarylocallyandregionally,sosurveillancedataneedsto
becollectedfromselectedsentinelsources.Patternscanchangerapidlyandtheyneedtobe

monitoredcloselybecauseoftheirimplicationsforpublichealthandasanindicatorof
appropriateorinappropriateantibioticusagebyphysiciansinthatarea.

Theresultsofinvitroantibioticsusceptibilitytesting,guidecliniciansintheappropriate
selectionofinitialempiricregimensand,drugsusedforindividualpatientsinspecific
situations.Theselectionofanantibioticpanelforsusceptibilitytestingisbasedonthe
commonlyobservedsusceptibilitypatterns,andisrevisedperiodically.

2.Principle
Theprinciplesofdeterminingtheeffectivityofanoxiousagenttoabacteriumwerewell
enumeratedbyRideal,Walkerandothersattheturnofthecentury,thediscoveryof
antibioticsmadethesetests(ortheirmodification)toocumbersomeforthelargenumbers
oftestsnecessarytobeputupasaroutine.Theditchplatemethodofagardiffusionused
byAlexanderFlemingwastheforerunnerofavarietyofagardiffusionmethodsdevised
by workersinthisfield.TheOxfordgroupusedthesemethods initiallytoassaythe
antibioticcontainedinbloodbyallowingtheantibioticstodiffuseoutofreservoirsinthe
mediumincontainersplacedonthesurface.
Withtheintroductionofavarietyofantimicrobialsitbecamenecessarytoperformthe
antimicrobialsusceptibilitytestasaroutine. Forthis,theantimicrobialcontainedina
reservoir was allowed to diffuse out into the medium and interact in a plate freshly
seeded with the test organisms. Even now a variety of antimicrobial containing
reservoirsareusedbuttheantimicrobialimpregnatedabsorbentpaperdiscisbyfarthe
commonesttypeused.ThediscdiffusionmethodofASTisthemostpracticalmethod
andisstillthemethodofchoicefortheaveragelaboratory.Automationmayforcethe
method outofthediagnosticlaboratorybutinthiscountryaswellasinthesmaller
laboratoriesofevenadvancedcountries,itwillcertainlybethemostcommonlycarried
out microbiological test for many years to come. It is, therefore, imperative that
microbiologists understand the principles of the test well and keep updating the

information as and when necessary. All techniques involve either diffusion of

antimicrobialagentinagarordilutionofantibioticinagarorbroth.
Evenautomatedtechniquesarevariationsoftheabovemethods.

3.
FactorsInfluencingAntimicrobialSusceptibilityTesting
pH
ThepHofeachbatchofMellerHintonagarshouldbecheckedwhenthemediumis
prepared.Theexactmethodusedwilldependlargelyonthetypeofequipmentavailablein
thelaboratory.TheagarmediumshouldhaveapHbetween7.2and7.4atroomtemperature
after gelling. If the pH is too low, certain drugs will appear to lose potency (e.g.,
aminoglycosides, quinolones, and macrolides), while other agents may appear to have
excessiveactivity(e.g.,tetracyclines). IfthepHistoohigh,theoppositeeffectscanbe
expected.ThepHcanbecheckedbyoneofthefollowingmeans:
*

MacerateasufficientamountofagartosubmergethetipofapHelectrode.

AllowasmallamountofagartosolidifyaroundthetipofapHelectrodeinabeaker
orcup.

Useaproperlycalibratedsurfaceelectrode.

Moisture
If,justbeforeuse,excesssurfacemoistureispresent,theplatesshouldbeplacedinan
incubator(35C)oralaminarflowhoodatroomtemperaturewithlidsajaruntilexcess
surfacemoistureislostbyevaporation(usually10to30minutes).Thesurfaceshouldbe
moist,butnodropletsofmoistureshouldbeapparentonthesurfaceofthemediumoronthe
petridishcoverswhentheplatesareinoculated.
EffectsofThymidineorThymine
Mediacontainingexcessiveamountsofthymidineorthyminecanreversetheinhibitory
effectofsulfonamidesandtrimethoprim,thusyieldingsmallerandlessdistinctzones,or
5

evennozoneatall,whichmayresultinfalseresistancereports.MellerHintonagarthatis
aslowinthymidinecontentaspossibleshouldbeused.ToevaluateanewlotofMeller
Hintonagar,EnterococcusfaecalisATCC29212,oralternatively,E.faecalisATCC33186,
shouldbetestedwithtrimethoprim/sulfamethoxazoledisks.Satisfactorymediawillprovide
essentiallyclear,distinctzonesofinhibition20mmorgreaterindiameter.Unsatisfactory
mediawillproducenozoneofinhibition,growthwithinthezone,orazoneoflessthan20
mm.
EffectsofVariationinDivalentCations
Variation indivalent cations,principally magnesium andcalcium, willaffectresults of
aminoglycosideandtetracyclinetestswithP.aeruginosastrains.Excessivecationcontent
willreducezonesizes,whereaslowcationcontentmayresultinunacceptablylargezonesof
inhibition.Excesszincionsmayreducezonesizesofcarbapenems.Performancetestswith
eachlotofMellerHintonagarmustconformtothecontrollimits.
Testingstrainsthatfailtogrowsatisfactorily
OnlyaerobicorfacultativebacteriathatgrowwellonunsupplementedMellerHintonagar
shouldbetestedonthatmedium.CertainfastidiousbacteriasuchasHaemophilusspp.,
N.gonorrhoeae,S.pneumoniae,andviridansandhaemolyticstreptococcidonotgrow
sufficientlyonunsupplementedMellerHintonagar.Theseorganismsrequiresupplements
ordifferentmediatogrow,andtheyshouldbetestedonthemediadescribedinseparate
sections.

4.MethodsofAntimicrobialSusceptibilityTesting
Antimicrobialsusceptibilitytestingmethodsaredividedintotypesbasedontheprinciple
appliedineachsystem.Theyinclude:
Diffusion
Stokesmethod

Dilution

Diffusion&Dilution

MinimumInhibitoryConcentration

KirbyBauermethod i)Brothdilution

ETestmethod

ii)AgarDilution

4.1DiskDiffusion
ReagentsfortheDiskDiffusionTest
1.MellerHintonAgarMedium
Ofthemanymediaavailable,MellerHintonagarisconsideredtobethebestforroutine
susceptibilitytestingofnonfastidiousbacteriaforthefollowingreasons:
*

Itshowsacceptablebatchtobatchreproducibilityforsusceptibilitytesting.

Itislowinsulphonamide,trimethoprim,andtetracyclineinhibitors.

Itgivessatisfactorygrowthofmostnonfastidiouspathogens.

Alargebodyofdataandexperiencehasbeencollectedconcerningsusceptibility
testsperformedwiththismedium.

AlthoughMellerHintonagarisreliablegenerallyforsusceptibilitytesting,resultsobtained
withsomebatchesmay,onoccasion,varysignificantly. Ifabatchofmediumdoesnot
supportadequategrowthofatestorganism,zonesobtainedinadiskdiffusiontestwill
usuallybelargerthanexpectedandmayexceedtheacceptablequalitycontrollimits.Only
MellerHintonmediumformulationsthathavebeentestedaccordingto,andthatmeetthe
acceptance limits described in, NCCLS document M62A7 Protocols for Evaluating
DehydratedMellerHintonAgarshouldbeused.
PreparationofMellerHintonAgar
MellerHintonagarpreparationincludesthefollowingsteps.
1.

MellerHintonagarshouldbepreparedfromacommerciallyavailabledehydrated
baseaccordingtothemanufacturer'sinstructions.

2.

Immediatelyafterautoclaving,allowittocoolina45to50Cwaterbath.

3.

Pourthefreshlypreparedandcooledmediumintoglassorplastic,flatbottomed
petridishesonalevel,horizontalsurfacetogiveauniformdepthofapproximately4
mm.Thiscorrespondsto60to70mlofmediumforplateswithdiametersof150
mmand25to30mlforplateswithadiameterof100mm.

4.

Theagarmediumshouldbeallowedtocooltoroomtemperatureand,unlessthe
plateisusedthesameday,storedinarefrigerator(2to8C).

5.

Plates should be used within seven days after preparation unless adequate
precautions,suchaswrappinginplastic,havebeentakentominimizedryingofthe
agar.

6.

Arepresentativesampleofeachbatchofplatesshouldbeexaminedforsterilityby
incubatingat30to35Cfor24hoursorlonger.

2.Preparationofantibioticstocksolutions
Antibitiotics maybereceived aspowders ortablets. Itisrecommended toobtainpure
antibioticsfromcommercialsources,andnotuseinjectablesolutions. Powdersmustbe
accuratelyweighedanddissolvedintheappropriatediluents(AnnexureIII)toyieldthe
requiredconcentration,usingsterileglassware.Standardstrainsofstockculturesshouldbe
usedtoevaluatetheantibioticstocksolution.Ifsatisfactory,thestockcanbealiquotedin5
mlvolumesandfrozenat20Cor60C.
Stocksolutionsarepreparedusingtheformula(1000/P)XVXC=W,whereP+potencyof
the anitbiotic base, V=volume in ml required, C=final concentration of solution and
W=weightoftheantimicrobialtobedissolvedinV.
Preparationofdriedfilterpaperdiscs
Whatmanfilterpaperno.1isusedtopreparediscsapproximately6mmindiameter,which
areplacedinaPetridishandsterilizedinahotairoven.
Theloopusedfordeliveringtheantibioticsismadeof20gaugewireandhasadiameterof
2mm.Thisdelivers0.005mlofantibioticstoeachdisc.
Storageofcommercialantimicrobialdiscs
Cartridges containing commercially prepared paper disks specifically for susceptibility
testingaregenerallypackagedtoensureappropriateanhydrousconditions.Discsshouldbe
storedasfollows:

Refrigeratethecontainersat8Corbelow,orfreezeat14Corbelow,inanonfrost
freefreezeruntilneeded.Sealedpackagesofdisksthatcontaindrugsfromthe
lactamclassshouldbestoredfrozen,exceptforasmallworkingsupply,whichmay
berefrigeratedforatmostoneweek.Somelabileagents(e.g.,imipenem,cefaclor,
andclavulanicacidcombinations)mayretaingreaterstabilityifstoredfrozenuntil
thedayofuse.

Theunopeneddisccontainersshouldberemovedfromtherefrigeratororfreezer
onetotwohoursbeforeuse,sotheymayequilibratetoroomtemperaturebefore
opening.Thisprocedureminimizestheamountofcondensationthatoccurswhen
warmaircontactscolddisks.

Onceacartridgeofdiscshasbeenremovedfromitssealedpackage,itshouldbe
placed in a tightly sealed, desiccated container. When using a discdispensing
apparatus, it should be fitted with atight cover and supplied withan adequate
desiccant. Thedispensershouldbeallowedtowarmtoroomtemperaturebefore
opening.Excessivemoistureshouldbeavoidedbyreplacingthedesiccantwhenthe
indicatorchangescolor.

Whennotinuse,thedispensingapparatuscontainingthediscsshouldalwaysbe
refrigerated.

Onlythosediscsthathavenotreachedthemanufacturer'sexpirationdatestatedon
thelabelmaybeused.Discsshouldbediscardedontheexpirationdate.

Turbiditystandardforinoculumpreparation
Tostandardizetheinoculumdensityforasusceptibilitytest,aBaSO4 turbiditystandard,
equivalent to a 0.5 McFarland standard or its optical equivalent (e.g., latex particle
suspension),shouldbeused.ABaSO40.5McFarlandstandardmaybepreparedasfollows:
1. A0.5mlaliquotof0.048mol/LBaCl2(1.175%w/vBaCl2.2H2O)isaddedto99.5
mlof0.18mol/LH2SO4(1%v/v)withconstantstirringtomaintainasuspension.
2. The correct density of the turbidity standard should be verified by using a
spectrophotometerwitha1cmlightpathandmatchedcuvettetodeterminethe

10

absorbance. The absorbance at 625 nm should be 0.008 to 0.10 for the 0.5
McFarlandstandard.
3. TheBariumSulfatesuspensionshouldbetransferredin4to6mlaliquotsinto
screwcaptubesofthesamesizeasthoseusedingrowingordilutingthebacterial
inoculum.
4. Thesetubesshouldbetightlysealedandstoredinthedarkatroomtemperature.
5. Thebariumsulfateturbiditystandardshouldbevigorouslyagitatedonamechanical
vortexmixerbeforeeachuseandinspectedforauniformlyturbidappearance. If
largeparticlesappear,thestandardshouldbereplaced.Latexparticlesuspensions
shouldbemixedbyinvertinggently,notonavortexmixer
6. Thebariumsulfatestandardsshouldbereplacedortheirdensitiesverifiedmonthly.

Discdiffusionmethods
The KirbyBauer and Stokes' methods are usually used for antimicrobial susceptibility
testing,withtheKirbyBauermethodbeingrecommendedbytheNCCLS.Theaccuracy
andreproducibilityofthistestaredependentonmaintainingastandardsetofproceduresas
describedhere.
NCCLS is an international, interdisciplinary, nonprofit, nongovernmental organization
composedofmedicalprofessionals,government,industry,healthcareproviders,educators
etc.Itpromotesaccurateantimicrobialsusceptibilitytesting(AST)andappropriatereporting
bydevelopingstandardreferencemethods,interpretativecriteriafortheresultsofstandard
ASTmethods,establishingqualitycontrolparametersforstandardtestmethods,provides
testingandreportingstrategiesthatareclinicallyrelevantandcosteffective
InterpretativecriteriaofNCCLSaredevelopedbasedoninternationalcollaborative
studiesandwellcorrelatedwithMICsandtheresultshavecorroboratedwithclinical
data.BasedonstudyresultsNCCLSinterpretativecriteriaarerevisedfrequently.NCCLS
isapprovedbyFDAUSAandrecommendedbyWHO.

11

ProcedureforPerformingtheDiscDiffusionTest
InoculumPreparation
GrowthMethod
Thegrowthmethodisperformedasfollows
1.

Atleastthreetofivewellisolatedcoloniesofthesamemorphologicaltypeare
selectedfromanagarplateculture.Thetopofeachcolonyistouchedwithaloop,
andthegrowthistransferredintoatubecontaining4to5mlofasuitablebroth
medium,suchastrypticsoybroth.

2.

Thebrothcultureisincubatedat35Cuntilitachievesorexceedstheturbidityofthe
0.5McFarlandstandard(usually2to6hours)

3.

Theturbidityoftheactivelygrowingbrothcultureisadjustedwithsterilesalineor
broth to obtain a turbidity optically comparable to that of the 0.5 McFarland
standard.Thisresultsinasuspensioncontainingapproximately1to2x10 8CFU/ml
forE.coliATCC25922.Toperformthisstepproperly,eitheraphotometricdevice
canbeusedor,ifdonevisually,adequatelightisneededtovisuallycomparethe
inoculum tube and the 0.5 McFarland standard against a card with a white
backgroundandcontrastingblacklines.

DirectColonySuspensionMethod
1.

Asaconvenientalternativetothegrowthmethod,theinoculumcanbepreparedby
makingadirectbrothorsalinesuspensionofisolatedcoloniesselectedfroma18to
24houragarplate(anonselectivemedium,suchasbloodagar,shouldbeused).
Thesuspensionisadjustedtomatchthe0.5McFarlandturbiditystandard,using
salineandavortexmixer.

2.

Thisapproachistherecommendedmethodfortestingthefastidious organisms,
Haemophilusspp.,N.gonorrhoeae,andstreptococci,andfortestingstaphylococci
forpotentialmethicillinoroxacillinresistance.

12

InoculationofTestPlates
1.

Optimally, within 15 minutes after adjusting the turbidity of the inoculum

suspension,asterilecottonswabisdippedintotheadjustedsuspension.Theswab
shouldberotatedseveraltimesandpressedfirmlyontheinsidewallofthetube
abovethefluidlevel.Thiswillremoveexcessinoculumfromtheswab.

2.

ThedriedsurfaceofaMellerHintonagarplateisinoculatedbystreakingtheswab
overtheentiresterileagarsurface. Thisprocedureisrepeatedbystreakingtwo
more times, rotating the plate approximately 60 each time to ensure an even
distributionofinoculum.Asafinalstep,therimoftheagarisswabbed.

3.

Thelidmaybeleftajarfor3to5minutes,butnomorethan15minutes,toallowfor
anyexcesssurfacemoisturetobeabsorbedbeforeapplyingthedrugimpregnated
disks.

NOTE:Extremesininoculumdensitymustbeavoided. Neveruseundilutedovernight
brothculturesorotherunstandardizedinoculaforstreakingplates.

ApplicationofDiscstoInoculatedAgarPlates
1.

Thepredeterminedbatteryofantimicrobialdiscsisdispensedontothesurfaceofthe
inoculatedagarplate.Eachdiscmustbepresseddowntoensurecompletecontact
withtheagarsurface.Whetherthediscsareplacedindividuallyorwithadispensing
apparatus,theymustbedistributedevenlysothattheyarenocloserthan24mm
fromcentertocenter.Ordinarily,nomorethan12discsshouldbeplacedonone150
mmplateormorethan5discsona100mmplate. Becausesomeofthedrug
diffusesalmostinstantaneously,adiscshouldnotberelocatedonceithascomeinto
contactwiththeagarsurface.Instead,placeanewdiscinanotherlocationonthe
agar.

2.

Theplatesareinvertedandplacedinanincubatorsetto35Cwithin15minutes
afterthediscsareapplied.WiththeexceptionofHaemophilusspp.,streptococciand

13

N.gonorrhoeae,theplatesshouldnotbeincubatedinanincreasedCO2atmosphere,
becausetheinterpretivestandardsweredevelopedbyusingambientairincubation,
andCO2willsignificantlyalterthesizeoftheinhibitoryzonesofsomeagents.

ReadingPlatesandInterpretingResults
1.

After 16 to 18 hours of incubation, each plate is examined. If the plate was

satisfactorily streaked, and the inoculum was correct, the resulting zones of
inhibitionwillbeuniformlycircularandtherewillbeaconfluentlawnofgrowth.If
individualcoloniesareapparent,theinoculumwastoolightandthetestmustbe
repeated. Thediameters ofthezonesofcompleteinhibition (asjudgedbythe
unaidedeye)aremeasured,includingthediameterofthedisc.Zonesaremeasured
tothenearestwholemillimeter,usingslidingcalipersoraruler,whichisheldonthe
backoftheinvertedpetriplate.Thepetriplateisheldafewinchesaboveablack,
nonreflectingbackgroundandilluminatedwithreflectedlight.Ifbloodwasadded
totheagarbase(as withstreptococci), thezones aremeasuredfromtheupper
surfaceoftheagarilluminatedwithreflectedlight,withthecoverremoved.Ifthe
testorganismisaStaphylococcusorEnterococcusspp.,24hoursofincubationare
requiredforvancomycinandoxacillin,butotheragentscanbereadat16to18
hours.Transmittedlight(platehelduptolight)isusedtoexaminetheoxacillinand
vancomycinzonesforlightgrowthofmethicillinorvancomycinresistantcolonies,
respectively,withinapparentzonesofinhibition. Anydiscernablegrowthwithin
zoneofinhibitionisindicativeofmethicillinorvancomycinresistance.

2.

Thezonemarginshouldbetakenastheareashowingnoobvious,visiblegrowth
thatcanbedetectedwiththeunaidedeye.Faintgrowthoftinycolonies,whichcan
bedetectedonlywithamagnifyinglensattheedgeofthezoneofinhibitedgrowth,
isignored. However,discretecoloniesgrowingwithinaclearzoneofinhibition
should be subcultured, reidentified, and retested. Strains of Proteus spp. may
swarmintoareasofinhibitedgrowtharoundcertainantimicrobialagents. With
14

Proteus spp.,thethinveilofswarminggrowthinanotherwiseobviouszoneof
inhibitionshouldbeignored.Whenusingbloodsupplementedmediumfortesting
streptococci,thezoneofgrowthinhibitionshouldbemeasured,notthezoneof
inhibitionofhemolysis.Withtrimethoprimandthesulfonamides,antagonistsinthe
mediummayallowsomeslightgrowth;therefore,disregardslightgrowth(20%or
lessofthelawnofgrowth),andmeasurethemoreobviousmargintodeterminethe
zonediameter.
3. ThesizesofthezonesofinhibitionareinterpretedbyreferringtoTables2Athrough
2I (Zone Diameter Interpretative Standards and equivalent Minimum Inhibitory
ConcentrationBreakpoints)oftheNCCLSM100S12:PerformanceStandardsfor
AntimicrobialSusceptibilityTesting: TwelfthInformationalSupplement,andthe
organismsarereportedaseithersusceptible,intermediate,orresistanttotheagents
thathavebeentested.Someagentsmayonlybereportedassusceptible,sinceonly
susceptiblebreakpointsaregiven.

4.2DilutionMethods
Dilutionsusceptibilitytestingmethodsareusedtodeterminetheminimalconcentration
ofantimicrobialtoinhibitorkillthemicroorganism.Thiscanbeachievedbydilutionof
antimicrobial in either agar or broth media. Antimicrobials are tested in log 2 serial
dilutions(twofold).
MinimumInhibitoryConcentration(MIC)
Diffusiontestswidelyusedtodeterminethesusceptibilityoforganismsisolatedfrom
clinical specimens have their limitations; when equivocal results are obtained or in
prolonged serious infection e.g. bacterial endocarditis, the quantitation of antibiotic
actionvisavisthepathogenneedstobemoreprecise.AlsothetermsSusceptibleand

15

Resistantcanhavearealisticinterpretation.Thuswhenindoubt,thewaytoaprecise
assessmentistodeterminetheMICoftheantibiotictotheorganismsconcerned.
TherearetwomethodsoftestingforMIC:
(a)Brothdilutionmethod
(b)Agardilutionmethod.
BrothDilutionMethod
TheBrothDilutionmethodisasimpleprocedurefortestingasmallnumberofisolates,
evensingleisolate. Ithas theaddedadvantagethatthesametubescanbetakenfor
MBCtestsalso:
Materials
Sterilegraduatedpipettesof10ml,5ml,2mland1mlSterilecapped7.5x1.3cmtubes/
smallscrewcappedbottles,Pasteurpipettes,overnightbrothcultureoftestandcontrolor
ganisms(sameasfordiscdiffusiontests),requiredantibioticinpowderform(eitherfrom
the manufacturer or standard laboratory accompanied by a statement of its activity in
mg/unit or perml.Clinical preparations shouldnotbeusedforreference technique.),
required solventfortheantibiotic,sterileDistilledWater 500mlandsuitablenutrient
brothmedium.
Trimethoprimandsulphonamidetestingrequiresthymidinefreemediaoradditionof4%
lysedhorsebloodtothemedia
Asuitableracktohold22tubesintworowsie11tubesineachrow.
Stocksolution
Stocksolutioncanbepreparedusingtheformula
1000
xVxC=W

WhereP=Potencygivenbythemanufacturerinrelationtothebase
16

V=Volumeinmlrequired
C=Finalconcentrationofsolution(multiplesof1000)
W=WeightoftheantimicrobialtobedissolvedinthevolumeV
Example:Formaking10mlsolutionofthestrength10,000mg/lfrompowderbasewhose
potencyis980mgpergram,thequantitiesoftheantimicrobialsrequiredis

W=1000
x10x10=102.04mg

980

Note:the stock solutions are made in higher concentrations to maintain their keeping
qualities and stored in suitable aliquots at 20oC .Once taken out,they should not be
refrozenorreused.
SuggesteddilutionrangesofsomeantimicrobialsareshowninAnnexureII.
Method
Preparestockdilutionsoftheantibioticofconcentrations1000and100g/Lasrequired
fromoriginalstocksolution(10,000mg/L).Arrangetworowsof12sterile7.5x1.3cm
cappedtubesintherack.Inasterile30ml(universal)screwcappedbottle,prepare8mlof
brothcontainingtheconcentrationofantibioticrequiredforthefirsttubeineachrow
fromtheappropriatestocksolutionalreadymade.Mixthecontentsoftheuniversalbottle
usingapipetteandtransfer2mltothefirsttubeineachrow.Usingafreshpipette,add4
mlofbrothtotheremaining4mlintheuniversalbottlemixandtransfer2mltothe
secondtubeineachrow.Continuepreparingdilutionsinthiswaybutwhereasmanyas
10ormorearerequiredtheseriesshouldbestartedagainhalfthewaydown.Place2ml
ofantibioticfreebrothtothelasttubeineachrow.Inoculateonerowwithonedropofan
overnight brothcultureofthetestorganismdiluted approximately to1in1000in a
suitable broth and the second row with the control organism of known sensitivity
similarly diluted. The result of the test is significantly affected by the size of the
inoculum.Thetestmixtureshouldcontain106 organism/ml.Ifthebrothcultureusedhas
grownpoorly,itmaybenecessarytousethisundiluted.Incubatetubesfor18hoursat
17

37oC.Inoculateatubecontaining2mlbrothwiththeorganismandkeepat+4oCina
refrigeratorovernighttobeusedasstandardforthedeterminationofcompleteinhibition.
Calculationsforthepreparationoftheoriginaldilution.
Thisoftenpresentsproblemstothoseunaccustomedtoperformingthesetests.Thefol
lowingmethodadvocatedbyPamelaMWaterworthispresented.Calculatethetotalvol
umerequiredforthefirstdilution.Twosetsofdilutionarebeingprepared(oneforthe
testandoneforthecontrol),eachin2mlvolumesieatotalof4mlforeachconcentra
tionas4mlisrequiredtomaketheseconddilution,thetotalrequirementis8ml.Nowcal
culate the total amount of the antibiotic required for 8ml. For 64 g/l concentration,
8x64mg/l=512gin8ml.Placeadecimalpointafterthefirstfigure(5.12)andtakethis
volumeinml(i.e5.12ml)ofthedilutionbelow512mg/landmakeupto8mlwithbroth.
Inthisexamplegivenabove,theserieshastobestartedagainmidwayat2mg/lwhich
wouldbeobtainedinthesameway:
8mlof2mg/l=16gin8ml.
1.6mlof10mg/l+6.4mlofbroth.

Readingofresult
MIC is expressed as the lowest dilution, which inhibited growth judged by lack of
turbidityinthetube.
Becauseveryfaintturbiditymaybegivenbytheinoculumitself,theinoculatedtubekept
in the refrigerator overnight may be used as the standard for the determination of
completeinhibition.
StandardstrainofknownMICvaluerunwiththetestisusedasthecontroltocheckthe
reagentsandconditions.

18

MinimumBactericidalConcentrations(MBC)
ThemainadvantageoftheBrothdilutionmethodfortheMICdeterminationliesinthe
factthatitcanreadilybeconvertedtodeterminetheMBCaswell.
Method
Dilutions and inoculations are prepared in the same manner as described for the
determination of MIC. The control tube containing no antibiotic is immediately
subcultured(Beforeincubation)byspreadingaloopfulevenlyoveraquarteroftheplate
on a medium suitable for the growth of the test organism and incubated at 37oC
overnight.Thetubesarealsoincubatedovernightat37oC.ReadtheMICofthecontrol
organismtocheckthatthedrugconcentrationsarecorrect.Notethelowestconcentration
inhibitinggrowthoftheorganismsandrecordthisastheMIC.Subculturealltubesnot
showingvisiblegrowthinthesamemanner as thecontroltubedescribedaboveand
incubateat37oCovernight.Comparetheamountofgrowthfromthecontroltubebefore
incubation,whichrepresentstheoriginalinoculum.Thetestmustincludeasecondsetof
thesamedilutionsinoculatedwithanorganismofknownsensitivity.Thesetubesarenot
subcultured;thepurposeofthecontrolistoconfirmbyitsMICthatthedruglevelis
correct,whetherornotthisorganismiskilledisimmaterial.

19

Readingofresult
Thesesubculturesmayshow

Similarnumberofcoloniesindicatingbacteriostasisonly.

Areducednumberofcoloniesindicatingapartialorslowbactericidalactivity.

Nogrowthifthewholeinoculumhasbeenkilled

Thehighestdilutionshowingatleast99%inhibitionistakenasMBC

Microbrothdilutiontest
This test uses doublestrength MellerHinton broth, 4X strength antibiotic solutions
preparedasserialtwofolddilutionsandthetestorganismataconcentrationof2x10 6/ml.In
a96wellplate,100 lofdoublestrengthMHB,50 leachoftheantibioticdilutionsand
theorganismsuspensionaremixedandincubatedat35Cfor1824hours.Thelowest
concentrationshowinginhibitionofgrowthwillbeconsideredtheMICoftheorganism.

Readingofresult
MIC is expressed as the highest dilution which inhibited growth judged by lack of
turbidityinthetube.Becauseveryfaintturbiditymaybegivenbytheinoculumitself,the
inoculatedtubekeptintherefrigeratorovernightmaybeusedasthestandardforthe
determinationofcompleteinhibition.StandardstrainofknownMIC,runwiththetestis
usedasthecontroltocheckthereagentsandconditions.

TheAgardilutionMethod
Agar dilutions are most often prepared in petri dishes and have advantage that it is
possibletotestseveralorganismsoneachplate.Ifonlyoneorganismistobetestede.g
M.tuberculosis,thedilutionscanbepreparedinagarslopesbutitwillthenbenecessaryto
prepareasecondidenticalsettobeinoculatedwiththecontrolorganism.Thedilutionsare
madeinasmallvolumeofwaterandaddedtoagarwhichhasbeenmeltedandcooledto

20

notmorethan60oC.Bloodmaybeaddedandifchocolateagarisrequired,themedium
mustbeheatedbeforetheantibioticisadded.
Itwouldbeconvenienttouse90mmdiameterpetridishesandadd
onemlofdesireddrugdilutionsto19mlofbroth.Thefactorofagardilutionmustbe
allowedforinthefirstcalculationasfollows.
finalvolumeofmediuminplate

=20ml

Topantibioticconcentrations

=64mg/l

Totalamountofdrug

= 1280g to be added to

1mlofwater

2mlof1280g/mlwillberequiredtostartthedilution

=2560gin2ml
= 1.28ml of 2000g /ml
0.72mlof

water.

1mlofthiswillbeaddedto19mlagar.
(Notestockdilutionof2000g/mlisrequiredforthisrangeofMIC)
Thequickestwaytopreparearangeofdilutionsinagarisasfollows:
Labelasterilepetridishonthebaseforeachconcentrationrequired.Preparethedilutions
inwaterplacing1mlofeachintheappropriatedish.Onemlwaterisaddedtoacontrol
plate. Pipette 19 ml melted agar, cooled to 55oC to each plate and mix thoroughly.
Adequatemixingisessentialandifsufficienttechnicalexpertiseisnotavailableforthe
skilled manipulation, it is strongly recommended that the agar is first measured into
stopperedtubesoruniversalcontainersandthedrugdilutionaddedtotheseandmixedby
inversionbeforepouringintopetridishes.Aftertheplateshavesettheyshouldbewell
driedat37oCwiththeirlidstippedfor20to30minutesinanincubator.Theyarethen
inoculatedeitherwithamultipleinoculatorasspotsorwithawirelooporaplatinum
loopcalibratedtodeliver0.001mlspreadoverasmallarea.Ineithercasetheculture
shouldbedilutedtocontain105 to106 organismsperml.Withordinaryfastgrowing
organisms, this can be obtained approximately byadding 5 l ofan overnight broth
cultureto5mlbrothorpeptonewater.

21

Itispossibletotestspreadingorganismsuchas P.mirabilis bythismethodeitherby

cuttingditchesintheagarbetweentheinocula,orbyconfiningeachwithsmallglassor
porcelain cylinders pressed into the agar. Although swarming of P.mirabilis can be
prevented by the use of higher concentration of agar in the medium, this is not
recommendedfordeterminationofMICbecauseofthedifficultyofensuringadequate
mixingofthedrugwiththisveryviscousmedium.Selectivemediashouldnotbeused
andelectrolytedeficientmediawillgivefalseresultsbecauseoftheeffectofvariationin
thesaltcontentontheactionofmanyantibiotics.
Readingofresults
Theantibioticconcentrationofthefirstplateshowing 99%inhibitionistakenastheMIC
fortheorganism.

4.3DilutionandDiffusion

22

Etestalsoknownastheepsilometertestisanexponentialgradienttestingmethodology
whereEinEtestreferstothe Greeksymbolepsilon( ).TheEtest(ABBiodisk)
whichisaquantitativemethodforantimicrobialsusceptibilitytesting appliesboththe
dilutionofantibioticanddiffusionofantibioticintothemedium..Apredefinedstable
antimicrobialgradientispresentonathininertcarrierstrip.WhenthisEteststripis
applied onto an inoculated agar plate, there is an immediate release of the drug.
Followingincubation,asymmetricalinhibitionellipseisproduced.Theintersectionof
theinhibitoryzoneedgeandthecalibratedcarrierstripindicatestheMICvalueover a
wideconcentrationrange(>10dilutions)withinherentprecisionandaccuracy.
EtestcanbeusedtodetermineMICforfastidiousorganismslikeS.pneumoniae,
hemolyticstreptococci,N.gonorrhoeae,Haemophilussp.andanaerobes.Itcanalsobe
usedforNonfermentingGramNegativebacilli(NFGNB)foregPseudomonas sp.and
Burkholderiapseudomallei.

Resistance ofmajor consequence may bedetected fore.g.,thetest is veryuseful in

detecting glycopeptide resistant Enterococci (GRE) and glycopeptide intermediate
S.aureus (GISA) and slow growing pathogens such as Mycobacterium tuberculosis.
Furtheritcanbeusedfordetectionofextendedspectrumbetalactamases(ESBL).In
conclusionEtestisasimple,accurateandreliablemethodtodeterminetheMICfora
widespectrumofinfectiousagents.

5.SusceptibilityofFastidiousBacteria
DISCDIFFUSIONFORFASTIDIOUSORGANISMS

AntibioticsusceptibilitytestingofS.pneumoniae
Mediafordiscdiffusion
MellerHintonSheepbloodagar

23

Standardizationofinoculum.
TheinoculaforseedingthesusceptibilitymediawithS.pneumoniaeispreparedfrom
fresh pure cultures (grown overnight on Chocolate agar). Cell suspensions of the
bacteria to be tested are prepared in sterile saline or MellerHinton broth. The cell
suspension is prepared by transferring a portion of the fresh growth with a swab or
inoculatinglooptothesuspendingmedium,usingcautionwhenmixingthecells
with the suspending medium so as not to form bubbles. The suspension is then
comparedtotheMcFarlandstandardbyholdingthesuspensionandMcFarlandstandard
in front of a light against a white background with contrasting black lines and
comparingtheturbidity.Iftheturbidityistooheavy,thesuspensionshouldbediluted
withadditionalsuspendingmedium.Iftheturbidityistoolightadditionalcellsshould
beaddedtothesuspension.
For S.pneumoniae Directcolonysuspensionismadeinnormalsalineand turbidity
adjustedto0.5McFarlandstandard.Within15minutesafteradjustingtheturbidityofthe
suspensiontheplateshouldbeinoculated.
Inoculationofthesusceptibilitytestmedia
Afterproperturbidityisachieved,anewsterileswab(cottonordacron)issubmergedin
thesuspension,liftedoutofthebroth,andtheexcessfluidisremovedbypressingand
rotatingtheswabagainstthewallofthetube.Theswabisthenusedtoinoculatethe
entiresurfaceofthesupplementedMellerHintonagarplatethreetimes,rotatingthe
plate60degreesbetweeneachinoculation. Theinoculumisallowedtodry(usually
takingonlyafewminutesbutnolongerthan15minutes)beforethediscsareplacedon
theplates. Thediscsshould be placedontheagarwith sterile forceps andtapped
gentlytoensuretheadherencetotheagar.Theplatescontainingthedisksareincubated
at35oCfor16to18hinaninvertedpositionina5%CO2incubator.Acandleextinction
jarmaybeusedifaCO2incubatorisnotavailable.

24

Estimatingthesusceptibilityofthestrains
Afterovernightincubation,thediameterofeachzoneofinhibitionismeasuredwitha
rulerorcalipers.Thezonesofinhibitiononthemediacontainingbloodaremeasured
fromthetopsurfaceoftheplatewiththetopremoved.Itisconvenienttousearuler
withahandleattachedforthesemeasurements,holdingtheruleroverthesurfaceofthe
diskwhenmeasuringtheinhibitionzone.Careshouldbetakennottotouchthediskor
surfaceoftheagar.Sterilizetheruleroccasionallytopreventtransmissionofbacteria.
Inallmeasurements,thezonesofinhibitionaremeasuredfromtheedgesofthelast
visiblecolonyforminggrowth.Therulershouldbepositionedacrossthecenterofthe
disctomakethesemeasurements. Theresults arerecordedinmillimeters(mm)and
interpretationofsusceptibilityisobtainedbycomparingtheresultstothestandardzone
sizes.ForS.pneumoniaethezonemeasurementisfromtopofplatewiththelidremoved.
Faintgrowthoftinycoloniesthatmayappeartofadefromthemoreobviouszoneshould
beignoredinthemeasurement.

Interpretation
EachzonesizeisinterpretedbyreferencetotheTable2G(ZoneDiameterInterpretative
StandardsandequivalentMinimumInhibitoryConcentrationBreakpointsforS.pneumoni
ae)oftheNCCLSM100S12:PerformanceStandardsforAntimicrobialSusceptibilityTest
ing:TwelfthInformationalSupplementassusceptible,intermediateandresistant.

AntibioticsusceptibilityofHaemophilusspecies

25

ThemediumofchoicefordiscdiffusiontestingofHaemophilus sp.isHaemophilusTest
Medium(HTM).MellerHintonchocolateagarisnotrecommendedforroutinetestingof
Haemophilusspp.
Initsagarform,HaemophilusTestmediumconsistsofthefollowingingredients.
*

MellerHintonagar,

15 g/mlNAD,

15 g/mlbovinehematin,and

5mg/mlyeastextract.

TomakeHTM,firstafreshhematinstocksolutionispreparedbydissolving50mgof
bovinehematinpowderin100mlof0.01mol/LNaOHwithheatandstirringuntilthe
powderisthoroughlydissolved.Thirtymlofthehematinstocksolutionareaddedto1Lof
MHAwith5gofyeastextract.Afterautoclavingandcoolingto45to50C,3mlofan
NAD stock solution (50 mg of NAD dissolved in 10 ml of distilled water and filter
sterilized)arealsoasepticallyadded.ThepHshouldbe7.2to7.4.
TestProcedure
1.

ThedirectcolonysuspensionprocedureshouldbeusedwhentestingHaemophilus
sp. Usingcoloniestakendirectlyfromanovernight(preferably20to24hour)
chocolate agar culture plate, a suspension of the test organism is prepared in
MellerHinton broth or 0.9% saline. The suspension should be adjusted to a
turbidityequivalenttoa0.5McFarlandstandardusingaphotometricdevice.This
suspensionwillcontainapproximately1to4x108CFU/ml.Caremustbeexercised
inpreparingthissuspension,becausehigherinoculumconcentrationsmayleadto
falseresistantresultswithsomelactamantibiotics,particularlywhenlactamase

26

producingstrainsofH.influenzaearetested.Within15minutesafteradjustingthe
turbidityoftheinoculumsuspension,itshouldbeusedforplateinoculation.
2.

Theprocedureforthedisctestshouldbefollowedasdescribedfornonfastidious
bacteria, except that,ingeneral,nomorethan9discsshouldbeappliedtothe
surfaceofa150mmplateornomorethan4discsona100mmplate.

3.

Platesareincubatedat35Cinanatmosphereof5%CO2for16to18hoursbefore
measuringthezonesofinhibition.

4.

Thezonemarginshouldbeconsideredastheareashowingnoobviousgrowth
visiblewiththeunaidedeye.Faintgrowthoftinycoloniesthatmayappeartofade
fromthemoreobviouszoneshouldbeignoredinthemeasurement.

ZoneDiameterInterpretiveCriteria
TheantimicrobialagentssuggestedforroutinetestingofHaemophilussp.areindicatedin
AnnexureI.EachzonesizeisinterpretedbyreferencetotheTable2E(ZoneDiameterIn
terpretativeStandardsandequivalentMinimumInhibitoryConcentrationBreakpointsfor
Haemophilussp.)oftheNCCLSM100S12:PerformanceStandardsforAntimicrobialSus
ceptibilityTesting:TwelfthInformationalSupplementassusceptible,intermediateandre
sistant.DiscdiffusiontestingofHaemophilusspp.withotheragentsisnotrecommended.

27

AntibioticsusceptibilitytestingforNeisseriagonorrhoeae
TherecommendedmediumfortestingN.gonorrhoeaeconsistsofGCagartowhicha1%
definedgrowthsupplementisaddedafterautoclaving.Cysteinefreegrowthsupplementis
notrequiredfordisctesting.Enrichedchocolateagarisnotrecommendedforsusceptibility
testingofN.gonorrhoeae.
TestProcedure
1.

Thedirectcolonysuspensionprocedureshouldbeusedwhentesting

N.gonorrhoeae.Usingcoloniestakendirectlyfromanovernightchocolateagar
culture plate, a suspension equivalent to that of the 0.5 McFarland standard is
preparedineitherMellerHintonbrothor0.9%saline. Within15minutesafter
adjusting the turbidity of the inoculum suspension, it should be used for plate
inoculation.
2.

The disc diffusion test procedure steps, as described for nonfastidious bacteria,
shouldbefollowed.Nomorethan9antimicrobialdiscsshouldbeplacedontothe
agarsurfaceofa150mmagarplatenotmorethan4discsontoa100mmplate.
However,whentestingsomeagents(e.g.,quinolones)whichproduceextremely
largezones,fewerdiscsmayneedtobetestedperplate.

3.

Theplatesareincubatedat35Cinanatmosphereof5%CO2for20to24hours
beforemeasuringthezonesofinhibition.

ZoneDiameterInterpretiveCriteria
Theantimicrobialagentssuggestedforroutinetestingof N.gonorrhoeaeareindicatedin
AnnexureI.EachzonesizeisinterpretedbyreferencetotheTable2F(ZoneDiameter
InterpretativeStandardsandequivalentMinimumInhibitoryConcentrationBreakpoints

28

forN.gonorrhoeae)oftheNCCLSM100S12:PerformanceStandardsforAntimicrobial
Susceptibility Testing: Twelfth Informational Supplement as susceptible, intermediate
and resistant. Disc diffusion testing of N. gonorrhoeae with other agents is not
recommended.
NOTE: Organisms with 10 gpenicillin disc zone diameters of< 19mm generally
producelactamase.However,lactamasetestsarefasterandarethereforepreferredfor
recognition of this plasmidmediated resistance. Organisms with plasmidmediated
resistancetotetracyclinealsohavezonesofinhibition(30 gtetracyclinediscs)of<19
mm.Chromosomalmechanismsofresistancetopenicillinandtetracyclineproducelarger
zonediameters,whichcanbeaccuratelyrecognizedusingtheinterpretivecriteriaindicated
inTable2F(ZoneDiameterInterpretativeStandardsandequivalentMinimumInhibitory
ConcentrationBreakpointsforN.gonorrhoeae)oftheNCCLSM100S12:Performance
StandardsforAntimicrobialSusceptibilityTesting:TwelfthInformationalSupplement.

DeterminationofMICforFastidiousorganisms
TheAgardilutionmethod
Standardizationofinoculum.
The inoculum should be an actively growing culture diluted in saline to 10 4 to 105
microorganismperml.
For S.pneumoniae Directcolonysuspensionfroma1215hourculturefromTSBA
mediumistobeused.Thecoloniesaresuspendedin0.5mlofnormalsalineandthe
opacityadjustedtoMcFarland0.5.A1/10dilutionofthissuspensionismadeandwithin
15minutesofmakingthedilutedsuspensionthetestplatesshouldbeinoculatedwith
eitheraplatinumloopcalibratedtodeliver0.001mlormultipointinoculator.
For N.gonorrhoeae and H. influenzae Similar to the procedure described above for
S.pneumoniae

29

Inoculationoftestplate
Ingeneraltheinoculumshouldbeappliedasaspotthatcoversacircleabout58mmin
diameter.Aplatinumloopcalibratedtodeliver0.001mloftheinoculumisusedtospot
inoculatethecultures.AppropriateATCCqualitycontrolorganism(s)shouldbeincluded
alongwitheachtest.Inoculatedplatesareleftundisturbeduntilthespotsofinoculum
havedried.

Incubation
Afterthespotsofinoculumhavedried,theplatesareincubatedat35oCfor16to18hin
aninvertedpositionina5%CO2incubator.Acandleextinctionjarmaybeusedifa
CO2incubatorisnotavailable.
Reading
ThecontrolplateshouldshowthegrowthoftheQCtestorganism.TheMICofthe
qualitycontrolstrainshouldbeintheexpectedqualitycontrolrange.Theendpointisthe
lowest concentration of antibiotic that completely inhibits growth. A barely visible
haziness or single colony shouldbe disregarded. Results are reported as the MIC in
microgramsorunits/ml.Interpretationismadeinaccordancetotheguidelineslaiddown
in the NCCLS M100S12: Performance Standards for Antimicrobial Susceptibility
Testing: Twelfth Informational Supplement (MIC Interpretative Standards) as
susceptible,intermediateandresistant.

30

6.ErrorsinInterpretationandreportingresults
Intheinterpretationoftestresultstherearepossibilitiesforerrorstooccur.Basedonimpact
oferrorsintreatmentofpatienttheyareclassifiedasminorerrors,majorerrorsandvery
majorerrors.Thisisachievedbycomparingdiskdiffusion,whichiswidelyusedtoreport
withtheMIC,whichisreferencemethod.Thefollowingflowchartshowstheerrors
inreporting.(adoptedfromManualofClinicalMicrobiology,7thedition)

Resistant

MIC
( g/ml)

MinorError

VeryMajorError

MinorError

Intermediate

MinorError

MajorError

MinorError

Susceptible

DiskDiffusionDiameter(mm)

31

7.QualityControlinAntibioticSusceptibilityTesting
QCisperformedtocheckthequalityofmedium,thepotencyoftheantibiotic,to
checkmanualerrors.Qualitycontrolstrainsshouldbeincludeddailywiththetest.Not
morethan1in20resultsshouldbeoutsideaccuracylimits.Nozoneshouldbemorethan4
standarddeviationsawayfrommidpointbetweenthestatedlimits.
If,forreasonsofexpenseormanpowerconstraints,itisnotpossibletoincludeallstrainson
adailybasis,thenthefollowingguidelinesshouldbefollowed.
Thefrequencycanbedecreasedtoonceweeklyifproficiencyhasbeendemonstratedby
1. PerformingQCdailyfor30dayswithlessthan10%inaccuracyforeachdrug
2. Proficiencytestingisrepeatedforeachnewdrugincludedinthetesting
3. Alldocumentationismaintainedindefinitely
4. Proficiencytestingisrepeatedforeachnewbatchofmediaorreagents
AlltestsmustbewithinaccuracylimitsifQCisdoneonceweekly.

Referencestrainsforqualitycontrol

EscherichiacoliATCC25922(betalactamasenegative)
EscherichiacoliATCC35218(betalactamasepositive)
StaphylococccusaureusATCC25923(betalactmasenegative,oxacillinsusceptible)
StaphylococccusaureusATCC38591(betalactmasepositive)
PseudomonasaeruginosaATCC27853(foraminoglycosides)
EnterococcusfaecalisATCC29212(forcheckingofthymidineorthyminelevelofMHA)
HaemophilusinfluenzaeATCC49766(forcephalosporins)
HaemophilusinfluenzaeATCC10211(formediumcontrol)
NeissseriagonorrheaeATCC49226

32

Stockculturesshouldbekeptat70CinBrucellabrothwith10%glycerolforupto3years.
BeforeuseasaQCstrain,thestrainshouldbesubculturedatleasttwiceandretestedfor
characteristicfeatures.WorkingculturesaremaintainedonTSAslantsat28Cforupto2
weeks.

8.StandardMethodsForTheDetectionOfAntibacterialResistance.
DetectionofOxacillin/MethicillinresistantStaphylococcusaureus(MRSA)
Strainsthatareoxacillinandmethicillinresistant,historicallytermedmethicillinresistant
S.aureus (MRSA),areresistanttoallbetalactamagents,includingcephalosporinsand
carbapenems.MRSAisolatesoftenaremultiplyresistanttocommonlyusedantimicrobial
agents, including erythromycin, clindamycin, and tetracycline. Since 1996, reports of
MRSA strains with decreased susceptibility to vancomycin (minimum inhibitory
concentration[MIC],>8 g/ml)havebeenpublished.
Glycopeptides,vancomycinandTeicoplaninaretheonlydrugofchoicefortreatmentof
severe MRSAinfections,althoughsomestrainsremainsusceptible tofluoroquinolones,
trimethoprim/sulfamethoxazole,gentamicin,orrifampin.Becauseoftherapidemergenceof
rifampin resistance, this drug should never be used as a single agent to treat MRSA
infections.
TheNationalCommitteeforClinicalLaboratoryStandards(NCCLS)hasrecommended
"Screening Test for Oxacillinresistant S. aureus and uses an agar plate containing 6
microg/mlofoxacillinandMellerHintonagarsupplementedwithNaCl(4%w/v;0.68
mol/L).Theseplatescanbestoredrefrigeratedforupto2weeks.
Theinoculumispreparedbymatchinga0.5McFarlandtube.Twomethodscanbefollowed
forinoculation:

33

1. Dilutethesupension1:100,andinoculate10microLontheplate,togetaninoculumof
104CFU.
2. Dipaswabinthesuspensionandexpressexcessfluidbypressingswabagainstthewall
ofthetube.Streakswabovera11.5incharea.

Inbothmethods,anygrowthafter24hoursincubationat35Cdenotesoxacillinresistance,
ifcontrolsaresatisfactory.
Accuratedetectionofoxacillin/methicillinresistancecanbedifficultduetothepresenceof
two subpopulations (one susceptible and the other resistant) that may coexist within a
culture.Allcellsinaculturemaycarrythegeneticinformationforresistancebutonlya
small number can express the resistance in vitro. This phenomenon is termed
heteroresistanceandoccursinstaphylococciresistanttopenicillinasestablepenicillins,such
asoxacillin.
Heteroresistanceisaproblemforclinical laboratorypersonnelbecausecells expressing
resistancemaygrowmoreslowlythanthesusceptiblepopulation.Thisiswhyisolatesbeing
testedagainstoxacillin,methicillin,ornafcillinshouldbeincubatedat35Cforafull24
hoursbeforereading.ThebreakpointsforS.aureusaredifferentfromthoseforcoagulase
negativestaphylococci(CoNS).

MICs OxacillinSusceptible

OxacillinIntermediate

OxacillinResistant

S.aureus

<2 g/ml

nointermediateMIC

MIC>4 g/ml

CoNS

<0.25 g/ml

nointermediateMIC

MIC>0.5 g/ml

34

Zonesizes

OxacillinSusceptibleOxacillinIntermediate

OxacillinResistant

S.aureus

>13mm

1112mm

<10mm

CoNS

>18mm

nointermediatezone

<17mm

Whenusedcorrectly,brothbasedandagarbasedtestsusuallycandetectMRSA.Oxacillin
screenplatescanbeusedinadditiontoroutinesusceptibilitytestmethodsorasabackup
method.Amplificationtestslikethosebasedonthepolymerasechainreaction(PCR)detect
the mecA gene. These tests confirm oxacillin/methicillin resistance caused by mecA in
Staphylococcusspecies.
DetectionofOxacillinresistantCoagulasenegativeStaphylococcussp.
Althoughthereareabout20CoNSspecies,theyoftenareconsideredtobeasinglegroup.
Some species are more resistant to commonly used antimicrobial agents than others.
Identification to species level can aid in the recognition of outbreaks and in tracking
resistancetrends.S.epidermidisisthemostcommonCoNSisolatedinclinicallaboratories.
Usually,S.epidermidis,S.haemolyticus and S.hominis aremorelikelytobemultiply
resistanttoantimicrobialagentsthanareotherCoNSspecies.However,resistancepatterns
ofCoNSmaydifferbetweenhospitalsandwards.
Oxacillinresistant CoNS isolates are resistant to all betalactam agents, including
penicillins,cephalosporins,andcarbapenems.Inaddition,oxacillinresistantCoNSisolates
are often resistant to other commonly used antimicrobial agents, so vancomycin is
frequentlythedrugofchoicefortreatmentofclinicallysignificantinfections.
Accuratedetectionofoxacillinresistancecanbedifficult.ColonysizesofCoNSareoften
smallerthanthoseofS.aureus,makinggrowthmoredifficulttoread.Inaddition,like
S.aureus,twosubpopulations(onesusceptibleandtheotherresistant)maycoexistwithina
culture. When studies were performed to evaluate oxacillin breakpoints for CoNS, the
35

currentbreakpointsforS.aureusfailedtodetectmanyCoNSthatcontainedthemecAgene.
Ingeneral,thenewbreakpointsforCoNScorrelatebetterwithmecAproductionforCoNS.

36

DetectionofExtendedSpectrumBetaLactamases(ESBLs)
ESBLs are enzymes that mediate resistance to extendedspectrum (third generation)
cephalosporins (e.g., ceftazidime, cefotaxime, and ceftriaxone) and monobactams (e.g.,
aztreonam)butdonotaffect cephamycins(e.g.,cefoxitinandcefotetan)orcarbapenems
(e.g., meropenem or imipenem). ESBLs can be difficult to detect because they have
different levels of activity against various cephalosporins. Thus, the choice of which
antimicrobialagentstotestiscritical.Forexample,oneenzymemayactivelyhydrolyze
ceftazidime, resultinginceftazidime minimum inhibitory concentrations (MICs)of256
microg/ml,buthavepooractivityoncefotaxime,producingMICsofonly4microg/ml.Ifan
ESBL is detected, all penicillins, cephalosporins, and aztreonam should be reported as
resistant,evenifinvitrotestresultsindicatesusceptibility.
Therearestandardbrothmicrodilutionanddiscdiffusionscreeningtestsusingselected
antimicrobialagents.EachK.pneumoniae,K.oxytoca,orEscherichiacoliisolateshouldbe
consideredapotentialESBLproducerifthetestresultsareasfollows:
Diskdiffusion

MICs

cefpodoxime <22mm

cefpodoxime >2 g/ml

ceftazidime

<22mm

ceftazidime

>2 g/ml

aztreonam

<27mm

aztreonam

>2 g/ml

cefotaxime

<27mm

cefotaxime

>2 g/ml

ceftriaxone

<25mm

ceftriaxone

>2 g/ml

ThesensitivityofscreeningforESBLsinentericorganismscanvarydependingonwhich
antimicrobialagentsaretested.Theuseofmorethanoneofthefiveantimicrobialagents
suggested for screening will improve the sensitivity of detection. Cefpodoxime and
ceftazidimeshowthehighestsensitivityforESBLdetection.
Phenotypic confirmation of potential ESBLproducing isolates of K. pneumoniae, K.
oxytoca,or E.coli canbedonebytestingbothcefotaximeandceftazidime,aloneandin

37

combinationwithclavulanicacid. Testingcanbeperformedbythebrothmicrodilution
methodorbydiskdiffusion.ForMICtesting,adecreaseof>3doublingdilutionsinan
MICforeithercefotaximeorceftazidimetestedincombinationwith4 g/mlclavulanic
acid,versusitsMICwhentestedalone,confirmsanESBLproducingorganism.Fordisc
diffusiontesting,a>5mmincreaseinazonediameterforeitherantimicrobialagenttested
incombinationwithclavulanicacidversusitszonewhentestedaloneconfirmsanESBL
producingorganism.

Discscanbemadebyadding10 lofa1000 g/mlstocksolutionofclavulanicacidto

cefotaximeandceftazidimediskseachdayoftesting.Infuture,commercialmanufacturers
of antimicrobial discs may produce discs containing cefotaxime and ceftazidime with
clavulanicacid.Untilcommercialdiscsareavailable,SmithKlineBeechamcanprovide
clinicallaboratorieswithclavulanicacidpowderforroutineuse.
K.pneumoniaeATCC700603(positivecontrol)andE.coliATCC25922(negativecontrol)
shouldbeusedforqualitycontrolofESBLtests.
SomeorganismswithESBLscontainotherbetalactamasesthatcanmaskESBLproduction
in the phenotypic test, resulting in a falsenegative test. These betalactamases include
AmpCsandinhibitorresistantTEMs(IRTs).HyperproductionofTEMand/orSHVbeta
lactamases in organisms with ESBLs also may cause falsenegative phenotypic
confirmatorytestresults.Currently,detectionoforganismswithmultiplebetalactamases
thatmayinterferewiththephenotypicconfirmatorytestcanonlybeaccomplishedusing
isoelectricfocusingandDNAsequencing,methodsthatarenotusuallyavailableinclinical
laboratories.
IfanisolateisconfirmedasanESBLproducerbytheNCCLSrecommendedphenotypic
confirmatory test procedure, all penicillins, cephalosporins, and aztreonam should be
reportedasresistant.Thislistdoesnotincludethecephamycins(cefotetanandcefoxitin),
which should be reported according to their routine test results. If an isolate is not
38

confirmedasanESBLproducer,currentrecommendationssuggestreportingresultsasfor
routinetesting.Donotchangeinterpretationsofpenicillins,cephalosporins,andaztreonam
forisolatesnotconfirmedasESBLs.
OtherisolatesofEnterobacteriaceae,suchasSalmonellaspeciesandProteusmirabilis,and
isolatesofPseudomonasaeruginosaalsoproduceESBLs.However,atthistime,methods
for screening and phenotypic confirmatory testing of these isolates have not been
determined.
DetectionofImipenemorMeropenemResistanceinGramnegativeOrganisms
Withinahealthcaresetting,increasesinspeciesspecificcarbapenemresistanceshouldbe
monitoredandsuddenincreasesinvestigatedtoruleoutanoutbreakofresistantorganisms
orspurioustestresults.
PublishedreportsindicatesomeresistanceinavarietyofclinicalGramnegativeorganisms,
includingPseudomonasaeruginosa,Burkholderiacepacia,Acinetobacterspecies,Proteus
species,Serratiamarcescens,Enterobacterspecies,andK.pneumoniae.Stenotrophomonas
maltophilia isolatesareintrinsicallyresistanttoimipenem.Organismscanproducemore
than one hydrolyzing enzyme and may show modifications in more than one porin,
producing highlevel resistance to the carbapenems (minimum inhibitory concentration
[MIC]>16 g/ml).Organismswithdecreasedsusceptibilityproducedbyporinchanges
aloneoftenhavelowerMICs(28microg/ml).
OrganismswithMICsnearinterpretationbreakpointshavegreaterpotentialforreporting
errors.Forexample,isolates of P.aeruginosa oftenhaveMICsthatareatornearthe
carbapenemintermediate(8 g/ml)andresistant(>16 g/ml)breakpoints.Somespecies,
suchasP.mirabilis,P.vulgaris,andMorganellamorganii,oftenhaveMICs(14 g/ml)
just below the carbapenem intermediate breakpoint of 8mg/ml. Mostother species of
Enterobacteriaceaeareverysusceptible(<0.5 g/ml).

39

Broth microdilution methods usually detect carbapenem resistance when the tests are
performed properly. However, studies have shown false resistance to imipenem in
commerciallypreparedtestpanelsduetodegradationofthedrugortoamanufacturing
problemwhereconcentrationsofimipenemweretoolow.Whenperformedproperly,disc
diffusionandagargradientdiffusionalsoareacceptablemethodsforcarbapenemtesting.
Imipenem degrades easily. Studies suggest that meropenem may be more stable than
imipenem. However, for either antimicrobial agent, storage conditions of susceptibility
panels,cards,anddiscsmustbemonitoredcarefullyandqualitycontrolresultschecked
frequently.Ifpossible,storesuppliescontainingcarbapenemsatthecoldesttemperature
range stated in the manufacturer's directions. An additional test method, such as agar
gradientdiffusion(i.e.,Etest),canbeusedtoverifyintermediateorresistantresults.
Quinolonesandresistance
The number and location of mutations affecting critical sites determine the level of
resistance.Organismsmayhavealterationsinmorethanoneenzymetargetsiteand,in
gramnegative organisms, may contain more than one porin change. Many resistant
organismshavemultipleenzymetargetsite,porin,andeffluxmutations,producinghigh
levelresistancetoquinolones.Incontrast,organismswithdecreasedsusceptibilityproduced
onlybyporinchangesusuallyhavelowerminimuminhibitoryconcentrations(MICs).
The fluoroquinolone susceptibility profile foreachclinical isolate isdetermined bythe
numberandlocationofmutationalchangesinspecificenzymetargetsites,porinproteins,
andeffluxmechanisms.Theeffectofeachmutationinanisolateisnotequivalentforall
fluoroquinolones,duetovariationsofthechemicalstructuresamongthisclassofagents.
Therefore,anorganismwithoneormoremutationsmayhaveresistantMICs/zonesizesto
onequinolonebuthaveintermediateorsusceptibleMICs/zonesizestoanotherquinolone.
Duringtherapy,thepotentialexistsforanorganismwithasingle mutationtoacquirea

40

second mutation, leading to highlevel resistance. After multiple mutations occur, an

organismisgenerallyhighlyresistanttoallquinolones.
Resistancetoquinoloneshasbeenreportedinavarietyofimportantbacterialpathogens,
including E.coli, K.pneumoniaeandotherentericorganisms; P.aeruginosa; Chlamydia
trachomatis, Mycoplasmapneumoniae; Campylobacterjejuni, B.cepacia; S.maltophilia,
N.gonorrhoeae,S.aureus(especiallyoxacillinresistantstrains),Enterococcusfaeciumand
S.pneumoniae.

41

DetectionofResistantEnterococci
Penicillin/AmpicillinResistance
Enterococcimayberesistanttopenicillinandampicillinbecauseofproductionoflow
affinity,penicillinbindingproteins(PBPs)or,lesscommonly,becauseoftheproductionof
lactamase.ThediscdiffusiontestcanaccuratelydetectisolateswithalteredPBPs,butit
willnotreliablydetectlactamaseproducingstrains. Therarelactamaseproducing
strains are detected best by using a direct, nitrocefinbased, lactamase test. Certain
penicillinampicillinresistantenterococcimaypossesshighlevelresistance(i.e.,penicillin
MICs>128 g/mlorampicillinMICs>64 g/ml).Thedisctestwillnotdifferentiate
thosewithnormalresistancefromthishighlevelresistance. Forenterococcirecovered
from blood and CSF, the laboratory should consider determining the actual MIC for
penicillin or ampicillin since E. faecium strains with normal lower level resistance
(penicillinMICs<64 g/mlandampicillin<32 g/ml)shouldbeconsideredpotentially
susceptibletosynergywithanaminoglycoside(intheabsenceofhighlevelaminoglycoside
resistance)whereasstrainswithhigherlevelresistancemayberesistanttosuchsynergy.

VancomycinResistance
Accuratedetectionofvancomycinresistantenterococcibythediscdiffusiontestrequires
thatplatesbeincubatedforafull24hours(ratherthan16to18hours)andthatanyzone
surroundingthevancomycindiscbeexaminedcarefullywithtransmittedlightforevidence
ofsmallcoloniesoralightfilmgrowingwithinthezone.Anintermediatecategoryresult
bythediscdiffusiontestshouldbeverifiedbydeterminingthevancomycinMIC.
HighlevelAminoglycosideResistance
Highlevelresistancetoaminoglycosidesisanindicationthatanenterococcalisolatewill
notbeaffectedsynergisticallybyacombinationofapenicillinorglycopeptideplusan
aminoglycoside. Special,highcontentgentamicin(120 g)andstreptomycin(300 g)

42

discscanbeusedtoscreenforthistypeofresistance. Nozoneofinhibitionindicates
resistance,andzonesof>10mmindicatealackofhighlevelresistance.Strainsthatyield
zonesof7to9mmshouldbeexaminedusingadilutionscreentest.Otheraminoglycosides
neednotbetested,becausetheiractivitiesagainstenterococciarenotsuperiortogentamicin
orstreptomycin.
AminoglycosideResistanceinEnterobacteriaceaeandPseudomonasaeruginosa
Resistance to one aminoglycoside may not predict resistance to the others. In general,
resistanceisrelativelycommonin P.aeruginosa butlesscommoninEnterobacteriaceae.
Enterobacteriaceaeresistanttogentamicinandtobramycincanbesusceptibletoamikacinor
netilmicinbecausethesedrugsarenotaffectedbymanyoftheaminoglycosidemodifying
enzymes(AMEs).Therefore,theprevalenceofamikacinresistancecanbelowerthanthe
prevalence of resistance to gentamicin and tobramycin, depending upon the resistance
mechanismspresentatahealthcarefacility.
Aminoglycosides are not clinically effective against Salmonella species and Shigella
species,althoughtheymayappearsusceptible,aminoglycosides shouldnotbetestedor
reported.

9.ApplicationofComputersinAntibacterialSusceptibilityTesting
Antimicrobialresistanceisaglobalproblem.
Emergenceofmultidrugresistancehaslimitedthetherapeuticoptions,
hencemonitoringresistanceisofparamountimportance.
Antimicrobial resistance monitoring will help to review the current status of
antimicrobialresistancelocally,nationallyandgloballyandhelpfulinminimizingthe
consequence of drug resistance, limit the emergence and spread of drug resistant
pathogens.
Thousandsoflaboratoriesaredistributedworldwideandneedtobelinkedtointegrate
dataonmostclinicallyrelevantorganismonadailybasistoobtainanaccuratepictureof

43

real resistance. For this purpose a software was developed for the management of
routinelaboratoryresultsbyWHOcalledWHONET.Thissoftwarehasfocusedondata
analysisparticularlyontheresultsofAST.

WHONET5.1isadatabasesoftwarecompatiblewith,Windows95,
Windows98,
WindowsNTandlaterversionsofWindows.Installationcapacityofthesoftwareis
9.34mb,thissoftwareisavailableinwebsite
http://www.who.int/emc/WHONET/Instructions.html.
Thisprogrammeisusefulinsupplyingcurrentguidelines,protocolstolocallaboratories,
inidentifyingtheclustersofresistantisolatesandemergingoutbreaks.UseofWHONET
software may aid the local laboratory in networking efficiently with the national
referencelaboratory,indevelopingguidelinesforuseofantimicrobialagentsandreview
the sameperiodically, in studyingthe effect ofantimicrobial control programmes on
resistantbacteria,tonetworkefficientlywithinternationalbodiessuchasWHO,NCCLS
etc.Thisisalsousedforresearchstudies.

44

10.Bibliography

DoernG.V.Susceptibilitytestsoffastidiousbacteria.ManualofClinical
Microbiology,6thedition,MurrayP.R,BaronE.J,PfallerM.A,TenoverF.C,
YolkenR,AmericanSocietyforMicrobiology,WashingtonDC,1995,P.
13421349.

IraR.Bacteriology,StandardOperativeproceduremanualformicrobiology
laboratories,NationalInstituteofBiologicals.1995,P7397

JohnD.TandJamesH.JAntimicrobialSusceptibilitytesting:General
Considerations.ManualofClinicalMicrobiology7thedition,MurrayP.R,Baron
E.J,PfallerM.A,TenoverF.C,YolkenR,AmericanSocietyforMicrobiology,
WashingtonDC,1999,P.14691473.

NationalCommitteeforClinicalLaboratoryStandards.PerformanceStandards
forantimicrobialsusceptibilitytesting.8thInformationalSupplement.M100S12.
NationalCommitteeforClinicalLaboratoryStandards,2002.Villanova,Pa.

ThruppL.D.SusceptibilityTestingofAntibioticinLiquidMedia.AntibioticsIn
LaboratoryMedcine2ndEdition,VictorLorian,WilliamsandWilkins,Balitimore.
1986;P.93150.

WaterworthP.M.Quantitativemethodsforbacterialsensitivitytesting.
Laboratorymethodsinantimicrobialchemotherapy.ReevesD.S,PhilipsI,
WilliamsJ.D,WiseR.ChurchillLivingstone,Baltimore.1978;P.3141.

KramerJ.andKirshbaumA.Effectofpaperontheperformanceassayinthe
controlofantibioticsensitivitydiscs.ApplMicrobiol1961:9;P.334336.

45

RippereR.A.Effectsofpaperontheperformanceofantibioticimpregnateddiscs.
JPharmaSci1978:67;P.367371.

LalithaM.K,.ManayaniD.J,PriyaL,JesudasonM.V,ThomasK,SteinhoffM.C.
EtestasanalternativetoconventionalMICdeterminationforsurveillanceof
drugresistantS.pneumoniae.IndianJ.MedRes.1997:106;P.500503.

MorleyD.C.Asimplemethodfortestingthesensitivityofwoundbacteriato
penicillinandsulphathiazolebyuseofimpregnatedblottingpaperdisc.J.Pathol
Bacteriol.1945:57;P.379382.

Aguidetosensitivitytesting:Reportofworkingpartyonantibioticsensitivity
testingoftheBritishSocietyforAntimicrobialChemotherapy.J.Antimicrob
Chemothrap1991;27:SupplementD,P.150.

46

AnnexureI.
GuidelinesForAntimicrobialSusceptibilityTesting.
SuggestedBatteryOfAntibioticsForSusceptibilityTesting.

Staphylococcus
sp.
Penicillin
Oxacillin
Cephalothin
Gentamicin
Netilmicin
Amikacin
Chloramphenicol
Tetracycline
Erythromycin
Cotrimoxazole
Clindamycin
Ofloxacin
Rifampicin
Vancomycin
Teicoplanin

Gram negative Streptococcus

(Enterococcus,
bacilli
Pneumococcus)
Ampicillin
Penicillin
Piperacillin
Oxacillin
Cephalothin
Cefotaxime
Ceftazidime
Gentamicin
Netilmicin
Amikacin
Chloramphenicol
Tetracycline
Cotrimoxazole
NalidixicAcid
Ciprofloxacin
Ofloxacin
Nitrofurantoin
Imipenem
Meropenem

Ampicillin
Cefotaxime
Erythromycin
Chloramphenicol
Tetracycline
Vancomycin

Haemophilussp

N.gonorrhoeae

Ampicillin
Amoxycillin/

Penicillin
Cefazolin

Clavulanicacid
Cefuroxime
Ceftriaxone
Cefotaxime
Chloramphenicol
Tetracycline
Ciprofloxacin
Erythromycin
Chloramphenicol

Note:

Thechoiceofantibioticdependsonthepatternexhibitedlocally.

Theselectionofantibioticsvariesbasedonspecimenandtheisolatesunder
consideration.

47

AnnexureII.
SuggestedAntibioticDilutionRangesForMICTesting

ANTIMICROBIALAGENT
PenicillinG
Methicillin
Naficillin/Oxacillin
Ampicillin(Gram+ve)
Ampicillin(Gramve)
Cephalosporin(Gram+ve)
CephalosporinIgeneration(Gramve)
CephalosporinII&IIIgeneration(Gramve)
CephalosporinIIIgeneration

CONCENTRATIONRANGE(g/ml)
0.01532
0.00364
0.00364
0.1532
0.13256
0.01532
0.13256
0.0364
0.13256

(Pseudomonassp.)
Vancomycin
0.01516
Amikacin
0.06128
Gentamicin/Tobramycin
0.0364
Erythromycin
0.01532
Clindamycin
0.01532
Rifampicin
0.01532
Chloramphenicol
0.06256
Tetracycline
0.06256
Trimethoprim
0.0364
Sulphamethoxazole
0.61216
Trimethoprim(UTI,Gramve)
0.564
SourceAguidetosenstivitytesting:Reportofworkingpartyonantibioticsensitivity
testingoftheBritishsocietyforAntimicrobialChemotherapy.J.AntimicrobChemotherap
1991;(27):SupplementD,150.

48

AnnexureIII.
SolventsandDiluentsforAntibiotics

49

FootNote:
a. Cephalosporinsandcephemsnotlistedaboveorinfootnotedaresolublized(un
lessthemanufacturerindicatesotherwise)inphosphatebuffer,pH6.0,0.1mol/L,
andfurtherdilutedinsteriledistilledwater.
b. The diammonium salt of moxalactam is very stable, but it is almost pure R
isomer. Maxolactam for clinical use is a 1:1 mixture of R and S isomers.
Therefore,thesaltisdissolvedin0.04mol/LHCLandallowedtoreactfor1.5to
2.0hourstoconvertittoequalpartsofbothisomers.
c. Alternatively,nitrofurantoinisdissolvedindimethylsulfoxide.
d. Thesesolventsanddiluentsareformakingstocksolutionsofantimicrobialagents
that require solvents other than water. They should be diluted further, as
necessary,inwaterorbroth.Theproductsknowntobesuitableforwatersolvents
and diluents are amikacin, azlocillin, carbencillin, cefaclor, cefemandole,
cefonicid, cefotaxime, cefoperazone, cefoxitin, ceftizoxime, ceftriaxzone,
ciprofloxacin,clindamycin,gatifloxacin(withstirring),gemifloxacin,gentamicin,
kanamycin, linezolid, mecillinam, meropenem, methicillin, metronidazole,
mezlocillin,minocyclin,moxifloxacin,nafcillin,netilmycin,oxacillin,penicillin,
piperacillin, quinupristindalfopristin, sparfloxacin, sulbactam, tazobactam,
teicoplanin,tetracyclines,tobramycin,trimethoprim (iflactate),trospectomycin
andvancomycin.
e. Anhydroussodiumcarbonateisusedataweightofexactly10%oftheceftazidme
tobeused.Thesodiumcarbonateisdissolvedinsolutioninmostoftherequired
water.Theantibioticisdissolvedinthissodiumcarbonatesolutionandwateris
addedtodesiredvolume.Thesolutionistobeusedassoonaspossible,butcanbe
storeduptosixhoursatnotmorethan25C.

50

f. These compounds are potentially toxic. Consult the material safety datasheets
(MSDS) available from the product manufacturer before using any of these
materials.
g. Usevolumeofwater,thenaddglacialaceticaciddropwiseuntildissolvednot
toexceed2.5 l/ml.

Annexure III: Reproduced with permission, from NCCLS publication

M100S12 Performance Standards for Antimicrobial Testing: Twelfth
InformationalSupplement(ISBN1562384546). Copies ofthecurrent
editionmaybeobtainedfromNCCLS,940WestValleyRoad,Suite1400,
Wayne,Pennsylvania190871898,USA.

51