MICROBIOLOGYSTANDARDOPERATINGPROCEDURE

TITLE:CULTUREOFPUS, SKIN SWABS,AND NORMALLY STERILE FLUIDS

Document no.: / Reviewed and Approved by:
Replaces document: / Date of original:14thDecember2012
Applies to: Bacteriology
Created by: / Date of revision:
Date for review: / Page1/7

------

1. Aim

Todescribetheprocessingof fluids fromnormallysterilesites(excludingcerebrospinal fluid), pus,andskin/pusswabs.

2. Principle

Therangeoforganismscausingsuperficialwoundinfectionsand superficial or deep tissueinfections/abscessesiswide.Gramstaining ofclinicalmaterialmay guideempiric antimicrobialtreatmentandaidselectionofcultureplates.Selectivemediamay be required,especially forspecimens fromsuperficialornon-sterilesites.Enrichment culturesmay berequired forspecimens fromcertainsitesandtoidentify particular pathogens(e.g.Burkholderiapseudomallei).Culture results must always beinterpreted inconjunctionwithclinical detail, especiallyfor superficial swabs.

3. Method

3.1.Specimen collection

Whereverpossibleaspecimenofpus/fluidcollected aseptically intoasterileuniversal containeris preferred overaswab.However,aswabcollectedintoasuitabletransport medium(e.g.Amie’s+/-charcoal)isacceptableifapus/fluidspecimenisnotavailable. Itisimportanttoaccurately recordtheanatomicalsiteofaswabspecimen,since culturesfromswabsofadeeppuscollectionwillrequireadifferentinterpretationto thosefromasuperficial site.

3.2.Specimen transport and storage

Specimensshouldideally bestoredand transportedinsealedplasticbags.Laboratory processing should occurassoon as possibleafter specimencollection.Specimens shouldberefrigeratedif delaysin processingover two hours are unavoidable.

3.3.Specimen processing

3.3.1.Reception

Log thespecimenintheappropriatespecimenbook/databaseandassignaspecimen number.

3.3.2.Microscopic examination

Cell count(fluids fromnormally sterilesite only)

MICROBIOLOGYSTANDARDOPERATINGPROCEDURE

TITLE:CULTUREOFPUS, SKINSWABS, ANDNORMALLYSTERILEFLUIDS

Document no.: Page2/7

------Usingacountingchamber(e.g.KovachamberorFuchs-Rosenthal)performacell countonthespecimenbeforeit has beencentrifuged.

Gramstain(allspecimens)

PrepareasmearofthespecimenandGramstain.ConsidertheneedforZN/auramine staining(mandatoryifTB is suspected).

Ifaswabis received, prepare theslideafter performingculture.

Forpus/fluidspecimens,centrifugationshouldbecarriedoutpriortoGramstaining

unlessthespecimen is frank pus or clotted:

Centrifugethespecimeninasterileconicalbottomcontainerat3,000gfor10 minutes;

Discard allbut0.5mlofthesupernatantwithasterile pipette;

Resuspendthepelletin theremaining0.5mlfluid and usethisfor preparationofa smearandinoculation ofculturemedia.

Wetprep(selectedspecimens)

Awetpreparationmayberequiredifamoebae,fungi,orparasites(e.g.paragonimus: forimage,seefaecalparasitologyMOPSOP-002) aresuspected.Placea drop of specimenontoacleandry slide,coverwithacoverslip,andexamineusingthex10 objective.

3.3.3.Culture

InoculateandincubateculturemediaasindicatedinTable1.

TITLE:CULTUREOFPUS, SKIN SWABS,AND NORMALLY STERILE FLUIDS

Document no.: Reviewed and Approved by:

Replaces document:Date of original:14thDecember2012

Applies to: BacteriologyDate of revision:

MICROBIOLOGYSTANDARDOPERATINGPROCEDURE

Created by:Date for review:Page3/7

------

Table1.Culturemedia, conditions,and target organisms

Clinical/Gram
strain / Standardmedia / Incubation / Culturesread / Targetorganism(s)
Temp(°C) / Atmosphere / Time
Swabs
Alla / Bloodagar / 35–37 / 5–10%CO2 / 48h / Daily / β-haemolyticstreptococci
Pasteurellaspecies(bitewounds)
S.aureus
Vibriospecies
Chocolateagar / 35–37 / 5–10%CO2 / 48h / Daily / Haemophilussp(cellulitisinchildren/
humanbitewounds)
MacConkeyagar / 35–37 / Air / 48h / Daily / Enterobacteriaceae
Pseudomonads
ASW
SBCT / 35–37 / Air / 5 days / Daily / B.pseudomallei
Ifyeastsseen,
burns,diabetic patient,intertrigo, paronychia / Sabouraudagar / 35–37 / Air / 5days / Daily / Fungi
If Gramstain
indicatesmixed infection(optional) / CNA-bloodagar / 35–37 / Air / 48h / Daily / β-haemolyticstreptococci
S.aureus

MICROBIOLOGYSTANDARDOPERATINGPROCEDURE

TITLE:CULTUREOFPUS, SKINSWABS, ANDNORMALLYSTERILEFLUIDS

Document no.: Page4/7

------

Pus/ Sterilefluids
All / Bloodagar / 35–37 / 5–10%CO2 / 48h / Daily / β-haemolyticstreptococci
Pasteurellaspecies
S.aureus
Vibriospecies
Chocolateagar / 35–37 / 5–10%CO2 / 48h / Daily / Haemophilussp
MacConkeyagar / 35–37 / Air / 48h / Daily / Enterobacteriaceae
Pseudomonads
Sabouraudagar / 35–37 / Air / 5days / Daily / Fungi
TSB / 35–37 / Air / 48h / Daily / Gramstainandsubcultureifturbid
If Gramstain
indicatesmixed infection(optional) / CNA-bloodagar / 35–37 / Air / 48h / Daily / β-haemolyticstreptococci
S.aureus
?Melioid/diabetic/
parotitisa / ASW
SBCT / 35–37 / Air / 5 days / Daily / B.pseudomallei
?Nocardia / Bloodagar
LJslope / 35–37 / Air / 14days / D3,D7,and
D14 / Nocardiasp
?TB / LJslope/liquid medium / 35–37 / Air / 9weeks / Weekly / M.tuberculosis

aRoutinecultureforB. Pseudomalleimaynotberequired outsideof highlyendemic areas

MICROBIOLOGYSTANDARDOPERATINGPROCEDURE

TITLE:CULTUREOFPUS, SKIN SWABS,AND NORMALLY STERILE FLUIDS

Document no.: / Reviewed and Approved by:
Replaces document: / Date of original:14thDecember2012
Applies to: Bacteriology
Created by: / Date of revision:
Date for review: / Page5/7

------

4.Interpretation

Record semi-quantitativegrowth of eachcolonytype.

4.1.Minimum levelofidentification in thelaboratory

Skin/pusswabsandpus specimens

Aeromonasspecieslevel Anaerobes"anaerobes"level Bacillus specieslevel ifpossible β-haemolytic streptococci Lancefield Grouplevel Burkholderiapseudomallei specieslevel

Coagulase-negativestaphylococci“coagulase-negative” level

Corynebacteria“diphtheroids” level (specieslevel if

diphtheriasuspected)

Eikenellacorrodensspecieslevel

Enterobacteriaceae“coliforms” level (specieslevel in pus)

Haemophilus specieslevel Neisseria specieslevel Pasteurella specieslevel

Pseudomonads“pseudomonads” level (specieslevel in pus)

S.aureusspecieslevel

S.milleri(S.anginosusgroup)Lancefieldgrouplevel

S.pneumoniaespecieslevel

Yeasts“yeasts” level (specieslevel for

Cryptococcusneoformans)

Vibriospecieslevel

Fluids fromnormally sterilesites

Anaerobes“anaerobes”level

β-haemolytic streptococciLancefieldgrouplevel Coagulase-negativestaphylococci “coagulase-negative” level Mycobacteriumsp. genus level (andrefer for speciesID) Allotherorganisms specieslevel

4.2.Antimicrobialsusceptibility testing

Allsignificantisolatesshouldhaveantimicrobialsusceptibilitiesdetermined,according toMOPSOP-004.

4.3.Reporting

Cell counts(if done).

MICROBIOLOGYSTANDARDOPERATINGPROCEDURE

TITLE:CULTUREOFPUS, SKINSWABS, ANDNORMALLYSTERILEFLUIDS

Document no.: Page6/7

------

Gramstainresults:WBC andorganismsdetected.

Wet prepresults(ifdone):presenceorabsenceofnamedorganisms(e.g.paragonimusova notseen).

Culture:

Swabs:presenceofsignificantisolates(e.g.S.aureus);nosignificantgrowth/

mixedgrowth of doubtful significancemaybeused;absence ofgrowth.

Pus: presenceofsignificantisolates orabsenceofgrowth.

Normallysterilefluids:organism(s)isolateorabsenceofgrowth.

5.Qualityassurance

Mediaandidentificationtestsshouldbequalitycontrolledaccordingtotherelevant

MOPSOP.

6.Limitations

Prior antimicrobial usemayresult innegativecultures

7.References

StandardOperatingProceduresfromLOMWRU,SMRUand AHC. Health Protection agency,UK SOPs

(

BSOP11:Investigationofskin,superficial and non-surgical woundswabs. BSOP14:Investigationof abscessesanddeep-seatedwoundinfections. BSOP26:Investigationoffluidsfrom normallysterilesites.

MICROBIOLOGYSTANDARDOPERATINGPROCEDURE

TITLE:CULTUREOFPUS, SKINSWABS, ANDNORMALLYSTERILEFLUIDS

Document no.: Page7/7

------

Safetyconsiderations

COSHHriskassessment-UniversityofOxfordCOSHHAssessmentForm
Descriptionofprocedure:
Culture of skinorwound swabs/pus /body fluids / Substancesused:
Variable,depending onorganismcultured (mayinclude Gram stainreagents;3%hydrogen peroxide (catalasetest); N,N,N',N'-tetramethyl-1,4-phenylenediamine(oxidase test); sodiumdeoxycholate(bile solubility test);bioMerieuxAPI reagents).
Quantitiesused:
Small / Frequencyof use:
Daily
Hazardsidentified:
Infection risk fromspecimens / culture plates Chemical exposure frombacterialidentification test kits / Couldaless hazardoussubstance be used instead?
No
Whatmeasureshaveyoutakentocontrolrisk?
Training ingoodlaboratory practices
Appropriate PPE (labcoat,gloves, eye protection)
Use of biosafetycabinet forreading ofplates, follow-up ofBSL-3 organisms(e.g.Bpseudomallei)
Checks oncontrolmeasures:
Observation andsupervision bysenior staff
Is healthsurveillance required?
No / Trainingrequirements:
GLP
Emergencyprocedures:
Reportallincidents tolaboratory safety officer
Eye wash forsplashes / Waste disposalprocedures:
Sharpsdiscarded intoappropriaterigid containers for incineration.
Infectiouswaste discarded intoautoclave bags or 1% Virkonsolutionprior toautoclaving andsubsequentincineration. Chemicalwaste disposed ofaccording tomanufacturer’s instructions.