Download Biosafety Manual - Collin College Faculty Website Directory

BIOSAFETYMANUALFOR
BIOSAFETYLEVEL2(BSL‐2)LABS
COLLINCOLLEGE
PrestonRidgeCampus
MICROBIOLOGY
RoomLH201|LawlerHall
DateofLastRevision:July29,2015
ThisBiosafetyManualhasbeenpreparedfortheCollinCollegelaboratorieslocatedat
thePrestonRidgeCampus.Roomnumbers,laboratorydescriptionsandlocationsof
safetyrelatedequipmentandproceduresoutlinedinthismanualarespecifictothe
PrestonRidgeCampuslaboratoryfacilities.Thismanualhasbeenadaptedfromthe
2012AppendixtotheGuidelinesforBiosafetyinTeachingLaboratoriespublishedby
theAmericanSocietyforMicrobiology(ASM).
2 | P a g e I.AuthorityforMicrobiologyLabandPrepAreaRegulations
ThePrestonRidgeCampusmicrobiologylabswillfollowtheguidelinespostedbytheU.S.
DepartmentofHealthandHumanServices,CentersforDiseaseControlandPrevention
(CDC),andNationalInstitutesofHealth(NIH).Theseguidelinesdescribeacceptable
biosafetypracticesinbiomedicalandmicrobiologicallaboratoriesandcanbefoundat:
http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm.
RoomsLH205andLH208atthePrestonRidgeCampusaremultipurposelaboratory
rooms(prepareas).BiosafetyLevel1(BSl‐1)precautionswillbefollowedduringroutine
mediaprep,autoclaving,andsubculturing.
AnytimeanagentrequiringBiosafetyLevel2(BSL‐2)containmentisinuse,biohazard
signswillbepostedonthedoorsandtheentireroomwillfollowBSL‐2practices.
Room LH201 is the only room on the Preston Ridge Campus where work with BSL‐
2 agents is permitted. Generally, work with all cultures for the microbiology program at PRC should be performed in Room LH201. SummaryofRecommendedBiosafetyLevelsforInfectiousAgents
Biosafety
Level
1
Agents
Notknowntocause
diseaseinhealthy
adults.
Practices
SafetyEquipment
(PrimaryBarriers)
StandardMicrobiologicalPractices
Nonerequired
(SMP)
Facilities
(SecondaryBarriers)
Openbench‐topsink
required
BSL‐1practices(SMP)plus:
2
Associatedwithhuman
disease,hazard=
autoinoculation,
ingestion,mucous
membraneexposure




Limitedaccess
Biohazardwarningsigns
Sharpsprecautions
Biosafetymanual
defininganyneeded
wastedecontamination
ormedicalsurveillance
policies
Primarybarriers=ClassIorII
BSCsorotherphysical
containmentdevicesusedforall
manipulationsofagentsthat
BSL‐1plusensurethatan
causesplashesoraerosolsof
autoclaveisavailable
infectiousmaterialsaswellas
PPEsuchaslaboratorycoats,
gloves,faceprotectionasneeded
BSL‐2plus:
3
BSL‐2practiceplus:
Indigenousorexotic
agentswithpotential
foraerosol
transmission;disease
mayhaveseriousor
lethalconsequences.
PrestonRidgeCampus



Controlledaccess
Decontaminationofall
waste
Decontaminationoflab
clothesbefore
laundering

Primarybarriers=ClassIorII
BSCsorotherphysical
containmentdevicesusedforall
manipulationsofagentsaswell
asPPEsuchasprotectivelab
clothing,glovesandrespiratory
protection,asneeded
BiosafetyManual(BSL2)|Fall2015



Physical
separationfrom
accesscorridors
Self‐closing,
doubledoor
access
Exhaustairnot
recirculated
Negativeairflow
intolaboratory
CollinCollege
3 | P a g e ThePrestonRidgeCampusMicrobiologyLabsarelimitedtoworkingwithorganismsrequiringup
toBiosafetyLevel2containmentonly.
OrganismsrequiringBiosafetyLevel3orBiosafetyLevel4containmentarenotpermittedto
bepurchased,storedorotherwiseacquiredbyCollinCollege.
StandardMicrobiologicalPractices,asdefinedbytheCDC’sBiosafetyinMicrobiologicaland
Biomedicallaboratories,5thEditionalongsideourcurrentmicrobiologylaboratorypracticesatthe
PrestonRidgeCampus:
CDCRecommendationsfor
StandardMicrobiologicalPractices
1.Thelaboratorysupervisormustenforcetheinstitutional
policiesthatcontrolaccesstothelaboratory.
2.Personsmustwashtheirhandsafterworkingwith
potentiallyhazardousmaterialsandbeforeleavingthe
laboratory.
3.Eating,drinking,smoking,handlingcontactlenses,
applyingcosmetics,andstoringfoodforhuman
consumptionmustnotbepermittedinlaboratoryareas.
Foodmustbestoredoutsidethelaboratoryareain
cabinetsorrefrigeratorsdesignatedandusedforthis
purpose.
4.Mouthpipettingisprohibited;mechanicalpipetting
devicesmustbeused.
5.Policiesforthesafehandlingofsharps,suchasneedles,
scalpels,pipettes,andbrokenglasswaremustbedeveloped
andimplemented.Wheneverpractical,laboratory
supervisorsshouldadoptimprovedengineeringandwork
practicecontrolsthatreduceriskofsharpsinjuries.
Precautions,includingthoselistedbelow,mustalwaysbe
takenwithsharpitems.Theseinclude:
a.Carefulmanagementofneedlesandothersharpsareof
primaryimportance.Needlesmustnotbebent,sheared,
broken,recapped,removedfromdisposablesyringes,or
otherwisemanipulatedbyhandbeforedisposal.
b.Useddisposableneedlesandsyringesmustbecarefully
placedinconvenientlylocatedpuncture‐resistant
containersusedforsharpsdisposal.
c.Non‐disposablesharpsmustbeplacedinahardwalled
containerfortransporttoaprocessingareafor
decontamination,preferablybyautoclaving.
d.Brokenglasswaremustnotbehandleddirectly.Instead,
itmustberemovedusingabrushanddustpan,tongs,or
forceps.Plasticwareshouldbesubstitutedforglassware
wheneverpossible.
PrestonRidgeCampus
CurrentPracticesatthePrestonRidgeCampus
1.Allsciencelaboratoryclassroomsandprepareasare
secureareaswithaccesscontrolledbykeypadlocks.Access
codesareonlyprovidedtoauthorizedfacultyandstaff.
Realorperceivedbreachesinsecurityarereportedtothe
CampusPoliceDepartment.
2.Handwashingisrequiredbyallstudentsfacultyand
staff.WASHyourhandswithsoapandwarmwaterbefore
leavingthelaband/orafterremovingcontaminatedgloves.
Donotwearpersonalprotectiveequipment(gloves,lab
coats,etc.)outofthelaboratoryareaunlessyouare
transportinghazardousmaterialsorwastes.
3.DONOTeat,drink,smoke/usetobaccoproducts,store
food,orapplycosmeticsinthelaboratory.Facultyandstaff
arepermittedtohavedrinksinCLOSEDcontainersatPREP
AREAcomputerworkstationsonly.Foodanddrinkarenever
permittedinanyofthelaboratoryclassrooms.
4.MOUTHPIPETTINGisstrictlyprohibited;usemechanical
pipettingdevices.
5.SeeWasteManagementPlanandBiologicalWasteTable
forcurrentpractices.
BROKENGLASS:Donothandlebrokenglasswarewithbare
hands.Disposeofallcrackedorbrokenglasswareina
puncture‐resistantcontainer–NOTtheregulartrash.
Discusswithinstructorwhatwasbrokenandwhereto
discardit.Discardbrokenglasswarecontaminatedwith
hazardouschemicalsinanappropriatelylabeledcontainer.
Seeyourinstructororamemberofthelabstaffifa
hazardouswaste/brokenglasscontainerisneeded.
Lubricateglasspiecesbeforeinsertingintocorks,stoppers,
etc.Topreventglassbreakage,insertglasswithatwisting
motion.
MedicalWasteContainers:Redplasticmedicalwaste
containersaretobeusedforanyglassorsharpobjectthat
hascomeincontactwithpotentiallybiohazardmaterial
suchasbacteria,blood,urine,salivaand/orotherhumanor
animalfluids.
BiosafetyManual(BSL2)|Fall2015
CollinCollege
4 | P a g e 6.Performallprocedurestominimizethecreationof
splashesand/oraerosols.
7.Decontaminateworksurfacesaftercompletionofwork
andafteranyspillorsplashofpotentiallyinfectious
materialwithappropriatedisinfectant.
8.Decontaminateallcultures,stocks,andotherpotentially
infectiousmaterialsbeforedisposalusinganeffective
method.Dependingonwherethedecontaminationwillbe
performed,thefollowingmethodsshouldbeusedpriorto
transport.
a.Materialstobedecontaminatedoutsideoftheimmediate
laboratorymustbeplacedinadurable,leakproof
containerandsecuredfortransport.
b.Materialstoberemovedfromthefacilityfor
decontaminationmustbepackedinaccordancewith
applicablelocal,state,andfederalregulations.
9.Asignincorporatingtheuniversalbiohazardsymbol
mustbepostedattheentrancetothelaboratorywhen
infectiousagentsarepresent.Thesignmayincludethe
nameoftheagent(s)inuse,andthenameandphone
numberofthelaboratorysupervisororotherresponsible
personnel.Agentinformationshouldbepostedin
accordancewiththeinstitutionalpolicy.
10.Aneffectiveintegratedpestmanagementprogramis
required.
11.Thelaboratorysupervisormustensurethatlaboratory
personnelreceiveappropriatetrainingregardingtheir
duties,thenecessaryprecautionstopreventexposures,
andexposureevaluationprocedures.Personnelmust
receiveannualupdatesoradditionaltrainingwhen
proceduralorpolicychangesoccur.Personalhealthstatus
mayimpactanindividual’ssusceptibilitytoinfection,
abilitytoreceiveimmunizationsorprophylactic
interventions.Therefore,alllaboratorypersonneland
particularlywomenofchildbearingageshouldbeprovided
withinformationregardingimmunecompetenceand
conditionsthatmaypredisposethemtoinfection.
Individualshavingtheseconditionsshouldbeencouraged
toself‐identifytotheinstitution’shealthcareproviderfor
appropriatecounselingandguidance.
Preparingculturesinvolumesthatexceedthecapacityofa
13mmODx100mmtesttube(approximately8milliliters)
arenotpermitted.Theaddeduseofafaceshieldis
requiredwhenworkinvolvinganticipatedaerosolsor
splashesmustbeperformed.
7.CLEANanddisinfectworksurfacesatthebeginningand
endofeverylabperiod.Keepextrabooks,bagsandclothing
indesignatedplacessoyourworkareaisuncluttered.
9.SeeWasteManagementPlanforafulldescriptionof
ourbiologicalwastemanagementpractices.Seethe
BiologicalWasteTableforbiologicalwastestream
profilesandcurrentdisposalmethods.
1)Biohazardbags(fromstepcans):bagsaresterilizedon‐
siteinaccordancewiththeprovisionsof25TAC§1.136and
disposedofassolidwaste.
2)RedSharpsContainer:regulatedmedicalwasteis
disposedofthroughacontractedvendor.
3)PipetteTray(forGLASSserologicalpipettesonly):glass
pipettesaredisinfectedandreused.
4)WhiteBuckets(fortubes):tubesandcapsareautoclaved
andreused.Sterilizedliquidmediaisdisposedofviasanitary
sewer.Sanitizedsolidmediaiscollectedinacontainer,
labeledinaccordancewiththeprovisionsof25TAC§1.136
anddisposedofassolidwaste.
5)RegularTrash:itemsthrownintotheregulartrashcans
areforuncontaminatedwasteonly.Thiswastegoestothe
dumpsters.
6)BrokenGlassBucket:uncontaminatedbrokenglassis
disposedofinapunctureresistantcontainer.
7)Biohazard+HazardousWaste:Tubesarefirstdisinfected
toremovebiohazard.Remainingwasteistreatedand
disposedofasHazardousWaste.
8)Animalwastes(carcasses,tissues,bones,skin)are
collectedforincinerationthroughacontractedvendor.
9.Asignincorporatingtheuniversalbiohazardsymbolis
postedattheentranceoftheinstructionallaboratory.
10.IPMisundertheauthority ofFacilities/Plant
Operations.
11.Alllaboratorypersonnelaretrainedannuallyinthe
followingareas:















PrestonRidgeCampus
RCRAHazardousWasteGeneratorTraining
DOTHazmatTraining
BiosafetyTraining
OrientationtoLaboratorySafety
SafetyShowersandEyeWashesintheLaboratory
FlammablesandExplosivesintheLaboratory
TheFormaldehydeStandard
ElectricalSafetyintheLaboratory
LaboratoryErgonomics
UsingMSDSsintheLaboratory
LaboratoryHoods
PreventingContaminationintheLaboratory
SafeHandlingofLaboratoryGlassware
PlanningforLaboratoryEmergencies
TheBasicsofFirstAid
BiosafetyManual(BSL2)|Fall2015
CollinCollege
5 | P a g e II.Regulations
A.Access,TrainingandResponsibilities
1.AccesstoLH201islimitedtoindividualsinvolveddirectlyinmediaprep,clean‐up,
laboratoryprep,facultyfacilitatedresearch(CASMNS),andbuildingmaintenance.CASMNS
studentsparticipatinginresearchrelatedprojectsmustcompleteadditionalsafetytraining
priortoworkinginLH201.
2.ThelabandpreproomdoorswillbeclosedwhenaBSL‐2agentisinuse.
3.Allstaffandstudentsarerequiredtoread,understand,andfollowtheseregulations
beforeworkinginLH201.
4.AllstaffandstudentsworkinginLH201willreceivetrainingfromtheirinstructoror
supervisorconcerninguseoftheequipment.
5.TheLabManagerwilltrainstaffandfacultywilltrainstudentsonaseptictechniques
appropriateforhandlingpathogenicagents.Thiswillincludethepotentialhazards
associatedwiththeworkinvolved,thenecessaryprecautionstopreventexposures,and
theexposureevaluationprocedures.
6.Laboratorystaffmembersreceiveannualupdatesoradditionaltrainingasnecessaryfor
proceduralorguidelinechanges.BiosafetyTrainingisperformedanddocumented
annuallyforallmicrobiologyfacultyandstaff.Studentsmustdemonstrateproficiencyin
BSL‐1laboratorysafetytechniquespriortobeingpermittedtoworkwithBSL‐2organisms.
Facultymembersareresponsiblefortrainingandtestingtheirstudentsinallareasof
laboratorysafetyandbiosafetypractices.
7.Laboratorypersonnelandstudentsareadvisedofspecialhazardsandarerequiredto
readandfollowinstructionsonpracticesandprocedures.
8.Anystafforstudentsfoundinviolationoftheregulationsmayhavetheiraccesstothe
laboratoryareasterminated.
9.TheLaboratoryManagerandteachingfacultyareresponsibleforseeingthatthe
consequencesofstudentorstaffactionsarerectified,includingcorrectionofdamagesand
violationsandtake‐downofexperiments.
B.Apparel
1.PersonnelenteringroomLH205orLH201willberequiredtowearclosed‐toeshoesand
havelonghairtiedback.
PrestonRidgeCampus
BiosafetyManual(BSL2)|Fall2015
CollinCollege
6 | P a g e 2.PersonnelworkinginLH201atBSL‐2mustwearlabcoatsatalltimeswhenculturesare
inuse.Thisprotectiveclothingisremovedandleftinthelaboratorybeforeleavingfornon‐
laboratoryareas(e.g.,restroom,cafeteria,library,oradministrativeoffices).
3.Glovesmustbewornwhenhandlingmicroorganismsorhazardouschemicals.Glovesare
disposedofwhencontaminatedandremovedwhenworkwithinfectiousmaterialsis
completedorwhentheintegrityofthegloveiscompromised.Glovesareplacedina
biohazardbagandautoclavedpriortodisposal.Disposableglovesarenotwashed,reused,
orusedfortouching“clean”surfaces(keyboards,telephones,etc.),andtheyshouldnotbe
wornoutsidethelab.Handsaretobewashedfollowingtheremovalofgloves.
4.Duringworkwithanylivecultures,chemicalsplashgogglesmustbewornfornormallab
proceduresinvolvingliquidculturesthatdonotgenerateasplashhazard(e.g.,proper
pipetting,spreadplates,etc.).Chemicalsplashgogglesandfaceshieldsaretobeworn
whenperformingproceduresthatmaycreateasplashhazard.Ataminimum,chemical
splashgogglesmustbewornwhenworkingwithbacterialcultures(forbothBSL‐1and
BSL‐2organisms).
5.Labcoatsshouldneverbesharedandmustalwaysstayinthelaboratory.Students
shouldstoretheirlabcoatinaziptopbagthatisthenkeptinadesignatedspaceinthe
laboratory.Attheendofthesemester,itemskeptinthelabmustbedecontaminatedbefore
studentsareallowedtotakethemhome.Ziptopbagsareavailablefromamemberofthe
labstaff.Disposablelabcoatsmustbedisposedofinaredbagandautoclaved.
6.Shortsandsleevelessshirtsareneverrecommendedforthelaboratorysetting.
C.StandardLaboratoryPractices
1.Eating,drinking,smoking,handlingcontactlenses,andapplyingcosmeticsarenot
permittedinthelab.Cellphones,tablets,andlaptopsshouldnotbeusedinLH201.Food
anddrinkforhumanconsumptionisnevertobestoredthelab.
2.Anorangebiohazardsignmustbepostedontheentrancetothelaboratorywhen
biologicalagentsareinuse.Informationtobepostedincludestheagent(s)inuse,
biohazardsymbol,biosafetylevel2,anypersonalprotectiveequipmentthatmustbeworn
inthelaboratory,andanyproceduresrequiredforexitingthelaboratory.
3.Personsmustwashtheirhandsuponenteringthelab,aftertheyfinishworkinginthe
lab,afterremovinggloves,andbeforeleavingthelaboratory.
4.WorksurfacesmustbedecontaminatedpriortobeginninganyworkinLH201,on
completionofworkorattheendofthedaywith10%bleachsolution,preparedLysol
solutionor70%ethanolsolution.Anyspillorsplashofviablematerialshouldbe
PrestonRidgeCampus
BiosafetyManual(BSL2)|Fall2015
CollinCollege
7 | P a g e decontaminatedwith25%bleachorLysolconcentrate.NOTE:Materialsthathavebeen
soakedinbleachshouldneverbeautoclaved.
5.Allproceduresareperformedcarefullytominimizethecreationofsplashesoraerosols.
6.Mouthpipettingisprohibited;mechanicalpipettingdevicesareused.
7.Needlesandsyringesmaybeusedforreconstitutingreagents.Afteruse,thesematerials
areplacedinapuncture‐proofredsharpscontainer.Donotrecapneedles.
8.Allcultures,swabs,andwastecontainersmustbedecontaminatedbeforedisposalby
autoclaving.Materialstobedecontaminatedoutsideoftheimmediatelaboratoryare
placedinadurable,leak‐proofcontainerfortransportfromthelaboratory.Allredsharps
containersaretransportedoffsitefordecontaminationandfinaldisposal.Fullredsharps
containersshouldbebroughttoRoomJ223Fforfinalpackagingpriortoshippingoffsite.
9.Microincineratorsarerecommendedfortheteachinglaboratoryduetothedangers
posedbyopenflamesinthelaboratory.Themicroincineratorreachesaninternal
temperatureof1500F,easilysterilizingloopsandneedlesinjustafewseconds.
Microincineratorsalsoeliminatethepossibilityofculturescreatingsplatteroraerosolizing
duringthesterilizationprocess.
Heat‐fixingcanbeaccomplishedbyholdingtheslideovertheopeningofthe
microincinerator.MicroincineratorsareusedintheLH205andLH201laboratoriesinstead
ofthetraditionalBunsenBurners.Therearetwoincineratorsateachlabbench.DONOT
leaveloopsorneedlesunattendedintheincineratoratanytime.AlwaysturnOFFand
UNPLUGtheincineratoratthecompletionofthelab.
D.SpecialPractices
1.LaboratoryAccess
AccesstothelaboratoryislimitedorrestrictedbytheLaboratoryManagerwhenwork
withinfectiousagentsisinprogress.Personswhoareatincreasedriskofacquiring
infection–e.g.,thosewhoareimmunocompromisedorimmunosuppressed–orforwhom
infectionmayhaveseriousconsequences,shouldconsultwiththeirphysiciantodetermine
theappropriatelevelofparticipationinthelab.Employeesthathaveamedicalcondition
thatmightputthematagreaterriskforinfectionshouldconsultwiththeirsupervisor
priortoconductinganyworkintheBSL‐2laboratory.Studentsthathaveamedical
conditionthatmightputthematagreaterriskforinfectionshouldconsultwiththeir
instructorpriortoconductinganyworkintheBSL‐2laboratory.
PrestonRidgeCampus
BiosafetyManual(BSL2)|Fall2015
CollinCollege
8 | P a g e 2.LaminarFlowHood/CleanBench
RoomLH205isequippedwithaLabconcoCleanBenchthatistobeusedforthe
preparationofmicrobiologicalmediaandothertasksnotinvolvingbiologicalagentsor
hazardouschemicals.Aspictured,themotor/blowerassemblypullsairthroughthepre‐
filtersonthetopofthebench,andflowsthroughtheHEPAfilter,whichthenflows
horizontallythroughtheworkarea.
Becauseairfromtheworkareaisdisperseddirectlyintothe
laboratory,thePurifierCleanBenchshouldneverbeusedin
conjunctionwithbiohazardousmaterial.
TheCleanBenchinLH205isrecertifiedannually.Thepre‐
filters,locatedatthetopoftheunitshouldbereplacedevery
12weeks.
UseoftheUVlightisnotpermittedsincethisunitislocated
inasharedpreparea,makingitdifficulttorestrictaccess
whentheUVlightisinuse.
ReadtheUser’sManuallocatedinLH205priortooperating
theCleanBench.Reportanyproblemswithoperationtothe
LabManager.
Followtheweekly,monthlyandquarterlyroutinemaintenanceschedulepostedontheside
oftheunit.
3.BiologicalSafetyCabinet
ThePrestonRidgecampusisnotequippedwithaBiologicalSafetyCabinet.Procedures
thatcreatethepotentialforcreatinginfectiousaerosolsorsplashesshouldbeavoided.
Preparingculturesinvolumesthatexceedthecapacityofan18mmODx150mmtesttube
(approximately35milliliters)arenotpermitted.Theaddeduseofafaceshieldisrequired
whenworkinvolvinganticipatedaerosolsorsplashesmustbeperformed.
E.TransferofMaterials
1.Culturesandpotentiallyinfectiouswastesareplacedinacontainerthatpreventsleakage
duringcollection,handling,processing,storage,andtransport.
PrestonRidgeCampus
BiosafetyManual(BSL2)|Fall2015
CollinCollege
9 | P a g e 2.Ifculturesorbiologicalwastesmustbetransferredbetweenbuildings,materialsmustbe
carriedwithinsecondarycontainmentwhileenroute.Alwaystransportwastesonacart.
F.DisposalofMaterialsandDecontamination
1.Laboratoryequipmentandworksurfacesshouldbedecontaminatedwith10%bleach,
preparedLysolsolutionor70%ethanolsolutiononaroutinebasisandafterworkwith
infectiousmaterialsisfinished.Overtspills,splashes,orothercontaminationbyinfectious
materialsshouldbedecontaminatedwith25%bleachorLysolconcentrate.Alcoholbased
disinfectantsshouldneverbeusednearanopenflameorotherheatsource.NOTE:
Materialsthathavebeensoakedinbleachshouldneverbeautoclaved.
2.Spillsandaccidentsthatresultinobviousexposurestoinfectiousmaterialsaretobe
immediatelyreportedtotheLabManager.Medicalevaluation,surveillance,andtreatment
areprovidedasappropriateandwrittenrecordsaremaintained.
3.Brokenglasswarethatdoesnotcontainliveculturesshouldbesweptupwiththebroom
anddustpananddiscardedintheglassdisposalbucket.Brokenglasswareshouldneverbe
handledwithbarehands.
4.BrokenglasswarethatcontainsliveculturesshouldbesaturatedwithLysolconcentrate.
After15minutes,thedebrisshouldbecarefullycollectedfordisposalinaredsharps
container.Smallglassfragmentsshouldbepickedupwithforceps.Papertowelsusedfor
clean‐upshouldbediscardedintheautoclavestepcanforfinalsterilizationanddisposal.
5.ThePRCMicrobiologyLabsadheretotheTexasRegulationsonMedicalWasteas
outlinedin30TAC330,SubchapterY.Autoclaveefficacytestingisperformedonaweekly
basis.
6.AllbiologicalwastethatistreatedonsiteissterilizedbyautoclavinginRoomLH205.
Eachredbagissterilizedataminimumof121Cat15psifor60minutes.Eachloadof
tubestobedecontaminatedissterilizedataminimumof121Cat15psifor60minutes.
Baggedandtubedwasteisnevertobesterilizedinthesameload.Runonebagpercycle
andruntubesseparatefrombags.
RecordKeepingandAutoclaveTesting
TheTexasCommissiononEnvironmentalQuality(TCEQ)regulatesthehandlingandon‐
sitetreatmentofallregulatedmedicalwaste(RMW).Thesewastestreamsinclude
microbiologywaste,bulkblood(volumesover100milliliters),sharpsandpathological
waste.PerTCEQregulations,thefollowingdocumentationmustbekeptforallRMWthatis
treatedon‐site.CollinCollegetreatsRMWbysteamsterilizationorchemicaldisinfection
whensteamsterilizationisnotpractical.
PrestonRidgeCampus
BiosafetyManual(BSL2)|Fall2015
CollinCollege
10 | P a g e Ageneratorofmorethan50poundspercalendarmonthofmedicalwastethattreatsallor
partofthewasteson‐siteandpersonsthattreatmedicalwastesoff‐siteshallmaintaina
writtenrecordthat,ataminimum,containsthefollowinginformationforeachbatchof
wastetreated:
(A)thedateoftreatment;
(B)theamountofwastetreated;
(C)themethod/conditionsoftreatment;
(D)thename(printed)andinitialsoftheperson(s)performingtreatment;and
(E)awrittenprocedurefortheoperationandtestingofanyequipmentusedanda
writtenprocedureforthepreparationofanychemicalsusedintreatment.
Theoperatorshalldemonstrateaminimumfourlogtenreduction(asdefinedin25TAC
'1.132(relatingtoDefinitions)onroutineperformancetestingusingappropriateBacillus
speciesbiologicalindicators(asdefinedin25TAC'1.132).Theoperatorshallconduct
testingatthefollowingintervals:
(I)forgeneratorsofmorethan50poundsbutlessthanorequalto100poundsper
month,testingshallbeconductedatleastoncepermonth;
(II)forgeneratorsofmorethan100poundsbutlessthanorequalto200pounds
permonth,testingshallbeconductedatleastbiweekly;and
(III)forgeneratorsofmorethan200poundspermonthandpersonsthattreat
medicalwastesoff‐site,testingshallbeconductedatleastweekly.
ThePrestonRidgeCampuscangenerateover150poundsofautoclavedredbagsaloneina
singlemonth.Inordertoassurevalidationtestingcompliance,autoclavetestingis
performedonaweeklybasiswithGeobacillusstearothermophilus,sporepopulation105.
Instructionsfortheuseofself‐containedbiologicalindicators:
(VWRItem#95029‐702)
http://www.namsa.com/Portals/0/Documents/SCS‐05‐technicaldatasheet.pdf
1)TobecompletedbyfulltimestaffmemberorLabManager:OnTuesdayor
Wednesdayofeachweek,labeloneunusedself‐containedbiologicalindicator
(SCBI)withthedateandwasteloadnumber.Recorddateoftest,SCBIlotnumber,
expirationdate,wasteloadnumberandusernameonAutoclaveTestLog.
PrestonRidgeCampus
BiosafetyManual(BSL2)|Fall2015
CollinCollege
11 | P a g e 2)PlacelabeledSCBIinsidetheloadtobetested.Donottesttheautoclaveonan
emptyload.Theindicatorshouldbepositionedinsidetheautoclavebagoramong
theracksinthemostinteriorpartoftheload.Securetheindicatortoastripof
autoclavetapeoralongglassrodinordertosafelyretrievethecapsuleafter
sterilization.
3)Allowcapsuletocoolsoitmaybesafelyhandled.Immediatelyfollowing
sterilizationandcooldown,sealthecapbypressingdownfirmlyuntilthemedia
ampouleiscrushed.Thisactivatestheampoule.
4)PlaceactivatedSCBIintoa55–60°Cincubatorforaminimumof24hours,maxof
30hours.
5)Tobecompletedbyasecondstaffmemberorlabmanager:Examineforgrowth
after24hours,recorddate/time,results(Color&Pass/Fail)andyourname.
Prolongedincubationmayhaveadversepositiveeffects.
6)InterpretingValidationResults:Theacidproductionassociatedwithbacterial
growthcausesashiftinthecoloroftheculturemediumfrompurpletoyellow.
Purple=Negativeforgrowth;Yellow=Positiveforgrowth*
7)Discard/Disposal:AutoclaveallactivatedSCBIsfor45minutesat121°Cpriorto
discard.SterilizedSCBIsmaybedisposedofinaredsharpscontainer.
8)StoreunusedSCBIsintheiroriginalbox,atroomtemperature.Donotfreezeor
refrigerate.
*ANYINSTANCESOFAFAILEDVALIDATIONTEST(YELLOWRESULT)MUSTBE
REPORTEDTOTHELABMANAGERIMMEDIATELY.ANYFUTHERUSEOFTHE
AUTOCLAVEFORWASTETREATMENTPURPOSESMUSTBEHALTEDUNTILTHEUNIT
CANBESERVICEDANDRE‐VALIDATED.ANYMEDIAPREPTHATISPERFORMED
BEFORETHEUNITCANBEINSPECTEDMAYBECOMPROMISED.
BiologicalWaste:Anywastethatispotentiallybiohazardous,infectiousorpathological
Biologicalwasteincludes:




humanandanimalblood,bloodproducts,bodyfluids,tissue,andbodyparts;
culturesandstocksofinfectiousagents;
dissectionwastefrompreservedorfreshspecimens;
itemssuchasgloves,papertowelsandplasticthataresoiledwithinfectious
materials.
PrestonRidgeCampus
BiosafetyManual(BSL2)|Fall2015
CollinCollege
12 | P a g e  Biologicalwastemayalsoincludesharpsmaterials(slides,scalpels,pipettes,
etc.).
PackagingandLabelingBiologicalWasteforDisposal
BiohazardCans/AutoclaveBags
RedbiohazardbagsinsideoftheBiohazardCansareusedforthecollectionofbiological
wastethatistobeautoclaved.Sharpitems(loops,swabs,slides,glasspipettes)should
neverbeplacedinanautoclavebagsincetheseitemscanpuncturethebag.Autoclavebags
areusedtocollectcontaminateddisposablepetridishes,gloves,papertowels.Sharpsof
anykindareneverpermittedinanautoclavebag.
BiohazardSharpsContainers
Theredsharpscontainerslocatedthroughoutthelaboratoryclassroomsandprepareas
aretocollectonlythefollowingitems:glassslidesandcoverslips,contaminatedswabsor
disposableloops,brokenglassthatiscontaminatedwithabiologicalorinfectiousagent,
smallglassbottles/vialsofanimalblood(10millilitersorless.Fullsharpscontainersareto
betransportedtoJ223Fforcollection.
PathologicalWasteContainers(LocatedinJ223F)
These31galloncollectionboxesarereservedspecificallyforthefollowingitems:
dissectionwaste(carcasses,skin,bone,tissue,etc.)fromallpreservedandfreshspecimens.
Dissectionwasteistobeplacedintoaredbag,sealedandthenplacedinthePathological
WasteContainerforpickupanddisposal.PathWasteContainersaredisposedofby
incinerationthroughacontractedvendor.Microbiologylabsdonotgeneratedissection
waste.
G.HygieneandHousekeeping
1.Benchtopsareimpervioustowaterandareresistanttomoderateheatandtheorganic
solvents,acids,alkalis,andbleachusedtodecontaminatetheworksurfaces.Allbenchtop
surfacesinLH205andLH201shouldbecleanedandsanitizedonadailybasis.
2.Laboratoryfurnitureiscapableofsupportinganticipatedloadinganduses.Spaces
betweenbenches,cabinets,andequipmentaretoremainaccessibleforcleaning.Chairs
usedinlaboratoryworkarecoveredwithanonporousmaterialthatcanbeeasily
decontaminated.Allseatingcoversshouldbecleanedandsanitizedonaweeklybasis.
3.SafetyDataSheets(SDS)arelocatedinLH201ontopoftheincubatornearthe
laboratoryentrance.
PrestonRidgeCampus
BiosafetyManual(BSL2)|Fall2015
CollinCollege
13 | P a g e 4.FirstaidkitsarelocatedinLH201attherearoftheroomnearthemiddlesink.
5.EyewashstationandsafetyshowerarelocatedinLH201nearthelaboratoryclassroom
door.
6.Fireextinguishersaremountedonthewallnearthelaboratoryclassroomdoorin
LH201.
7.RefrigeratorsandIncubatorsshouldbecleanedandsanitizedonamonthlybasiswitha
freshlyprepared10%bleachsolution.
H.BiologicalSpillResponse
Thefollowingbiologicalspillkitmaterialsshouldbekeptineachlaboratorywherework
withmicroorganismsisconducted.Basicmaterialsare:disinfectant(householdbleachor
Lysol I.C. Quaternary disinfectant), paper towels, nitrile gloves, autoclave bags, sharps
container,andforcepstopickupbrokenglass.
For any biological spill, first notify others in the laboratory so they will not unknowingly
spreadthecontamination.Forbiologicalorchemicalspillsofalargevolume,evacuatethe
areaandcallforoutsidehelp.
CollinPoliceDepartment:5555
CityofFriscoFireDepartment:911
Forsmall,commonlaboratoryspills:
1.Weargloves,labcoatandgoggles.Ifsplashingislikely,alsowearfaceshield.
2.Useforcepstopickupbrokenglassanddiscardintoaredsharpscontainer.Donotpickup
brokenglasswithglovedorbarehands.
3.Coverspilledmaterialwithpapertowels.
4.Carefullypour(donotspray)dilutedLysolI.C.Quaternarydisinfectant(fromspraybottlesor
carboys)ontopapertowelsinsufficientquantitytoensureeffectivemicrobialinactivation.
5.Allowa20‐minutecontactperiod.
6.Pickuppapertowelsanddisposeinbiohazardwastecontainer.
7.Re‐wipespillareawithLysoldisinfectant.Placepapertowelsinbiohazardcontainer.
8.MistareawithLysoldisinfectantorafreshlyprepared10%bleachsolution.Allowtreatedareato
airdry.Keepareafreefromtrafficuntilallsurfaceshavebeenproperlydisinfected,driedandspill
materialscleanedup.
PrestonRidgeCampus
BiosafetyManual(BSL2)|Fall2015
CollinCollege
14 | P a g e 9.Placeallcontaminatedmaterials,includinganycontaminatedPersonalProtectiveEquipment,
intobiohazardwastecontainerandautoclave.DONOTautoclavewastematerialsthathavesoaked
inbleachsolutions.
10.Washhandswithsoapandwater.
I.ExposuresintheLaboratory
AllsuspectedorknownexposuresmustbeappropriatelydocumentedonanIncidentReportform
andsubmittedtotheLabManagerpromptlyaftertheincident.
BiologicalExposure
Removeanycontaminatedclothingorjewelryandwashtheskinexposedtotheagentwith
antibacterialsoap.
Autoclaveanycontaminatedclothingbeforedisposalorfurtherlaundering.Decontaminateany
surfacesbysprayingtheareawithLysoldisinfectantorfreshlyprepared10%bleachsolution.
Ifyoubelieveyoumayhavebeenexposedtoabiologicalagent,notifyyourinstructorortheLab
Managerandseekimmediatemedicalattention.
Ifyoususpectyouhavealab‐acquiredillnessregardlessofarecentexposure,seekmedical
attention.
ChemicalExposure
Formostchemicalexposurestotheskin,removeanycontaminatedclothingandwashthechemical
offwithwaterforaminimumof15minutes.Safetyshowersshouldbeutilizedintheeventoflarger
exposures.
J.MicrobiologyCultureList(BIOL2420/2421)
BiosafetyLevel1
Alcaligenesfaecalis
Bacillusbrevis
Bacilluscoagulans
Bacilluslicheniformis
Bacillusmegaterium
Bacilluspolymyxa
Bacillussphaericus
Bacillusstearothermophilus
Bacillussubtilis
Citrobacterfreundii
Clostridiumsporogenes
Enterobacteraerogenes
Enterobactercloacae
Escherichiacoli
Flavobacteriumcapsulatum
Klebsiellaoxytoca
Micrococcusluteus
Mycobacteriumsmegmatis
Providenciaalcalifaciens
Pseudomonasfluorescens
Pseudomonasputida
Saccharomycescerevesiae
Serratiamarcescens
Staphylococcusepidermidis
Staphylococcusintermedius
Staphylococcussaprophyticus
Staphylococcusxylosus
Streptococcusbovis
Streptococcusmutans
BiosafetyLevel2
Bacilluscereus
Edwardsiellatarda
Enterococcusfaecalis
Klebsiellapneumonia
Morganellamorganii
Proteusmirabilis
Proteusvulgaris
Pseudomonasaeruginosa
Shigellaboydii
Staphylococcusaureus
Streptococcusagalactiae
Streptococcuspyogenes
Streptococcusequi
Streptococcusequisubspecies
zooepidemicus
PrestonRidgeCampus
BiosafetyManual(BSL2)|Fall2015
CollinCollege
15 | P a g e Resources
BiosafetyinMicrobiologicalandBiomedicalLaboratories(BMBL)5thEdition
http://www.cdc.gov/biosafety/publications/bmbl5/index.htm
GuidelinesforBiosafetyinTeachingLaboratories
http://www.asm.org/images/asm_biosafety_guidelines‐FINAL.pdf
AppendixtotheGuidelinesforBiosafetyinTeachingLaboratories
http://www.asm.org/images/Education/FINAL_Biosafety_Guidelines_Appendix_Only.pdf
MicrobiologicalandChemicalExposureAssessment
http://www.epa.gov/nerlcwww/microbiology.html
LaboratoryBiosafetyManual‐ThirdEdition
http://www.who.int/csr/resources/publications/biosafety/WHO_CDS_CSR_LYO_2004_11/
en/
CultureSelection
http://www.atcc.org/
LaboratoryTrainingResources
LaboratoryBiosafetyLevels(FOCUS,Volume5.1)
http://cdc.train.org/DesktopModules/eLearning/CourseDetails/CourseDetailsForm.aspx?
courseId=1030020
CoreMicrobiologySkills
http://cdc.train.org/DesktopModules/eLearning/CourseDetails/CourseDetailsForm.aspx?
courseId=1035232
RoutineMicroscopy
http://cdc.train.org/DesktopModules/eLearning/CourseDetails/CourseDetailsForm.aspx?
courseId=1046095
AutoclaveSafetyhttp://www.youtube.com/watch?v=T901F2W7wks
PrestonRidgeCampus
BiosafetyManual(BSL2)|Fall2015
CollinCollege