1. Biosafety Level 3
BiosafetyLevel 3 is applicable to clinical,diagnostic,teaching,research,or production facilitieswhere
work is performedwith indigenousorexoticagents that may cause seriousor potentiallylethal
disease through the inhalationroute of exposure.Laboratory personnel mustreceive specifictraining
in handlingpathogenicand potentiallylethal agents,and must be supervisedbyscientistscompetent
in handlinginfectiousagentsand associated procedures.
All proceduresinvolvingthe manipulationof infectiousmaterialsmustbe conductedwithinBSCsor
otherphysical containmentdevices.
A BSL-3 laboratoryhas special engineeringanddesignfeatures.
The followingstandard and special safety practices,equipment,and facilityrequirementsapplyto
BSL-3.
A. Standard Microbiological Practices
1. The laboratorysupervisormustenforce the institutional policiesthatcontrol accesstothe
laboratory.
2. Personsmustwashtheirhandsafterworkingwithpotentiallyhazardousmaterialsandbefore
leavingthe laboratory.
3. Eating,drinking,smoking,handlingcontactlenses,applyingcosmetics,andstoringfoodfor
humanconsumptionmustnotbe permittedinlaboratoryareas.Foodmustbe storedoutside
the laboratoryarea incabinetsor refrigeratorsdesignatedandusedforthispurpose.
4. Mouth pipettingisprohibited;mechanicalpipettingdevicesmustbe used.
5. Policiesforthe safe handlingof sharps,suchas needles,scalpels,pipettes,andbrokenglassware
mustbe developedand implemented.Wheneverpractical,laboratorysupervisorsshouldadopt
improvedengineeringandworkpractice controlsthat reduce riskof sharpsinjuries.
Precautions,includingthose listedbelow,must always be takenwith sharp items.These include:
a. Careful managementof needlesandothersharpsare of primaryimportance.Needles
mustnot be bent,sheared,broken,recapped,removedfromdisposablesyringes,or
otherwise manipulatedbyhandbefore disposal.
b. Useddisposable needlesandsyringesmustbe carefullyplacedinconvenientlylocated
puncture-resistantcontainersusedforsharpsdisposal.
c. Non-disposablesharpsmustbe placedina hardwalledcontainerfortransporttoa
processingareafordecontamination,preferablybyautoclaving.
d. Brokenglassware mustnotbe handleddirectly.Instead,itmustbe removedusinga
brushand dustpan,tongs,or forceps.Plasticware shouldbe substitutedforglassware
wheneverpossible.
6. Performall procedurestominimize the creationof splashesand/oraerosols.

2. 1. Decontaminate worksurfacesaftercompletionof workandafterany spill orsplashof
potentiallyinfectiousmaterialwithappropriatedisinfectant.
2. Decontaminate all cultures, stocks,andotherpotentiallyinfectiousmaterialsbefore
disposal usinganeffective method.A methodfordecontaminatingall laboratorywastes
shouldbe available inthe facility,preferablywithinthe laboratory(e.g.,autoclave,
chemical disinfection, incineration,orothervalidateddecontaminationmethod).
Dependingonwhere the decontaminationwill be performed,the followingmethods
shouldbe usedpriorto transport:
a.Materialsto be decontaminatedoutside of the immediate laboratorymustbe
placedina durable,leakproof containerandsecuredfortransport.
b.Materialsto be removedfromthe facilityfordecontaminationmustbe packed
inaccordance withapplicable local,state,andfederal regulations.
3. A signincorporatingthe universalbiohazardsymbol mustbe postedatthe entrance to
the laboratorywheninfectiousagentsare present.Postedinformationmustincludethe
laboratory’sbiosafetylevel,the supervisor’sname (orotherresponsiblepersonnel),
telephonenumber,andrequiredproceduresfor enteringandexitingthe laboratory.
Agentinformationshouldbe postedinaccordance withthe institutional policy.
4. An effective integratedpestmanagementprogramisrequired.(See AppendixG.)
5. The laboratorysupervisormustensure thatlaboratorypersonnel receiveappropriate
trainingregardingtheirduties,the necessaryprecautionstopreventexposures,and
exposure evaluationprocedures.Personnel mustreceive annual updatesoradditional
trainingwhenprocedural orpolicychangesoccur.Personal healthstatusmayimpactan
individual’ssusceptibilitytoinfection,abilitytoreceive immunizationsorprophylactic
interventions.Therefore,all laboratorypersonnel andparticularlywomenof
childbearingage shouldbe providedwithinformationregardingimmune competence
and conditionsthatmaypredispose themtoinfection.Individualshavingthese
conditionsshouldbe encouragedtoself-identifytothe institution’shealthcare provider
for appropriate counselingandguidance.
B. Special Practices
1. All personsenteringthe laboratorymustbe advisedof the potential hazardsandmeetspecific
entry/exitrequirements.
2. Laboratory personnel mustbe providedmedical surveillance andofferedappropriate
immunizationsforagentshandledorpotentiallypresentinthe laboratory.
3. Each institutionshouldconsiderthe needforcollectionandstorage of serumsamplesfromat-
riskpersonnel.
4. A laboratory-specificbiosafetymanual mustbe preparedandadoptedaspolicy.The biosafety
manual mustbe available andaccessible.
5. The laboratorysupervisormustensure thatlaboratorypersonnel demonstrateproficiencyin
standardand special microbiological practicesbefore workingwithBSL-3agents.

3. 6. Potentiallyinfectiousmaterialsmustbe placedinadurable,leakproof container during
collection,handling,processing,storage,ortransportwithinafacility.
7. Laboratory equipmentshouldbe routinelydecontaminated,aswell as,afterspills,splashes,or
otherpotential contamination.
a. Spillsinvolvinginfectiousmaterialsmustbe contained,decontaminated,andcleanedup
by staff properlytrainedandequippedtoworkwithinfectiousmaterial.
b. Equipmentmustbe decontaminatedbefore repair,maintenance,orremoval fromthe
laboratory.
8. 8. Incidentsthatmay resultinexposure toinfectiousmaterialsmustbe immediatelyevaluated
and treatedaccordingto proceduresdescribedinthe laboratorybiosafetymanual.All such
incidentsmustbe reportedtothe laboratorysupervisor.Medical evaluation,surveillance,and
treatmentshouldbe providedandappropriate recordsmaintained.
9. 9. Animalsandplantsnotassociatedwiththe workbeingperformedmustnotbe permittedin
the laboratory.
10. 10. All proceduresinvolvingthe manipulationof infectiousmaterialsmustbe conductedwithin
a BSC, or otherphysical containmentdevices.Noworkwithopenvesselsisconductedonthe
bench.Whena procedure cannotbe performedwithinaBSC,a combinationof personal
protective equipmentandothercontainmentdevices,suchasa centrifuge safetycuporsealed
rotor mustbe used.
C. Safety Equipment (Primary Barriers and Personal Protective Equipment)
1. All proceduresinvolvingthe manipulationof infectiousmaterialsmustbe conductedwithina
BSC (preferablyClassII or Class III),or other physical containmentdevices.
2. Workersinthe laboratorywhere protectivelaboratoryclothingwithasolid-front,suchastie-
back or wrap-aroundgowns,scrubsuits,orcoveralls.Protectiveclothingisnotwornoutside of
the laboratory.Reusable clothingisdecontaminatedbeforebeinglaundered.Clothingis
changedwhencontaminated.
3. Eye and face protection(goggles,mask,face shieldorothersplashguard) isusedforanticipated
splashesorspraysof infectiousorotherhazardousmaterials.Eye andface protectionmustbe
disposedof withothercontaminatedlaboratorywaste ordecontaminatedbefore reuse.
Personswhowearcontact lensesinlaboratoriesmustalsoweareye protection.
4. Glovesmustbe wornto protect handsfromexposure tohazardousmaterials.Glove selection
shouldbe basedonan appropriate riskassessment.Alternativestolatex glovesshouldbe
available.Glovesmustnotbe wornoutside the laboratory.Inaddition,BSL-3laboratory
workers:
a. Changesgloveswhencontaminated,glove integrityiscompromised,orwhenotherwise
necessary.Weartwopairsof gloveswhenappropriate.
b. Remove glovesandwashhandswhenworkwithhazardousmaterialshasbeen
completedandbefore leavingthe laboratory.

4. c. Do not washor reuse disposablegloves.Dispose of usedgloveswithother
contaminatedlaboratorywaste.Handwashingprotocolsmustbe rigorouslyfollowed.
5. Eye,face,and respiratoryprotectionmustbe usedinroomscontaininginfectedanimals.
D. Laboratory Facilities (Secondary Barriers)
1. Laboratory doors must be self-closing and have locks in accordance with the institutional
policies.The laboratorymustbe separatedfromareasthat are open to unrestricted traffic flow
withinthe building.Laboratoryaccessisrestricted.Accesstothe laboratory is through two self-
closingdoors.A clothingchange room(anteroom) maybe includedinthe passageway between
the two self-closing doors.
2. Laboratories must have a sink for hand washing. The sink must be hands-free or automatically
operated.Itshouldbe located near the exit door. If the laboratory is segregated into different
laboratories,asinkmustalsobe available for hand washing in each zone. Additional sinks may
be required as determined by the risk assessment.
3. The laboratorymust be designed so that it can be easily cleaned and decontaminated. Carpets
and rugs are not permitted.Seams,floors, walls, and ceiling surfaces should be sealed. Spaces
around doors and ventilation openings should be capable of being sealed to facilitate space
decontamination.
a. Floors must be slip resistant, impervious to liquids, and resistant to chemicals.
Considerationshouldbe giventothe installationof seamless,sealed,resilientor poured
floors, with integral cove bases.
b. Walls should be constructed to produce a sealed smooth finish that can be easily
cleaned and decontaminated.
c. Ceilings should be constructed, sealed, and finished in the same general manner as
walls.
d. Decontamination of the entire laboratory should be considered when there has been
gross contamination of the space, significant changes in laboratory usage, for major
renovations, or maintenance shut downs. Selection of the appropriate materials and
methodsusedtodecontaminate the laboratory must be based on the risk assessment.
4. Laboratory furniture mustbe capable of supportinganticipatedloadsanduses.Spacesbetween
benches, cabinets, and equipment must be accessible for cleaning.
a. Benchtops mustbe impervioustowaterandresistanttoheat,organicsolvents,acids,
alkalis,andotherchemicals.
b. Chairsusedin laboratoryworkmust be coveredwitha non-porousmaterialthatcan be
easilycleanedanddecontaminatedwithappropriate disinfectant.
5. All windows in the laboratory must be sealed.
6. BSCs mustbe installedsothat fluctuations of the room air supply and exhaust do not interfere
with proper operations. BSCs should be located away from doors, heavily traveled laboratory
areas, and other possible airflow disruptions.

5. 7. Vacuumlinesmustbe protectedwithHEPA filters,ortheirequivalent. Filters must be replaced
as needed. Liquid disinfectant traps may be required.
8. An eyewash station must be readily available in the laboratory.
9. A ducted air ventilation system is required. This system must provide sustained directional
airflowbydrawingairintothe laboratoryfrom“clean” areastoward“potentiallycontaminated”
areas.The laboratoryshall be designedsuchthatunderfailure conditionsthe airflow will not be
reversed.
a. Laboratory personnel must be able to verify directional airflow. A visual monitoring
device, which confirms directional airflow, must be provided at the laboratory entry.
Audible alarms should be considered to notify personnel of air flow disruption.
b. The laboratory exhaust air must not re-circulate to any other area of the building.
c. The laboratorybuildingexhaust air should be dispersed away from occupied areas and
from building air intake locations or the exhaust air must be HEPA filtered.
10. HEPA filter housings should have gas-tight isolation dampers, decontamination ports, and/or
bag-in/bag-out (with appropriate decontamination procedures) capability. The HEPA filter
housing should allow for leak testing of each filter and assembly. The filters and the housing
should be certified at least annually.
11. HEPA filtered exhaust air from a Class II BSC can be safely re-circulated into the laboratory
environment if the cabinet is tested and certified at least annually and operated according to
manufacturer’srecommendations.BSCscanalsobe connectedtothe laboratoryexhaustsystem
by either a thimble (canopy) connection or directly exhausted to the outside through a hard
connection. Provisions to assure proper safety cabinet performance and air system operation
mustbe verified.BSCsshouldbe certifiedatleastannuallyto assure correct performance. Class
III BSCs must be directly (hard) connected up through the second exhaust HEPA filter of the
cabinet.Supplyairmust be provided in such a manner that prevents positive pressurization of
the cabinet.
12. A method for decontaminating all laboratory wastes should be available in the facility,
preferably within the laboratory (e.g., autoclave, chemical disinfection, or other validated
decontamination method).
13. Equipmentthatmay produce infectious aerosols must be contained in primary barrier devices
that exhaustairthroughHEPA filtrationorotherequivalenttechnologybefore being discharged
into the laboratory. These HEPA filters should be tested and/or replaced at least annually.
14. Facility design consideration should be given to means of decontaminating large pieces of
equipment before removal from the laboratory.
15. Enhanced environmental and personal protection may be required by the agent summary
statement,risk assessment, or applicable local, state, or federal regulations. These laboratory
enhancementsmayinclude,for example, one or more of the following: an anteroom for clean
storage of equipmentandsupplieswithdress-in, shower-out capabilities; gas tight dampers to
facilitate laboratory isolation; final HEPA filtration of the laboratory exhaust air; laboratory
effluent decontamination; and advanced access control devices, such as biometrics.

6. 16. The BSL-3 facility design, operational parameters, and procedures must be verified and
documentedpriortooperation.Facilitiesmustbe re-verifiedanddocumentedatleast annually.
 Reference
 Book Name
 Biosafety in Microbiologicaland Biomedical Laboratories
5th Edition
 From Page No 38 to page 45