Biosafety Practices and Procedures

Hierarchy of Controls

Administrative ControlAdministrative Control Engineering ControlEngineering Control Work PracticesWork Practices Personal Protective Clothing or Equipment Personal Protective Clothing or Equipment

Administrative Controls SubstitutionSubstitution Authorization/ApprovalAuthorization/Approval Written biosafety procedures required for the Written biosafety procedures required for the

experimental procedures and equipment including experimental procedures and equipment including inventory of biological agents or materialsinventory of biological agents or materials

Laboratory personnel biosafety trainingLaboratory personnel biosafety training Medical Surveillance (BSL 2 and above)Medical Surveillance (BSL 2 and above)

Health historyHealth history Medical screeningMedical screening ImmunizationImmunization Serum storageSerum storage Post-exposure prophylaxisPost-exposure prophylaxis

Engineering Controls

Biological safety cabinets, glove boxesBiological safety cabinets, glove boxes Animal containment caging systemsAnimal containment caging systems Safety equipment (filtered or sealed Safety equipment (filtered or sealed

equipment)equipment) Ventilation systemVentilation system Containment facilitiesContainment facilities

Personal Protective Clothing and Equipment

Provides barrier against skin, mucous Provides barrier against skin, mucous membrane or respiratory exposure to membrane or respiratory exposure to infectious agents during proceduresinfectious agents during procedures

Prevent spread of contaminationPrevent spread of contamination Does not eliminate the hazardDoes not eliminate the hazard Integrity wanes with use (i.e., change Integrity wanes with use (i.e., change

gloves frequently)gloves frequently)

BSL 1: Work Practices and Procedures ApplicationsApplications

Non-infectious agent and tissue culture, media Non-infectious agent and tissue culture, media preparationpreparation

Prevent Cross ContaminationPrevent Cross Contamination Keep cultures coveredKeep cultures covered Flame instruments and containersFlame instruments and containers Use sterile media and equipmentUse sterile media and equipment Keep hands or face away from culturesKeep hands or face away from cultures

Biosafety ProceduresBiosafety Procedures Work with agents may be conducted on open Work with agents may be conducted on open

benchbench Wash hands oftenWash hands often No mouth pipettingNo mouth pipetting No eating or drinking in labNo eating or drinking in lab Minimize aerosol generationMinimize aerosol generation Decontaminate work surfacesDecontaminate work surfaces Wear applicable PPEWear applicable PPE

BSL 1: Work Practices and BSL 1: Work Practices and ProceduresProcedures

BSL 2: Work Practices and Procedures Increasing emphasis on safety Increasing emphasis on safety

procedures and practicesprocedures and practices Increasing need for staff trainingIncreasing need for staff training Increasing need for competent Increasing need for competent

supervisionsupervision Biohazard sign posted at entry doorBiohazard sign posted at entry door Biohazard labels affixed on regulated Biohazard labels affixed on regulated

waste containerswaste containers Use of personal protective equipment Use of personal protective equipment

as a barrier to exposure: lab coat, as a barrier to exposure: lab coat, gloves, eye and face protectiongloves, eye and face protection

Some work on open bench allowedSome work on open bench allowed

BSL 2: Work Practices and Procedures Aerosol generating procedures Aerosol generating procedures

performed in a biosafety cabinet:performed in a biosafety cabinet: HomogenizingHomogenizing VortexingVortexing Vigorous mixingVigorous mixing Pipetting infectious liquidsPipetting infectious liquids SonicationSonication PouringPouring

If breach occurs:If breach occurs: Evacuate lab, post spill signEvacuate lab, post spill sign With appropriate PPE and With appropriate PPE and

disinfectant, decontaminate disinfectant, decontaminate centrifuge, buckets, other centrifuge, buckets, other items or areasitems or areas

Correct Use of Biosafety Cabinets Start UpStart Up

Turn off ultraviolet light (if so Turn off ultraviolet light (if so equipped) as soon as you enter the equipped) as soon as you enter the room. room.

Turn on all blowers and BSC Turn on all blowers and BSC illumination lights. illumination lights.

Allow five minutes of operation to Allow five minutes of operation to purge system; check flow alarm purge system; check flow alarm system audio and visual alarm system audio and visual alarm function (if so equipped).function (if so equipped).

Decontaminate readily accessible Decontaminate readily accessible interior surfaces and items with a interior surfaces and items with a disinfectant (appropriate for the disinfectant (appropriate for the agents or suspected agents present) agents or suspected agents present) before loading and wait at least 10 before loading and wait at least 10 minutes prior to start of work.minutes prior to start of work.

Correct Use of Biosafety Cabinets During UseDuring Use

Load supplies prior to Load supplies prior to work. work.

Do not overload cabinet. Do not overload cabinet. Separate clean and dirty Separate clean and dirty

side.side. Work in center of work Work in center of work

area. area. Do not block front or rear Do not block front or rear

grills.grills. Minimize disruption of Minimize disruption of

airflow (turbulence).airflow (turbulence). Clean up spill promptly.Clean up spill promptly. Discard waste within the Discard waste within the

cabinet.cabinet.

Correct Use of Biosafety Cabinets Shut DownShut Down

Decontaminate and remove all Decontaminate and remove all items from interior work area. items from interior work area.

Decontaminate readily Decontaminate readily accessible interior surfaces with accessible interior surfaces with a disinfectant appropriate for a disinfectant appropriate for the agents or suspected agents the agents or suspected agents present.present.

Turn on ultraviolet light (if so Turn on ultraviolet light (if so equipped). equipped).

Allow five minutes of Allow five minutes of operation to purge system. operation to purge system. Then wait at least 10 Then wait at least 10 minutes.minutes.

Turn off BSC blower.Turn off BSC blower.

Correct Use of Biosafety Cabinets Moving/InstallationMoving/Installation

BSCs must be decontaminated prior to moving. In order to BSCs must be decontaminated prior to moving. In order to ensure filter integrity, the equipment must be recertified after the ensure filter integrity, the equipment must be recertified after the BSC is installed at its final new location. Arrangements need to BSC is installed at its final new location. Arrangements need to be made well in advance in order for contractors to meet your be made well in advance in order for contractors to meet your schedule. The PI is responsible for contacting the contractor or to schedule. The PI is responsible for contacting the contractor or to schedule this work.schedule this work.

DecontaminationDecontaminationDecontamination is usually performed by certified professionals. Decontamination is usually performed by certified professionals.

CertificationCertificationAll BSCs that are used for handling biohazardous materials must All BSCs that are used for handling biohazardous materials must be recertified annually. SDSU has contracted with a specific be recertified annually. SDSU has contracted with a specific contractor to provide a consistent level of certification and contractor to provide a consistent level of certification and maintenance service. Contact EHS at (619) 594-6778 to obtain maintenance service. Contact EHS at (619) 594-6778 to obtain contractor information.contractor information.

DO NOT use Bunsen burners or open flamesDO NOT use Bunsen burners or open flames Fire hazardFire hazard Can damage HEPA filterCan damage HEPA filter Interferes with proper air flowInterferes with proper air flow Microincinerator preferredMicroincinerator preferred Burner with pilot light not a good alternative Burner with pilot light not a good alternative Open flames react with disinfectants (flammables)Open flames react with disinfectants (flammables)

Correct Use of Biosafety Cabinets – Open Flames

Use sealed tubes, rotors, and safety cups/buckets that are sealed with Use sealed tubes, rotors, and safety cups/buckets that are sealed with O-ringsO-rings

Inspect tubes, O-rings and rotors for wear, and buckets for cracks, Inspect tubes, O-rings and rotors for wear, and buckets for cracks, chips, erosion, etc.chips, erosion, etc.

Do not use aluminum foil to cap centrifuge tubesDo not use aluminum foil to cap centrifuge tubes Clean and maintain gaskets and O-ringsClean and maintain gaskets and O-rings Change O-rings if compromisedChange O-rings if compromised Load/unload centrifuge tubes, rotors and accessories in BSCLoad/unload centrifuge tubes, rotors and accessories in BSC Small, low speed centrifuges may be placed in a BSC; however, high Small, low speed centrifuges may be placed in a BSC; however, high

speed centrifuges pose additional hazards speed centrifuges pose additional hazards Do not overfill tubesDo not overfill tubes Balance buckets, tubes and rotors properly before centrifugationBalance buckets, tubes and rotors properly before centrifugation Wait 5 minutes (or 30 mins. for high speed centrifuge) after each run Wait 5 minutes (or 30 mins. for high speed centrifuge) after each run

before openingbefore opening Do not decant or pour off supernatant. Use a vacuum system with Do not decant or pour off supernatant. Use a vacuum system with

appropriate in-line reservoirs and filtersappropriate in-line reservoirs and filters

Safe Use of Centrifuge

Avoid use of glass blender jars, unless covered Avoid use of glass blender jars, unless covered with polypropylene jarwith polypropylene jar

Place disinfectant-moistened towel over the top of Place disinfectant-moistened towel over the top of the blender during usethe blender during use

Before opening the blender jar, allow the unit to Before opening the blender jar, allow the unit to rest at least 1 minute for aerosols to settle and then rest at least 1 minute for aerosols to settle and then open in a BSCopen in a BSC

Decontaminate promptly after useDecontaminate promptly after use

Safe Use of Blenders

Use careful pipetting practicesUse careful pipetting practices Avoid drops onto hard surfacesAvoid drops onto hard surfaces Wipe up spills promptly with appropriate Wipe up spills promptly with appropriate

disinfectantdisinfectant For ejection of liquid from micropipetteFor ejection of liquid from micropipette

No blowoutNo blowout No pressure ejectionNo pressure ejection Use wall contactUse wall contact

Use capped tubes when mixing, blending, or Use capped tubes when mixing, blending, or vortexingvortexing

Pour liquids carefullyPour liquids carefully Avoid bubblesAvoid bubbles

Minimizing Aerosols

Careful Pipetting Techniques Never blow out last drop in Never blow out last drop in

pipettepipette Use pipetting aids with filtersUse pipetting aids with filters Never mix by suction and Never mix by suction and

expulsion (mix by sonication)expulsion (mix by sonication) Discharge liquid down side Discharge liquid down side

of container, using tip-to-wall of container, using tip-to-wall contactcontact

Deliver as close as possible Deliver as close as possible to contentsto contents

Work over plastic-backed Work over plastic-backed absorbent matting (ensure it absorbent matting (ensure it doesn’t slide forward or doesn’t slide forward or backward blocking air grill)backward blocking air grill)

Use Extreme Care with Sharps

Use sharps if only absolutely Use sharps if only absolutely required as part of a processrequired as part of a process

Percutaneous exposure riskPercutaneous exposure risk Employ safe work practicesEmploy safe work practices Utilize safe sharp devicesUtilize safe sharp devices

Aerosol exposure riskAerosol exposure risk Use biosafety cabinet for Use biosafety cabinet for

removal of air from needleremoval of air from needle Use mechanical methods for Use mechanical methods for

needle removalneedle removal Never bend, recap or manipulate Never bend, recap or manipulate

sharps by handsharps by hand Keep hands away from needleKeep hands away from needle

Vacuum System Protection

In-line filter and disinfectant in collection and In-line filter and disinfectant in collection and overflow flasksoverflow flasks