East Carolina University Brody School of Medicine Office of Prospective Health/Biological Safety Biological Safety.

East Carolina University Brody School of Medicine

Office of Prospective Health/Biological Safety

http://www.ecu.edu/prospectivehealth/

Biological Safety Training




Introduction to Biological Safety

Biological Hazards are divided into 4 Biosafety Levels– BSL 1– BSL 2– BSL 3– BSL4

Biosafety levels define the lab requirements, protective clothing, and work practices




BioSafety Definitions

BSL 1 (BMBL)– Not known to consequently cause disease in

healthy human adults– Pose minimal hazards under ordinary

conditions of handling




BioSafety Level 1

• Standard Practices– Use Mechanical Pipetting devices

– No Eating, Drinking, Smoking in Lab

– Minimize splashes and aerosols

– Decontaminate work surfaces

– Safe handling of sharps

– Wash Hands before leaving lab




BioSafety Level 1

• Protective Clothing– Lab Coat– Gloves– Eye Protection





• BSL 2 (BMBL)– suitable for work involving well-characterized

agents not known to cause disease in healthy adult humans, and of minimal potential hazard to laboratory personnel and the environment

Examples: Measles Virus, Salmonella,

Hepatitis B Virus





• Other Materials Handled at BSL 2(Requires compliance with OSHA BBP Standard)

– Human Blood

– Human Blood Components

– Human Tissues

– Human Cell Lines




BioSafety Level 2

• Standard Practices– All requirements for BSL 1 plus:

• Access to laboratory is limited or restricted when work is being conducted

• Personnel have specific training in handling pathogenic agents

• Biohazard Sign posted on the door• Extreme precautions are taken with contaminated

sharp items




BioSafety Level 2

• Standard Practices cont.• Written Biosafety Procedures

• Report Spills

• Minimize aerosol generation

• Personnel receive appropriate immunizations or test (e.g. Hep B vaccine or TB skin Testing)




BioSafety Level 2

Written Biosafety Procedures– Prepared specifically for lab by PI– Incorporated into Lab Safety Plan– Personnel are advised of special hazards– Personnel are required to read and follow

procedures




BioSafety Level 2

• Personal Protective Equipment– Lab Coats– Gloves

• Double Gloving when appropriate

– Eye Protection - Safety Glasses/Shield




BioSafety Level 3

• BSL 3 (BMBL)– applicable to clinical, diagnostic, teaching,

research, or production facilities in which work is done with indigenous or exotic agents which may cause serious or potentially lethal disease as a result of exposure by the inhalation route.




BioSafety Level 3

• Standard Practices– All requirements for BSL 2 plus:

• Limited lab access

• 2 doors in a series to access lab

• Able to wash entire lab

• Special exhaust ventilation (Not re circulated, negative pressure)




BioSafety Level 3

• Standard Practices –cont.– High level of training– Personnel receive vaccinations if available– Work in Biosafety cabinets




BioSafety Level 3

• Examples– Mycobacterium Tuberculosis– Hanta virus– SARS




BioSafety Level 4

• BSL 4 (BMBL)– required for work with dangerous and exotic

agents which pose a high individual risk of aerosol-transmitted laboratory infections and life-threatening disease.




BioSafety Level 4

• Requirements– All requirements for BSL3 plus:

• Class III Biosafety cabinet or positive pressure suits

• Shower/Change rooms

• Clothing Autoclaved before laundering

• Air Locks




BioSafety Level 4

• Examples– Ebola Virus

– Monkey B Virus

– Marburg Virus




Containment

• Primary– Protection of personnel and immediate lab from

exposure• Good Techniques• Safety equipment

– Biological Safety Cabinet– Engineering Controls– PPE– Serologic surveillance




Containment

• Secondary– Protection of environment external to lab

• Facility Design

• Operational practices




Safe Work Practices for all Levels

• Wash hands after work; when removing gloves; before leaving lab

• No eating, drinking, applying cosmetics, handling contact lenses in the lab.

• Maintain labs in clean, orderly fashion.




Safe Work Practices for all Levels

• Limit access to lab when work with organisms is in progress

• Use good microbiological techniques

• No mouth pipetting

• When possible use plastic instead of glass




Sharps Safety

• Sharps include needles, syringes, razor blades, lancets, slides, scalpels, pipettes, micropipettes, pipette tips, broken plastic or glassware, and other devices capable of cutting or piercing the skin.




Sharps Safety

• Contaminated needles shall not be bent, recapped, or removed unless there is no feasible alternative.

• If required, use a mechanical device or a one handed technique.




Sharps Safety

• Safety devices or alternatives to needles should be used when available.




Sharps Disposal

• Sharps containers for disposal of these items should be conveniently located and easily accessible in all work places where sharps are used.




Sharps Disposal

• Syringes and syringes without a needle attached go into a sharps container

• Contaminated micropipettes, pipette tips, and Pasteur pipettes are discarded in a puncture-resistant container or a sharps container for disposal.




Sharps Disposal

• Don’t place needles or sharps in office waste containers




Laminar Flow Equipment

BioSafety Cabinets (BSCs)contain infectious agents to protect personnel and the environment

Laminar Flow Clean Benches (LFBs)Non Hazardous work onlyProtect work from contamination




BioSafety Cabinets

• 3 Classes

• All exhaust is HEPA filtered before leaving the cabinet

• Class I– Do not protect the work from contamination– Air entering cabinet is not filtered




Biosafety Cabinets

• Class II (4 types – A, B1, B2, and B3)– Each type recirculates different amount of air– Some are hard ducted, and some exhaust into

the room.

Class IIITotally enclosed, ventilated cabinetsWork through portals with attached gloves




BioSafety Cabinets

All equipment is laid out to not restrict airflow in the cabinet




Laminar Flow Benches

• Do not protect personnel or the environment

• Discharges HEPA filtered air across the work surface toward the user.




Use of Laminar Flow Equipment

Minimize airflow disturbances (moving in/out of cabinet, people walking by, opening doors, blocking grills with equipment)

Wipe down surfaces with approved disinfectant before and after work.




Use of Laminar Flow Equipment

• Minimize use of flames in cabinet

• Try not to use chemicals in Class I or II hoods that are ducted

• Do not store excess equipment in cabinet

• All laminar flow equipment must be certified annually.




Centrifuges




Centrifuge Hazards

• Mechanical failure of Machine

• Lab equipment failure (tubes etc.)

• Aerosol generation• Operator error




Centrifuge Operation

1. Check tubes for cracks/chips2. Use matched sets of tubes, buckets, etc. Make

sure the rotor is balanced properly3. Tightly seal all tubes and safety cups4. Ensure that rotor is locked to spindle and the

bucket is seated5. Close lid during operation6. Allow to come to complete stop before opening




Centrifuge Safety

• Use sealed tubes, safety buckets or rotors when possible

• When possible fill and open centrifuge tubes or buckets in the biological safety cabinet.

• Lubricate O-rings and rotor threads weekly

• Disinfect weekly and after all spills or breaks




Biohazardous Waste

– All cultures, stocks, and other regulated waste are decontaminated before disposal by an approved decontamination method such as autoclaving.




Biohazardous Waste

• Examples– stock cultures

– materials containing or contaminated with blood

– blood and body fluids

– sharps

– pipette tips

– animal waste

– other contaminated lab material




Blood and Body Fluids Waste

• Serum, • Plasma • Other blood components• Semen• Vaginal secretions• Cerebrospinal fluid• Pleural fluid• Peritoneal fluid• Pericardial fluid• Amniotic fluid• Any other body fluid visibly

contaminated with blood




Infectious Sharps Waste

• Hypodermic needles• Scalpels• Pipettes /pipette tips• Breakable containers• Glass products (i.e.,

slides or cover skips)




Biohazardous Waste

• Inappropriate materials

• Dispose of noninfectious waste in regular trash




Biohazardous Waste

• Inappropriate materials

• These include: aerosol cans, chemical waste, flammable material




Biohazardous Waste

• Inappropriate packaging

• Use puncture resistant package if needed




2. Pull bag edges up and twist bag into single braid.

Waste must be placed in a red bag in the tub to comply with DOT regs.

1. Only Biohazardous Waste should be placed in these containers -items contaminated with pourable/dripable or dried, crusted blood or body fluids, sharps (in sharps containers), etc.

PROPER HANDLING OF BIOHAZARDOUS WASTE




Biohazardous Waste Containers

• Biohazardous waste containers shall be clearly marked with the universal biohazard symbol.

East Carolina University Brody School of Medicine Office of Prospective Health/Biological Safety Biological Safety.

Documents

lab slide

biosafety definitions

biosafety levels bsl

biosafety cabinets

procedures slide

lab safety plan personnel

written biosafety procedures

bsl4 biosafety levels