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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


– 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


– 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 only

Protect 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 III

Totally enclosed, ventilated cabinets

Work 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/chips

2. Use matched sets of tubes, buckets, etc. Make sure the rotor is balanced properly

3. Tightly seal all tubes and safety cups

4. Ensure that rotor is locked to spindle and the bucket is seated

5. Close lid during operation

6. 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





Biohazardous Waste Containers

• Biohazardous waste

containers shall be

clearly marked with

the universal

biohazard symbol.

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