Biosafety in Microbiological and Biomedical Laboratories

S. Wongratanacheewin, Ph.D.Melioidosis Research Center, Faculty of

Medicine, KKU.E-mail: sura_wng@kku.ac.th

September 11, 2001

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Biosafety

Personal protection Work place practices Administrative Engineering

All play an equal role in protecting workers from occupational exposures in laboratories

Biosafety

Personal protection PPE, respirotors, gloves, booties, eye and ear

protections Work place practices

Entry and exit practices, decontamination, emergency procedure, handle sharp.

Biosafety

Administrative Training Risk management Immunization Security, supervision

Engineering System, Hepa filter, Accessible

CDC Bioterrorism Agents

Category A Category B Category C

Category A Diseases/Agents

can be easily disseminated or transmitted from person to person;

result in high mortality rates and have the potential for major public health impact;

might cause public panic and social disruption; and

require special action for public health preparedness.

Category B Diseases/Agents

Second highest priority agents include those that

are moderately easy to disseminate; result in moderate morbidity rates and

low mortality rates; and require specific enhancements of CDC's

diagnostic capacity and enhanced disease surveillance.

Category C Diseases/Agents

Third highest priority agents include emerging pathogens that could be engineered for mass dissemination in the future because of

availability; ease of production and dissemination; and potential for high morbidity and mortality

rates and major health impact.

Containment Primary containment, the protection of

personnel and the immediate laboratory environment from exposure to infectious agents, is provided by both good microbiological technique and the use of appropriate safety equipment.

Secondary containment, the protection of the environment external to the laboratory from exposure to infectious materials,

Laboratory Practice and Technique

Develop or adopt a biosafety or operations manual that identifies the hazards that will or may be encountered, and that specifies practices and procedures designed to minimize or eliminate exposures to these hazards.

Principles of Biosafety

Laboratory personnel Safety practices, and

techniques must be supplemented

Appropriate facility design and engineering features

Safety equipment, and management practices.

Safety Equipment (Primary Barriers)

Biological safety cabinets (BSCs) Enclosed containers, Engineering controls designed to remove

or minimize exposures to hazardous biological materials.

The biological safety cabinet (BSC)

Device used to provide containment of infectious splashes or aerosols.

Three types of biological safety cabinets (Class I, II, III) used in microbiological laboratories.

Open-fronted Class I and Class II biological safety cabinets are primary barriers which offer significant levels of protection to laboratory personnel.

Safety centrifuge cup, an enclosed container designed to prevent aerosols.

Personal Protection

gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles.

Facility Design and Construction (Secondary Barriers)

Depend on the risk of transmission of specific agents.

Separation of the laboratory work area from public access, availability of a decontamination facility (e.g., autoclave), and handwashing facilities.

If an infectious aerosol is present, higher levels of primary containment and multiple secondary barriers may become necessary to prevent infectious agents from escaping into the environment.

Biological Safety

Biological Hazards Divided into 4 biosafety levels

Levels define the lab requirements, protective equipment, and work practices

4 animal biosafety levels also

Biosafety Level 1

Definition Work with agents not known to cause disease

in healthy adults Minimal potential hazard to personnel and

environment

Biosafety Level 1

Requirements Wash hands before leaving lab No eating, drinking, etc. in lab Follow safe sharps procedures Decontaminate waste and work surfaces

Biosafety Level 1

Examples Escherichia coli K12 Klebsiella oxytoca Lactobacillus Saccharomyces

Biosafety Level 2

Definition Work with agents of moderate potential

hazard to personnel and the environment. Agents associated with human disease which is rarely serious and for which preventative or therapeutic interventions are often available.

Biosafety Level 2

Requirements All requirements for BSL1 plus: Training to handle organisms Personal protective equipment (PPE)- lab

coats, eyewear, and gloves Minimize aerosol generation Biohazard sign posted on door

Biosafety Level 2

Examples Pathogenic E. coli Salmonella sp. Streptococcus sp. Hepatitis B and C Viruses

Biosafety Level 3

Definition Agents associated with serious or lethal

disease for which preventative or therapeutic intervention may be available. High individual risk, low community risk. Often risk is by inhalation route.

Biosafety Level 3

Requirements All requirements for BSL2 plus: Limited lab access 2 doors in series to access lab Able to wash entire lab Special exhaust ventilation- not recirculated

Biosafety Level 3

Requirements All requirements for BSL2 plus: High level of training Personnel receive vaccinations if available Work in biosafety cabinets

Biosafety Level 3

Examples HIV Mycobacterium tuberculosis

BSL 3 cabinet

Class IHigh Efficiency Particulate Air filter Removes over 99.95% of particles 0.3 m in diameter or larger

HEPA Filters

Class II A Class II B1

Connection to building exhaust system required.

Class II B2

Connection to building exhaust system required.

Class II B3

Biosafety Level 4

Definition Agents likely to cause serious or lethal

disease for which preventative or therapeutic intervention is usually NOT available. Dangerous or exotic agents.

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 No Bacteria or Fungi Ebola Virus Monkey B Virus Marburg Virus

Safe Work Practices Wash hands after work; before leaving lab;

when removing gloves No eating, drinking, applying cosmetics,

handling contact lenses in lab Maintain labs in clean, orderly fashion 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

Personal Protective Equipment

Contaminated personal protective equipment (PPE) should remain in the work area- do not wear in any "clean" areas

PPE that is contaminated must be discarded as infectious waste or disinfected prior to routine laundering

Use of Equipment

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

Wipe down surfaces with alcohol before and after work. Let run 10 minutes to clean it

Minimize use of flames in cabinet Try not to use chemicals in cabinets- only

use in Class I or II hoods that are ducted- contact DOHS

Do not store excess equipment in cabinet

Centrifuge Safety

Aerosol generation from broken tubes, opening tubes, decanting supernatant, resuspending materials

Balance load Use sealed tubes, safety buckets or

rotors when possible When possible fill & open centrifuge

tubes or buckets in BSC

Centrifuge Accidents

If a tube breaks- Turn off centrifuge Leave the lab Call Officers If you find a tube broke when you open the

centrifuge- Close the lid Leave the lab Call Officers

Shipping/ Transporting

Who regulate transportation of biologicals????

Includes infectious materials, diagnostic specimens, dry ice

Program being developed- to include mandatory training

Disinfection

All equipment, environmental, and working surfaces must be cleaned and decontaminated after work with infectious materials

Broken glass or sharps shall be cleaned up using mechanical means- NOT by hand

Different levels of cleaning- decontamination, sanitization, disinfection, sterilization

Decontamination

Use appropriate disinfectant for agents in use

Follow instructions, paying attention to dilution, shelf life, and contact time to assure effective kill

Care must be used to ensure mixing of incompatible materials does not occur

Infectious Waste

What is infectious waste Procedures for disposal Solid waste Liquid waste Sharps Autoclaving waste Containers and pick-ups Mixed waste

Emergencies

Safety equipment in labs Showers Eye washes

BSL2 Spills Small spills- Wipe up with disinfectant-soaked paper towel then clean surface

with suitable disinfectant Large BSL2 Spill in BSC Keep cabinet running during and 10 minutes after cleanup Don PPE Cover spill with disinfectant- let set- wipe up Clean catch basin under work surface if spill ran there Remove PPE when done and wash Leave the lab, close door, post lab off-limits Decon people if necessary After 30 minutes, put on PPE and assemble supplies

(building name & room number, list subrooms, if any)

Hazards Within:

List hazards such as Flammables, Acids, Bases, General Chemicals, high pressure compressed gases, Biohazards, Radioactive materials, etc

Primary Contact: (name & office phone)

Second Contact: (name & office phone)

Building Monitor/Safety: (name & office phone)

Department Head: (name & office phone)

Fire/Police/Ambulance: 911Envir. Health & Safety (or RSO, if needed): 646-3327

Acknowledgements T2 (Thailand Tropical Diseases

Research Program). TRF (Thailand Research Fund). NSTDA ONSITE, Emory University Faculty of Science, MU The Virology Association of Thailand Faculty of Medicine, KKU.