Dr. Malathi M20/11/2014

BIOSAFETY LEVELS &

BIOSAFETY CABINET

Topics to be discussed

:

• What is biosafety?• Why we need ?• Levels of biosafety• Biosafety cabinet• Types• Decontamination

Biosafety is the application of safety precautions that reduce a laboratorians risk of exposure to a potentially infectious material and limit contamination of the work environment and ultimately the community { CDC }

Why we need biosafety ????

1. Lab has hazards of processing infectious agents

2. Accidental threat to workers and environment

3. To have adherence with safety regulations while dealing with highly infectious agents

BIOSAFETY LEVEL 1

• Microbes not known consistently to cause disease in healthy adults and present minimal potential hazard to lab and environment

• Eg : non pathogenic strain of E.coli

BSL – 1 practices:

• Standard microbiological practices are followed

• Work can be performed on an open table or bench

• PPE{Personal protective equipment} needed• Sink – hand washing• Lab – doors seperate

Biosafety level 2

• Microbes that possess moderate hazards to laboratorians

• Eg: Staphylococcus aureus

BSL – 2 practices:

• Access to lab is restricted when work is being conducted

• PPE , face shields, eye goggles• Biosafety cabinet • Autoclave/Decontamination proper• Self closing doors• Sink with eyewash apparatus readily available

Biosafety level 3

• Serious / potentially lethal disease through respiratory transmission

• Eg: Mycobacterium tuberculosis

BSL 3 - practices

• Laboratorians – under medical surveillance and receive immunisation

• Access to lab restricted & controlled• PPE with respirators• BSC• Sink with eyewash• Exhaust air – not recirculated• Self closing doors with automatic locking

Biosafety level 4

• Highest level of biological safety

• Dangerous and exotic microbes

• Eg: Ebola , marburg viruses

BSL 4

BSL 4 - practices

• Change clothes before entering• Shower upon exiting• Decontaminate all materials before exiting• Class III BSC• Separate building for lab• Vacuum lines and decontamination systems

BSL 4 lab

BIOSAFETY CABINET

Introduction • Biosafety cabinets (BSCs) are primary means of

containment, developed for working safely with infectious micro-organisms

• BSCs are only one overall part of biosafety program, which requires consistent use of – good microbiological practices– primary containment equipment– primary containment facility design

To be precise,

“BSCs are designed to provide personnel, environmental and product protection when appropriate practices and procedures are followed”

Adapted from CDC-BMBL- 5th Edition/1999Appendix A – Primary Containment for Biohazards: Selection, Installation and Use of Biological Safety Cabinets

Historical perspective1. Early prototype clean air cubicles (clean filtered air

was blown directly at the working surface inside a cubicle – this places the personnel in a contaminated air stream)

2. Concept of small workstation (non-ventilated cabinets – wood/stainless steel)

3. Ventilated cabinets (lack of controlled/ adequate air flow leading on to mass airflow) Class I

4. HEPA filter were introduced (undergoing modifications till date)

• HEPA – High efficiency particulate air filter

• It removes the most penetrating particle size (MPPS) of 0.3 μm with an efficiency of at least 99.97 %

• The typical HEPA filter is a single sheet of borosilicate fibers treated with a wet-strength water-repellant binder

HEPA FILTER

• The filter medium is pleated to increase the overall surface area, with pleats being separated by corrugated aluminum tubes

• This separation is mainly to prevent collapse

• It removes particulate matter by three mechanisms interception, impaction, diffusion

• The filtering efficiency depends upon fiber diameter, filter thickness and face velocity

• These filters are fitted either in the exhaust or air supply system to remove particulate matter

http://en.wikipedia.org/wiki/HEPA

Importance of a Biosafety cabinet

• Provide protection to the

– personnel handling infectious material

– environment by preventing the release of microbes

– product (e.g. in handling cell cultures)

BIOSAFETY CABINET - I

• Provides personnel and environmental protection, but no product protection

• Exhaust system – HEPA filter

• Class I BSC – unfiltered room air is drawn in through the work opening and across the work surface

• Inward airflow – Minimum velocity – 75 linear feet / minutes

• To enclose equipment (Eg. Centrifuges, harvesting equipment, small fermenters)

• For procedures with potential to generate aerosols ( tissue homogenation, culture aeration)

• Class I BSC is hard-ducted

• Cabinet air is drawn through a HEPA filter as it enters the cabinet exhaust plenum.

REQUIREMENTS:• Open fronted• Glass in the upper front• An integral tray to contain spills and splashes• Inward airflow – 0.7 to 1 m/sec• Protection factor – 1.5 * 105

• Protection factor = number of particles which, if liberated into the air of the cabinet will not escape into the room

• Filtration from the exhaust air - HEPA

CLASS I cabinet

Biosafety cabinet class II

• Product protection

• Predictable particle behaviour

• Laminar air flow principle (1960)

• Particle barrier systems

• Risk of contaminant release into the lab and risk of product contamination

Class II

A1 A2 B1 B2

CLASS II – Type A1

• Internal fan – draws room air – 75lfm velocity

• Supply air flows through HEPA – particulate free air to the work surface

• Reduced turbulence

• Reduced cross contamination

• Downward moving air – splits into two1) To the front grille2) To the rear grille

• 30% of the air – exhaust HEPA filter• 70% of the air – recirculates through HEPA

filter back into the work zone of the cabinet

• Not to be used for work involving volatile toxic chemicals

• Exhaust the air outside the building ( through use of canopy hood and filter housing )

• CLASS II A1 and A2 – never be hard ducted to the building exhaust system

CLASS II A1

CLASS II A2 ( formerly B3)

• Inflow air velocity 100lfm• all positive pressure contaminated plenums

within the cabinet are surrounded by a negative air pressure plenum ensures leakage

CLASS II A2

CLASS II B1

• For hazardous chemicals and carcinogens

• Designed and originated with the National cancer institute type 212 ( later called Type B)

• Definition of Type B1 cabinets:• Classic NCI design Type B, and cabinets without

supply HEPA filters located immediately below the work surface, or those with exhaust/recirculation down flow splits other than exactly 70/30%

• Cabinet supply blowers draw room air through the front grille and through HEPA

• Inflow velocity 100lfm

• Split in the down flowing air stream just above the work surface

• 70% air through the rear grille exhaust HEPA filter discharge through building

• 30% air Down flow air front grille

CLASS II B1

CLASS II B2• Total exhaust cabinet

• No air recirculation

• Simultaneous biological and chemical containment

• Inflow air velocity 100lfm

• Exhaust 1200 cubic feet/min of room air expensive cabinet high cost of heavier gauge and higher capacity exhaust fan hence only for research

CLASS II B2

CLASS III

• Highly infectious agents, hazardous operations• Gas tight no leak greater than 1 *10-7 cc/sec

with 1% test gas at 3 inches pressure water gauge

• Non opening view window• Passage of materials through a dunk tank• Double door pass through box with autoclave

• Supply and exhaust air HEPA

• Negative pressure cabinet

• No exhaust through the general lab exhaust

• Long heavy duty rubber gloves attached in a gas tight manner to port in the cabinets

Class III

Work practices and procedures

• Checklist of materials and work activity protocol

• Arm movement slowly• Minimum persons• Lab coats buttoned fully• Proper Stool height

Check list

• Daily check of airflow by airflow indicator and monthly or weekly with an anemometer

• Ideal air flow – 0.7 to 1 m/s

• All procedures should be done atleast four inches in from the front grille

• Only the materials needed for work should be kept inside

• Wait for minimum of four minutes to switch off the blowers after the work is over

Decontamination• Disinfectant selection EPA registration number

in the label and list of infectious agents that the disinfectant is effective

• BSC – ethanol not used as decontamination as it evaporates – no proper contact time – ethanol can be used as a rinsing agent

• Formaldehyde vapour sterilisation to be done to kill spores

Disinfection method A

• Cabinets with an internal electric power supply

• Place 25 ml formalin(cabinet with internal volume of 0.38cu.m) to a vaporizer, or into a beaker on a hotplate

• Close the cabinet and ensure that the exhause blow back valve is closed

• Boil away formalin

Disinfection method B

• 35ml formalin in a 100ml beaker inside the cabinet add 10g potassium permanganate

seal the cabinet

• Leave the cabinet at least 5 hours , preferably overnight and label DANGER – FUMIGATION IN PROGRESS

• Open next day and work after 30 min for residual formaldehyde to exhaust

• Use of Ultraviolet lamps for BSC is not advisable { NIH, CDC }

Summary

Class I

Class IIIClass II

References

• Appendix A – primary containment for biohazards – CDC article

• Koneman`s color atlas and T.B of diagnostic microbiology

• Diagnostic microbiology – Bailey and scott – 13th edition

• Practical medical microbiology – Mackie and Mccartney – 14th edition

Thank you……….

Questions pls