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
 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/1999

Appendix A – Primary Containment for Biohazards: Selection, Installation and
Use of Biological Safety Cabinets
 Historical perspective
1. 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
• 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 two
1) To the front grille
2) 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
II
Class
III