Biosafety is the application of safety precautions that reduce a laboratorians risk of expossyre to a potentially infectious
material amd limit comtamination of the work environment and ultimately the community {CDC}
WHY WE NEEED 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 higjhly 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:
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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 separate
BIOSAFETY LEVEL 2
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Microbes that possess moderate hazard to laboratorians
EG: STAPHYLOCOCCUS AUREUS
BSL- 2 PRACTICES:
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Access to lab is restricted when work is being conducted
PPE, face shield, eye goggles
Biosafety cabinet
Autoclave /Decontamination proper
Self-closing doors
Sink with eyewash apparatus readily available
BIOSAFETY LEVEL 3
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Serious/potentially lethal disease through respiratory transmission
EG: MYCOBACTERIUM TUBERCULOSIS
BSL-3 PRACTICES:
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Laboratorians- under medical surveillance and receive Immunization
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
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Highest level of biological safety
Dangerous and exotic microbes
EG: EBOLA, MARBURG VIRUSES
BSL-4 PRACTICES:
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Change clothes before entering
Shower upon exiting
Decontaminate all materials before exiting
Class III BSC
Separate building for lab
Vacuum lines and decontamination systems
BIOSAFETY CABINET
INTRODUCTION
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Biosafety cabinets (BSCs) are primary means of containment, developed for working safely
with infectious micro-organism
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 environment and product protection when appropriate
practices and procedures are followed”
HISTORICAL PERSPECTIVE
1) Early prototype clean air cubicles (clean filtered air was blown directly at the working
surface inside a cubicle – this place 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 FILTER
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HEPA- high efficiency particular air filter
It removes the most penetrating particle size (MPPS) of 0.3 um 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 blinder
The filter medium is pleated to increase the overall surface area, with pleats being
separated by coggurated 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 particular
matter
IMPORTANCE OF A BIOSAFETY CABINET
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PROVIDE PROTECTION TO THE
- personnel handing infectious material
- environment by preveting the release of microbes
- product (e.g in handing cell cultures)
BIOSAFETY CABINET-I
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Provides personnel and environment protection, but no product protection
Exhaust system – HEPA FILTER
Class l 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 encose 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 HEPS filter as it enters the cabinet exhaust plenum.
REQUIREMENTS:
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Open fronted
Glass in the upper front
An integral tray to contain spills and splashes
Inward airflow – 0.7 to 1m/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
BIOSAFETY CABINET CALL II
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Product protection
Predictable particle behaviour
Laminar air flow principle (1960)
Particle barrier systems
Risk of contamination 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
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through 30% of the air – exhaust HEPA filter
70% of the air – recirculation 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)
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Inflow air velocity 100lfm
All positive pressure conntaminated plenums within the cabinet are sorrounded by a negative air
pressure plenum > ensures leakage
CLASS II A2
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Foe 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 spilts other than exactly 70/30%
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Cabinet supply blowers draw room air through the front grille and through HEPA
Inflow velocity 100lfm
Spilt 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
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Total exhaust cabinet
No air recirculation
Simultaneous biologiical and chemical containment
Inflow air velocity 100lfm
Exhaust 1200 cubic feet/min of room air > expensive cabinet >high cost of heavier gauge and higher
capcoty exhaust fan > hence only for research
CLASS II B2
CLASS III
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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 drunk 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
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WORK PRACTICE 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 anenometer
 Ideal air flow – 0.7 to 1 m/s
 All procedures should be done atleast four inches in form 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 agenst that the
disinfectant is effective
 BSC- ethanol not used as decontamination as it evaporates- no proper contact time – ethanol can be
used as ransing as a ransing agent
DECONTAMINATION METHOD A
 Cabinets with an internal electric power sopply
 Place 25ml formalin (cabinet with internal volume of 0.38cu.m) to a vaporiizer, or into a beaker
on a hotplate
 Close the cabinet and ensure that the exhause blow black valve is closed
 Biol away formalin
DECONTAMINATION METHOD B
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35ml formalin in a 1000ml beaker inside the cabinet > add 10g potassium permanganate > seal
the cabinet
Leave the cabinet at keast 5 hours, preferably overnight and label DANGER- FUMIGATION IN
PROGRESS
Open next day and work after 30 mins. For residual formaldehyde to exhaust
USE OD ULTRAVIOLET LAMPS FOR BSC IS NOT ADVISABLE {NIH, CBC}
REFERENCES
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Appendix A- primary containment for biohazards- CDC article
Koneman’s color atlas and T.B of diagnostic microbiology
Diagnostic microbiology-Bailey and scott 13th editon
Practical medical microbiology- Mackie and Mccartney- 14 th editon