L12 – DETAILED NOTES IN BIOSAFETY LEVELS & BIOSAFETY CABINETS

Biosafety Levels

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.

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


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

- Sserious / potentially lethal disease through respiratory transmission

Eg: Mycobacterium tuberculosis

BSL-3 practices:

 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

- Highest level of biological safety

- Dangerous and exotic microbes

Eg: Ebola, Marburg viruses

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
Biosafety Cabinet

 Biosafety (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 design

To be precised,

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

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- woods/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

 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
 The separation is mainly to prevent collapse
 It removes particulate matter by three mechanisms; interception, impaction, diffusion
 These filters are fitted either in the exhaust or air supply system to remove particulate matter
Importance of 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-deducted
 Cabinet air is drawn through a HEPA filter as t 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 1m/sec
 Protection factor- 1.5*10 5
 Protection factor= number of particles which, if liberated into the air of the cabinet will not
escape into the room
 Filtration from exhaust air- HEPA
Biosafety Cabinet Class II

 Product protection
 Predictable particle behavior
 Laminar air flow principle (1960)
 Particle barrier system
 Risk of contaminant release into the lab and risk of product
contamination

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 sone 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 system
Class II A2 (formerly B3)

 Inflow air velocity 100lfm

 All positive pressure contaminated plenums within cabinet are surrounded by a negative air
pressure plenum -> ensures leakage

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 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 BII 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 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 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
Work practices and procedures

 Checklist of materials and work activity

 Arm movement slowly
 Minimum persons
 Lab coats buttoned fully
 Proper stool height

Checklist

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

 Ideal airflow – 0.7 to 1m/s
 All procedures should be done at least 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 solution -> EPA registration number in the label and list of infectious agents that the
disinfectant id 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 ina 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)