Biosafety Levels

Biological Safety Cabinets

and
Biosafety Laboratory Construction

Dr Ravi Kant Agrawal, MVSc, PhD

Senior Scientist (Veterinary Microbiology)
Food Microbiology Laboratory
Division of Livestock Products Technology
ICAR-Indian Veterinary Research Institute
Izatnagar 243122 (UP) India
 Biosafety Level 1 (BSL-1 and ABSL-1)
 Suitable for work involving well-characterized agents not
known to consistently cause disease in immuno-competent
adult humans.
 Prophylactic treatment available.
 Minimal potential hazard to laboratory personnel and the
environment.
 Laboratories are not necessarily separated from the general
traffic patterns in the building.
 Work is typically conducted on open bench tops using standard
microbiological practices.
 Special containment equipment or facility design is not
required.
 Animals in open cage system or open environment (outdoors)
 Laboratory personnel must have specific training in the
procedures conducted in the laboratory and must be
supervised by a scientist with training in microbiology or a
related science.
 BSL-1 Practices & Procedures
Only standard practices required at this level:
 Good laboratory practices
 Frequent hand washing, after removing gloves & before leaving lab
 Door that can be kept closed when working
 Limits on access to the lab space when working
 No smoking, eating, drinking, storage of food in laboratory
 Open bench-top work allowed
 Biosafety cabinet not required (unless creating aerosols)
 Care to minimize splashes and actions that may create aerosols
(tiny droplets)
 Daily De-contamination (Decontamination of work surfaces after
every use after any spills)
 Red bag waste
 Decontamination of laboratory wastes
 Use of Mechanical pipetting only (no mouth pipetting)
 “Sharps" precautions, including special containers for disposing of
needles and other sharp objects
 Maintenance of insect/ rodent control program
 Use of personal protective equipment (lab coats, latex gloves, eye
protection or face shields)
 Open bench top sink for hand washing
Biosafety Level 1
Biosafety Level 1
 Risk Group 1 Agents
 E. coli K-12, Bacillus subtilis, Adenoasociated viruses 1-4, T4
bacteriophages, Saccharomyces cerevisiae, Rhizopus stolonifer,
Candida albicans, Pseudomonas, Infectious canine hepatitis
 Transgenic Plants
 Plasmids
 Fungi
 Mold
 Yeast
 BSL-1 Containment Overview

 RG-1 Agents
 Not known to cause disease in healthy/immunocompetent
adult humans
 Practices
 Standard microbiological practices
 Primary Barrier (Safety equipment)
 Minimal requirements
 Secondary Barrier (Facilities)
 Open bench top work
 Biosafety Level 2
 Builds upon BSL-1
 BSL-2 is suitable for work involving agents that pose
moderate hazards to personnel and the environment.
 Agents associated with human disease
 Treatment for disease available (Antibiotics/Vaccines)
 Laboratory personnel have specific training in handling
pathogenic agents
 Personnel are supervised by scientists competent in
handling infectious agents and associated procedures
 Access to the laboratory is restricted when work is being
conducted
 All procedures in which infectious aerosols or splashes
may be created are conducted in biological safety
cabinets (BSCs) or other physical containment
equipment.
 Biosafety manual with definitions of needed waste
decontamination or medical surveillance policies
 Biosafety Level 2 (BSL-2)
 In working with BSl-2 agents, th eprimary
hazards to personnel are –
 Ingestion of infectious materials
 Direct contact or exposure
 Accidental Needle sticks or per-cutaneous
exposure by Scratch, Puncture
 Potential infection through exposure to
Eyes, Mouth, nose, open cut (Mucus
membrane)
 BSL-2 agents do not cause lethal infections,
are not transmissible via airborne route (do
not cause infection if tiny droplets become
airborne and are inhaled, which might occur if
the material were spattered)
 Extreme care should be taken with
contaminated needles and sharp lab
instruments when they are contaminated with
agents.
 BSL-2 Practices &Procedures
Include BSL-1 plus
 Limited access to lab when work
in progress
 Daily de-contamination
 Mechanical pipetting
 Lab coat, safety glasses and
gloves required
 Red bag & sharps containers
required
 Biohazard warning sign posted at
entrance to lab with contact
information: MANDATORY
 Label all equipment (incubators,
freezers, etc.)
 TC room – negative air flow
 Documented training
 Special Entry Procedures:
Baseline serology or pre-
vaccination/Immunizations may
be required
 Risk Group 2 Agents

 S. aureus, Bordetella pertussis,

Corynebacterium diphthriae, Other E
coli, Nisseria gonorrhoea,
Streptococcus pyogenes, Vibrio
cholerae, Klesiella spp., Proteus,
Serratia marcescens, Salmonella, L.
monocytogenes
 Rabies,
 Hepatitis A, B, C
 Cryptococcus neoformans
 Most parasitic agents
 Human or Primate Cells
 Herpes Simplex Virus
 Replication Incompetent Attenuated
Human Immunodeficiency Virus
 Patient specimens
 BSL-2 Containment Overview
 RG-2 Agents
 Associated with mild to moderate disease in humans
 Practices
 BSL-1 plus limited access.
 Primary Barrier (Safety equipment and PPE)
 biosafety cabinets or other approved containment devices
 Personal protective equipment: lab coats, gloves, face
protection as needed
 Protective clothing removed when personnel leave laboratory
area
 Secondary Barrier (Facilities)
 BSL-1 plus the availability of a mechanism for
decontamination (autoclave).
Biosafety Level 2
Biosafety Level 2
 Biosafety Level 3: Working in High Containment
 Is applicable to clinical, diagnostic, teaching, research,
or production facilities where work is performed with
indigenous or exotic agents that may cause serious or
potentially lethal disease through inhalation route
exposure.
 Primary hazards: needle sticks, ingestion, exposure to
infectious aerosols
 Treatment may or may not exist
 Laboratory personnel must receive specific training in
handling pathogenic and potentially lethal agents
 Must be supervised by scientists competent in handling
infectious agents and associated procedures.
 Biosafety Level 2 plus all procedures involving the
manipulation of infectious materials must be conducted
within BSCs, or other physical containment devices
 Personnel wear additional appropriate personal
protective equipment including respiratory protection as
determined by risk assessment
 A BSL-3 laboratory has special engineering and design
features.
 Directional air flow
 BSL-3 Practices & Procedures
 Standard practices include BSL-2 plus:
 Strictly controlled access to the lab;
 Specific training for lab personnel in
handling potentially lethal agents;
 Decontaminating all waste;
 Changing contaminated protective lab
clothing,
 Decontaminating lab clothing before
laundering;
 Daily decontamination upon completion of
experiment and after spill
 Autoclave required and waste is disposed
at the end of day
 Required foot activated hand washing sink
and controls
 No sharps unless absolutely necessary
 Aerosol minimization procedures required
 Wrap around disposable clothing is
required.
 Specialized equipment may be required
depending upon procedures
 BSL-3 Practices & Procedures (contd…)

 Biohazard Signs and labels posted

 Air flow from low hazard to high hazard-“Pressure
Mapping”
 Bench top work not permitted
 Documented training and personnel competency
certification (for BSL-3 procedures)
 Baseline serology
 Spills – report immediately and treat accordingly
 Vaccinations/ post exposure protocols
 SOP’s
 Biosafety Manual
 Biosafety Officer
Risk Group 3 Agents
 SARS
 Rift valley fever
 Human Immunodeficiency Virus
 Yellow fever virus
 VEE virus
 Hanta virus
 Prions
 M. tuberculosis, M. bovis
 Coxiella burnetii
 Franciella tulerensis
 B. abortus
 Bacillus anthracis,
 Pasteurella multocida
 Yersinia pestis
 Coccidiodes immitis
 Plasmodium
 Trypanosoma
 No parasitic agents
BSL-3 Containment Overview

 RG-3 Agents
 Associated with serious or potentially lethal disease in humans
 Practices
 BSL-2 plus controlled access.
 Primary Barrier (Safety equipment)
 Biological Safety Cabinet and personal protective equipment
required similar to BSL-2.
 Respiratory equipment if risk of infection through inhalation
 Secondary barrier (Facilities): All BSL-2 barriers with
 Access through self-closing double doors
 Corridors separated from direct access to lab
 Single-pass negative directional airflow- Air handling systems
to ensure negative air flow (air flows into the lab)
 Air pumped into lab not re-circulated in building
BSL-3
Biosafety Level 3
Biosafety Level 3
Biosafety Level 4
 Required for work with dangerous and exotic agents that pose a
high individual risk of life-threatening disease, aerosol transmission
or related agent with unknown risk of transmission.
 Agents with a close or identical antigenic relationship to agents
requiring BSL-4 containment must be handled at this level until
sufficient data are obtained either to confirm continued work at this
level, or re-designate the level.
 Dangerous/exotic agents
 Life threatening disease
 No known treatment available
 Aerosol transmission/mucous membrane exposure/accidental prick
 Agents of unknown risk of transmission or health affects
 Laboratory staff must have specific and thorough training in
handling extremely hazardous infectious agents.
 Laboratory staff must understand the primary and secondary
containment functions of standard and special practices,
containment equipment, and laboratory design characteristics.
 All laboratory staff and supervisors must be competent in handling
agents and procedures requiring BSL-4 containment.
 Access to the laboratory is controlled by the laboratory supervisor
in accordance with institutional policies
Biosafety Level-4: Working in High Containment
 Maximum containment facilities
 Builds on BSL-3/ ABSL-3 practices
 Standard practices include BSL-3 plus:
 strictly controlled access to the laboratory;
 changing clothing before entering and exiting lab
(showering upon exiting recommended-Chemical
decontamination showers)
 decontaminating all material exiting facility (Liquid
effluent collection / decontamination)
 Personnel must receive specialized training in
handling extremely dangerous infectious agents,
containment equipment and functions
 Immunocompromised persons are never allowed to
enter the lab
 Two types of laboratory providing absolute separation
of the worker from the infectious agents
 Suit Laboratory
 Cabinet Laboratory
 Pressurized Containment Suite
 BSL-3 + Class III Biosafety Cabinet
 BSL-4: High Safety Animal Disease Laboratory,
Bhopal, IVRI
 Risk Group 4 agents

 Ebola Hemmorrhagic Fever Virus

 Marburg Virus
 Lassa Fever Virus
 Machupo virus
 Crimean congo Haemorrhagic
viruses, Bolivian and Argentine
Haemorrhagic fever viruses
 Some encephalitis viruses
 Herpesvirus simiae
 No bacterial agents
 No fungal agents
 No parasitic agents
 BSL-4 Containment Overview

 RG-4 Agents
 Associated with high risk of life-threatening disease in
humans and/or animals
 Practices
 BSL-3 plus controlled access
 Primary Barrier (Safety equipment)
 Biological Safety Cabinet
 Full-body air-supplied, positive pressure personnel suit
 Secondary Barrier (Facilities)- BSL-3 plus
 dedicated air and exhaust,
 decontamination procedures for exit,
 separate building
 a recommended absence of windows (or sealed and
resistant to breakage)
Biosafety Level 4
 CDC/NIH Guidelines
Biosafety Levels
(www.cdc.gov)

BSL-1
BSL-3

Lower Risk
BSL-2

Higher Risk
Animal Biosafety Level-4: Working in High Containment
Biosafety Level Summary
Risk Groups and Biosafety Levels

Risk Biosafety Level Laboratory Type Laboratory Safety

Group Practices Equipment

Basic – Basic teaching, GMT None; open

Biosafety Level research bench work
1 1

Basic – Primary health GMT plus Open bench

Biosafety Level services; protective plus BSC for
2 diagnostic clothing, aerosols
services, research biohazards sign
2

World Health National Institutes

Organization of Health
Risk Groups and Biosafety Levels

Risk Biosafety Level Laboratory Type Laboratory Safety Equipment

Group Practices

Containment- Special Level 2 + special BSC and/or

Biosafety diagnostic clothing, access other primary
3 Level 3 services, control, directed devices for all
research airflow activities

Maximum Dangerous Level 3 + airlock Class III BSC, or

Containment – pathogen units entry, shower positive
Biosafety Level exit, special pressure suites
4 waste disposal with class II
4
BSCs, double
ended autoclave

World Health National Institutes

Organization of Health
 Biocontainment
 The principle of holding or being capable of holding or
including within a fixed limit or area
 Preventing the unintentional release of biological agents
through a combination of laboratory practices,
containment equipment (primary barrier) and laboratory
facility design (secondary barrier)
Primary Barrier
 Primary barriers contain the agent at the source
 Equipment/Engineering Control
 Biological safety cabinet, fume hood, glove box, animal
housing, centrifuge, fermenter
Secondary Barrier
 Secondary barrier is the structure surrounding the
primary barrier
 Facility/Engineering Control
 Rooms, building
 Types of Facilities
 Basic laboratory
 Containment laboratory
 Primary Barriers - Equipment
 Personnel Protection
 Any aerosol generated within the cabinet is contained and
kept away from the researcher
 Product Protection
 Air within the work space of the cabinet has been filtered
so that it is virtually free of airborne particles and
organisms; thus protecting the work from outside
contamination
 Environmental Protection
 Aerosols generated within the unit are removed from the
air before the air is discharged
Ventilation Equipment
Classes and Types
Chemical Fume Hood
 Offer only personnel protection
 Do not offer protection to the product or the environment, as there
is no filtration of intake and exhaust air (Sometimes air cleaning
treatment is added to the exhaust).
 Always exhaust air to the outside
 Do draw contaminants in the laboratory air directly over the product
being worked on
 Used for work with chemical hazards
 100 fpm face velocity
 Any Comments?

Fumehood - keep hood clean, sash should be closed

when hood is not in use, equipment should be 9” from
slash
 Clean Bench / Laminar Flow Hoods
 Provide product protection only
 Product protection is provided by creating a unidirectional airflow
generated through a HEPA filter
 Discharge air goes directly into workroom

Applications
 Any application where the product is not hazardous but must be
kept contaminant free
 Preparation of non-hazardous intravenous mixtures and media
 Particulate free assembly of sterile equipment and electronic
devices

 Eliminate Clean Bench in containment laboratory

Biological Safety Cabinets
• Biological Safety Cabinets (BSC):
primary means of containment
developed for working safely with
infectious microorganisms
 Designed to contain biological
hazards
 Supply air HEPA filter for product
protection (except Class I)
 Inward airflow for personnel
protection
 HEPA filtered exhaust air for
environmental protection
 Separated into Classes and Types
– Class I
– Class II
• Type A1, A2
• Type B1, B2
– Class III
 Microbiological studies, cell
cultures, pharmaceutical research
etc.
Class I Cabinet
 Provides personnel and environmental protection
 No product protection
 In fact, the inward flow of air can contribute to contamination of
samples
 An exhaust blower to pull the air through - usually to the outdoors
 BSCs of this class are either ducted (connected to the building
exhaust system) or un-ducted (re-circulating filtered exhaust back
into the laboratory
 Inward airflow is maintained at 75 fpm velocity

Applications
– Housing centrifuges, fermenters
– Cage dumping in an animal lab
– Aerating cultures that potentially generate aerosols
Class I BSCs

 Provides personnel and environment protection only.

 No product protection.
 Suitable for low to moderate risk (biosafety level 1, 2, and 3)
 HEPA filter protects environment by filtering air before it is exhausted
Class II Cabinets

 Provides personnel, product, and environmental protection

 Widely used in clinical, hospital, life science, research and
pharmaceutical laboratories.
 Have 3 main features:
 Downward HEPA filtered unidirectional/laminar airflow for product
protection
 Open front with careful maintained inward airflow for personnel
protection
 HEPA filtered exhaust air to the room or to a facility exhaust system
for environmental protection
Class II BSCs
Type A1 (Previously A): Face Velocity
of >0.38 m/s or 75 lfpm
 The filtered makeup air is divided
equally over the work surface at
about two to six inches above the
work surface.
 Exhaust is drawn at the bottom of
the cabinet where it rises to the
top.
 At the top of the cabinet, 70% of
the air re-circulates through the
supply HEPA filter, the other 30%
of air exhausted through the
exhaust HEPA filter into the room.
 This is due to the relative sizes of
the two filters, and dampers
typically allow the adjustment of
this ratio.
 This type is not safe for work with
hazardous chemicals except when
ducted, usually with a "thimble" or
canopy hood to avoid disturbing
internal air flow.
Class II A1/A2 BSC
Class II BSCs
Type A2 (A/B3): Face Velocity
of >0.51m/s or >100lfpm
 HEPA filtered exhaust air
may be re-circulated into
the room or released
outside
 70% of air is re-circulated,
30% of air filtered through
HEPA and exhausted into
the room.
 A negative air pressure
plenum surrounds all
contaminated plenums that
are under positive
pressure.
 In other respects, the
specifications are identical
to those of a Type A1
cabinet
Class II BSCs
Type B1 Face Velocity of >0.51m/s
or >100lfpm
 These cabinets must be hard-
ducted to an exhaust system
rather than exhausted through
a thimble connection
 30% of air is re-circulated to
work area through HEPA
supply filter and exhausts 70%
of circulated air through HEPA
exhaust filter
 Since exhaust air is drawn
from the rear grille, Type B1
offers more protection to the
personnel if vapor source is at
rear of work area.
 Suitable for work with low
levels of volatile toxic
chemicals and trace amounts
of radionuclides.
Class II BSCs
Type B2 Face Velocity of >0.51m/s or
>100lfpm
 0% air re-circulated, 100% exhausted
from cabinet
 These cabinets must be hard-ducted
to an exhaust system rather than
exhausted through a thimble
connection
 Widely used in toxicology labs and
similar labs where clean air is
essential.
 Additionally, there is the risk that
contaminated air would flow into the
laboratory if the exhaust system for
a Type B1 or B2 cabinet were to fail.
 To mitigate this risk, cabinets of
these types generally monitor the
exhaust flow, shutting off the supply
blower and sounding an alarm if the
exhaust flow is insufficient.
 Any Comments?
BSC - remove unnecessary objects, keep grill at front
of cabinet unobstructed
Class III BSC
 Used to work with microbiological agents assigned to biosafety level 4
 Provides maximum protection to personnel and environment
 Generally only installed in maximum containment laboratories
 The enclosure is gas-tight, and all materials enter and leave through a
chemical DUNK TANK OR PASS THROUGH BOX
 ClassIII BSC may be connected to a double-door autoclave.
 Heavy duty rubber Gloves attached to the front prevent direct contact with
hazardous materials (Class III cabinets are sometimes called glove box).
 Supply air is HEPA filtered and exhaust air is double HEPA filtered.
 Air flow is maintained by a dedicated exhaust system exterior to the cabinet,
which keeps cabinet interior under negative pressure (ABOUT 124.5PA).
 These custom-built cabinets often attach into a line, and the lab equipment
installed inside is usually custom-built as well
 Applications for Cabinet:
 Working with high risk agents
 Working with highly infectious or hazardous experimental materials
 Working with emerging diseases
 Working with diseases that are near eradication
 Weighing and diluting chemical carcinogens
Class III
Comparison of BSCs
Personnel Product Environment

Chemical Fumehood x ---- ----

(X)
Laminar Flowhood ---- x ----

Class I Biosafety x ---- x

Cabinet
Class II Biosafety x x x
Cabinet
Class III Biosafety x x x
Cabinet
Isolators x x x
Types of Biosafety Cabinets
NSF/ANSI Standard 49 – 2002
Face Bio-
Type velocity Airflow Pattern Radionuclides/ safety Product
(lfpm) Toxic Chemicals Level(s) Protection

Class I 75 In at front; rear and top No 2, 3 No

through HEPA filter

Class II 70% recirculated through

Type A1 75 HEPA; No 2, 3 Yes
30% Exhaust through HEPA

30% recirculated through

Class II 100 HEPA; No 2, 3 Yes
Type A2 70% Exhaust via HEPA and
hard ducted

Class II 30% recirculated through Yes

Type B1 100 HEPA; (Low 2, 3 Yes
70% Exhaust via HEPA and levels/volatility)
hard ducted
Class II 100 No recirculation; total exhaust Yes 2, 3 Yes
Type B2 via HEPA and hard ducted

Class III NA Supply air inlets and exhaust Yes 3, 4 Yes

through 2 HEPA filters
Biological Safety Cabinet Certification
 First Certification
 Annually
 When moved
 When filter is changed
 When repaired or modified

Note: Certification is paid by the researcher.

Other Primary Barriers- Engineering Control
 Gasketed blenders, homogenizers
 Cotton plugs, filters for flasks in shakers
 Filtered pipette tips
 HEPA and hydrophobic vacuum line filters
 Plasticware substituted for glassware
 Gas burners with shield, microincinerator
 Centrifuges
 Interlock, solid cover, safety buckets, O-rings
Secondary Barrier- Facilities

Laboratory Biosafety Level 2

 Lockable doors (a must for restricted agents)

 Sink

 Bench tops impervious and easily cleaned

 Biological safety cabinet (if applicable)

 Eyewash

 Inward airflow (desirable)

BSC Operating Procedures
Ready Work Area
 turn off UV lamp, turn on fluorescent
 check air grilles for obstructions, switch on blower
 allow air to purge work space- 5-10 minutes

Pre-disinfect
 spray or swab all interior surfaces with appropriate disinfectant
 allow to air dry

Assemble material
 introduce only material required to perform procedure
 place material such that clean and contaminated items do not
meet
 place contaminated material container at right rear
 ensure view screen is properly located and secured

Pre-purge cabinet
 allow air purge period with no activity inside (leave blower on!)

Prepare self - don protective clothing, gloves, mask, etc. as

appropriate
 BSC Operating Procedure
Do the procedures
DO NOT remove hands from work space until procedures
are complete and all critical material is secured, remove gloves
into contaminated material container

Post-purge cabinet
allow air purge period with no activity inside (leave blower
on!)

Finish personally
remove protective clothing, mask, and wash hands

Post-disinfect
don gloves, remove materials to incubator, to biohazard
bag, autoclave as appropriate, spray or swab all interior
surfaces with appropriate disinfectant

Shutdown cabinet
turn off blower and fluorescent lamp, turn on UV lamp
 Safe Work Practices for BSC Use

 Do not use the top of the cabinet for storage. The

HEPA filter could be damaged and the airflow
disrupted.
 Make sure the cabinet is level. If the cabinet
base is uneven, airflow can be affected.
 Never disengage the alarm. It indicates improper
airflow and reduced performance which may
endanger the researcher or the experiment.
 Never completely close the window sash with the
motor running as this condition may cause motor
burnout.
 Cabinets should be placed away from doors,
windows, vents or high traffic areas to reduce air
Safe Work Practices for BSC Use

 For BSC without fixed exhaust, the cabinet exhaust

should have a twelve inch clearance from the ceiling for
proper exhaust air flow. Also, allow a twelve inch
clearance on both sides of the cabinet for maintenance
purposes.
 Never operate a cabinet while a warning light or alarm
is on.
 The operator should be seated with shoulders level with
the bottom of the sash.
 Perform all work using a limited number of slow
movements, as quick movements disrupt the air barrier.
 Try to minimize entering and exiting your arms from the
cabinet, but if you need to, do it directly, straight out
and slowly.
 Keep all materials at least four inches inside the sash
opening.
 To avoid excessive movements in and out of the
Safe Work Practices for BSC Use
 If a bunsen burner must be used, place it at the rear of
the work area where the air turbulence from the flame
will have the least possible effect on the air stream.
Often the use of a flame is redundant in what should be
a germ free work space.
 All equipment which has come in contact with the
biological agent should be decontaminated. The cabinet
should be allowed to run for at least three minutes with
no activity so that the airborne contaminants will be
purged from the work area before removing equipment.
 After all items have been removed, wipe the interior
surfaces with disinfectant.
Biological Safety Cabinet Certification

 A cabinet must be certified when first installed and then

annually.
 It must be recertified anytime it is moved even within the
same room.
 Before certification personnel arrive, remove all items
from the cabinet and wipe it down with a disinfectant.
This will expedite the certification.
 Any decontaminations, certifications, repairs or
adjustments are to be made by qualified personnel.
 Questions???

Thanks
National Sanitation Foundation
American National Standards Institute

Acknowledgement: All the material/presentations available online on

the subject are duly acknowledged.
Disclaimer: The author bear no responsibility with regard to the source
and authenticity of the content.