BIOSAFETY

AND
BIOETHICS

Course no: Biotech-622

Contents
ROUTES/WAYS OF CONTAMINATION FROM A SUSPECTED PERSON TILL LABORATORY PERFORMANCE.. .3
Collection of sample................................................................................................................................3
Specimen containers...............................................................................................................................3
Labelling..................................................................................................................................................3
Transport of specimens...........................................................................................................................3
Receipt of specimens...............................................................................................................................3
Opening packages....................................................................................................................................3
Safe handling of specimens in the laboratory.........................................................................................4
Preservation of specimen........................................................................................................................4
BIOSAFETY...................................................................................................................................................4
Definition.................................................................................................................................................4
BIOSAFETY LEVELS.......................................................................................................................................4
BIOSAFETY LEVEL 3:.....................................................................................................................................4
Standard Microbiological Practices.........................................................................................................5
Safety Practices.......................................................................................................................................5
Safety Equipment....................................................................................................................................5
Uses.........................................................................................................................................................6
Organisms................................................................................................................................................6
BIOSAFETY LEVEL 4:.....................................................................................................................................6
Requirements..........................................................................................................................................6
Standard microbiological practices..........................................................................................................6
Safety Practices.......................................................................................................................................7
Safety equipment....................................................................................................................................7
Uses.........................................................................................................................................................7
Organisms................................................................................................................................................8
CHEMICAL HAZARDS....................................................................................................................................8
Flammable chemicals..............................................................................................................................8

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 Reactive chemicals..................................................................................................................................8
Explosive chemicals.................................................................................................................................8
Toxic, harmful and irritating chemicals....................................................................................................8
Oxidizing chemicals.................................................................................................................................9
Environmental Hazards............................................................................................................................9
Routes of exposure................................................................................................................................10
Storage of chemicals..............................................................................................................................10
Toxic effects of chemicals......................................................................................................................10
Following Precautions should be followed to prevent Chemical hazards..............................................11
DUTIES OF A BIOSAFETY OFFICER..............................................................................................................12
Evaluation of Laboratory-Associated Hazards.......................................................................................13
Policy and Procedure Manuals..............................................................................................................13
Accident Reports and Investigations.....................................................................................................14
Waste Management..............................................................................................................................14
Registry of Agents..................................................................................................................................14
BIOSAFETY CABINETS.................................................................................................................................14
BIOSAFETY CABINET I:...............................................................................................................................15
BIOSAFETY CABINET II................................................................................................................................15
a. Type A1..............................................................................................................................................16
b. Type A2..............................................................................................................................................16
c. Type B1..............................................................................................................................................16
d. Type B2..............................................................................................................................................16
e. Type C1..............................................................................................................................................17
BIOSAFETY CABINET III...............................................................................................................................17

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Question # 1:

Briefly explain the routes/ways of contamination from a suspected person till laboratory
performance.

ROUTES/WAYS OF CONTAMINATION FROM A SUSPECTED PERSON TILL

LABORATORY PERFORMANCE.
It is critical that the laboratory provide complete guidelines for the proper collection and
transport of specimens to ensure quality patient care. Following are the routes of sample
contamination if proper guidelines as described below are not followed:

Collection of sample
Sample can be contaminated if proper SOP’s are not followed by laboratory staff. Collect an
adequate amount of specimen. Inadequate amounts of specimen may yield false-negative results.

Specimen containers
Specimen containers may be of glass or preferably plastic. They should be robust and should not
leak when the cap or stopper is correctly applied. No material should remain on the outside of the
container. Containers should be correctly labelled to facilitate identification. Specimen request or
specification forms should not be wrapped around the containers but placed in separate,
preferably waterproof envelopes.

Labelling
Clearly label the specimen container with the patient’s name and identification number or date of
birth (DOB). Always include date and time of collection and your initials.

Transport of specimens
To avoid accidental leakage or spillage, secondary containers, such as boxes, should be used,
fitted with racks so that the specimen containers remain upright. The secondary containers may
be of metal or plastic, should be autoclavable or resistant to the action of chemical disinfectants,
and the seal should preferably have a gasket. They should be regularly decontaminated.

Receipt of specimens
Laboratories that receive large numbers of specimens should designate a particular room or area
for this purpose.

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Opening packages
Personnel who receive and unpack specimens should be aware of the potential health hazards
involved, and should be trained to adopt standard precautions , particularly when dealing with
broken or leaking containers. Primary specimen containers should be opened in a biological
safety cabinet. Disinfectants should be available.

Safe handling of specimens in the laboratory

Improper collection, transport and handling of specimens in the laboratory carry a risk of
infection to the personnel involved.

Preservation of specimen
Specimen should be preserved at appropriate temperature before further processing to avoid
contamination.

Question # 02

Discuss Biosafety level 3 and 4 in detail.

BIOSAFETY
Definition
Biosafety is the prevention of risk to human health and safety, and the conservation of the
environment and the pathogen, as a result of the use for research and commerce of infectious or
genetically modified organisms.
Biosafety is an important concept in microbiology as bio-related research activities may involve
manipulation of microbial, animal, or plant cells which might potentially be pathogenic.

BIOSAFETY LEVELS
As per the CDC, biosafety levels are of four types depending on the risk associated with the
microorganism and the facilities available. The levels of containment range from the biosafety
level 1 (BSL-1), which is the lowest to the level 4 (BSL-4), which is the highest.
These levels are defined by the Central for Disease Control and Prevention (CDC), where each
of these levels is outlined with specific practices and safety requirements.
Biosafety level designations are based on the combination of the design features, equipment,
practices, and procedures required while working with agents from the various risk groups.

BIOSAFETY LEVEL 3:
o Biosafety level 3 (BSL-3) is the level where work is performed with agents that may
cause severe or potentially lethal disease through inhalation or aerosol formation, to the
personnel, and may even contaminate the environment.

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 o The tasks performed in the BSL-3 laboratories involve indigenous or exotic agents where
the potential for infection by aerosols is high, and the disease may have lethal
consequences.
o BSL-3 laboratories are also called the containment laboratory as they require containment
equipment to protect the personnel, the microbial agent, and the environment.
o The requirements for BSL-3 include all the requirements of the BSL-1 and BSL-2
laboratories, along with some additional design features and special equipment.

Standard Microbiological Practices

o The entry to the BSL-3 laboratories is limited to individuals with appropriate training in
handling BSL-3 organisms, all of whom are selected by the laboratory supervisor.
o Besides the general procedures and laboratory practices, the supervisor also formulates
additional policies to limit the entry to the laboratory.
o All the procedures to be conducted in the BSL-3 must be conducted within a biosafety
cabinet to prevent the exposure of the aerosols to the laboratory personnel.
o Personnel working in the laboratory must wear personal protective equipment before
entering the laboratory and then remove them before leaving.
o The work surfaces and sinks should be decontaminated once every work shift or after any
spills or splashes.
o The BSL-3 laboratories should be separated from the general traffic in a building to limit
entry into the laboratories at all times.

Safety Practices
o The doors of the BSL-3 laboratories are closed at all times with appropriate BSL-3 signs
outside the suite, along with a universal biohazard sign and emergency contact
information.
o Laboratory personnel must have medical surveillance and offered appropriate
immunizations for agents handled or potentially present in the laboratory.
o Potentially hazardous materials must be placed in a durable, leak-proof container or vial
during collection, processing, storage, or transport within a facility.
o All laboratory equipment should be routinely decontaminated after work or after any
spills or splashes.
o The laboratory biosafety manual must define procedures t be adopted in the case of
exposure to infectious materials, and these should be treated accordingly.
o No work in the BSL-3 laboratories should be conducted on an open bench or an open
vessel. All the activities involving the infectious agents must be conducted within
Biosafety cabinets or other physical containment devices.

Safety Equipment
o Biosafety cabinets are to be used for the manipulation of all infectious agents.

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 o Individual protection gears like personal protective equipment, coats, gloves, and
respiratory protection should be worn while entering the laboratories and then removed
before leaving.
o The air flowing in the laboratory shouldn’t be recirculated to any area of the laboratory
and should be HEPA-filtered prior to being discharged to the outside.
o The filters, manuals, equipment, vacuum pipes, autoclaves, etc. should be revised and
reviewed annually.

Uses
o BSL-3 laboratories are used for clinical, diagnostic, teaching, research, or production
facilities.
o These laboratories are used for the handling and manipulation of highly infectious agents
that prose direct severe effects on the health of the personnel.
o These are used for the studies regarding the effects of infectious agents and various toxins
and their effects.

Organisms
o The pathogens that require BSL-3 laboratories include HIV, H1N1 flu, Yersinia pestis,
Mycobacterium tuberculosis, SARS, Rabies Virus, West Nile Virus, Ricketts, etc.
o The placement of the organisms in different Biosafety levels, however, might defer and
should also be determined after risk assessment.

BIOSAFETY LEVEL 4:
o Biosafety level 4 is the highest level that is employed while working with dangerous
infectious agents that present a high individual as well as environmental risk in the form
of life-threatening disease, aerosol transmission, or unknown risk of transmission.
o The BSL-4 laboratories are often used while handling and manipulating Risk Group 4
pathogens those are extremely dangerous, with no known vaccines or therapies, and
require extreme precautions during work.
o The BSL-4 laboratories are of two types; cabinet laboratory where all the work is
performed in a Class III biosafety cabinet or similar physical containment with very
carefully formulated precautions and suit laboratory where all the laboratory personnel
are required to wear full-body, air-supplied suits protective gears in the form of PPEs.

Requirements
o The requirements of BSL-4 laboratories are extensive with specific laboratory design,
training procedures, and highly protective equipment and personal gears.
o These laboratories should be certified for use before initial operation and subsequently on
an annual schedule or after a program change, renovation, or replacement of system
components that may affect the operating environment of the laboratory.
o BSL-4 laboratories are also termed the maximum containment laboratories as they have
secondary barriers to prevent hazardous materials from escaping into the environment.

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 o The BSL-4 laboratories should follow the requirements of all BSL-1, BSL-2, and BSL-3,
along with additional specific precautions.

Standard microbiological practices

o No work conducted within the BSL-4 should be done on an open bench or an open
vessel.
o The work stations, equipment, and sinks should be sterilized post work.
o The laboratory personnel should be in protective gear that might include full-body PPEs,
gloves, masks, and coats.
o The doors of the laboratories should be closed at all times with the laboratory placed
away from the general traffic in the building.
o Activities like drinking, eating, mouth pipetting should be avoided at all costs.
o Only people that are trained in handling the BSL-4 organisms and the equipment in the
laboratory should be allowed into the laboratory.

Safety Practices
o Viable or intact biological materials to be removed from the Class III cabinet in a BSL-4
are transferred in a nonbreakable, sealed primary container with a nonbreakable, sealed
secondary container.
o No materials, except the biological materials that are to remain in a viable or intact state,
are removed from the BSL-4 laboratory unless they have been autoclaved or
decontaminated before they leave the facility.
o Only individuals whose presence in the facility is required for microbiological processes
or support purposes are authorized to enter. Individuals that are at increased risk of
acquiring an infection or for whom infection may be unusually hazardous are not allowed
in the laboratory.
o Personnel can enter and leave the facility only after the clothing change and through the
shower rooms.
o When the BSL-4 laboratory is at work or when infectious materials or infected animals
are present in the laboratory, a hazard warning sign, along with the universal biohazard
symbol, is placed on all access doors.
o A system is set up for reporting laboratory accidents, exposures, and the medical
surveillance of potential laboratory-associated illnesses.

Safety equipment
o A Class III biological safety cabinet or Class I or II biological safety cabinets used in
conjunction with one-piece personnel suits ventilated by a life support system are to be
present in a BSL-4 while conducting all procedures within the facility.
o Walls, floors, and ceilings of the laboratories must form a sealed internal shell which
facilitates fumigation and is animal and insect-proof.
o A double-doored autoclave is placed for decontaminating materials passing out of the
facility.

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 o The exhaust air from the facility is filtered through HEPA filters before being discharged
to the outside so as to prevent its entry into occupied buildings and air intakes.

Uses
o BSL-4 laboratories are used for diagnostic and research work on easily transmitted
pathogens, causing fatal diseases.
o These laboratories are used for new and unknown pathogenic microbes, for which no
vaccines or therapies are available.
o They are also used for clinical and production facilities that require highly sophisticated
techniques and advanced processes.

Organisms
o The BSL-4 level pathogens include the risk group IV organisms like Ebola virus, SARS-
CoV-2, Central European Encephalitis virus, Hemorrhagic viruses, etc.

Question # 03:
Discuss chemical hazards in detail.

CHEMICAL HAZARDS
o The use of chemicals in research laboratories is inevitable, and the potential for harm or
injury could be significant if they are misused or mishandled.
o Hazards associated with the transport, storage, and handling of chemicals include fire,
explosion and the effects from toxic (poisonous), harmful, irritating, and corrosive
chemicals. In district laboratories the risks associated with the use of chemicals can be
minimized by laboratory staff knowing which chemicals are hazardous and how to handle
and store them correctly.
o Chemical hazards can be of following types:

Flammable chemicals are those that readily catch fire and burn in air, and they may be
solid, liquid, or gaseous. Proper use of flammable substances requires knowledge of their
tendencies to vaporize, ignite, or burn under the variety of conditions in the laboratory.
Preventing the coexistence of flammable vapors and an ignition source is the best way to deal
with the hazard.

Reactive chemicals are substances that react violently in combination with another
substance. They include water-reactive substances, such asalkali metals; pyrophoric materials,
such as finely divided metals; and incompatible chemicals, such as pure liquid or gaseous
hydrocyanic acid and bases.

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 Explosive chemicals include a variety of substances that can explode under certain
conditions. They include explosives, organic azo compounds and peroxides, oxidizing agents,
and certain powders and dusts.

Toxic, harmful and irritating chemicals A toxic substance is one that can cause serious
acute or
Chronic effects, even death, when inhaled, swallowed, or absorbed through the skin. The term
very, or highly, toxic is used if the substance is capable of causing serious effects.
Toxic chemicals include potassium cyanide, mercury (e.g. from broken thermometers), mercury
11 (mercuric) nitrate, sodium azide, sodium nitroprusside.

Oxidizing chemicals
An oxidizing substance is one that produces heat or evolves oxygen in contact with other
substances causing them to burn strongly or become explosive or spontaneously combustible.
Once a fire is started the oxidizing substance promotes it and impedes fire-fighting.
Oxidizing chemicals include hydrogen peroxide and other strong peroxides, nitric acid,
ammonium nitrate, sodium nitrite, perchloric acid, sodium chlorate, chromic acid.

Environmental Hazards Materials that are toxic and/or cause harm to the environment at
large, particularly aquatic animals.
Examples of an Environmental Hazard: Anthrax, Arsenic, Asbestos, Lead, Mercury, Oil.

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Routes of exposure
Exposure to hazardous chemicals may occur by:
1. Inhalation
2. Contact
3. Ingestion
4. Needle-sticks
5. Through broken skin.

Storage of chemicals
o Only amounts of chemicals necessary for daily use should be stored in the laboratory.
o Bulk stocks should be kept in specially designated rooms or buildings.
o Chemicals should not be stored in alphabetical order.
o General rules regarding chemical incompatibilities
o To avoid fire and/or explosions, substances in the left-hand column of Table 13 should
be stored and handled so that they cannot come into contact with the corresponding
substances in the right-hand column of the table.

Toxic effects of chemicals

o Some chemicals adversely affect the health of those who handle them or inhale their
vapors.

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 o Apart from overt poisons, a number of chemicals are known to have various toxic
effects.
o The respiratory system, blood, lungs, liver, kidneys and the gastrointestinal system, as
well as other organs and tissues may be adversely affected or seriously damaged.
o Some chemicals are known to be carcinogenic or teratogenic.

Following Precautions should be followed to prevent Chemical hazards

o Identify and list all chemicals kept in the laboratory and stores. Maintain an up to date
inventory and information sheets on each chemical including its use, storage
requirements, any known risk, and instructions for its safe use. Request safety data
sheets from manufacturers.
o Before opening a chemical, always read the safety (S) and risk (R) phrases written on the
label. Know the hazard symbols for chemicals.
o Label clearly all reagents with their name, date of preparation, expiry date (if of limited
shelf-life), and hazard symbol if indicated. Prepare written instructions on how to
prepare each reagent safely.
o Store each chemical and reagent correctly, making sure chemicals that can react
dangerously together, i.e. incompatible chemicals, are not stored together.
o Store stock containers of liquid acids, alkalis, corrosive and flammable chemicals at
floor or low level in drip trays to contain any spillage.
o Keep chemicals and reagents out of direct sunlight and do not allow them to overheat.
o Ensure the caps of containers are airtight and tightly closed, particularly of chemicals
that are volatile, flammable, hygroscopic, deliquescent or have a toxic or irritating
vapor.
o Do not use rubber liners in the caps of bottles containing iodine, ether, xylene or other
chemical which attacks rubber. Do not use ground glass stoppers in bottles containing
potassium or sodium hydroxide or other chemical that absorbs carbon dioxide from the
air.
o When opening a corrosive chemical or one with an irritating vapour, use a cloth over
the cap and neck of the container and wear chemical resistant protective gloves.
o Keep the laboratory well ventilated when using hazardous chemicals particularly those
that are flammable or have a toxic or irritating vapour. Whenever possible use a fume
cupboard.
o When preparing reagents, always mix chemicals with care. Wear appropriate footwear
and protective clothing, e.g. laboratory overall, gloves, dust mask, eye goggles or face
visor.
o Ensure an eyewash bottle is available and accessible.
o Never mouth-pipette, taste, or inhale a chemical or reagent.
o Always wash the hands immediately after handling chemicals.
o Use appropriate hazard symbols on the doors of cupboards storing dangerous
chemicals.

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 o Extinguish all open flames when using flammable and oxidizing chemicals and reagents.
Ensure fire fighting equipment is available and accessible.
o Understand how accidents involving chemicals and reagents can occur and know what
to do to control and extinguish a chemical fire, treat a chemical spillage (see later text),
and apply First Aid.
o Use plenty of water when washing toxic, corrosive, flammable and volatile reagents
down the plumbing system, e.g. during staining.
o Keep stores and cupboards containing stock and dangerous chemicals securely locked.
Do not allow unauthorized persons to enter the laboratory and never leave it unlocked
when unoccupied.

Question # 04
Suppose you will be appointed as Biosafety officer, what your duties will be as this post?
Explain in detail.

DUTIES OF A BIOSAFETY OFFICER

The job of a Laboratory Biosafety Officer (BSO) has been expanded to include virtually all
aspects of safety in the clinical laboratory. Some main duties of Biosafety officers are:

o Performs risk assessments of the lab and mitigates the risks.

o Receives reports of safety issues, and works to correct any issues identified.
o Writes biosafety policies and procedures including a Biosafety Manual.
o Enforces the biosafety policies, rules and regulations on the lab staff.
o Responsible for safe shipping of hazardous material to and from the laboratory.
o Available on-call 24 hours a day, 7 days a week as needed for emergencies, spills, etc.
o Prepares reports for the hospital-wide safety committee.
o Ensures compliance with all biosafety related regulatory requirements.
o Provides technical advice as needed to the lab staff, Lab Supervisor, Lab Director and/or
Medical Staff
o Biosafety, biosecurity and technical compliance consultations.
o Periodic internal biosafety audits on technical methods, procedures and protocols,
biological agents, materials and equipment.
o Discussions of violation of biosafety protocols or procedures with the appropriate
persons.
o Verification that all staff have received appropriate biosafety training.
o Provision of continuing education in biosafety.
o Investigation of incidents involving the possible escape of potentially infectious or toxic
material, and reporting of findings and recommendations to the laboratory director and
biosafety committee.

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 o Coordination with medical staff regarding possible laboratory-acquired infections.
o Ensuring appropriate decontamination following spills or other incidents involving
infectious material(s).
o Ensuring proper waste management.
o Ensuring appropriate decontamination of any apparatus prior to repair or servicing.
o Maintaining awareness of community attitudes regarding health and environmental
considerations.
o Establishment of appropriate procedures for import/export of pathogenic material
to/from the laboratory, according to national regulations.
o Reviewing the biosafety aspects of all plans, protocols and operating procedures for
research work involving infectious agents prior to the implementation of these
activities.
o Institution of a system to deal with emergencies.

These duties are further elaborated below:

Evaluation of Laboratory-Associated Hazards

The dangers to personnel and the environment from biohazardous laboratory activities should
be assessed by biosafety officer in a systematic fashion. A number of factors should be
considered, but the two most important factors are (1) the agents and (2) the potential
consequences of infection. The characteristics of the microbiological agents being used are
particularly important; i.e., their virulence, pathogenicity, communicability, and route of spread
are properties affecting the potential danger for laboratory workers and the environment. The
individuals working in a laboratory should be trained adequately to understand the hazards of
their work, to become proficient in the procedures that should be followed to minimize
personal danger, and to be aware of the possibility that they might expose others to the
organisms.

Policy and Procedure Manuals

It is essential that biosafety policies and procedures be clearly spelled out in a manual, by a
biosafety officer including the information the laboratory worker should know for day-to-day
activities as well as for handling emergencies. The laboratory biosafety manual should include
the following subjects:

o Policy and goals

o Safety organization
o Medical program
o Procedures for general laboratory operations, including: labeling and handling of
specimens; methods to minimize hazards of aerosols and droplets; proper uses of
needles, syringes, and "sharps"; appropriate discard of working materials; sterilization

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 and disinfection; cleanup of spills; use and maintenance of biological safety cabinets;
control of insects and other pests; work with animals; and waste disposal;
o Safety equipment location and proper use
o Emergencies

Accident Reports and Investigations

Each organization should have a defined system for reporting laboratory injuries and accidents,
as well as for investigating them. These events should be documented and reported to the
appropriate supervisory personnel and to the employee health service. Biosafety officer should
make sure that these events are properly reported.

Waste Management
It is the responsibility of the biosafety officer to see that waste is properly handled and, when
necessary, that it has been made noninfectious before being discharged into the environment.

Registry of Agents
Biosafety officer should also take care of registry of infectious agents.In any large organization,
a central registry should be maintained of the identity and location of the various infectious
agents being handled throughout the facility, particularly for those agents requiring Biosafety
Level 3 or 4 operations. A central registry is essential for dealing with emergencies. For
example, a fire in a laboratory might cause excessive damage if the firemen were unwilling to
enter an area marked only with a conventional biohazardous or other type of warning sign if no
one was readily available who could explain the nature of the potential hazard within that
laboratory. It may be helpful to list, on the hazard warning signs on the laboratory doors, the
agents, the common names of the diseases caused by them, and the names and telephone
numbers of persons to be contacted in the event of an emergency.

Question # 05
Discuss in detail about Biosafety cabinets.

BIOSAFETY CABINETS
Biosafety Cabinets (BSCs) are enclosed workspaces with a ventilated hood that is designed to
contain pathogenic microorganisms during microbiological processes.
o The primary purpose of biosafety cabinets is to protect the laboratory personnel and the
environment from the pathogenic microorganism as aerosols might be formed during the
processing of such microorganisms.

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 o Biosafety cabinets are only used for certain risk group organisms and for processes that
might result in aerosol formation.
o These cabinets are provided with HEPA-filters that decontaminate the air moving out of
the cabinet.
o Biosafety cabinets might be confused with the laminar hood as both of these pieces of
equipment work as enclosed workspaces. But, laminar hood only provides protection to
the sample and not to the personnel and the environment, whereas biosafety cabinets
protect all three.
o The use of biosafety cabinets or other such physical containment is not required in the
biosafety level 1, but depending on the risk assessment, some processes might require
such containment.
o BSCs are an essential part of biosafety as they minimize the formation of aerosol,
protecting the environment, the pathogen, and the laboratory personnel.
o Besides, most BSCs also function to sterilize biological materials that are kept inside the
cabinets.

BIOSAFETY CABINET I:
o Class I is the most basic biosafety cabinet that provides protection to the environment
and the laboratory personnel.
o It doesn’t, however, provide protection to the product as the unsterilized room air is
drawn over the work surface.
o Class I biosafety cabinets are typically used to either enclose specific equipment like
centrifuges or for procedures like aerating cultures that might potentially generate
aerosols.
o Biosafety cabinets of this class are either ducted (connected to the building exhaust
system) or inducted (recirculating filtered exhaust back into the laboratory).
o In the Class I BSC, the room air is drawn in through the opening that also allows the
entry of the operator’s arm during work.
o The air inside the cabinet then takes in the aerosol particles that may have been
generated and moves it away from the operator towards the HEPA filter.
o The air moving out of the cabinet is thus, sterilized via the HEPA filters before its
discharge to the environment.
o In this way, the cabinets protect the operator and the environment from the aerosol but
not the sample.

BIOSAFETY CABINET II
o BSC-Class II cabinets provide both kinds of protection (of the samples and the
environment) since makeup air is also HEPA-filtered.

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 o The principle of operation of Class II cabinets involves a fan mounted in the top of the
cabinet that draws a curtain of sterile air over the workstation where the biological
products are being handled.
o The air then moves underneath the work station and back up to the top of the cabinet
before passing through the HEPA filters.
o The air drawn in acts as a barrier against the potentially contaminated air coming back
out to the operator.
o Class II BSCs are further divided into five types depending on the exhaust system and
the mechanism of work (recirculation of the exhaust air); Type A1, Type A2, Type B1,
Type B2, and Type C1.

a. Type A1
o The type A1 cabinets have a minimum inflow velocity of 75ft/min where the
contaminated divided just above the work station and mixes with the inflow air.
o The mixed air is then drawn through a duct network so that it reaches the back of the
cabinet.
o After this, air might be either recirculated after passing through the HEPA filters or
exhausted out of the cabinet, also through a HEPA filter.
o This type of cabinet is not as widely used as it is not safe to work with hazardous
chemical substances.

b. Type A2
o The type A2 cabinets have a minimum inflow velocity of 100 ft/min.
o In Class II, Type A2 BSC air enters the chamber through the front aperture, which
provides operator protection.
o The inflow air mixes with the downflow air (from the top of the cabinet)and enters the
front intake grille and then passes over the workstation where the air splits.
o Approximately 60% to 70% of the contaminated air is recycled and pushed back into
the workstation in the chamber through the downflow HEPA filter, while the remaining
30% to 40% is exhausted through the exhaust HEPA filters.

c. Type B1
o Type B cabinets are different from Type A cabinets as they use single-pass airflow to
control the flow of hazardous vapors.
o Type B1 cabinets divide the airflow so that the contaminated air is directed towards the
exhaust system while the air between the operator and the workstation mixes with the
inflow and is recirculated.
o The exhaust air dispersed out of the facility should be passed through the HEPA filters to
provide protection to the environment.
o These cabinets have a dedicated duct system which allows the release of the
contaminated air out of the facility.

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 o In the case of Type B1 cabinets, 40% of the air is recirculated,
whereas the remaining 60% is exhausted out of the facility.

d. Type B2
o For a Type B2 BSC, like in Type A cabinets, the air is drawn in from the front opening
creating an air barrier that protects the operator.
o Air is also drawn in from an opening at the top of the cabinet that supplies the down flow
of air in the cabinet.
o The air then passes through a HEPA filter, where 100% of the air is exhausted through a
dedicated duct system with an exhaust fan motor. The air moving out of the facility is
thus sterilized before its release into the atmosphere.
o The advantage of this system is the removal of toxic vapors that are generated in the
cabinet with no recirculation within the BSC.
o All of the contaminated airflow (100%) in a Type B2 cabinet is externally exhausted
which means the air drawn into the cabinet is 100% exhausted into the atmosphere.
o As a result, none of the air drawn into the B2 from either inflow or down flow is recycled
within the airflow system.
o Because none of the air is recirculated, these cabinets are the best to be used for tasks
involving the release of chemical vapors.
o Type B2 cabinets, however, are expensive, and their use is limited to toxicology
laboratories where protection against hazardous chemicals is imperative.

e. Type C1
o Type C1 cabinets are similar to Type B cabinets in their working mechanism, but these
are designed to reduce operating costs add flexibility to the laboratories.
o These cabinets work by using the single-pass airflow system where the cabinets move the
air by mixing it with the down flow air separated into columns for recirculation.
o The air above the workstation is drawn with a second fan which moves the contaminated
air out through the exhaust system with a HEPA filter.
o In this way, the cabinets provide protection to the environment, the operator, and the
workstation or the biological material.
o Type C cabinets are different from Type A cabinets as they use a single-pass airflow
mechanism where the air is not circulated.
o These differ from Type B cabinets in that they don’t require a dedicated ducted exhaust
system, can work for an extended duration to increase operator protection in the case of
exhaust failure, and can even run without the exhaust at all.

BIOSAFETY CABINET III

o Class III cabinets are leak-tight, totally enclosed but ventilated cabinets, where all air that
either enters or leaves through the facility pass through a HEPA filter.
o The cabinets are provided with rubber gloves that are attached to the system to be used
during operations in the cabinet. This is why these cabinets are also termed ‘glove boxes’.
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o The cabinet even has a transfer chamber that facilitates the sterilization of materials
before they leave the glove box.
o Even though the gloves restrict the hand movement of the operator inside the cabinet, it
prevents direct contact between the operator and the samples.
o The exhaust air is treated with double HEPA filters or HEPA filters in combination with
incineration.
o These cabinets can be used for all four Biosafety levels (1, 2, 3, and 4). But these are the
most important for the manipulation of biological materials in the Biosafety level 4.
o These cabinets are mostly custom-built for specific laboratories with lab equipment built
inside the chamber.
o All of these structural and design features provide maximum protection to the operator,
the environment, and the sample against the high-risk group 4 pathogenic organisms.

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