HISTORY OF BIOSAFETY:

The history of biosafety can be traced back to the late 19th century, when scientists began to
understand the risks of exposure to pathogenic microorganisms. The field of biosafety has evolved
through many areas of science, including microbiology, molecular biology, and veterinary science.
[1]

Early developments [1]

• 1890s: Scientists like Pasteur and Koch began to recognize the need for safety measures to
protect against pathogenic microorganisms [1]

• 1905: Robert Koch designed the first biological safety cabinets (BSCs) to protect workers
from aerosols [2]

• 1908: Winslow developed a method for counting bacteria in the air [3]

• 1919: Fricke published a laboratory safety manual in Germany [2]

• 1941: A survey highlighted the risk of laboratory-acquired brucellosis [3]

Later developments [3]

1947

The National Institutes of Health (NIH) built the first peacetime research laboratory designed for
microbiological safety [3]

1974

The United States Postal Service and Department of Transportation introduced regulations for
shipping etiologic agents [4]

1975

The Asilomar Conference on Recombinant DNA Molecules was held, where scientists decided to
impose strict guidelines for research involving recombinant DNA [5]

1980s

The American Biological Safety Association (ABSA) focused on international safety issues and
individual or groups of agents [4, 6]

Today, biosafety is regulated through national and international policies. [6]

 Biosafety cabinet

A microbiologist performing influenza research within a

biosafety cabinet

Acronym BSC

Other names Biological safety cabinet, microbiological

safety cabinet

Uses Biocontainment

Related Laminar flow cabinet

items Fume hood
Glove box

A biosafety cabinet (BSC)—also called a biological safety cabinet or microbiological safety

cabinet—is an enclosed, ventilated laboratory workspace for safely working with materials
contaminated with (or potentially contaminated with) pathogens requiring a defined biosafety level.
Several different types of BSC exist, differentiated by the degree of biocontainment they provide.
BSCs first became commercially available in 1950.[1]

Purposes
[edit]

The primary purpose of a BSC is to serve as a means to protect the laboratory worker and the
surrounding environment from pathogens. All exhaust air is HEPA-filtered as it exits the biosafety
cabinet, removing harmful bacteria and viruses.[2] This is in contrast to a laminar flow clean bench,
which blows unfiltered exhaust air towards the user and is not safe for work with pathogenic
agents.[3]: 13 [4] Neither are most BSCs safe for use as fume hoods.[2] Likewise, a fume hood fails to
provide the environmental protection that HEPA filtration in a BSC would provide.[5] However, most
classes of BSCs have a secondary purpose to maintain the sterility of materials inside (the "product").

Classes
[edit]

The U.S. Centers for Disease Control and Prevention (CDC) classifies BSCs into three
classes.[3]: 6 These classes and the types of BSCs within them are distinguished in two ways: the level
of personnel and environmental protection provided and the level of product protection provided.[6]

Class I
[edit]

Class I cabinets provide personnel and environmental protection but no product protection. In fact,
the inward flow of air can contribute to contamination of samples.[7] Inward airflow is maintained at
a minimum velocity of 75 ft/min(0.38 m/s). These BSCs are commonly used to enclose specific
equipment (e.g. centrifuges) or procedures (e.g. aerating cultures) that potentially generate
aerosols. BSCs of this class are either ducted (connected to the building exhaust system) or unducted
(recirculating filtered exhaust back into the laboratory).[3]: 6

Class II
[edit]

Class II cabinets provide both kinds of protection (of the samples and of the environment) since
makeup air is also HEPA-filtered. There are five types: Type A1 (formerly A), Type A2 (formerly A/B3),
Type B1, Type B2 and Type C1. Each type's requirements are defined by NSF International Standard
49,[3]: 31 which in 2002 reclassified A/B3 cabinets (classified under the latter type if connected to an
exhaust duct) as Type A2,[5] and added the Type C1 in the 2016 standard.[8] About 90% of all
biosafety cabinets installed are Type A2 cabinets.[7]

Principles of operation use motor driven blowers (fans) mounted in the cabinet to draw directional
mass airflow around a user and into the air grille - protecting the operator. The air is then drawn
underneath the work surface and back up to the top of the cabinet where it passes through the
HEPA filters. A column of HEPA filtered, sterile air is also blown downward, over products and
processes to prevent contamination. Air is also exhausted through a HEPA filter, and depending on
the Type of Class II BSC, the air is either recirculated back into the laboratory or pulled by an exhaust
fan, through ductwork where it is expelled from the building.[9]

Class II Biosafety Cabinet Types using color to illustrate where

it is safe to handle hazardous chemistry with microbiology. (Blue is recirculated air where it is NOT
safe to use hazardous chemistry. Red is single pass air and is safe for chemical use. Purple is
uncertainty due to location of the BSCs smoke-split)
The Type A1 cabinet, formerly known as Type A, has a minimum inflow velocity of 75 ft/min. The
downflow air, considered contaminated, splits just above the work surface (the BSCs smoke split)
and mixes with the inflow. This air is drawn, through ductwork, up the back of the cabinet where it is
then blown into a positive pressure, contaminated plenum. Here, the air is either recirculated,
through a HEPA filter, back down over the work zone, or exhausted out of the cabinet (also through
a HEPA filter). Sizing of HEPA filters and an internal damper are used to balance these air volumes.
This type is not safe for work with hazardous chemicals even when exhausted with a "thimble" or
canopy to avoid disturbing internal air flow.[3]: 8–9

The Type A2 cabinet, formerly designated A/B3, has a minimum inflow velocity of 100 ft/min. A
negative air pressure plenum surrounds all contaminated positive pressure plenums. In other
respects, the specifications are identical to those of a Type A1 cabinet.[3]: 9–11 [5]

Type B1 and B2 cabinets have a minimum inflow velocity of 100 ft/min, and these cabinets must be
hard-ducted to an exhaust system rather than exhausted through a thimble connection. Their
exhaust systems must also be dedicated (one BSC per duct run, per blower). In contrast to the type
A1 and A2 cabinets, Type B BSCs use single pass airflow (air that does not mix and recirculate) in
order to also control hazardous chemical vapors. Type B1 cabinets split the airflow so that the air
behind the smoke-split is directed to the exhaust system, while air between the operator and the
smoke-split mixes with inflow air and is recirculated as downflow. Since exhaust air is drawn from
the rear grille, the CDC advises that work with hazardous chemistry be conducted in the rear of the
cabinet.[3]: 10 This is complicated, since the smoke split (demarking the "rear of the cabinet") is an
invisible line that extends the width of the cabinet (approximately 10-14 inches from the front grille)
and drifts as the internal HEPA filters load with particulate.[9]

The Type B2 cabinet (also known as a Total Exhaust BSC) is expensive to operate because no air is
recirculated within.[3] Therefore, this type is mainly found in such applications
as toxicology laboratories, where the ability to safely use hazardous chemistry is
important.[5] 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.

Class III
[edit]A researcher observing a specimen through the built-in microscope in a Class III biosafety
cabinetAerosol control platform inside a Class III Biosafety Cabinet

The Class III cabinet, generally only installed in maximum containment laboratories, is specifically
designed for work with BSL-4 pathogenic agents, providing maximum protection. The enclosure is
gas-tight, and all materials enter and leave through a dunk tank or double-door autoclave. Gloves
attached to the front prevent direct contact with hazardous materials (Class III cabinets are
sometimes called glove boxes). These custom-built cabinets often attach into a line, and the lab
equipment installed inside is usually custom-built as well.[3]: 12–13
Ergonomics
[edit]

Biosafety cabinets are used on a daily basis for hours. Besides protection of user and sample
material, the human design factors (ergonomics) of the work become more and more important.
This includes reduction of the noise level (for a more convenient working atmosphere), a height
adjustable stand or stool and footrest (for optimized sitting position), panorama side windows (more
light within cabinet), 10° angled front sash (enables better sitting position), strong light sources
(better view within cabinet), forearm support for comfort and safety, as well as extended access
openings and sloped viewing windows to improve the working conditions.[10]

Ultraviolet lamps
[edit]

The CDC does not recommend the installation of UV lamps in BSCs.[3]: 12–13 The American Biological
Safety Association supports this position, citing the safety risk to personnel, shallow penetration,
reduced effectiveness in high relative humidity, and the frequent need to clean and replace the
bulb.[11] UV lamps should not be used as a primary source of surface decontamination within a
BSC.[12] However, these assertions have been formally disputed in at least one peer-reviewed article
which points out that:[13]

• There is no cited basis for the need to remove dust and dirt from bulbs

• Properly functioning biosafety cabinets have very clean air so dust is less likely to build up

• Laboratories are generally air-conditioned which eliminates the concern over humidity
inhibition of UV effectiveness

• With proper use UV exposure risk to users is very low

• UV disinfection is effective for germicide and virucide as well as inhibiting DNA

contamination for PCR

• UV disinfection has the advantage of not leaving residues like physical disinfectants

• The relative safety and risks of UV versus other disinfection techniques (which also entail
risks) should be considered

Maintenance and service

[edit]

Biological safety cabinets are generally required to comply with that country or region's standards.
This requirement may be governed by an institutional body such as the TGA, FDA or WHO. Within
Australia, for example, Class II BSCs are required to meet construction standards entitled AS2252.2.
Those standards reference several other standards, such as AS2243.3. AS2243.3 classifies the level of
risk that microorganisms pose based on their pathogenicity, mode of transmission, and host range,
along with current preventative measures and effective treatments.[14]

There are specific field test requirements for class II BSC's. The United States' base for field testing is
NSF49; Europe relies on EN12469; and Australia has the AS1807 series of test methods (referenced
within AS2252.2).[15] The field test requirements may include:
 • Air velocity within the work zone.

• Air barrier testing (barrier between operator and product; some standards utilise inward
velocity testing instead)

• Filter integrity (leak testing or the amount of aerosols that a filter allows to pass through it)

• Particle counting within the work zone

• Gas tightness

• Leak testing of the work zone (work zone integrity testing)

• Illuminance within work zone

• UV light effectiveness

• Sound level

In general terms, a regular service maintenance schedule may include the following tasks:

• Airflow and filter capacities are verified. The filters have a limited lifespan, determined by
the air quality within the laboratory space, the amount of particles & aerosols generated
inside the BSC work zone, and the volume of air passing through the filters. As these filters
load, the internal fan is required to do more work to push/pull the same volume of air
through them. Newer cabinets measure the airflow constantly and self-compensate fan
performance to ensure constant volumes of air moving through the filters and the cabinet.
However, self-adjusting cabinets should be cross-verified by calibrated instruments to
ensure that performance is maintained to the correct levels. If the flow drops below desired
performance, an audio and visual alarm will alert the operator. Changing the filter should be
limited to trained persons as the filter is potentially contaminated. This can be done either
after the cabinet has been decontaminated using a gaseous procedure
(using formaldehyde, chlorine dioxide, or vaporized hydrogen peroxide) or a "bag-in/bag-
out" procedure.

• UV lights are checked and changed. UV lights decrease in power over time, resulting in
diminished disinfection of the working area.

Work practices
[edit]

As with work on open bench tops, work performed within a BSC must be performed carefully and
safely. To avoid contamination and the risk of personnel exposure, the CDC advises investigators to
follow best practices to reduce and control splatter and aerosol generation, such as keeping clean
materials at least 12 inches (30 cm) from aerosol-generating activities and arranging the work flow
"from clean to contaminated".[3]: 22 In particular, open flames, not necessary within the clean
environment of a Class II or III BSC, cause disruption of the airflow inside.[3]: 22 Once work inside a BSC
has been completed, it is necessary to decontaminate the surfaces of the BSC as with other lab
equipment and materials.[3]: 24
Primary Containment For Biohazards
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Containment-for-Biohazards
Biosafety Levels 1, 2, 3 & 4
Biological Safety Levels (BSL) are a series of protections relegated to the activities that take
place in particular biological labs. They are individual safeguards designed to protect
laboratory personnel, as well as the surrounding environment and community.
These levels, which are ranked from one to four, are selected based on the agents or
organisms that are being researched or worked on in any given laboratory setting. For
example, a basic lab setting specializing in the research of nonlethal agents that pose a
minimal potential threat to lab workers and the environment are generally considered BSL-
1—the lowest biosafety lab level. A specialized research laboratory that deals with
potentially deadly infectious agents like Ebola would be designated as BSL-4—the highest
and most stringent level.
The Centers for Disease Control and Prevention (CDC) sets BSL lab levels as a way of
exhibiting specific controls for the containment of microbes and biological agents. Each BSL
lab level builds upon on the previous level—thereby creating layer upon layer of constraints
and barriers. These lab levels are determined by the following:
• Risks related to containment
• Severity of infection
• Transmissibility
• Nature of the work conducted
• Origin of the microbe
• Agent in question
• Route of exposure
The reason biosafety levels are so important is because they dictate the type of work
practices that are allowed to take place in a lab setting. They also heavily influence the
overall design of the facility in question, as well as the type of specialized safety equipment
used within it.
The following is an explanation of each biosafety level—what they mean and how they differ
in safety measures and best practices.

BSL–1
As the lowest of the four, biosafety level 1 applies to laboratory settings in which
personnel work with low-risk microbes that pose little to no threat of infection in healthy
adults. An example of a microbe that is typically worked with at a BSL-1 is a
nonpathogenic strain of E. coli.
This laboratory setting typically consists of research taking place on benches without the
use of special contaminant equipment. A BSL-1 lab, which is not required to be isolated
from surrounding facilities, houses activities that require only standard microbial
practices, such as:
• Mechanical pipetting only (no mouth pipetting allowed)
• Safe sharps handling
• Avoidance of splashes or aerosols
• Daily decontamination of all work surfaces when work is complete
• Hand washing
• Prohibition of food, drink and smoking materials in lab setting
• Personal protective equipment, such as; eye protection, gloves and a lab coat or
gown
• Biohazard signs
BSL-1 labs also requires immediate decontamination after spills. Infection materials are
also decontaminated prior to disposal, generally through the use of an autoclave .

BSL–2
This biosafety level covers laboratories that work with agents associated with human
diseases (i.e. pathogenic or infections organisms) that pose a moderate health hazard.
Examples of agents typically worked with in a BSL-2 include equine encephalitis viruses
and HIV, as well as Staphylococcus aureus (staph infections).
BSL-2 laboratories maintain the same standard microbial practices as BSL-1 labs, but also
includes enhanced measures due to the potential risk of the aforementioned microbes.
Personnel working in BSL-2 labs are expected to take even greater care to prevent injuries
such as cuts and other breaches of the skin, as well as ingestion and mucous membrane
exposures.
In addition to BSL 1 expectation, the following practices are required in a BSL 2 lab setting:
• Appropriate personal protective equipment (PPE) must be worn, including lab
coats and gloves. Eye protection and face shields can also be worn, as needed.
• All procedures that can cause infection from aerosols or splashes are performed
within a biological safety cabinet (BSC).
• An autoclave or an alternative method of decontamination is available for proper
disposals.
 • The laboratory has self-closing, lockable doors.
• A sink and eyewash station should be readily available.
• Biohazard warning signs
Access to a BSL-2 lab is far more restrictive than a BSL-1 lab. Outside personnel, or those
with an increased risk of contamination, are often restricted from entering when work is
being conducted.
List of common BSL-2 level agents
Searchable Database
• Risk Group Classification for Infectious Agents (American Biological Safety
Association)
• Material Safety Data Sheets for Biological Materials (Health Canada)

BSL-3
Again building upon the two prior biosafety levels, a BSL-3 laboratory typically includes work
on microbes that are either indigenous or exotic, and can cause serious or potentially lethal
disease through inhalation. Examples of microbes worked with in a BSL-3 includes; yellow
fever, West Nile virus, and the bacteria that causes tuberculosis.
The microbes are so serious that the work is often strictly controlled and registered with the
appropriate government agencies. Laboratory personnel are also under medical surveillance
and could receive immunizations for microbes they work with.
Common requirements in a BSL-3 laboratory include:
• Standard personal protective equipment must be worn, and respirators might be
required
• Solid-front wraparound gowns, scrub suits or coveralls are often required
• All work with microbes must be performed within an appropriate BSC
• Access hands-free sink and eyewash are available near the exit
• Sustained directional airflow to draw air into the laboratory from clean areas
towards potentially contaminated areas (Exhaust air cannot be re-circulated)
• A self closing set of locking doors with access away from general building corridors
Access to a BSL-3 laboratory is restricted and controlled at all times.
List of Biological Safety Level III agents.

Searchable Database
• Risk Group Classification for Infectious Agents (American Biological Safety
Association)
 • Material Safety Data Sheets for Biological Materials (Health Canada)

BSL-4
BSL-4 labs are rare. However some do exist in a small number of places in the US and
around the world. As the highest level of biological safety, a BSL-4 lab consists of work with
highly dangerous and exotic microbes. Infections caused by these types of microbes are
frequently fatal, and come without treatment or vaccines. Two examples of such microbes
include Ebola and Marburg viruses.
In addition to BSL-3 considerations, BSL-4 laboratories have the following containment
requirements:
• Personnel are required to change clothing before entering, shower upon exiting
• Decontamination of all materials before exiting
• Personnel must wear appropriate personal protective equipment from prior BSL
levels, as well as a full body, air-supplied, positive pressure suit
• A Class III biological safety cabinet
A BSL-4 laboratory is extremely isolated—often located in a separate building or in an
isolated and restricted zone of the building. The laboratory also features a dedicated supply
and exhaust air, as well as vacuum lines and decontamination systems.
Knowing the difference in biosafety lab levels and their corresponding safety requirements
is imperative for anyone working with microbes in a lab setting.
Figure 1. Summary of Biological Agents.

Biosafety
BSL-1 BSL-2 BSL-3 BSL-4
Level

• Max
• No
• Containment containment
containment • High containment
• Moderate • "Exotic,"
• Defined • Aerosol
risk high-risk
Description organisms Transmission
agents
• Disease of
• Unlikely to • Serious/potentially
varying • Life-
cause lethal disease
severity threatening
disease
disease

Sample Influenza, HIV, Lyme

E.Coli Tuberculosis Ebola Virus
Organisms disease
 Dangerous and
Agents associated Indigenous or exotic exotic agents that
Agents that present
with human disease agents, agents that pose a high risk of
minimal potential
Pathogen and pose moderate present a potential for aerosol-transmitted
hazard to personnel
Type hazards to aerosol transmission, and laboratory
and the
personnel and the agents causing serious or infections and life
environment.
environment. potentially lethal disease. threatening
disease.

Pass-thru autoclave
Pass-thru autoclave with
Autoclave with bioseal
None None bioseal required in
Requirements required in
laboratory room.
laboratory room

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program/biosafety-levels/index.htm
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The biosafety level (BSL) of a microorganism depends on the level of risk it

poses to humans and the environment. The four BSLs are BSL-1, BSL-2, BSL-3, and BSL-4, with BSL-4
being the highest level.

BSL-1

• The lowest level, for microbes that pose a minimal risk

• Standard microbiological practices are used

BSL-2

• For microbes that pose a moderate health risk, such as those associated with human
disease

• Standard microbiological practices are used, plus additional precautions like limited access
and biohazard warning signs

BSL-3

• For microbes that can cause serious or potentially lethal disease, such as those that can be
transmitted through the air

• Additional precautions include decontamination of all waste and laboratory clothing, and
controlled access

BSL-4
 • The highest level, for microbes that are highly dangerous and exotic, and pose a high risk of
life-threatening disease

• Additional precautions include a completely sealed cabinet, positive pressure personnel

suits, and showers

The recommended biosafety level for infectious agents depends on the risk of the agent to people
and the environment. The four biosafety levels are BSL-1, BSL-2, BSL-3, and BSL-4, with BSL-4 being
the highest level.

BSL-1

• Suitable for work with agents that are unlikely to cause disease in healthy adults

• Minimal potential hazard to people and the environment

• Laboratory is not separated from the general traffic patterns in the building

BSL-2

• Suitable for work with agents that pose moderate risk to people and the environment

• Laboratory personnel must be trained and supervised

• Access to the laboratory is restricted

• Procedures that may create infectious aerosols or splashes must be conducted in Biosafety
Cabinets (BSCs)

BSL-3

• Suitable for work with agents that may cause serious or potentially lethal disease

• Laboratory must have physical separation from access corridors

 • Laboratory must have negative airflow

BSL-4

• Suitable for work with dangerous agents that pose a high risk of life-threatening disease

• Laboratory must be in a separate building or isolated zone

• Laboratory must have dedicated supply and exhaust, vacuum, and decontamination systems

The recommended biosafety levels for infected animals are Animal Biosafety Levels (ABSLs) 1–
4. These levels are based on the risk of the infectious agents and the potential for exposure to
personnel and the environment.

ABSL 1

• Suitable for work with agents that pose minimal or no known risk

• The laboratory is not separated from the general traffic patterns in the building

ABSL 2

• Suitable for work with agents that pose a moderate risk

• Laboratory personnel have specific training in handling pathogenic agents

• Access to the laboratory is limited when experiments are being conducted

ABSL 3

• Suitable for work with agents that pose a serious or potentially lethal risk

• Facilities require restricted access, specific training for personnel, and primary containment
equipment

ABSL 4

• Suitable for work with agents that pose a high risk of life-threatening disease
 • Facilities require maximum containment equipment and are often in a separate building or
isolated

Biosafety guidelines are a set of policies, rules, and procedures that personnel must follow in various
facilities handling microbiological agents, such as bacteria, viruses, parasites, fungi, prions, and other
related agents and microbiological products.

Biosafety guidelines are a set of rules and procedures that ensure the safe handling of
microbiological agents. These guidelines are important in healthcare institutions, biomedical
research facilities, and teaching laboratories.

Purpose

• Protect laboratory workers, the environment, and clinical specimens

• Reduce or eliminate exposure to potentially hazardous agents

Components

• Risk assessment: Identify and assess the risk of the work being done

• Containment: Use safe methods, equipment, and facilities to manage infectious materials

• Training: Ensure staff are trained and proficient in handling hazardous materials

• Safety protocols: Follow safety protocols for fire, radiation, and electricity

• Laboratory design: Ensure laboratories are designed for the appropriate biosafety level

• Equipment: Use the appropriate equipment for the work being done
 • Personal protective equipment (PPE): Wear the appropriate PPE, such as gloves, face shield,
and safety glasses or goggles

Examples of biosafety guidelines

• Mechanical pipetting aids should be used instead of mouth pipetting

• Eating, drinking, and storing food in laboratory areas is not permitted

• Use a biological safety cabinet (BSC) when working with potentially aerosol-generating
specimens

• Perform proper handwashing procedures

The Government of India regulates biosafety through the Environment (Protection) Act, 1986 and
the Rules for the Manufacture, Use, Import, Export and Storage of Hazardous
Microorganisms/Genetically Engineered Organisms or Cells, 1989. The Department of Biotechnology
(DBT) and the Ministry of Environment, Forest and Climate Change (MoEF&CC) are the primary
regulatory bodies for biosafety in India.

Biosafety regulations

• The Rules, 1989 govern the manufacture, import, research, and release of genetically
engineered organisms (GEOs).

• The rules also cover the handling of hazardous microorganisms and products made from
them.

• The rules are intended to ensure the safety of laboratory personnel and the environment.

Biosafety committees

• The Recombinant DNA Advisory Committee (RDAC) and the Review Committee on Genetic
Manipulation (RCGM) are two committees that operate under the DBT.

• Institutional Biosafety Committees (IBSCs) are responsible for overseeing research activities
at their institutions.

Biosafety resources
• The Indian Biosafety Rules and Regulations website provides information on biosafety rules
and regulations, including guidelines for DNA safety, biotechnology safety, and food safety.

• The Biosafety Clearing House (BCH) is a mechanism for sharing information on LMOs.