2. Objectives of this Overview
Meaning of Biosafety
General safety practices
• Standard microbiological practices
• Safety Equipment and waste disposal
3. What is Biosafety?
• Biosafety are all steps taken to protect human, products or
environment from biological hazard that might occur from
research and commerce of infectious or from genetically
• The risks associated with the laboratory activities occur either
from the samples or the procedural requirements hence
application of the standard microbiological techniques and
employing facilities suitable for the risk level of the pathogen
helps to protect the researcher from laboratory-acquired
5. General safety practices.
Basic safety rules for laboratory conduct should be observed whenever
working in a laboratory. Many of the most common safety rules are
• Know locations of laboratory safety showers, eyewash stations, and
fire extinguishers. The safety equipment may be located in the
hallway near the laboratory entrance.
• Know emergency exit routes.
• Minimize all chemical exposures.
• No horseplay will be tolerated.
• Assume that all chemicals of unknown toxicity are highly toxic.
• Post warning signs when unusual hazards, hazardous materials,
hazardous equipment, or other special conditions are present.
• Do not use mouth suction for pipetting or starting a siphon.
• Wash exposed areas of the skin prior to leaving the laboratory.
6. Biosafety Laboratories (BSL).
Biosafety laboratory levels are designed to identify various protective
measures that are to be taken in a laboratory setting to protect the
researchers, the environment, and the microorganisms.
• 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.
• The allocation of a pathogenic agent to a biosafety level for laboratory
work must be based on the risk assessment.
• Such assessments take the risk group as well as other factors into
consideration while establishing the appropriate biosafety level. The
biosafety levels, thus, might differ from one region to another.
• 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.
8. Biosafety Level 1 (BSL-1)
• Biosafety Level 1 is the level appropriate for work involving well-characterized
agents not known to consistently cause disease in immune-competent adult
humans and cause a minimal potential hazard to the laboratory personnel and the
• Biosafety level 1 is the lowest safety level, and the precautions required for the
level are thus limited and not as extensive.
• These laboratories provide general space in which work is done with viable agents
that are not associated with disease in healthy adults.
• The BSL-1 laboratories are not necessarily separated from the general traffic in the
• Most of the work is typically conducted on open bench tops using general
• Unique laboratory design or containment equipment are not required but may be
used depending on the risk assessment.
• Laboratory personnel must be provided with specific training in the procedures to
be conducted in the laboratory, which is then supervised by a scientist with
training in microbiology or related sciences.
9. • Standard microbiological practices
• The laboratory supervisor should implement the policies regarding the access
control to the laboratory.
• Laboratory personnel must wash their hands after working with potentially
hazardous materials and before leaving the laboratory.
• Activities like eating, drinking, smoking, handling contact lenses, applying
cosmetics, and storing food are not be permitted in laboratory areas.
• Mouth pipetting is prohibited; mechanical pipetting devices must be employed.
• All procedures to be conducted in the laboratory should be performed while
avoiding the creation of splashes and aerosols.
• There are no safety specific safety practices required for BSL-1.
10. Safety equipment and waste disposal for BSL-1
• Special containment devices like the Bio-safety Cabinets are not required for BSL-1.
• In order to prevent the contamination of personal clothing, protective laboratory coats,
gowns, or uniforms are recommended.
• While conducting tests with a high possibility of aerosol formation, protective eyewear
can be used.
• Biosafety Level-1 is commonly used while performing tests on microbial agents that are
not known to cause diseases in immune-compromised individuals.
• These laboratories include the laboratories used for teaching purposes in colleges and
The common organisms that require Biosafety Level-1 containment include less
hazardous organisms like Agrobacterium radiobacter, Aspergillus niger, Bacillus
thuringiensis, Escherichia coli strain K12, Lactobacillus acidophilus, Micrococcus
leuteus, Neurospora crassa, Pseudomonas fluorescens, Serratia marcescens.
• However, the requirement of the biosafety level might differ depending on the risk
assessment of the pathogen.
11. Biosafety Level 2 (BSL-2)
• Biosafety level-2 laboratories are the laboratories that are used for the tasks involving
microbial agents of moderate potential hazards to the laboratory personnel, the
environment, and the agent.
• However, the infectious agents or the toxins might pose a moderate danger if
accidentally inhaled, swallowed, or exposed to the skin.
• The precautions associated with biosafety level-2 are comparatively more extensive than
BSL-1, but BSL-1 and BSL-2 laboratories are generally considered as basic laboratories.
• BSL-2 laboratories like BSL-1 laboratories are not necessarily separated from the general
traffic patterns in the building.
• However, access into the laboratory is limited while BSL-2 experiments are in progress.
• The annual inspection of the laboratories is also an important part of the BSL-2
requirements. These might include changing the filters or replacement of some devices.
• The work is mostly conducted on sterilized bench tops except for some processes that
might form aerosols. The latter is conducted in safety cabinets.
• The precautions to be followed in BSL-2 include all the precautions of the BSL-1 and
some additional precautions.
12. • Standard microbiological practices
• All the laboratory personnel must wash their hands after using viable microorganisms and before leaving the
• Eating, drinking, smoking, and handling contact lenses in the laboratory are strictly prohibited.
• Mechanical pipetting should be done instead of mouth pipetting.
• All contaminated cultures, glassware, plastic ware, and biologically contaminated waste must be treated as bio-
hazards and thus, autoclaved.
• Work surfaces must be decontaminated with disinfectant at the end of the day or after any spills or splashes.
• Used hypodermic syringes and needles, Pasteur pipettes, razor blades, contaminated broken glass, and blood
vials are treated as medical waste and discarded in puncture-resistant sharps disposal containers.
• People with increased risk of acquiring infections like the immune-compromised and pregnant individuals
should not be allowed to enter the BSL-2 laboratories while the laboratories are at work.
• An annual review of the BSL-2 manual should be done to update the guidelines.
• Documented policies and procedures should be established that limit the entrance to individuals who know of
the potential hazards and are appropriately trained.
• A biohazard symbol is placed on pieces of equipment where biohazardous materials are used or stored.
13. Safety Equipment and waste disposal for BSL-2
• Protective coats are to be worn while entering the laboratory and then removed and kept in the laboratory
• The laboratory design should be made such that it can be easily cleaned and decontaminated with minimum
nooks and corners.
• The laboratory doors should be closed whenever work with hazardous biomaterials is conducted.
• An autoclave must be available.
• Biosafety level-2 laboratories are mostly used for routine analysis and culture of moderately hazardous agents.
• Besides, some of the laboratories used for teaching and training purposes are also BSL-2 laboratories.
• The organisms that require BSL-2 laboratories include the pathogenic strains of E.
coli, Staphylococcus, Salmonella, Plasmodium falciparum, Toxoplasma, and Herpes Simples Viruses.
• The allocation of organisms to the laboratories, however, might differ depending on the risk assessment.
15. Biosafety Level 3 (BSL-3)
• 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.
• 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
• Autoinoculation and ingestion present primary hazards to personnel working with these
agents at this level.
• Working in such laboratories require laboratory personnel with specific training in
handling pathogenic and potentially lethal agents, along with supervisors scientists
competent in handling infectious agents and associated procedures.
• Biosafety Level 3 containment laboratories for animals and research are the most
challenging containment level facilities to design and operate.
• 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.
16. • BSL-3 laboratories are also called the containment laboratory as they require
containment equipment to protect the personnel, the microbial agent, and the
• 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
• 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.
• Besides the general procedures and laboratory practices, the supervisor also formulates
additional policies to limit the entry to the laboratory.
• 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.
• The work surfaces and sinks should be decontaminated once every work shift or after
any spills or splashes.
• The BSL-3 laboratories should be separated from the general traffic in a building to limit
entry into the laboratories at all times.
17. Safety Practices
• 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
• Laboratory personnel must have medical surveillance and offered appropriate
immunizations for agents handled or potentially present in the laboratory.
• Each institution should consider the collection and storage of serum samples from at-risk
• A laboratory-specific biosafety manual, which is available and accessible to all, must be
prepared and adopted as a policy.
• The laboratory supervisor must check for the demonstration of proficiency in standard
and special microbiological practices by all laboratory personnel before working with
• Potentially hazardous materials must be placed in a durable, leak-proof container or vial
during collection, processing, storage, or transport within a facility.
• All laboratory equipment should be routinely decontaminated after work or after any
spills or splashes.
• The laboratory biosafety manual must define procedures t be adopted in the case of
exposure to infectious materials, and these should be treated accordingly.
• 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.
18. Safety Equipment and waste disposal for BSL-3
• Biosafety cabinets are to be used for the manipulation of all infectious agents.
• Individual protection gears like personal protective equipment, coats, gloves, and respiratory
protection should be worn while entering the laboratories and then removed before leaving.
• 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.
• The filters, manuals, equipment, vacuum pipes, autoclaves, etc. should be revised and
• BSL-3 laboratories are used for clinical, diagnostic, teaching, research, or production facilities.
• These laboratories are used for the handling and manipulation of highly infectious agents that
prose direct severe effects on the health of the personnel.
• These are used for the studies regarding the effects of infectious agents and various toxins
and their effects.
• The pathogens that require BSL-3 laboratories include HIV, H1N1 flu, Yersinia
pestis, Mycobacterium tuberculosis, SARS, Rabies Virus, West Nile Virus, Ricketts, etc.
• The placement of the organisms in different Biosafety levels, however, might defer and should
also be determined after risk assessment.vg
20. Biosafety Level 4 (BSL-4)
• 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
• The BSL-4 laboratories are often used while handling and
manipulating Risk Group 4 pathogens that are extremely
dangerous, with no known vaccines or therapies, and require
extreme precautions during work.
• 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.
• The requirements of BSL-4 laboratories are extensive with specific
laboratory design, training procedures, and highly protective
equipment and personal gears.
• 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.
• BSL-4 laboratories are also termed the maximum containment
laboratories as they have secondary barriers to prevent hazardous
materials from escaping into the environment.
• The BSL-4 laboratories should follow the requirements of all BSL-1,
BSL-2, and BSL-3, along with additional specific precautions.
22. Standard microbiological practices
• No work conducted within the BSL-4 should be done on an open
bench or an open vessel.
• The work stations, equipment, and sinks should be sterilized post
• The laboratory personnel should be in protective gear that might
include full-body PPEs, gloves, masks, and coats.
• The doors of the laboratories should be closed at all times with the
laboratory placed away from the general traffic in the building.
• Activities like drinking, eating, mouth pipetting should be avoided at
• Only people that are trained in handling the BSL-4 organisms and the
equipment in the laboratory should be allowed into the laboratory.
23. Safety Practices
• 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.
• 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.
• 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.
• Personnel can enter and leave the facility only after the clothing change and
through the shower rooms.
• 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.
• A system is set up for reporting laboratory accidents, exposures, and the
medical surveillance of potential laboratory-associated illnesses.
24. Safety equipment and waste disposal
• 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.
• Walls, floors, and ceilings of the laboratories must form a sealed
internal shell which facilitates fumigation and is animal and
• A double-doored autoclave is placed for decontaminating
materials passing out of the facility.
• 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.
• BSL-4 laboratories are used for diagnostic and research work on
easily transmitted pathogens, causing fatal diseases.
• These laboratories are used for new and unknown pathogenic
microbes, for which no vaccines or therapies are available.
• They are also used for clinical and production facilities that
require highly sophisticated techniques and advanced
• The BSL-4 level pathogens include the risk group IV organisms
like Ebola virus, SARS-CoV-2, Central European Encephalitis
virus, Hemorrhagic viruses, etc.
• National Research Council (US) Committee on Hazardous Biological Substances in the
Laboratory. Biosafety In The Laboratory: Prudent Practices for the Handling and Disposal of
Infectious Materials. Washington (DC): National Academies Press (US); 1989. Appendix A,
Biosafety in Microbiological and Biomedical Laboratories. Available
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• Janosko, K., Holbrook, M. R., Adams, R., Barr, J., Bollinger, L., Newton, J. T., Ntiforo, C., Coe, L.,
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• Standard Microbiological Practices for Biosafety Level 1 Laboratories at the University of
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• Biosafety Manual. Boston University Research Support.