Recommended Biosafety level for Infectious Agents and Infected Animals

RECOMMENDED BIOSAFETY LEVEL FOR
INFECTIOUS AGENTS AND INFECTED
ANIMALS
JINO AFFRALD R
MSC MEDICAL BIONANOTECHNOLOGY
BIOETHICS AND IPR

INFECTION
An infection is the invasion of an organism's body tissues by
disease-causing agents, their multiplication, and the reaction of
host tissues to the infectious agents and the toxins they
produce.
INTRODUCTION FACTS
• Many infections are preventable through vaccination.
• The World Health Organization (WHO) estimates that
vaccinations prevent 2 to 3 million deaths each year.
• Antimicrobial resistance continues to be a public health
threat. Some organisms of concern include drug-resistant
strains of gonorrhea, staph bacteria, and Candida yeast.
• New causes of infection are always emerging Trusted
Source. Some examples you may be familiar with
include severe acute respiratory syndrome (SARS) and
Zika virus.
• You can acquire an infection in many different ways,
such as directly from a person with an infection, via
contaminated food or water, and even through the bite
of an insect.
According to the latest Revised National Tuberculosis
Control Programme Report, as many as 4.4 lakh people
died in 2018 of TB, which is 29 per cent of the total 1.5
million deaths due to TB in the world.
According to the WHO (2018), there have been three to
five million cases of severe illness due to influenza,
and about 290,000 to 650,000 respiratory deaths.
Already 1,469 people in India have been affected by the
communicable respiratory disease caused by a sub-
type of influenza A virus, called H1N1.

CAUSES OF INFECTION TRANSMISSION
Direct contact Indirect contact
Infections can spread when
you come directly into
contact with a person who
has an infection, whether
through touching, kissing.
Direct contact with the
bodily fluids of a person who
has an infection can also
spread infections in some
instances.
This can include things like:
• blood
• nasal secretions
• saliva
• semen
• vaginal secretions
Some infectious organisms can be
found throughout your
environment. You can come into
contact with these things and
then spread the infection to
yourself.
A common example of this is
when someone with the flu
coughs or sneezes. Influenza virus
can then be present in the air or
on objects such as door and
faucet handles.
If you touch a contaminated
object and then touch your face,
mouth, or nose, you may become
infected.
1) CONTACT
Direct contact
Indirect contact

Causes of infection transmission
2) Through contaminated food
or water
Food or water may be
contaminated with infectious
organisms. You can get these
infections by consuming things
like:
• Foods prepped or prepared in
unsanitary conditions.
• Raw or undercooked foods, such
as produce, meats, or seafood.
• Improperly canned foods.
• Unpasteurized milks or juices.
• Foods that have been
improperly stored or refrigerated.
3) From an infected
animal
4) From a bug bite
• Some infections are spread to people
from an infected animal.
• One example is the rabies virus,
which you can get if an infected
animal bites you.
• Another example is toxoplasmosis.
You can come down with this
parasitic disease from changing an
infected cat’s litter box.
There are many different types of biting
bugs, including ticks, mosquitoes, and
lice. In some cases, you can get an
infection if a bug carrying around an
infectious microorganism bites you.
Some examples include malaria, Lyme
disease, and West Nile Virus.
Through contaminated food or water
Infected animal Bug bite

IDENTIFICATION OF INFECTION PREVENTING INFECTION
Some infections have very characteristic symptoms.
diagnosis based on
• Symptoms
• Medical history
• Physical examination.
In other cases, it can be hard to determine what
type of organism may be causing your condition. For
example, some bacterial and viral infections can
have very similar symptoms.
In this cases sample is collected from depends on
your illness and the type of organism suspected.
Some sample types can include:
• Blood
• Urine
• Stool
• Nasal or Throat Sputum
• Cerebrospinal fluid (CSF)
Imaging tests: such as an X-ray, CT scan, or MRI
scan.
Biopsy of the affected tissue to examine it.
1. Practice good hand hygiene.
2. Get vaccinated.
3. Avoid sharing personal items.
4. Cover cuts or scrapes.
5. Use insect repellents.
6. Be careful with food.
7. Avoid wild animals.
8. Know before you go.
9. Cover mouth while coughing.
10. Stay home if you’re sick.

BIOSAFETY LEVEL FOR INFECTIOUS
AGENTS AND INFECTED ANIMALS

BIOSAFETY
• Microbiology laboratories are special, often unique,
work environments that may pose special infectious
disease risks to persons in or near them.
• Personnel have contracted infections in the
laboratory throughout the history of microbiology.
Four biosafety levels
are also described
for infectious
disease activities
utilizing small
laboratory animals.
Recommendations
for biosafety levels
for specific agents
are made on the
basis of the
potential hazard of
the agent and of the
laboratory function
or activity.
CONTAINMENT: Safe methods for managing infectious agents in the
laboratory environment where they are being handled or
maintained. The purpose of containment is to reduce exposure of
lab workers and other persons and to prevent escape into the
outside environment of potentially hazardous agents.
PRIMARY CONTAINMENT
Primary containment, the protection of personnel
and the immediate laboratory environment from
exposure to infectious agents, is provided by good
microbiological technique and the use of appropriate
safety equipment. The use of vaccines may provide
an increased level of personal protection.
SECONDARY CONTAINMENT
Secondary containment, the protection of the
environment external to the laboratory from
exposure to infectious materials, is provided
by a combination of facility design and
operational practices.

The three elements of containment include
1. Laboratory practice and technique
2. Safety equipment
3. Facility design.
ELEMENTS OF CONTAINMENT
1) LABORATORY PRACTICE AND TECHNIQUE
• Strict adherence to standard microbiological practices and
techniques.
• Persons working with infectious agents or infected materials must be
aware of potential hazards and must be trained and proficient in the
practices and techniques required for safely handling such material.
• The director or person in charge of the laboratory is responsible for
providing or arranging for appropriate training of personnel.
• When standard laboratory practices are not sufficient to control the
hazard associated with a particular agent or laboratory procedure,
additional measures may be needed. Each laboratory should develop
or adopt a biosafety or operations manual which identifies the
hazards that will or may be encountered and which specifies
practices and procedures designed to minimize or eliminate risks.
• Laboratory personnel, safety practices, and techniques must be
supplemented by appropriate facility design and engineering
features, safety equipment, and management practices.

ELEMENTS OF CONTAINMENT 2) SAFETY EQUIPMENT
• Safety equipment includes biological safety
cabinets and a variety of enclosed containers.
• The biological safety cabinet is the principal device
used to provide containment of infectious aerosols
generated by many microbiological procedures.
• Three types of biological safety cabinets (Class I,
II, III) used in microbiological laboratories are
described and illustrated in.
Open-fronted Class I and Class II
Biological safety cabinets are partial containment cabinets which
offer significant levels of protection to laboratory personnel and to
the environment when used with good microbiological techniques.
The gastight Class III
Biological safety cabinet provides the highest attainable level of
protection to personnel and the environment. An example of an
enclosed container is the safety centrifuge cup, which is designed
to prevent aerosols from being released during centrifugation.
Safety equipment also includes items for personal protection such as
• Gloves
• Coats
• Gowns
• Shoe covers
• Boots
• Respirators
• Face shields
• Safety glasses

In some situations in which it is impractical to work in
biological safety cabinets, personal protective devices
may form the primary barrier between personnel and
the infectious materials.
Examples of such activities include certain animal
studies, animal necropsy, production activities, and
activities relating to maintenance, service, or support
of the laboratory facility.
These personal protective devices are often used in combination
with biological safety cabinets and other devices which contain the
agents, animals, or materials being worked with
2) SAFETY EQUIPMENT
Biological safety cabinet Spatter shield Pipetting aids
Disposable transfer loops Micro incinerators Safe disposal
container

3) FACILITY DESIGN
• The design of the facility is important in providing a barrier to protect persons working in the facility but outside the
laboratory and those in the community from infectious agents which may be accidentally released from the laboratory.
• Laboratory management is responsible for providing facilities commensurate with the laboratory's function.
THE LEVEL OF CONTAINMENT
1) THE BASIC LABORATORY
This laboratory is also appropriate for work with infectious
agents or potentially infectious materials when the hazard
levels are low and laboratory personnel can be adequately
protected by standard laboratory practice.
While work is commonly conducted on the open bench, certain
operations are confined to biological safety cabinets.
Conventional laboratory designs are adequate. Areas known to
be sources of general contamination, such as animal rooms and
waste staging areas, should not be adjacent to patient care
activities. Public areas and general offices to which non
laboratory staff require frequent access should be separated
from spaces which primarily support laboratory functions.
Laboratory coats; decontamination of all infectious
wastes; limited access; protective gloves and biohazard
warning signs as indicated.

2) THE CONTAINMENT LABORATORY
• This laboratory has special engineering features which
make it possible for laboratory workers to handle
hazardous materials without endangering themselves,
the community, or the environment.
• The unique features which distinguish this laboratory
from the basic laboratory are the provisions for access
control and a specialized ventilation system.
• The containment laboratory may be an entire building or
a single module or complex of modules within a
building.
• In all cases, the laboratory is separated by a controlled
access zone from areas open to the public.
Special laboratory clothing; controlled access.

3) THE MAXIMUM CONTAINMENT LABORATORY
• This laboratory has special engineering and containment
features that allow activities involving infectious agents that
are extremely hazardous to the laboratory worker or that
may cause serious epidemic disease to be conducted safely.
• Although the maximum containment laboratory is
generally a separate building, it can be constructed as an
isolated area within a building.
• The laboratory's distinguishing characteristic is that it has
secondary barriers to prevent hazardous materials from
escaping into the environment.
• Such barriers include sealed openings into the laboratory,
airlocks or liquid disinfectant barriers, a clothing-change
and shower room contiguous to the laboratory ventilation
system, and a treatment system to decontaminate exhaust
air.
Entrance through a change room where street clothing is
removed and laboratory clothing is put on; shower on exit;
all wastes are decontaminated on exit from the facility.

BIOSAFETY LEVEL FOR INFECTIOUS
AGENTS
Summary of Recommended Biosafety
Levels for Infectious Agents

RECOMMENDED BIOSAFETY LEVEL FOR INFECTIOUS AGENTS
BIOSAFETY LEVEL 1
Agents:
Agents that not known to consistently
cause disease in healthy adults.
Practices :
1. Standard Microbiological Practices.
2. Persons must wash their hands after
working.
3. Eating, drinking, smoking, handling
contact lenses, applying cosmetics, and
storing food for human consumption
must not be permitted in laboratory
areas.
4. Mouth pipetting is prohibited; mechanical
pipetting devices must be used.
5. Perform all procedures to minimize the
creation of splashes and/or aerosols.
6. Proper disposal of waste.
Safety Equipment(Primary Barriers):
None Required
Facilities (Secondary Barriers):
• Laboratory bench
• Sink.
• Laboratories with windows
BSL-1 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,
but may be used as determined by appropriate risk assessment. 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.

BIOSAFETY LEVEL 1
Bacillus subtilis, Naegleria gruberi, and infectious canine hepatitis virus are representative of those microorganisms meeting
these criteria. Many agents not ordinarily associated with disease processes in humans are, however, opportunistic pathogens
and may cause infection in the young, the aged, and immunodeficient or immunosuppressed individuals.
E.coli K12 derivative Bacillus subtilis Naegleria gruberi Alcaligenes faecalis Aspergillus niger
Clostridium sporogenes Enterobacter cloacae Penicillium notatum Saccharomyces cerevisiae Rhizopus stolonifer

BIOSAFETY LEVEL 2
BSL 2 would cover work with agents associated with human disease, in other words, pathogenic or infectious organisms
posing a moderate hazard. Examples are the equine encephalitis viruses and HIV when performing routine diagnostic
procedures or work with clinical specimens. BSL 2 labs must also provide the next level of barriers, i.e., specialty safety
equipment and facilities. Preferably, this is a Class II cabinet or equivalent containment device for work with agents and an
autoclave or other suitable method for decontamination within the lab.
Practices :
• Class I or II Bio-safety cabinet
• Contaminated sharps are handled with
extreme caution.
• Use of disposable syringe-needle units and
appropriate puncture-resistant sharps
containers is mandatory.
• Direct handling of broken glassware is
prohibited and decontamination of all
sharps prior to disposal.
• Access to the lab is more controlled than
for BSL 1 facilities. Immunocompromised,
immunosuppressed and other persons with
increased risk for infection may be denied.
Agents:
Agents associated with human
diseases.
Routes of transmission:
percutaneous injury, ingestion, mucous
membrane exposure.
• Class I or II Bio-safety cabinet or
other physical containment devices
used for all manipulations of agents
that cause splashes or aerosols of
infectiousmaterials;
• PPEs: laboratory coats; gloves; face
protection as needed.
Facilities (Secondary
Barriers):
• Sink.
• A readily available
eyewash station is needed.
• Self closing lockable doors
and biohazard warning
signs are also required at
all access points.
• Autoclave

BIOSAFETY LEVEL 2
With good microbiological techniques, these agents can be used safely in activities conducted on the open bench, provided
the potential for producing aerosols is low. Hepatitis B virus, the salmonellae, and Toxoplasma spp. are representative of
microorganisms assigned to this containment level. Procedures with high aerosol potential that may increase the risk of
exposure of personnel must be conducted in primary containment equipment or devices.
Class I biological safety cabinets
are partially enclosed work stations that protect the worker and
the environment from contamination.
• Class I biological safety cabinets are used in microbiology labs,
in pharmaceutical research and development, and in cancer
research labs to protect the environment from bacteria,
viruses, and carcinogens.
• The class I safety cabinet has an open front, for access to the
materials inside of it, negative air pressure to pull air from
outside of the cabinet, and a HEPA filter that air from inside
the cabinet goes through, before returning to the lab or being
vented outside, to remove the contaminants.
• When purchasing a class I biological safety cabinet there are
several features to consider. The space it will take, its energy
efficiency and cost to run it, and the ergonomics are some
important ones.
•Internal Dimensions:
(WxDxH 87.3 x 61.0 x 71.1 cm( 34.375 x 24 x 28 in )
•Exterior Dimensions:
(WxDxH) 91.4 x 73.7 x 130.8 cm (36 x 29 x 51 ½ in )

BIOSAFETY LEVEL 2
Fusobacterium sp. Klebsiella sp.
Mycobacterium
tuberculosis
Salmonella typhi
Bacterial Agents Fungal Agents
Blastomyces
dermatitides
Candida albicans
Paracoccidioides
brasilienisis
Microsporum –
pathogenic sp.
Ascaris sp.
Trichinella spiralis
Parasitic Agents

BIOSAFETY LEVEL 3
BSL 3 practices, safety equipment, and facilities are applicable to clinical, diagnostic, teaching, research, or production
facilities in which work is done with indigenous or exotic agents where the potential for infection by aerosols is real and the
disease may have serious or lethal consequences. Autoinoculation and ingestion also represent primary hazards to personnel
working with these agents.
Practices :
• Controlled access
• Decontamination of all waste
• Decontamination of lab clothing before laundering;
• Baseline serum.
Agents:
Indigenous or exotic agents with
potential for aerosol
transmission; disease may have
serious or lethal consequences.
Safety Equipment(Primary
Barriers):
• PPEs: protective lab clothing;
gloves; face protection as
needed.
• Sink.
• A readily available eyewash station is needed.
• Self closing lockable doors and biohazard warning signs are also required at all access
points.
• Autoclave
• Physical separation from access corridors
• Self-closing, double-door access
• Exhausted air not recirculated
• Negative airflow into laboratory.

BIOSAFETY LEVEL 3
Class 2 biological safety cabinets
• A class II biological safety cabinet is a partially enclosed
workspace that has built in protection for the worker, the
environment, and the material inside of it.
• Class II biological safety cabinets have open fronts, a vertical
laminar air flow, so outside air is run through a filter before it
gets inside, and a HEPA filter for filtering air from inside before
it goes outside the cabinet.
• Used in microbiology labs, pharmaceutical labs, and cancer
research labs, class II biological safety cabinets protect the
bacteria, viruses, and carcinogens being manipulated inside the
cabinet while shielding them from outside contamination.
• There are four types of class II cabinets, A1, A2, B1, and B2,
each having different requirements for how much air must be
vented outside the building.
• The size, cost of running and maintenance, and the type of
material to be handled will all help decide which type of class II
biological safety cabinet is right for any given laboratory.
•Dimensions: Varies with model
•Class/Type: Class II, Type A2
•Air Inflow Rate: 105 fpm (0.53 m/s)
•Size(s): 3, 4 , 5 or 6 ft.
Biosafety cabinets Class 2 type A1

Biosafety cabinets Class 2 type
B1
BIOSAFETY LEVEL 3
Biosafety cabinets Class 2 type
B2
Biosafety cabinets Class 2 type A2

BIOSAFETY LEVEL 3
Standard Operating Procedures for the establishment of BSL 3
SARS coronavirus
Poxvirus monkey
pox
Venezuelan Equine
Encephalitis

BIOSAFETY LEVEL 4
BSL-4 practices, safety equipment, and facilities are applicable to work with dangerous and exotic agents which pose a high
individual risk of life-threatening disease. All manipulations of potentially infectious diagnostic materials, isolates, and
naturally or experimentally infected animals pose a high risk of exposure and infection to laboratory personnel. Lassa fever
virus is representative of the microorganisms assigned to Level 4.
Practices :
• Controlled access
• Decontamination of all waste
• Decontamination of lab clothing
before laundering;
• Baseline serum.
• Clothing change before entering
• Shower on exit
• All material decontaminated on exit
from facility.
Agents:
Dangerous/exotic agents which
pose high risk of life- threatening
disease, aerosol- transmitted lab
infections have occurred; or
relatedagents with unknown risk
of transmission.
Barriers):
• Class III Bio-safety cabinet
• PPE
• Sink.
• A readily available eyewash station is needed.
• Self closing lockable doors and biohazard warning signs are
also required at all access points.
• Autoclave
• Physical separation from access
corridors
• Self-closing, double-door access
• Exhausted air not recirculated
• Positive pressure Personal suit.
• Separate building or isolated zone.
• Dedicated supply/exhaust vacuum and
deacon systems.

BIOSAFETY LEVEL 4
• Class III biological safety cabinets, also known as glove boxes or
isolation glove boxes, are enclosed work areas designed to
protect the worker, the environment, and the sample from
contamination.
• The laboratory technician can manipulate materials inside the
class III biological safety cabinet by using rubber gloves that are
attached to the cabinet.
• Outside air is filtered through a HEPA filter prior to entry, and
air leaving the class III biological safety cabinet is filtered
through two HEPA filters before being vented outside.
• Anaerobic chambers are also available. These units allow
researchers to easily process, culture and examine samples
without exposure to atmospheric oxygen.
• Accessories for Class III cabinets include heating elements, filter
and ionizers.
• When choosing a class III biological safety cabinet consider
space available, price, lighting options, and energy efficiency.

BIOSAFETY LEVEL 4
Marburg virus Ebola virus
Lassa virus
Bolivian
haemorrhagic virus
Hendra virus
Nipah virus
Diseases that have high fatality rates and have no known treatments are considered level 4 diseases. An example of a level
4 disease is Ebola virus, a disease that causes headache, muscle pain, fever, impaired liver and kidney function, and in
some cases, death.
Russian spring summer
encephalitis
crimean congo hemorrhagic
fever virus

The specific scope and extent of the preventive maintenance and equipment inspection will be dependent on the facility
and its design. However, the following is a list of minimum recommendations:
1. Equipment maintenance
2. Biosafety cabinet maintenance
3. Emergency equipment testing
Certification of high containment laboratories will be performed by a team of professionals with experience and credentials in
engineering and biosafety/occupational safety and health.
• When appropriate, DOHS may delegate the responsibility for providing
certification of a laboratory or facility to a third party. As a part of the
laboratory certification process, the attached checklist must be
completed as a retained record document. DOHS will manage and
perform certification of NIH intramural laboratories and other high
containment facilities.
• Re-certification of the facility will be performed on an annual basis, as a
minimum. A comparison should be made to the baseline established
during initial certification. Detailed records of the certification process
and test results must be maintained to provide an accurate operations
history of the laboratory.

High containment laboratory certification - checklist
1. Review background materials that affect maintenance operations
2. Inspect and Evaluate
3. Inspect room layout, placement of equipment and equipment condition
4. Evaluate maintenance frequency and review maintenance logs
5. Validation of Engineering Controls
6. Assess HVAC equipment condition
7. Perform smoke tests to demonstrate directional airflow
8. Inspect and challenge door interlock systems and automatic door closers
9. Test all alarms
10. Discharge exhaust assessment (as a measure of performance)
11. Verification of air change rates (ACR) in containment spaces.
12. Review BSC certification data including serial number validation.
13. Validate MEP
14. Validate autoclave availability, operations and bio seal integrity.
15. Autoclave & Decontamination.
16. Safety SOPs
17. Occupational Health Monitoring (Policy and records of implementation),
if appropriate
18. Biohazardous Materials Use Authorization
(Human Pathogen Registration, Recombinant DNA Registration, Select Agent)

BIOSAFETY LEVEL 1 BIOSAFETY LEVEL 2 BIOSAFETY LEVEL 3 BIOSAFETY LEVEL 4
SAFETY SUITS

RECOMMENDED BIOSAFETY LEVEL
FOR INFECTIOUS ANIMALS
Summary of Recommended Biosafety
Levels for Activities in which
experimentally or naturally infected
vertebrate animals

RECOMMENDED BIOSAFETY LEVEL FOR INFECTIOUS ANIMALS
ANIMAL BIOSAFETY LEVEL
• Animal facilities, like laboratories, may be designated
according to a risk assessment and the risk group of the
microorganisms under investigation, as Animal facility
Biosafety Level 1, 2, 3 or 4.
• With respect to agents to be used in the animal laboratory,
factors for consideration include:
1. The normal route of transmission.
2. The volumes and concentrations to be used.
3. The route of inoculation.
4. Whether and by what route these agents may be excreted.
With respect to animals to be used in the animal
laboratory, factors for consideration include:
1. The nature of the animals, i.e. their aggressiveness
and tendency to bite and scratch.
2. Their natural ecto- and endoparasites
3. The zoonotic diseases to which they are susceptible.
4. The possible dissemination of allergens.
As with laboratories, the requirements for design features,
equipment and precautions increase in stringency
according to the animal biosafety level.

ANIMAL BIOSAFETY LEVEL 1
This is suitable for the maintenance of most stock animals after quarantine (except nonhuman primates, regarding which
national authorities should be consulted), and for animals that are deliberately inoculated with agents in Risk Group 1. GMT
are required. The animal facility director must establish policies, procedures and protocols for all operations, and for access
to the vivarium. An appropriate medical surveillance programme for the staff must be instituted. A safety or operations
manual must be prepared and adopted.
Practices :
Standard animal care and
management practices, including
appropriate medical surveillance
programs.
Agents:
Not known to consistently
cause diseases in healthy
human adults.
Barriers):
As required for normal care
of each species.
Standard animal facility:
• Non recirculation of exhaust air
• directional air flow
recommended.
• Hand washing sink is available

The following safety precautions apply:
1. All the requirements for animal facilities – Biosafety Level 1
must be met.
2. Biohazard warning signs should be posted on doors and other
appropriate places.
3. The facility must be designed for easy cleaning and
housekeeping.
4. Doors must open inwards and be self-closing.
5. Heating, ventilation and lighting must be adequate.
6. If mechanical ventilation is provided, the airflow must be
inwards. Exhaust air is discharged to the outside and should not
be recirculated to any part of the building.
7. Access must be restricted to authorized persons.
8. No animals should be admitted other than those for
experimental use.
9. There should be an arthropod and rodent control programme.
10. Windows, if present, must be secure, resistant to breakage
and, if able to be opened, must be fitted with arthropod-proof
screens.
This is suitable for work with animals that are deliberately inoculated with microorganisms in Risk Group 2.
11. Work space should be decontaminated after work.
12. Biological safety cabinets (Classes I or II) or isolator
cages with dedicated air supplies and HEPA-filtered
exhaust air must be provided for work that may involve
the generation of aerosols.
13. An autoclave must be available on site or in
appropriate proximity to the animal facility.
14. Animal bedding materials must be removed in a
manner that minimizes the generation of aerosols and
dust.
15. All waste materials and bedding must be
decontaminated before disposal.
16. Use of sharp instruments should be restricted
whenever possible. Sharps should always be collected in
puncture-proof/-resistant containers fitted with covers
and treated as infectious.
17. Material for autoclaving or incineration must be
transported safely, in closed containers.
18. Animal cages must be decontaminated after use.

19. Animal carcasses should be incinerated.
20. Protective clothing and equipment must be worn in the facility, and removed on leaving.
21. Hand-washing facilities must be provided. Staff must wash their hands before leaving the animal facility.
22. All injuries, however minor, must be treated appropriately, reported and recorded.
23. Eating, drinking, smoking and application of cosmetics must be forbidden in the facility.
24. All personnel must receive appropriate training.
Practices :
• ABSL - 1 practices plus: limited
access.
• biohazard warning signs.
• sharps precautions; biosafety
manual.
• Decontamination of all infectious
wastes and of animal cages prior to
washing.
Agents:
Associated with human
disease.
Hazard:- percutaneous
exposure, ingestion, mucous
membrane.
Barriers):
• ABSL - I equipment plus
primary barriers:
containment equipment
appropriate for animal
species;
• PPEs: laboratory coats
gloves, face and respiratory
protection as needed.
• ABSL - 1 facility plus.
• Autoclave available.
• Handwashing sink available in the
animal room.
• Mechanical cage washer
recommended.

The following safety precautions apply:
1. All the requirements for animal facilities – Biosafety Levels 1 and 2 must be
met.
2. Access must be strictly controlled.
3. The facility must be separated from other laboratory and animal house areas
by a room with a double-door entrance forming an anteroom.
4. Hand-washing facilities must be provided in the anteroom.
5. Showers should be provided in the anteroom.
6. There must be mechanical ventilation to ensure a continuous airflow through
all the rooms. Exhaust air must pass through HEPA filters before being
discharged to the atmosphere without recirculation. The system must be
designed to prevent accidental reverse flow and positive pressurization in any
part of the animal house.
7. An autoclave must be available at a location convenient for the animal house
where the biohazard is contained. Infectious waste should be autoclaved
before it is moved to other areas of the facility.
8. An incinerator should be readily available on site or alternative arrangements
should be made with the authorities concerned.
This is suitable for work with animals that are deliberately inoculated with agents in Risk Group 3, or when otherwise indicated
by a risk assessment. All systems, practices and procedures need to be reviewed and recertified annually.

9. Animals infected with Risk Group 3 microorganisms must be housed in cages in isolators or rooms with ventilation exhausts
placed behind the cages.
10. Bedding should be as dust-free as possible.
11. All protective clothing must be decontaminated before it is laundered.
12. Windows must be closed and sealed, and resistant to breakage.
13. Immunization of staff, as appropriate, should be offered.
Practices :
• ABSI -2 practices plus: controlled
access; decontamination of
clothing before laundering
• cages decontaminated before
bedding removed
• disinfectant foot bath as needed.
Agents:
Indigenous or exotic agents with
potential for aerosol transmission;
disease may have serious health
effects.
• ABSL -2, equipment plus.
• Containment equipment for
housinganimals and cage dumping
activities
• Class I or II or II BSCs available for
manipulative procedures
(inoculation, necropsy) that may
create infectious aerosols.
• PPEs: Appropriate respiratory
protection.
• ABSL -2, facility plus.
• physical separation from access
corridors.
• self-closing, double-door
access.
• sealed penetrations.
• sealed windows autoclave
available in facility.

1. All the requirements for animal facilities – Biosafety Levels 1, 2 and 3
must be met.
2. Access must be strictly controlled; only staff designated by the
director of the establishment should have authority to enter.
3. Individuals must not work alone: the two-person rule must apply.
4. Personnel must have received the highest possible level of training
as microbiologists and be familiar with the hazards involved in their
work and with the necessary precautions.
5. Housing areas for animals infected with Risk Group 4 agents must
maintain the criteria for containment described and applied for
maximum containment laboratories – Biosafety Level 4.
6. The facility must be entered by an airlock anteroom, the clean side
of which must be separated from the restricted side by changing and
showering facilities.
7. Staff must remove street clothing when entering and put on special,
protective clothing. After work they must remove the protective
clothing for autoclaving, and shower before leaving.
Work in this facility will normally be linked with that in the maximum containment laboratory – Biosafety Level 4, and
national and local rules and regulations must be harmonized to apply to both. If work is to be done in a suit laboratory,
additional practices and procedures must be used over.

8. A pass-through airlock with the clean end in a room outside the containment rooms must be provided for exchange of non-
autoclavable materials.
9. All manipulations with animals infected with Risk Group 4 agents must take place under maximum containment – Biosafety
Level 4 conditions.
10. All animals must be housed in isolators.
11. All animal bedding and waste must be autoclaved before removal from the facility.
12. There must be medical supervision of staff. The facility must be ventilated by a HEPA-filtered exhaust system designed to
ensure a negative pressure (inward directional airflow).
13. The ventilation system must be designed to prevent reverse flow and positive pressurization.
14. A double-ended autoclave with the clean end in a room outside the containment rooms must be provided for exchange of
materials.
Practices :
• ABSL - 3 practices plus
• entrance through change room
where personal clothing is
removed and laboratory
clothing is put on
• shower on exiting
• all wastes are decontaminated
before removal from the
facility.
Agents:
Dangerous/exotic agents that pose high
risk of life threatening disease; aerosol
transmission, or related agents with
unknown risk of transmission.
• ABSL- 3 equipment plus
• Maximum containment
equipment (i.e., Class III BSC or
partial containment equipment in
combination with full body, air-
supplied positive-pressure
personnel suit) used for all
procedures and activities.
• ABSL -3 facility plus
• separate building or isolated zone
• dedicated supply and exhaust,
vacuum and decontamination
systems.

Guidelines for animal laboratory / facility certification
In order to assure that adaptation and maintenance are undertaken promptly and in an appropriate and safe manner, all
biological research and clinical laboratories should be regularly certified.
Laboratory certification helps to ensure that:
1. Proper engineering controls are being used and are functioning adequately as designed
2. Appropriate site and protocol specific administrative controls are in place
3. Personal protective equipment is appropriate for the tasks being performed
4. Decontamination of waste and materials has been adequately considered and proper waste management procedures are
in place
5. Proper procedures for general laboratory safety, including physical, electrical and chemical safety are in place.
Laboratory certification differs from laboratory commissioning activities in several important ways. Laboratory certification is
the systematic examination of all safety features and processes within the laboratory (engineering controls, personal protective
equipment and administrative controls). Biosafety practices and procedures are also examined.
Laboratory certification is an on-going quality and safety assurance
activity that should take place on a regular basis. Adequately trained
safety and health or biosafety professionals may conduct laboratory
certification activities. Institutions may employ personnel having the
appropriate skill-set required for conducting audits, surveys or
inspections (these terms are used interchangeably) associated with
the certification process. However, institutions may consider engaging
or be required to engage a third party to provide these services.
WHO Biosafety programme
Certification of the laboratory should not be
completed, and the laboratory should not be
declared functional, until deficiencies have been
adequately addressed.
The complexity of Biosafety Level 4 laboratory
operations goes beyond the scope of this manual.

SUMMARY
INFECTION
CAUSES OF INFECTION
TRANSMISSION
IDENTIFICATION OF
INFECTION
PREVENTING INFECTION
BIOSAFETY
ELEMENTS OF CONTAINMENT
BIOSAFETY LEVEL FOR
INFECTIOUS AGENTS
BIOSAFETY LEVEL 1
BIOSAFETY LEVEL 2
BIOSAFETY LEVEL 3
BIOSAFETY LEVEL 4
BIOSAFETY LEVEL FOR
INFECTIOUS ANIMALS
Guidelines for laboratory /
facility certification
Guidelines for laboratory /
facility certification
SECONDARY CONTAINMENT
PRIMARY CONTAINMENT

Recommended Biosafety level for Infectious Agents and Infected Animals

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