Bio Safety Iitb Handout

BIOSAFETY
Introduction
Bio-related research activities may involve manipulation of

microbial, animal or plant cells.
The risks associated with these activities arise from the

samples and /or the procedural requirements.
Adherence to standard microbiological techniques and

using facilities suitable to the risk level of the pathogen
helps to protect the researcher from laboratory-acquired
infections.
Bio hazards
Hazards related to bio research can be classified into two

categories.
• hazards related with the pathogen

or human/animal cells being used
in research.
• related with the procedures and

practices followed in the lab.
Pathogenic risks
Cell cultures
• Researchers who handle or manipulate human or

animal cells are at risk of possible exposure to
potentially infectious pathogens that may be present in
those cells/ tissues.
• The human cell lines may contain blood borne

pathogens, which can be transmitted due to improper
handling.
Routes of entry for pathogen
The probable routes of entry are
• Inhalation of infectious aerosols.
• Contact of the agent with the skin, eyes or mucous

membrane.
• Inoculation by contaminated sharps.
• Bites from infected animals or contact with their body

fluids.
• Ingestion of infectious agent through mouth pipetting

or contaminated hands.
Aerosols
Aerosols generated during research activities remain

undetected and can spread easily and remain suspended
in the laboratory atmosphere for a long time.
They possess a serious hazard to the person performing

the task and also to others who are exposed to the air
from the laboratory.
Aerosols
Aerosols can be generated during the following activities
• Pipetting
• Blending
• Centrifugation
• Use of sonicators and
vortex mixers
These respirable size particles when inhaled are retained

in the lungs and can cause infection to the person.
Pathogenic risks
The risk from the pathogen handled depends on the

following factors.
• Capability to cause infection in the host and the severity

of the same.
• Preventive measures and treatment available.
• Route of entry
• Infective dose level
• Stability in the environment
• The range of cells/strains that can act as a host.
Based on the above factors the microorganisms are

classified into four risk groups.
Classification of pathogenic microorganisms
Risk group I
A pathogen that is unlikely to cause any disease in humans

or animals.
All bacterial, fungal and parasitic agents not included in

higher groups.
Risk group II
A pathogen that can cause disease in humans or animals

but is unlikely to be a serious hazard.
Effective treatment and preventive measures are available

and the risk of spread of infection is limited.
• Bacterial- Vibrio cholerae

• Fungal- Aspergillus fumigatus, Actinomycetes
• Parasitic- P.falciparum, Plasmodium thcilera
• Viral and Rickettssial -Vole rickettsia, Mumps virus
Risk group III
A pathogen that can cause serious human or animal

disease , but does not ordinarily spread from one infected
person to another.
Effective treatment and preventive measures are

available.
• Bacterial - Clostridium botulium, Francisella tularensis

• Fungal - Coccidioides immitis,Histoplasma capsulatum
• Parasitic- Schisistosoma mansomi
• Viral and Rickettssial - Foot-and- Mouth disease virus
Risk group IV
A pathogen that usually causes serious human or animal

disease and that can be readily transmitted from one
individual to another, directly or indirectly.
Effective treatment and preventive measures are not

usually available.
• Korean hemorrhagic fever

• Omsk hemorrhagic fever and
• Central European Encephalitis viruses
Containment
The term containment is used to describe the safe work

practices in handling infectious agents to reduce exposure
to laboratory personnel and others.
Types of containment
• Biological containment
• Physical containment
Biological containment (BC)
Any combination of vector and host which is to provide

biological containment must be chosen or constructed to
limit the infectivity of vector to specific hosts and control
the host-vector survival in the environment.
Physical Containment (PC)
Physical containment helps to confine the pathogenic

organisms being handled and prevent exposure to
personnel.
Physical containment is achieved by

• Laboratory practices Primary containment
• Containment equipment
• Special laboratory design Secondary containment
Primary containment offers protection to personnel and

immediate laboratory environment whereas secondary
containment offers protection to the environment outside
the laboratory.
Primary containment
Laboratory practices
Consists of adhering to standard microbiological practices

and techniques and awareness of potential hazards.
Primary containment
Containment equipment
This includes biological safety cabinets and enclosed

containers (e.g. safety centrifuge cup).
Secondary containment
Proper design of the facility helps in protecting personnel

inside the facility and also prevents the release of
pathogenic organisms outside the facility.
Facility designs are of three types
Basic Laboratory (for Risk Group I and II)

Containment Laboratory (for Risk Group III)
Maximum Containment Laboratory (for Risk Group IV)
Biosafety levels
It consists of a combination of laboratory practices,

equipment and facilities suitable to the procedures being
performed and hazards of the pathogen.
The four biosafety levels corresponds to four risk groups.
A lower risk group can be assigned a higher biosafety

level, if a biological risk assessment carried out requires
so.
Biosafety level I
Suitable for teaching laboratories and for facilities in

which work is done with defined and characterised strains
of agents not known to cause any disease.
Good microbiological techniques(GMT) to be followed.

Biosafety Level II
Applicable to facilities in which work is done with
indigenous moderate-risk agents present in the
community and associated with human disease of varying
severity.
BSL II is appropriate when work is done with any human-

derived blood, body fluids, tissues, or primary human cell
lines, in which presence of an infectious agent may be
unknown
BSL II requires
• Following GMT
• Use of personal protective equipment
• Use of BSC
• Use of autoclaves
Biosafety level III
Applicable to 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.
BSL III requires in addition to that of BSL II requirements

• Special clothing
• Directional airflow
• Controlled access
• Double door entry/Anteroom
• Supervision
Biosafety level IV
Applicable to work with dangerous and exotic agents which

pose a high individual risk of life-threatening disease.
BSL IV requires in addition to BSL III requirements

• Positive pressure personnel suits
• Strictly limited access
• Double ended autoclave
• Class III BSC
• Airlock with shower
• Supervision
Good microbiological techniques (GMT)
• Specimen containers must be correctly labelled for

easy identification.
• Use secondary containers (autoclavable) while

transporting specimens to contain spill.
• Specimen containers received from external agencies

must be opened in the biosafety cabinet.
• Use mechanical pipettes.

Good microbiological techniques
• Open flame must not be used in BSC as it can

distort the air flow pattern and damage the filters.
• Always use disposable gloves. Do not touch mouth, eyes

and face with contaminated hands.
• Food and drink must not be stored or consumed in the

laboratory.
• Glassware must be replaced with plasticware

wherever possible.
• Sharps(e.g., needle sticks, glass) must be avoided

wherever possible as it can transmit blood borne
pathogens in case of injury.
• Use engineered sharp-safety devices when syringes

and needles are necessary.
• Needles must not be recapped, to prevent needle stick

injury.
• Puncture-proof containers fitted with covers must be used

for disposing sharps.
• Tubes and specimen containers must always be

securely capped (screw-capped if possible) for
centrifugation.
• Refer to manufacturer’s instructions before operating

equipments.
• Work area must be decontaminated with a suitable

disinfectant at the end of the work.
• Hands must be thoroughly washed before leaving the lab.

Personal protective equipment
• Personal protective equipment act as a barrier to

minimize the risk of exposure to aerosols, splashes and
other injuries.
• Personal protective equipment must be selected on

the basis of the risks involved in the task performed.
• Lab coat, safety glasses and toe covered footwear is a

minimum requirement while working in the lab.
• Face shield must be used if there is any risk of

splashing of infectious materials.
Personal protective equipment
• Gloves must be worn for all procedures that may

involve direct contact with blood, infectious materials,
or infected animals.
• Gloves must be removed aseptically and autoclaved

with other laboratory wastes before disposal.
• If re-usable gloves are used, on removal they must be

cleaned and disinfected before re-use.
• Lab coats and other personal protective equipment used

must not be used outside the laboratory.
Biosafety cabinets(BSC)
Biological safety cabinets provide containment of

infectious aerosols generated during the laboratory
procedures.
Three types of BSCs are used in microbiological

laboratories.
These are
Class I
Class II
Class III
Biosafety Cabinets
Class I BSC
Offers protection to laboratory personnel and to the
laboratory environment .
It doesn’t protect the samples from external contamination.
Class II BSC
Provides protection to the samples in the cabinet from
external contamination in addition to personnel and
laboratory environment protection.
Class III BSC

Provides the maximum attainable level of protection to
personnel and the environment.
The following factors reduce the efficiency of the BSC
• Poor location
• Room air currents
• Decreased airflow
• Leakage in HEPA filters
• Working with raised sashes
• Overcrowding the work surface
• Improper user methodology

Emergency measures
In case of exposure to bio samples
• Remove the contaminated clothing.
• Wash the skin thoroughly with soap and water.
• In case of eye contact flush the eyes with water.
• Report the exposure to the Lab in charge.
• Get medical attention immediately.

Decontamination
• Decontamination renders an item (work bench,
equipment, etc.) safe to handle by reducing the number
of organisms to below the threshold infectious dose level
such that transmission is unlikely to occur.
• Decontamination requirements will depend on the

experimental work and the nature of the infectious agent
handled.
• Decontamination is usually accomplished by steam

sterilization or autoclaving.
• Sterilization and disinfection are different forms of

decontamination.
Decontamination
Sterilisation
• Sterilisation makes an item free from all living

microorganisms and viruses.
• The process of sterilization can be accomplished by

applying heat.
Decontamination
Disinfection
• Is not as effective as sterilization, as some organisms

such as bacterial endospores may survive.
• A disinfectant is a chemical or mixture of chemicals used

to kill microorganisms, but not spores. They are usually
applied to inanimate surfaces or objects.
Decontamination
Disinfectants
• Sodium hypochlorite and formaldehyde are the

disinfectants recommended for general laboratory use.
• For special purposes phenolic compounds, alcohols,

iodine etc., can be used effectively.
Biohazard waste disposal
Biohazard waste generated in laboratories must be

segregated into the following:
• Non-contaminated general waste
• “Sharps”-needles, glass pieces, etc
• Contaminated material for autoclaving and recycling
• Contaminated material for incineration

Biohazard waste disposal
• Biohazard waste for autoclaving must be collected in

red plastic bags and those for incineration in yellow non
chlorinated plastic bags.
• Biohazard waste of human and animal origin must be

incinerated.
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BIOSAFETY

Bio-related research activities may involve manipulation of

The risks associated with these activities arise from the

Adherence to standard microbiological techniques and

Hazards related to bio research can be classified into two

• hazards related with the pathogen

• related with the procedures and

• Researchers who handle or manipulate human or

• The human cell lines may contain blood borne

The probable routes of entry are

• Inhalation of infectious aerosols.

• Contact of the agent with the skin, eyes or mucous

• Inoculation by contaminated sharps.

• Bites from infected animals or contact with their body

• Ingestion of infectious agent through mouth pipetting

Aerosols generated during research activities remain

They possess a serious hazard to the person performing

Aerosols can be generated during the following activities

These respirable size particles when inhaled are retained

The risk from the pathogen handled depends on the

• Capability to cause infection in the host and the severity

Based on the above factors the microorganisms are

A pathogen that is unlikely to cause any disease in humans

All bacterial, fungal and parasitic agents not included in

A pathogen that can cause disease in humans or animals

Effective treatment and preventive measures are available

• Bacterial- Vibrio cholerae

Risk group III

A pathogen that can cause serious human or animal

Effective treatment and preventive measures are

• Bacterial - Clostridium botulium, Francisella tularensis

A pathogen that usually causes serious human or animal

Effective treatment and preventive measures are not

• Korean hemorrhagic fever

The term containment is used to describe the safe work

Any combination of vector and host which is to provide

Physical containment helps to confine the pathogenic

Physical containment is achieved by

Primary containment offers protection to personnel and

Consists of adhering to standard microbiological practices

This includes biological safety cabinets and enclosed

Proper design of the facility helps in protecting personnel

Facility designs are of three types

Basic Laboratory (for Risk Group I and II)

It consists of a combination of laboratory practices,

The four biosafety levels corresponds to four risk groups.

A lower risk group can be assigned a higher biosafety

Suitable for teaching laboratories and for facilities in

Good microbiological techniques(GMT) to be followed.

BSL II is appropriate when work is done with any human-

Applicable to facilities in which work is done with

BSL III requires in addition to that of BSL II requirements

Applicable to work with dangerous and exotic agents which

BSL IV requires in addition to BSL III requirements

• Specimen containers must be correctly labelled for

• Use secondary containers (autoclavable) while

• Specimen containers received from external agencies

• Use mechanical pipettes.

• Open flame must not be used in BSC as it can

• Always use disposable gloves. Do not touch mouth, eyes