CONTAINMENT AND SAFETY LEVELS

1. BIOSAFETY
Dr. Esther Shoba R
Assistant Professor
Kristu Jayanti College

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.

7. 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.

8. 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.

9. 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.

10. Classification of pathogenic microorganisms
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

11. Classification of pathogenic microorganisms
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

12. Classification of pathogenic microorganisms
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

13. 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

14. 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.

15. Physical Containment (PC)
Physical containment helps to confine the pathogenic
organisms being handled and prevent exposure to
personnel.
Physical containment is achieved by
Primary containment
• Laboratory practices
• 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.

16. Primary containment
Laboratory practices
Consists of adhering to standard microbiological practices
and techniques and awareness of potential hazards.

17. Primary containment
Containment equipment
This includes biological safety cabinets and enclosed
containers (e.g. safety centrifuge cup).

18. 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)

19. CONTAINMENT LEVELS
Biosafety containment levels have to be designated for a facility
depending on the level of risk associated with the biological and
chemical agents used and released from it. Following NIH
(National Institute of Health, USA) and DBT (Department of
Biotechnology, India) guidelines, different facilities for biological
research have been classified under three containment levels)

20. CONTAINMENT LEVELS - I
Viable organisms should be handled in a production system which physically
separates the process from the environment;
Exhaust gases should be treated to minimize (i.e. to reduce to the lowest
practicable level consistent with safety) the release of viable organisms;
Sample collection, addition of materials to the system and the transfer of viable
organisms to another system should be done in a manner which minimizes
release;
Bulk quantities of culture fluids should not be removed from the system unless
the viable organisms have been inactivated by validated means;
Effluent from the production facility should be inactivated by validated means
prior to discharge.

21. CONTAINMENT LEVELS - II
•Viable organisms should be handled in a production system which physically
separates the process from the environment;
•Exhaust gases should be treated to prevent the release of viable organisms;
•Sample collection, addition of materials to a closed system and the transfer of
viable organisms to another closed system should be done in a manner which
prevents release;

22. CONTAINMENT LEVELS – II - CONTD
•Culture fluids should not be removed from the closed system unless the viable
organisms have been inactivated by validated chemical or physical means;
•Seals should be designed to prevent leakage or should be fully enclosed in
ventilated housings;
•Closed systems should be located in an area controlled according to the
requirements;
•Effluent from the production facility should be inactivated by validated chemical
or physical means prior to discharge.

23. CONTAINMENT LEVELS – III
•Viable organisms should be handled in a production system which physically
separates the process from the environment;
•Exhaust gases should be treated to prevent the release of viable organisms;
•Sample collection, addition of materials to a closed system and the transfer of
viable organisms to another closed system should be done in a manner which
prevents release;
•Culture fluids should not be removed from the closed system unless the viable
organisms have been inactivated by validated chemical or physical means;
•Seals should be designed to prevent leakage or should be fully enclosed in
ventilated housings;

24. CONTAINMENT LEVELS – III - CONTD
•Production systems should be located within a purpose built controlled
area according to the requirements;
•Entry should be restricted in the laboratory area and only persons with
appropriate authority should be allowed access to the working area.
Effluent from the production facility should be inactivated by validated
chemical or physical means prior to discharge.
Different containment levels have been assigned for rDNA GILSP (Good
industrial large scale practice) micro-organisms.
Examples of containment approaches for recombinant organisms are
discussed

25. CONTAINMENT LEVELS - I

26. 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 levels

27. 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.

28. 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

29. 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

30. 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

31. BIOSAFETY GUIDE LINES - RDNA
Biosafety guidelines are designed and applied to
research involving recombinant DNA (rDNA) techniques.
Handling, production, storing and transportation of
genetically modified organism (GMOs) involve different
biosafety issues under different category.
Biosafety practices deal with the application of standard
safety principles handling hazardous material/agents to
minimize potential harmful effect on human health and
environment.

32. BIOSAFETY MANAGEMENT - RDNA
Biosafety regulatory principles and protocols regulates the
potential risk and allow access to the benefits of rDNA
technology.
RISK ASSESSMENT
RISK MANAGEMENT
Are the components of biosafety

33. BIOSAFETY MANAGEMENT - RDNA
The foundation of any safety program is the use of control measures
appropriate for the risk posed by the activities and the agents in use.
The process of analyzing and determining the risk associated with
recombinant DNA work is called as Risk analysis.
The principle behind biosafety regulations is to minimize the risk to
human health and safety, and the conservation of environment including
safe handling of hazardous material. Risk analysis consists of three
components: risk assessment, risk management and risk communication.

34. BIOSAFETY MANAGEMENT - RDNA
Risk Assessment: Estimation and determination of risk associated with
the handling and production of a recombinant DNA molecule.
Risk Management: The process of analyzing possible prevention
measures to minimize the risk and designing policies accordingly
including implementation of them.
Risk Communication: The exchange of information and opinions on risk
management between academic parties, industry, consumers and
policy makers.

35. RISK ASSESSMENT - RDNA
The biosafety level is determined based on the risk associated with the work.
The principle investigator is responsible for implementing the necessary safety
requirements in his/her laboratory.
Risk assessment process accounts the following criteria to determine
biosafety level:
i.Pathogenicity – The ability of an organism to cause disease in human
system.
ii.Virulence – The severity of the disease (lethal/non lethal, availability of cure
etc) in a healthy adult.
iii.Proliferation – the subsequent multiplication, genetic reconstruction,
growth, transport, modification and die-off of these micro-organisms in the
environment, including possible transfer of genetic material to other micro-
organisms.

36. RISK ASSESSMENT - RDNA
iii.Transmission route – The possible route of transmission (mucous
membrane, inhalation etc) to establish the disease in human or other
organism.
iv.Infectious dose (ID) – The amount of infectious agent required to
cause disease in healthy human.
vi.Antibiotic/disinfectant resistance – The resistance acquired by the
infectious agent to available antibiotic/disinfectant.

37. RISK MANAGEMENT - RDNA
Risk management in biosafety issues is related to the target site
where the practice is conducting (laboratory, industry,
agriculture field etc.).
Recommendations: General
i.Harmonization of approaches to rDNA techniques can be
facilitated by exchanging principles or guidelines for national
regulations; developments in risk analysis; and practical
experience in risk management. Therefore, information should
be shared as freely as possible.
ii.There is no scientific basis for specific legislation for the
implementation of rDNA techniques and applications.

38. RISK MANAGEMENT - RDNA
Member countries should examine their existing oversight and
review mechanisms to ensure that adequate review and control
may be applied while avoiding any undue burdens that may
hamper technological developments in this field.
iii. Any approach to implement guidelines should not impede
future developments in rDNA techniques. International
harmonization should recognize this need.

39. RISK MANAGEMENT - RDNA
iv. To facilitate data exchange and minimize trade barriers
between countries, further developments such as testing
methods, equipment design and knowledge of microbial
taxonomy should be considered at both national and
international levels.
Due account should be taken of ongoing work on standards
within international organizations.
v. Special efforts should be made to improve public
understanding of the various aspects of rDNA techniques.

40. RISK MANAGEMENT - RDNA
v.For rDNA applications in industry, agriculture and the
environment, it will be important for member countries to watch
the development of these techniques.
vi.For certain industrial applications and for environmental and
agricultural applications of rDNA organisms, some countries may
wish to have a notification scheme.
vii.Recognizing the need for innovation, it is important to consider
appropriate means to protect intellectual property and
confidentiality interests while assuring safety.

41. BIOSAFETY LEVELS - RDNA
All the facilities handling microorganisms and materials
containing recombinant DNA molecules have risk
assessment program.
Depending on the risk possessed by the samples, four
biosafety levels have been assigned to rDNA research
facilities.
Each BSL facility has requirement of unique design features
and safety equipments

42. BIOSAFETY LEVEL I (BSL –I) - RDNA
•Agents: Characterized strains of microorganisms known to cause
no disease in healthy adults. eg. E. coli,
S. cerevesiae, B. subtilis etc.
•Recombinant DNA based research activities involving non-
pathogenic micro-organisms for expression of genes using plasmid
vectors or low risk viral vectors.
•Work practice: Standard aseptic microbiological techniques.
•Safety equipment requirement: Lab coats and eye protection
recommended.
•Facilities: Bench top, sink etc.

43. BIOSAFETY LEVEL II (BSL - II) - RDNA
•Agents: Handling of micro-organisms which possess moderate
hazard to personal and environment.
•rDNA based research activities in micro-organisms using non-viral or
viral vectors.
•Work practice: Standard BSL-I practices with addition of limited
access, biohazard sign, defined procedure for disposal of “Regulated
Medical Waste”, proper training to lab personal and medical
surveillance.
•Safety equipment: Class-II biological safety cabinet, lab coats,
gloves, eye/face protection, physical containment equipment to
reduce infectious aerosol exposure or splashes.
•Facility: BSL-I facility with addition of autoclave, decontamination
facility and proper airflow.

44. BIOSAFETY LEVEL III (BSL -III) - RDNA
•Agents: Handling of micro-organisms which are designated as
hazardous or potentially lethal agents to personal and
environment.
•Laboratory personnel must have specific training in handling
infectious micro-organisms and should be supervised by
scientist competent in handling infectious agents.
•Work practices: BSL-2 practices, with the addition of:
controlled access, on-site decontamination of all waste and
lab clothing and medical surveillance.

45. BIOSAFETY LEVEL III (BSL -III) - RDNA
•Safety equipment: Class-III biological safety cabinet, lab
coats, gloves, eye/face protection, respiratory protection,
physical containment equipment to reduce infectious aerosol
exposure or splashes.
•Facility: BSL-III facility has specific criteria to meet. Lab should
have double door entry with physical separation of working
area from the access corridors, directional airflow in lab, and
no recirculation of exhaust air in the lab, sufficient
decontamination facility, in lab autoclave etc.

46. BIOSAFETY LEVEL IV (BSL -IV) - RDNA
•Agents: Hazardous and potentially lethal organisms that
posses high individual risk of laboratory transmitted disease
for which there is no vaccine or treatment, or a related
agent with unknown risk of transmission.
•Laboratory personnel must have specialized training in
handling BSL-IV agents and should be supervised by
scientist competent in handling infectious agents.

47. BIOSAFETY LEVEL IV (BSL -IV) - RDNA
•Safety equipment: Class-IV biological safety cabinet, lab
coats, gloves, eye/face protection, respiratory protection,
physical and containment equipment to reduce infectious
aerosol exposure or splashes.
•Facility: BSL-IV facility requires specialized design to
minimize the exposure to risk and only the authorized entry
should be permitted in laboratory area in BSL-IV labs.

48. 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.

49. 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.

50. Good microbiological techniques
• Food and drink must not be stored or consumed in the
laboratory.
• Glassware must be replaced with plasticware
wherever possible.

51. Good microbiological techniques
• Sharps(e.g., needle sticks, glass) must be avoided
wherever possible as it can transmit blood borne
pathogens in case of injury.

52. Good microbiological techniques
• 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.

53. Good microbiological techniques
• 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.

54. 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.

55. 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.

56. 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

57. 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.

58. 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

59. 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.

60. 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.

61. Decontamination
Sterilisation
•Sterilisation makes an item free from all living
microorganisms and viruses.
• The process of sterilization can be
accomplished by applying heat.

62. 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.

63. 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.

64. 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

65. • 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.
Biohazard waste disposal