This document provides guidelines on biosafety and biosecurity for veterinary microbiology laboratories. It defines key terms like biohazard, biosafety, risk assessment, biosecurity, and the biohazard symbol. It discusses the chain of infection and approaches to reduce risk of exposure like risk assessment, personal protective equipment, immunizations, and surveillance. The document also compares and contrasts biosecurity and biosafety. It provides guidance on developing a biosecurity program and addressing breaches. It discusses challenges of preventing interference while ensuring legitimate access.

1. Safety Considerations and Guidelines
for Veterinary Microbiology Laboratory
Dr Ravi Kant Agrawal, MVSc, PhD
Senior Scientist (Veterinary Microbiology)
Food Microbiology Laboratory
Division of Livestock Products Technology
ICAR-Indian Veterinary Research Institute
Izatnagar 243122 (UP) India

2. Biohazard:Biohazard:
An agent of biological origin that has the capacityAn agent of biological origin that has the capacity
to produce harmful effects on humans;to produce harmful effects on humans; i.e.i.e.
microorganisms, toxins and allergens derived frommicroorganisms, toxins and allergens derived from
those organisms, and allergens and toxins derivedthose organisms, and allergens and toxins derived
from plants or animals.from plants or animals.

3. Biosafety:Biosafety:
Laboratory biosafety refers to
containment principles,
technologies and practices to
prevent unintentional
exposure to pathogens and
toxins, or their accidental
release.
Applying a combination ofApplying a combination of
laboratory practices andlaboratory practices and
procedures, laboratoryprocedures, laboratory
facilities, and safetyfacilities, and safety
equipment when working withequipment when working with
potentially infectiouspotentially infectious
microorganisms.microorganisms.

4. Risk Assessment:Risk Assessment:
Addressing laboratory activities involvingAddressing laboratory activities involving
infectious or potentially infectious material andinfectious or potentially infectious material and
implementing measures to reduce the worker’simplementing measures to reduce the worker’s
and environment’s risk of exposure to an agent toand environment’s risk of exposure to an agent to
an absolute minimum.an absolute minimum.

5. Chain of InfectionChain of Infection
Reservoir of pathogenReservoir of pathogen
Portal of escapePortal of escape
TransmissionTransmission
Route of entry/Route of entry/
infectious doseinfectious dose
Susceptible hostSusceptible host
Incubation periodIncubation period
Risk AssessmentRisk Assessment
Practices/Equipm
ent
Practices/Equipm
ent
Personal Protective
Personal Protective
Equipm
ent (PPE)
Equipm
ent (PPE)
Im
m
unizations
Im
m
unizations
Surveillance
Surveillance
Risk
Assessm
ent
Risk
Assessm
ent

6. Biosecurity:Biosecurity:
Protection of high-consequenceProtection of high-consequence
microbial agents and toxins, or criticalmicrobial agents and toxins, or critical
relevant information, against theft orrelevant information, against theft or
diversion by those who intend todiversion by those who intend to
pursue intentional misuse.pursue intentional misuse.
Laboratory Biosecurity is Biorisk
Management
“Laboratory biosecurity” refers to
institutional and personal security
measures designed to prevent the
loss, theft, misuse, diversion or
intentional release of pathogens and
toxins.

7.  BiosecurityBiosecurity refers to ensuring therefers to ensuring the securitysecurity of biologicalof biological
materials to prevent theft, illicit use, or releasematerials to prevent theft, illicit use, or release
(Intentional)(Intentional)
 BiosafetyBiosafety focuses onfocuses on reducing exposurereducing exposure to andto and releaserelease
of biological materialsof biological materials (Un-intentional)(Un-intentional)
 Both involve conducting aBoth involve conducting a risk assessmentrisk assessment to mitigateto mitigate
risksrisks
Biosecurity vs. BiosafetyBiosecurity vs. Biosafety

8. The Biosecurity Programme
 Should be adapted to needs of the institution.
 Should include input from scientific personnel and
laboratory management, biosafety officers,
maintenance, IT, administrators, and law
enforcement.
 Should be based on a accountability for pathogens:
Storage location
Identification of personnel with access
Description of use
Documentation of transfers
Inactivation or disposal of materials

9. Laboratory Biosecurity Programs
 Are intended to protect pathogens, toxins and
security-related information from theft.
 Achieved by instituting a culture of responsibility
 Restricts access to dangerous pathogens and
toxins to authorized persons and
 Establishing accountability over these materials
based on assessment of security risks
 Graded security measures

10. Institutional Laboratory Biosecurity
Protocols Should Include How to Handle:
 Breaches or near-breaches in laboratory biosecurity
including:
 Incident notification
 Reporting protocols
 Investigation reports
 Recommendations and remedies
 Oversight and guidance through the Biosafety
Committee
 Should establish means to identify, report, investigate, and
remediate breaches in biosecurity.
 Should describe specific training for personnel
 Should become part of routine laboratory work

11. Biosecurity Challenges
 Preventing undue interference with day to day lab activities
 Ensuring efficient sharing of research and reference
materials, specimens and information
 Providing legitimate access to important materials and
research
 Assuring compliance with national and institutional
standards
 The WHO has published guidance on Laboratory Biosecurity
 Each institution will have a unique biosecurity programme
based on identified vulnerabilities

12. Biosafety?
Biosafety:Biosafety:
 Laboratory biosafety refers to
containment principles,
technologies and practices to
prevent unintentional exposure
to pathogens and toxins, or
their accidental release.
 Applying a combination ofApplying a combination of
laboratory practices andlaboratory practices and
procedures, laboratoryprocedures, laboratory
facilities, and safety equipmentfacilities, and safety equipment
when working with potentiallywhen working with potentially
infectious microorganisms.infectious microorganisms.

13. What is Biosafety?
Sunday, Sep. 20, 2009
Did the Plague Kill Illinois Scientist?
By AP
(AP / CHICAGO) — The University of Chicago Medical Center says the
infection that killed a scientist may be connected to bacteria he
researched that causes the plague.
The university said Saturday that its researcher studied the genetics
of harmful bacteria including Yersinia pestis, which causes the
illness. He died Sept. 13. His name and age haven't been released
The medical center says the bacteria he worked with was a weakened
strain that isn't known to cause illness in healthy adults. The
strain was approved by the Centers for Disease Control and
Prevention for laboratory studies.
An autopsy found no obvious cause of death but did find the
presence of the bacteria. More tests are planned. No other
illnesses have been reported.

14. Biosafety in Various Disciplines
Biosafety is related to several fields
 ECOLOGY: referring to imported life forms not
indigenous to the region (Reggie the alligator)
 AGRICULTURE: reducing the risk of alien viral or
transgenic genes, or prions such as
BSE/"MadCow“; reducing the risk of food
bacterial contamination
 MEDICINE: referring to organs or tissues from
biological origin, or genetic therapy products,
virus; levels of lab containment protocols BSL-1,
2, 3, 4 in rising order of danger
 CHEMISTRY: Nitrates in water, PCB
(polychlorinated biphenyls) levels affecting
fertility
 EXOBIOLOGY: i.e., NASA's policy for containing
alien microbes that may exist on space samples -
sometimes called "biosafety level 5"

15. Biosafety in Academic Research
 Research Universities:
Promoting safe
laboratory practices and
procedures; proper use
of containment
equipment and
facilities; provides
advice on laboratory
design and risk
assessment of
experiments involving
infectious agents, rDNA
in-vitro and in-vivo.

16. Biohazard Symbol
 The biohazard symbol was developed in 1966 by
Charles Baldwin, an environmental-health
engineer working for the Dow Chemical
Company on the containment products
 Symbol to be “memorable but meaningless” so it
could be learned.
 Striking in form in order to draw immediate
attention;
 Unique and unambiguous, in order not to be
confused with symbols used for other purposes;
 Quickly recognizable and easily recalled;
 Easily stenciled;
 Symmetrical, in order to appear identical from all
angles of approach;
 Acceptable to groups of varying ethnic
backgrounds.
The chosen symbol scored the best on nationwide
testing for memorability.
 Blaze orange – most visible under harsh
conditions

17. Biosafety Issues
 Laboratory Safety
 Bloodborne pathogens (BBP)
 Respiratory Protection
 Recombinant DNA (rDNA)
 Biological waste disposal
 Infectious substance and diagnostic specimen
shipping
 Bioterrorism and Select agents
 Mold, mould spores and indoor air quality
 Occupational safety and health in the use of research
animals
 Biohazards used in animal models

18. Biohazardous Materials
 Viruses
 Bacteria
 Fungi
 Chlamydiae/Rickettsiae
 Prions
 Recombinant DNA
 Transgenic Plants, Animals and Insects
 Human and Primate Cells, Tissues, and
Body Fluids
 Brain Tissue from Demented Patients
 Viral Vectors
 Replication deficient viruses

19. Biosafety In Microbiological and
Biomedical Laboratories “BMBL”
(acronym)
CDC/NIH Publication
Safety “Guidelines”
Regulations of Institution
receives
NIH funding
Code of Practice and “Gold”
Standard in Industry
“Guidelines” does not mean
“optional”
They are a term and condition of
NIH funding for recombinant DNA
research.
Clinical & Research Lab.
Biosafety Concepts
HHS Publication No. (CDC) 93-8395

20. Biosafety Concepts from the BMBL
Principles of Biosafety
1. Practice and Procedures
 Standard Practices
 Special Practices & Considerations
1. Safety Equipment
2. Facility Design and Construction
3. Increasing levels of protection

21. Biosafety Concepts: The BMBL
Standard Microbiological Practices
 Most important concept
 Strict adherence
 Aware of potential hazard
 Trained & proficient in techniques
 Supervisors responsible for:
 Appropriate Laboratory facilities
 Personnel & Training
Special practices & precautions
 Occupational Health Programs

22. Standard Lab Procedures
Hygienic Practices
 No Smoking, Eating, drinking, applying
cosmetics, lip balm, contacts in
laboratory work areas - Hand to mouth
transmission of disease is a common
route of exposure while handling
biological agents.
 Do not store or heat/chill food or
beverages in the lab.
 Prohibit mouth pipetting- Mechanical
pipetting devices should be used for
manipulating all liquids in the
laboratory
 Wash hands before/after procedures
with disinfectant soap.
 Decontaminate lab bench before and
after work
 Restrict or limit access when conducting
research experiments

23. General Operational Practices
 Proper attire
 Minimum – lab coat, safety glasses, gloves
 Plan your work
 Know in advance what you are working with
 Read available resources

24. Personal Protective Equipment
(PPEs)
 Use personal protective
equipment to prevent skin/
mucous membrane exposure
during agent use, such as:
Lab coat
Gloves
Safety glasses/ face shield
Closed toe shoes/ foot covers
Face Mask/ Respiratory
protection (BL-3 agents)

25. Minimize Sharps Usage
 Needles must not be re-
capped, re-used, bent, break,
sheared or removed from a
disposable syringe.
 All used sharps must be placed
in a rigid, hard-plastic,
puncture-resistant container
for disposal.
 DON’T place needles or sharps
in office waste containers.
 Use alternatives to needles
when available.
 Substitute plastic for glass
whenever possible.

26. Handwashing
 Hands should be washed:
 immediately and
thoroughly with a
disinfectant hand soap, if
contaminated with
biological agents.
 after gloves are
compromised or removed
 with water and soap after
a puncture wound
 If a sink is not available (e.g.
equipment room), consider
the use of disinfectant
towelettes in these areas.

27. Minimize Aerosol Generation
 To avoid inhalation exposure to agents, workers must minimize
the potential generation of aerosols.
 These procedures include but are not limited to:
 Grinding,
 Blending,
 Mixing
 Vortexing
 Sonicating
 Centrifuging
 Work on the open bench top should be limited whenever
conducting these procedures.
 Decontaminate work surfaces daily
 Decontaminate infectious waste
 Maintain an insect & rodent control program

28. Use Biosafety Cabinets
 Biological safety cabinets are:
 Used for product/personnel protection
 Used for aerosol generating procedures
 Disinfected after use
 Certified annually
 Not recommended for chemical or radioisotope usage.

29. Decontaminate Work Surfaces
Laboratory work surfaces should be decontaminated with an
appropriate chemical disinfectant.
 Before the work
 When work activities are completed.
 After a spill of biohazardous materials
Disinfectant Selection
Disinfectants must be selected on a case by case basis to ensure
efficiency.
 Quaternary Ammonia Compounds
 Chloride Compounds (Bleach)
 Iodophores (Wescodyne)
 Phenolics (Amphyl)
 Alcohols (70% Ethanol)
 Formaldehyde/ Glutaraldehyde

30. Infectious Waste Disposal
 Ensure that:
Infectious waste is placed in a red bag lined bin.
Sharps waste is placed in a sharps container
Mixed chemical-biological waste is handled as
chemical waste
Mixed radioactive-biological waste is handled as
radioactive waste

31. Hazard Communication
Biohazard labels shall be
placed on:
The surface of all equipment
(freezers, incubators,
refrigerators) which may be
containing/ contaminated with
biohazardous materials.
Sample transport outer
containers.
The outer door of BL 2 labs.
Medical waste bins
Medical waste storage areas

32. Biosafety Issues: The BMBL
(2) Safety Equipment
 Primary Containment Barrier
 Biological Safety Cabinets
 Minimize exposure to hazard
 Prevent contact / Contain aerosols
 Personal Protective Equipment (PPE)
 Gloves, gowns, Respirator, face mask,
Face shield, eye protective devices,
Booties
 Covered or ventilated animal cage
systems

33. Biosafety Concepts: The BMBL
(3) Facility Design and Construction
 Secondary Barrier/ Engineering controls
 Contributes to worker protection
 Protects outside the laboratory
 Environment & Neighborhood
 Ex. Building & Lab design, Ventilation, Autoclaves,
Cage wash facilities, etc.

34. Classification of Infectious Agents
 Classified into risk groups on the basis of risk to the individual
and to the community.
 Currently, 4 risk group levels have been designated: the least
risk (RG-1) to the most risk (RG-4). NIH Guidelines, Appendix B
 This classification based on:
 Capability to infect and cause disease
 Severity of disease
 Availability of preventive measures and effective treatments
Other characteristics to consider include:
 Infective dose
 Stability in environment
 Host range
 Probable routes of transmission
 Endemic nature
• This risk group classification meant for laboratory work only

35. RG-1 Agents
 Not associated with disease in healthy adult humans.
 No or low individual and community risk
 Generally only require a laboratory with minimal
containment
 Biosafety Level 1
 “Declaration of Dangerous Goods” is not generally
required for transportation by air.
 Classified as a diagnostic specimen

36. RG-2 Agents
 Associated with human disease which is rarely
serious.
 Moderate individual risk but low community risk
 Preventative and therapeutic interventions are
available.
 Generally require a laboratory with moderate
containment.
 Biosafety Level 2
 “Declaration of Dangerous Goods” required for
shipment.

37. RG-3 Agents
 Associated with serious or lethal disease.
 High individual risk but low community risk
 Preventative or therapeutic interventions may be
available.
 Generally require a laboratory with high level
containments
 Biosafety level 3
 “Declaration of Dangerous Goods” required for
shipment.

38. RG-4 Agents
 Likely to cause serious or lethal human disease
 High individual and high community risk
 Preventative or therapeutic interventions are
not usually available.
 Require a laboratory with extensive high-level
containment
 Biosafety level 4
 Research on these agents are allowed only in
those campus where a BSL-4 containment lab is
currently available.

39. W.H.O. Agent Risk Group Classification

40. Risk Group vs Biosafety Level
 Biosafety risk assessment
 To determine the risk group of a biological agent
 RG-2 organisms are generally handled in BSL-2
containment and RG-3 in BSL-3.
 Exceptions
 RG-2 agents used in large quantities may require BSL-3
containment
 RG-3 agents under some circumstances may be manipulated
at BSL-2 containment.
 Institutional Biosafety Committee (IBSC) approves the
level of laboratory containment required following an
IBC protocol review.

41. Biosafety Levels
Biosafety Level-1 (BSL-1 or ABSL-1)
 Agents not known to cause disease (in healthy
human adults; now healthy immunocompetent adults) i.e. Well
characterized agents.
 Prophylactic treatment available
 Open bench procedures
 Animals in open cage system or open
environment (outdoors)
 Good laboratory practices

42. BSL-1 Practices
 Bench-top work allowed
 Daily Decontamination
 Manual pipetting
 Required Handwashing
 Red bag waste
 Biosafety cabinet not
required (unless creating
aerosols)
 2˚ containment

43. Risk Group 1 Agents
 E. coli K-12, Bacillus subtilis, Adenoasociated viruses 1-
4, T4 bacteriophages, Saccharomyces cerevisiae,
Rhizopus stolonifer, Candida albicans, Pseudomonas
Infectious canine hepatitis
 Transgenic Plants
 Plasmids
 Fungi
 Mold
 Yeast

44. BSL-1 Containment Overview
 RG-1 Agents
 Not known to cause disease in healthy adult humans
 Practices
 Standard microbiological practices
 Safety equipment
 Minimal requirements
 Facilities
 Open bench top

45. Bio-safety Level-2
 Agents associated w/ human
disease
 Treatment for disease available
 Agent poses moderate hazard to
personnel and environment
 Direct contact or exposure
 Percutaneous exposure
 Scratch, Puncture, Needle
stick
 Mucus membrane exposure
 Eyes, Mouth, open cut

46. BSL-2 Practices & Procedures
 Limited access to lab when
work in progress
 Daily decontamination
 Mechanical pipetting
 Lab coat, safety glasses and
gloves required
 Red bag & sharps containers
required
 Biohazard Sign posted at
entrance to lab
 Label all equipment
(incubators, freezers, etc.)
 Documented training
 Baseline serology or pre-
vaccination may be required
 TC room – negative air flow

47. Risk Group 2 Agents
 S. aureus, Bordetella pertussis,
Corynebacterium diphthriae,
Other E coli, Nisseria gonorrhoea
 Streptococcus pyogenes, Vibrio
cholerae, Klesiella spp., Proteus,
Serratia marcescens,
 Rabies,
 Hepatitis ABC
 Cryptococcus neoformans,
 Most parasitic agents
 Human or Primate Cells
 Herpes Simplex Virus
 Replication Incompetent
Attenuated Human
Immunodeficiency Virus
 Patient specimens

48. BSL-2 Containment Overview
 RG-2 Agents
 Associated with mild to moderate disease in humans
 Practices
 BSL-1 plus limited access.
 Safety equipment
 Biological Safety Cabinet and personal protective
equipment as needed.
 Facilities
 BSL-1 plus the availability of a mechanism for
decontamination.

49. Biosafety Level 3: Working in High Containment
Biosafety Level-3 (BSL-3 or ABSL-3)
 Indigenous or exotic agents
 Aerosol transmission
 Serious health effects
 Treatment may or may not exist

50. BSL-3 Practices
 Public access NOT permitted
 Daily decontamination upon completion of
experiment and after spill
 Required foot activated hand washing sink
and controls
 No sharps unless absolutely necessary
 Aerosol minimization procedures required
 Wrap around disposable clothing is
required.
 Autoclave required and waste is disposed at
the end of day
 Specialized equipment may be required
depending upon procedures

51. BSL-3 Practices (con’t)
 Biohazard Signs and labels posted
 Air flow from low hazard to high hazard “Pressure
Mapping”
 Bench top work not permitted
 Documented training and personnel competency
certification (for BSL-3 procedures)
 Baseline serology
 Spills – report immediately and treat accordingly
 Vaccinations/ post exposure protocols and SOP’s,
Biosafety Manual, Biosafety Officer

52. Risk Group 3 Agents
 SARS
 Rift valley fever
 Human Immunodeficiency
Virus
 Yellow fever virus
 VEE virus
 Hanta virus
 Prions
 M. tuberculosis, M. bovis
 Coxiella burnetii
 B. abortus
 Bacillus anthracis,
 Yersinia pestis
 Pasteurella multocida
 Coccidiodes immitis
 Plasmodium
 Trypanosoma
 No parasitic agents

53. BSL-3 Containment Overview
 RG-3 Agents
 Associated with serious or potentially lethal disease
in humans
 Practices
 BSL-2 plus controlled access.
 Safety equipment
 Biological Safety Cabinet and personal protective
equipment required.
 Facilities
 BSL-2 with self-closing double door access and
single-pass negative directional airflow.

54. BSL-3

55. Biosafety Level-4: Working in High Containment
Biosafety Level-4
 Builds on BSL-3/ ABSL-3 practices
 Maximum containment facilities
 Pressurized Containment Suite
 BSL-3 + Class III Biosafety Cabinet
 Chemical decontamination showers
 Liquid effluent collection / decontamination
 BSL-4: High Safety Animal Disease Laboratory, Bhopal
(National Institute of High Security Animal Diseases)

56. Biosafety Level-4 (BSL-4 or ABSL-4) practices
 Dangerous/exotic agents
 Life threatening disease
 Aerosol transmission
 Agents of unknown risk of transmission or
health affects
 No known treatment

57. Risk Group 4 agents
 Ebola Hemmorrhagic Fever
Virus
 Marburg Virus
 Lassa Fever Virus
 Machupo virus
 Crimean congo Haemorrhagic
viruses, Bolivian and Argentine
Haemorrhagic fever viruses
 Some encephalitis viruses
 Herpesvirus simiae
 No bacterial agents
 No fungal agents
 No parasitic agents

58. Animal Biosafety Level-4: Working in High Containment

59. BSL-4 Containment Overview
 RG-4 Agents
 Associated with high risk of life-threatening disease in humans
and/or animals
 Practices
 BSL-3 plus controlled access
 Safety equipment
 Biological Safety Cabinet
 Full-body air-supplied, positive pressure personnel suit
 Facilities
 BSL-3 plus dedicated air and exhaust, decontamination
procedures for exit, separate building, etc.

60. BSL Agents Microbiology
Practices
Safety Equipment
(Primary Barriers)
Facilities
(Secondary Barriers)
1
Not known to
consistently cause
disease in healthy
adults
Standard Microbiological
Practices
None required Open bench top sink
required
2
Associated with
human disease,
hazard equals
percutaneous injury,
ingestion, mucous
membrane exposure
BSL-1 plus: Limited
access, biohazard warning
signs, “sharps”
precautions, biosafety
manual defining waste
decontamination &
medical surveillance
policies
Class I or II BSCs or
other physical
containment for
manipulations of
agents that cause
splashes or aerosols
of infectious materials,
PPE: lab coats, gloves,
face protection as
needed
BSL-1 plus: Autoclave
3
Indigenous or exotic
agents with potential
aerosol transmission;
may have serious or
lethal consequences
BSL-2 plus: Controlled
access; Decontamination
of all waste & lab clothing
before laundering;
Baseline serum
Class I or II BCSs or
other physical
containment for all
manipulations; PPE:
protective clothing;
gloves; respiratory
protection as needed
BSL-2 plus: Physical
separation from
corridors; Self-closing,
double-door access;
Exhausted air not
recirculated; Negative
airflow into laboratory
4
Dangerous/ exotic
agents with high risk
of life-threatening
disease, aerosol-
transmitted
infections; or related
agents with unknown
risk of transmission
BSL-3 practices plus:
Clothing change before
entering, shower on exit,
all material
decontaminated on exit
All procedures in
Class III BSCs or Class
I or II BSCs in
combination with full-
body, air-supplied,
positive pressure
personnel suit
BSL-3 plus: Separate
building or isolated
zone, dedicated
supply, exhaust,
vacuum, & decon
systems; other
requirements.
Recommended Biosafety Levels for Infectious Agents
“Biosafety in Microbiological and Biomedical Laboratories”,5th Ed

61. Type of hazard Image
Generic caution
Poison
Ionizing radiation
Radiation – high-level
source
Non-ionizing radiation
Biological hazard
Carcinogen
High voltage
Laser hazard
Chemical weapon
Hazard Symbols

62. Thanks
Acknowledgement: All the material/presentations available online on
the subject are duly acknowledged.
Disclaimer: The author bear no responsibility with regard to the source
and authenticity of the content.
Questions???