• Biohazards are:
– Materials of biological origin or synthetic material
which mimic biological entities and may induce
adverse conditions to humans, other animals,
plants, or the environment.
Biosafety
Is protection of research personnel, the
environment, and the
public health from potential adverse
consequences during the use of biological
agents in research
Working with biologic agents often pose risks
Death of a researcher from infection with an attenuated strain
of Yersinia pestis in Chicago [September 2009]
Infection of a few laboratory scientists with a wild-type
Francisella tularensis strain contaminating samples of an
attenuated tularemia (rabbit fever) vaccine strain.
Biosafety guidelines
It is a set of guidelines that is essential for further
advances in prevention, diagnosis, and treatment of
many diseases and health risks
{Biosafety: Protection of (research) personnel, the environment,
and the public health from potential adverse consequences
during the use of biological agents in research
Biohazard in Research
Any agent which may cause any
infection, allergy, toxicity or can
create any hazard to human health
and/or environment
Categories of biohazards or potentially
infectious materials
• Human, animal and plant pathogens:
–
–
–
–
–
–
Bacteria
Plasmids
Fungi
Viruses, including oncogenic viruses
Parasites
Prions
• All human blood, blood products, tissues and certain body fluids.
• Cultured cells (all human or certain animal) and potentially infectious
agents these cells may contain
• Allergens
• Toxins (bacterial, fungal, plant, etc.).
• Certain recombinant products
• Clinical specimens
• Infected animals and animal tissues
• Recombinant deoxyribonucleic acid (rDNA)
•rDNA -molecules that are constructed outside living cells by joining
natural or synthetic DNA segments to DNA molecules that can replicate in
a living cell, or molecules that result from the replication of those.
Synthetic DNA segments that are likely to yield a potentially harmful
polynucleotide or polypeptide (e.g., a toxin or a pharmacologically active
agent) are considered as equivalent to their natural DNA counterpart.
If the synthetic DNA segment is not expressed in vivo as a biologically
active polynucleotide or polypeptide product, it can be exempted from the
classification.
e.g. In Human Recombinant Vaccine Trials
Toxins
• These are toxic substances produced within living cells or organisms
[man-made substances created by artificial processes are excluded].
•
Toxin: the term was first used by organic chemist Ludwig Brieger (1849–1919).
• Toxins can be small molecules, peptides, or proteins that are capable of
causing disease on contact with or absorption by body tissues interacting
with biological macromolecules such as enzymes or cellular receptors.
• Deadly toxins - botulinum toxin.
• Hemotoxins – Causes destruction of RBCs
• Phototoxins –Causes photosensitivity
• Prion is an infectious agent composed of protein in a
misfolded form.
When a prion enters a healthy organism, it induces existing,
properly folded proteins to convert into the diseaseassociated, prion form
The prion acts as a template to guide the mis-folding of
more proteins into prion form.
Prion diseases –Fatal (affects brain and Neural tissues)
Prion diseases, collectively called Transmissible Spongiform
Encephalopathies (TSEs), are untreatable and fatal
• When cell cultures are known to contain an etiologic agent or an
oncogenic virus
• the cell line is classified as the same level as that recommended for
the agent.
– Infected cell lines, Infection models
– Drug testing, Drug development
– Disease
Established human cell lines which are characterized to be free of
contamination from human hepatitis viruses, HIV, and other recognized
bloodborne pathogens, are not considered as biohazardous agents
Established human or animal cell lines which are known to be or likely
infected/contaminated with human microbes or agents classed as
bloodborne pathogens, especially hepatitis viruses and HIV are
potentially hazardous.
Microorganisms
• Whole organisms of pathogenic microbes
(e.g. viruses, bacteria, fungi)
Other agents
• Insects, and animals, which may a harbour human pathogen(s)
• Ecto- and endo- parasites, their genetic material (natural or
synthetic)
• Gene products, Genetically modified organisms (GMOs, GMMs)
• Tissues or fluids derived from living organisms that harbor or may
harbor such material
Bio hazardous agents -classified for transportation by UN number
Category A, UN 2814- Infectious substances affecting humans and
animals: An infectious substance in a form capable of causing permanent
disability or life-threatening or fatal disease in otherwise healthy humans
or animals when exposure to it occurs.
Category B, UN 2900- Infectious substances affecting animals only: An
infectious substance that is not in a form generally capable of causing
permanent disability of life-threatening or fatal disease in otherwise
healthy humans and animals when exposure to themselves occurs.
Category B, UN 3373- Biological substance transported for diagnostic or
investigative purposes.
Regulated Medical Waste, UN 3291- Waste or reusable material derived
from medical treatment of an animal or human, or from biomedical
research, which includes the production and testing of biological
products.
Risk Groups: Biohazardous agents
• Risk Group 1 (RG1)
Agents that are not associated with disease in healthy adult humans
• Risk Group 2 (RG2)
Agents that are associated with human disease which is rarely serious and for
which preventive or therapeutic interventions are often available
• Risk Group 3 (RG3)
Agents that are associated with serious or lethal human disease for which
preventive or therapeutic interventions may be available (high individual
risk but low community risk)
• Risk Group 4 (RG4)
Agents that are likely to cause serious or lethal human disease for which
preventive or therapeutic interventions are not usually available (high
individual risk and high community risk)
Micro-organisms: Risk Groups
Risk
Definition
Group
1
Risk to
Individual
Risk to
Community
Low
Low
1
Microorganisms not associated with
disease in healthy adult humans
2
Microorganisms associated with disease
that is rarely serious and for which
treatment is available
Moderate
Low
3
MO associated with serious and/or fatal
disease for which treatments may be
available
High
low
4
MO associated with serious and/or fatal
disease for which treatments is often not
available
high
High
Experiments with Recombinant DNA (rDNA)
(DBT guidelines)
Category 1 experiments:
Involve self cloning, using strains, and interspecies cloning belonging to
same exchanger group – no serious risks
Category II experiments*:
Falling under contaminant levels II, III, and IV
Large scale recombinants made of self cloning, in systems belonging to
except category I etc.
Category III experiments**:
Involve toxin gene cloning. Cloning of genes for vaccine production, use of
infectious animals and plant viruses, self fusion experiments, field testing,
release of GMOs etc.
Cat II and III: either prior intimation or review and approval of the of institutional Biosafety
Committee prior to commencement
Risk Group I
• Low individual and community risk
• A microorganism unlikely to cause human or animal disease
Eg. E. coli K12, Bacillus subtilis, Lactobacilli, Pseudomonas ( except P. mallei, P.
psuedomallei)
- Most cell cultures
- [Refer to the reading material for detailed list]
Risk Group II
• Moderate individual risk, limited community risk
• A pathogen causing human or animal disease unlikely to be a serious hazard to
laboratory staff or the community
• May cause serious infections but effective treatment is available
risk of spread is limited
E.g. Staphylococcus, influenza virus, Acenetobacter, Listeria , Klebsiella
prion,
Risk Group III
• High individual risk, low community risk
•
A pathogen causing serious human disease but not readily transmitted to
others
• Effective treatment is available (vaccines are also available for some cases)
E.g. Mycobacterium tuberculosis, Shigella dysenteria, Brucella, Psuedomonas
mallei, P. psuedomalei
Risk Group IV
• High individual and community risk
•
A pathogen causing serious human or animal disease, readily
transmitted between individuals
Bacteria: None
Fungi: None
Parasites: None
• Effective treatment usually not available
E.g. Ebola virus (Ebola hemorrhagic fever -EHF)
•
•
Guanarito virus
Lassa Virus
Human Blood, Blood Products, Body Fluids and Tissues
• Bloodborne Pathogens (BBP) are pathogenic
microorganisms that are transmitted primarily
through blood and other infectious materials
• BBPs include human immunodeficiency virus (HIV),
Hepatitis B and C viruses and other microbial agents that
may contaminate human blood.
• BBPs may contaminate human blood, blood products,
organs, tissues, other bodily fluids and human cell lines.
• Universal precautions states that “all human blood or other
potentially infectious materials must be assumed to be
contaminated with one or more pathogens and must be
stored, processed and disposed of in accordance with
Biosafety guidelines”
Zoonoses
• Zoonoses are diseases that are communicable from animals (i.e., rats
and mice) to humans under natural conditions
• Two diseases of concern when working with mice are lymphocytic
choriomeningitis virus (LCMV) and hantavirus.
• LCMV - cause of nonbacterial or aseptic meningitis.
• Rat-bite fever is caused by two bacteria, Streptobacillus moniliformis
and Spirillum minor.
• These bacteria are present in the upper respiratory tract and mouths of
rats.
• Rats are asymptomatic as the bacteria do not cause disease in them.
•
Commercial vendors have virtually eliminated this bacterium from their animals
Biosafety: Women Researchers
Infectious organisms that are believed to have adverse effects on human embryo and fetal
development:
• Rubella Virus, Herpes Simplex Virus, Varicella Virus
• Toxoplasma gondii , (protozoa)
• Cytomegalovirus, Venezuelan Equine Encephalitis Virus
• Human Immunodeficiency Virus, Coxsackie virus type B, Human Parvovirus B 19
• Treponema pallidum
The Infectious organisms that are known to cause birth defects in animals, but have not YET
been shown to be teratogenic for humans:
e.g.
• Lymphocytic Choriomeningitis (LCMV), Feline Panleukopenia Virus
•
Influenza Newcastle Disease Virus
• Salmonella typhimurium and Salmonella enteriditis
• Bovine diarrhea-mucosal disease virus
• Bluetongue Virus Parainfluenza type 2
• Rodent Parvovirus (Minute Virus)
• Hog Cholera Virus
Routes of infection in the lab
•
•
•
Object to person
Contact with media/materials
Inhalation
Aerosol-related transmissions likely account for infections of laboratory
workers when there is no recognition of an exposure. Any procedure that
imparts energy to a microbial suspension can produce aerosols of respirablesized particles that may remain airborne for extended periods of time.
Inoculation
Laboratory exposure events that are readily recognized include
percutaneous inoculation with syringe needles or other contaminated sharps,
spills, and splashes onto skin or mucous membranes, and animal bites or
scratches.
Biosafety in Research laboratories
• Responsibilities
Biological agents
GMM/GMO
Blood or blood products
Biosafety measure
Responsibility
“Guanarito Virus Vial Missing From Galveston National Laboratory's Secure
Facility
[The Huffington Post | By Ryan Grenoble Posted: 03/25/2013 2:02 pm
EDT | Updated: 03/25/2013 3:04 pm EDT ]
The Centers for Disease Control and Prevention has referred to the FBI the
case of the laboratory where one of five vials of a deadly Venezuelan virus
went missing, an official from the CDC told ABCNews.com.
• "CDC reported the incident to the FBI and we understand that the FBI will
initiate an investigation concerning the reported incident," Dr. Rob
Weyant, director of the CDC's Division of Select Agents and Toxins, told
ABCNews.com in an email. "Since the investigation is just underway, the
agency will not comment further regarding details of this incident."
• The FBI would not confirm it was investigating the incident at the
Galveston National Laboratory.”
Responsibilities of a Researcher

Be fully aware of all risks associated with the
materials (biologic) used in the study

Be fully aware what protective measures are required

Comply fully with applicable rules

Report incidents and accidents

Educate co-workers

Be aware of emergency routines
Risk assessment
Is the first principle in maximizing safety while working with biologic agents.
This includes identification of potential hazards
(1)From exposure to or release of biologic agents
Not limited but also look into
(2) Related to laboratory procedures operation of equipment in laboratory settings.
Appropriate protective measures should be assigned to each specific task.
All investigators and research personnel share these responsibilities
(e.g. non decontaminated culture of a potentially pathogenic bacteria
Or a swab containing PPB Or a blood sample containing Pathogen )
Institutional Biosafety Committee (IBC)
Is responsible for the protection of research personnel, the
environment, and the public health from potential adverse
consequences during research uses of biologic agents
Guidelines for Institutional Biosafety Committee (IBSC) is available with DBT which
was developed in association with BCIL (India)
At the local level IBCs review research protocols involving biologic agents to
•
Determine potential risks of the work
•
Ensure adequate bio-containment and expertise of research personnel
•
Determine whether health surveillance of research personnel is necessary
Handling blood or blood products
All contacts with blood that has not been purified from infectious agents for example
by heat treatment is a potential risk.
Possible infections
Most common:
Hepatitis B, vaccine available, risk of infection by inoculation 10-30%
Other diseases with a risk are:
HIV, no vaccine, risk of infection by inoculation 0.3%
Hepatitis C, no vaccine, risk of infection by inoculation 3%
• Clinical laboratories, especially those in health care
facilities, receive clinical specimens with requests for a
variety of diagnostic and clinical support services.
– Typically, the infectious nature of clinical material
is unknown
•
•
Biosafety Level 2 practices and
procedures must be followed when
handling human blood, blood products,
body fluids and tissues because of the
infectious agents they may contain.
Biosafety Level 2 practices and
procedures requires all specimens of
human blood or other potentially
infectious materials to be treated as if they
Personal Protective Equipment (PPE) is a primary line of defense against BBP exposures
The following procedures are implemented during the handling of BBP:

•
•
•
•
•
•
•
•
•
A laboratory coat is worn whenever potential exposure is anticipated.
If any garments are penetrated by blood or other infectious materials, they are
removed immediately, or as soon as is feasible.
All PPE is removed prior to leaving a work area.
Gloves are worn when handling or touching contaminated items or surfaces.
Disposable gloves are replaced as soon as practical after contamination or if they are
torn, punctured, or otherwise lose their ability to function as an exposure barrier.
Utility gloves are decontaminated for reuse unless they are cracked, peeling, torn, or
exhibit other signs of deterioration.
Full-face protection, such as facemasks, face shields, and eye protection, is used
whenever splashes or sprays may generate droplets of infectious materials.
Head covers/hoods and/or shoe covers/boots are used in any instances where gross
contamination is anticipated, such as perfusion activities.
•
•
•
•
•
Wear required PPE at all times when working with animals.
Not wear PPE outside of the animal facility.
Wear gloves at all times when handling animals.
Not distribute animal bedding in the immediate work environment.
All cage cleaning procedures should be performed in a manner that
prevents bedding debris from entering the work environment. Change
bedding in a cage changing station, BSC, or fume hood.
• Ensure that animal cages are properly fitted into ventilated racks and
cage lids properly fit.
• Not overpopulate animal cages.
• Follow containment facilities required for different risk group
organisms
Guidelines for working with animals contd..
• Work with animals in a ventilated hood or BSC when required and
whenever possible.
• Work with animals in well ventilated areas when not working under
a hood/cabinet.
• Clean and disinfect all equipment after use.
• Wash hands with soap and water frequently and always after
handling animals (even when wearing gloves).
• Avoid touching the face when working with animals.
• Keep the work area clean.
• Keep animal cages and transport containers properly covered at all
times.
• Not handle common items (i.e., door knobs) with gloved hands that
have had animal contact.
Disinfection and Sterilization
• Disinfection is the planned reduction of the
concentration of microbial agents and is therefore a
relative term.
• Generally disinfection is used to describe the elimination
of microbial agents but not necessarily their spores;
• Ethanol is a commonly used disinfectant.
• Sterilization is an absolute term, which means the
planned alumination of all microbial agents. Sterilization
eliminates all biological agents, including their spores,
and can be chemical or physical;
• Autoclaving is the most commonly used sterilant within
the laboratory.
Risk Group Micro-organisms
• When genetic manipulation is to be
undertaken, possible impacts on cell tropism,
host range, virulence, or susceptibility to
antimicrobial agents or other treatments,
including protection from vaccines, must be
considered.
BIOSAFETY LEVELS (BSL)
Biologic containment is the important aspect of Biosafety
Includes enclosure of agents as much as possible during manipulation
and transport
4 Biosafety levels (BSL) represent
risk-related combinations of laboratory practices, safety
equipment,
and laboratory facilities necessary to work safely with
biologic agents.
BSL
Typical Lab Type*
Practices/Procedures
Typical PPE
Containment Equipment
BSL 1
Basic research,
teaching
Standard procedures
Lab coat, gloves
Open bench
BSL II
Clinical ,
diagnostic,
general research,
RG2 pathogens/
agents in Res.
controlled, increased
training requirements,
occupational health
program recommended
Lab coat, gloves,
respiratory
protection as
needed
Biologic safety cabinet
(class II BSC) for activities
with aerosol generation
potential
BSL III
Clinical, RG3
pathogens/agent
s in research
heightened security,
extensive training and
emergency response
procedures, occupational
health program required
Dedicated clothing,
gloves, overalls/
gowns respiratory
protection
Primary containment
devices (BSC, centrifuge
containment devices) are
utilized at all times.
BSL IV
Risk group 4
pathogens/agent
s in research
BSL 3 standards plus
extremely specialized
facility with extensive
training, medical
surveillance
Scrubs for glovebox lab, positive
pressure suit
(moon suit)
All work performed in a
class III BSC (glove box)
or use of positive-pressure
suit with class II BSC
• High-Efficiency Particulate Arresting or HEPA is a type of air
filter.
• To qualify as HEPA by government standards, an air filter must
remove 99.97% of all particles greater than 0.3 micrometer from
the air that passes through.
• HEPA filter was designed in the 1940s in the Manhattan Project
to prevent the spread of airborne radioactive contaminants
• This level is required for work with dangerous and exotic
agents that pose a high individual risk of aerosoltransmitted laboratory infections, agents which cause
severe to fatal disease in humans for which vaccines or
other treatments are not available [refer reading material]
–
–
–
–
–
Bolivian and Argentine hemorrhagic fevers, Marburg virus
Ebola virus
Lassa fever
Crimean-Congo hemorrhagic fever,
Smallpox, and various other hemorrhagic diseases
•
Use of a positive pressure personnel suit, with a segregated air supply, is mandatory.
•
The entrance and exit of a level four biolab shall contain multiple showers, a vacuum
room, an ultraviolet light room,
•
Other safety precautions designed to destroy all traces of the biohazard.
•
Multiple airlocks are employed and are electronically secured to prevent both doors
opening at the same time.
•
All air and water service going to and coming from a biosafety level 4 (or P4) lab will
undergo similar decontamination procedures to eliminate the possibility of an accidental
release.
•
Members of the laboratory staff have specific and thorough training in handling
extremely hazardous infectious agents
• BSL IV facility is either in a separate building or in a
controlled area within a building
• Completely isolated from all other areas of the building.
• A specific facility operations manual is prepared or
adopted.
• Building protocols for preventing contamination often
use negatively pressurized facilities
BSL IV facilities in India
1.
2.
3.
4.
High Security Animal Disease Laboratory (HSADL) India, Bhopal
Established in 1998
This facility deals especially to zoonotic organisms and emerging
infectious disease threats
Centre for Cellular and Molecular Biology Hyderabad, India
Established in 2009
National Bio-Safety Level-4 Containment Facility for Human
Infectious Diseases & Clinical Research Facility in Regenerative
Medicine
All India Institute of Medical Sciences India, New Delhi
BSL 1-4
Established in 1993
Conducts studies on major pathogenic organisms. Has contributed in
discovering new strains & vaccines
Microbial Containment Complex India, Pune: [National Institute of
Virology]
BSL-IV Laboratory
Established in 2012 : [ICMR +support from DST]
Good Laboratory Practice
A quality system concerned with the organisational process and the
conditions under which non-clinical health and environmental safety
studies are planned, performed, monitored, recorded, archived and
reported.
How does GLP relate to academic research?
1. Safety
2. Reproducibility
3. Reporting
Good Microbiological Techniques GMT
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Specimens are handled safely.
No mouth pipetting is permitted.
Pipettes and pipetting aids are used safely.
Dispersal of infectious materials is avoided.
Contact of infectious materials with skin and eyes is avoided.
Ingestion of infectious materials is avoided.
Separation of serum is carried out safely.
Centrifuges are used safely.
Homogenizers, shakers and sonicators are used safely.
Tissue grinders are used safely.
Refrigerators are maintained and used safely.
Ampoules containing infectious materials are opened safely.
Infectious materials are stored safely.
Precautions are taken with blood and other bodily fluids.
Specimens and infectious materials are shipped safely.
Appropriate disinfection and sterilization are carried out.
Gloves are worn for procedures that involves contact with blood or infectious material.
Hands are washed between procedures and prior to leaving laboratory.
Laboratory gowns are worn for work in laboratory.
Closed-toed shoes are worn for work in laboratory.
Storage of food or drink in the laboratory is prohibited.
Eating, drinking, or smoking in the laboratory is prohibited.
Personal protective equipments
•
•
Also be aware of hazards from your work
possibly affecting others working
around you
Use a fume cupboard, warn others, restrict
access to where you are working
GMM Genetically modified microorganism
GMM is defined as a microorganism whose genetic material has been changed in a way
that does not occur naturally by mating or natural recombination
Includes:
• Bacteria, virus, viroids, blue-green algae, molds, protozoa, etc.
• RNAi if inserted via a vector, but not if inserted by a liposome
• Cell lines from plants or animals
• Cells microinjected with DNA
• Cell fusions or hybridization techniques generating living cells with new combination
of genetic material that might be dangerous for health or environment.
Examples of what is not considered as GMM
• Microorganisms generated by mutagenesis
• Microorganisms generated by cell fusions of prokaryotes exchanging genetic materials
by known natural processes
• Cell fusions or hybridization techniques generating living cells with new combination
of genetic material that is not dangerous for health or environment.
Waste disposal
Waste disposal
Waste must be disposed of correctly:
 Autoclave bags or the yellow carton bin dedicated for incineration
 Stainless steel container
 Sharp bin containers
 Treatment of liquid waste with disinfectant
Storage of chemical products
No more chemicals than needed should be stored
Always read the chemical's label
Mark it with the date of receipt/opening before storage
Never store a chemical with an obscured or missing label
Define separate storage areas for highly toxic chemicals
Never store liquid hazardous chemicals above eye level
Risk Assessments
Hazardous Chemicals
Control of Substances Hazardous to Health Regulations 2002 (COSHH).
Biological Hazards
Genetic manipulation, handling pathogens, Crime & Security Act 2001
Radiochemicals
Contact the safety office for permission before use
Experimental risk assessments
Combination of hazards associated with a technique or protocol
EMERGENCY PROCEDURES -The safety signs to recognize
• Emergency exits and evacuation routes
• Fire extinguishers
• Spill kits and First aid kits
• Emergency showers
• Eye-wash fountains
• Know your nearest first aiders
Safety icons
Hazard icons