Transcript General Biosafety Training University of Idaho

Presenter
Ashish Sawant
Biosafety Associate
Training Objectives
Understand biosafety risks
To learn how to mitigate these risks
Biohazard Sign
International Symbol
for biohazard or
biological hazard
Unicode U+2623
In Microsoft word
Alt+2623(☣)
Developed at DOW chemicals
by Charles Baldwin
Laboratory accidents
Accidents involving infectious agents
Not always due to human error
Occurs in the best regulated laboratories
Even skilled workers are not immune
Common documented infection following
Spillage and splash
Needle stick injury
Sharp objects, broken glass
Bite scratch or ectoparasite from animal
Aspiration through pipette
Laboratory accquired infections
Causes for lab-associated infections
Improper Facility Design
Containment Equipment Failure
Improper laboratory Practices
1%
9%
90%
Misidentified culture sent to laboratory
Worker gets B. melitensis infection
Pregnant worker suffers a cut handling quality
control material
Develops Hepatitis B: material infected!
Conjunctiva: portal of entry
Spray on face while inoculating with syringe
gonococcal eye infection
Can cause mucopurulent conjunctivitis
Droplets in eye while grinding chicken tissue
Newcastle virus conjunctivitis
Needle fell off from syringe-droplets into eye
Acute hemorrhagic conjunctivitis by virus
Squirted HBsAg-positive blood into eye and
rubbed it with hand
Hepatitis B viral infection
>80% infections involve Aerosols
Principles of Biosafety
Standard Practices
Special Practices
Safety Equipment (Primary Barriers)
Laboratory Facilities (Secondary Barriers)
Biosafety Levels 1-4 Provide:
Increasing levels of personnel and environmental
protection
Guidelines for working safely in microbiological and
biomedical laboratories
References:
Biosafety in the Microbiological and Biomedical
Laboratories 5th Edition (BMBL). CDC/NIH:
http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm
Lab practices and techniques
Knowledgeable supervisor
Demonstrates appropriate lab techniques
Project specific training
Ensures personnel receive additional required training
Personnel
Aware of potential hazards
Proficient in practices/techniques
Obtain required training
Biosafety manual specific to lab
Specific to protocols in the lab
Routine safe practices
Emergency procedures
Safety equipment
Biosafety cabinets (BSCs)
Primary containment
Three types (Class I, II, and III)
Personal Protective Equipment (PPE)
Gloves
Gowns
Eye and face protection
Respiratory protection
Mechanical pipette devices
Safety centrifuge cups and enclosed rotors
enclosed to prevent release of aerosols
Risk Assessment
Risk assessment: shared responsibility
Principal investigators
Institutional Biosafety Committees (IBC)
Institutional Animal Care and Use committee(IACUC)
Biosafety professionals
Risk assessment is used to:
Identify the hazards associated with the agent
Activities that can result in an exposure
Likelihood that such an exposure will cause a LAI
Consequences of such an infection
Risk Assessment
Primary factors in the risk assessment process:
Agent hazards
Laboratory procedure hazards
Capability of lab personnel to control hazards
Training
Technical proficiency
Good habits
Pre-existing conditions
Immuno-compromised, pregnant, medications
Medical surveillance
Operational integrity of containment and facility safeguards
Risk Group
Classification
NIH Guidelines for Research
Involving Recombinant DNA
Molecules 2002
World Health Organization Laboratory Biosafety
Manual 3rd ed. 2004
Risk Group 1
Not associated with disease in healthy
humans
(No or low individual and community risk)
Unlikely to cause human or animal disease
Risk Group 2
Associated with human disease
rarely serious infection
Preventative or therapeutic interventions
are often available
(Moderate individual risk; low community risk) A
pathogen that can cause human or animal disease but is
unlikely to be a serious hazard to laboratory workers, the
community, livestock or the environment. Laboratory
exposures may cause serious infection, but effective
treatment and preventive measures are available and the
risk of spread of infection is limited
Risk Group 3
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)
(High individual risk; low community risk) A pathogen
that usually causes serious human or animal disease but
does not ordinarily spread from one infected individual to
another. Effective treatment and preventive measures are
available
Risk Group 4
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)
(High individual risk and high community risk) 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.
Biosafety levels
There are four biosafety levels that consist of combinations of:
Laboratory practices and techniques
Safety equipment
Laboratory facilities
Biosafety levels differ from risk groups described in the NIH
Guidelines and the WHO Biosafety Manual
Risk groups are defined by the classification of a potentially infectious
agent and association with, and severity of resulting disease in
humans
Biosafety levels include the
risk group of the agents along with mode of transmission, procedures,
experience of personnel, and other risk factors to determine the
appropriate containment level for the proposed project activities, staff ,
equipment and location.
Biosafety Levels: know the risks
BSL 1: Material not known to consistently cause disease in
healthy adults.
BSL 2: Associated with human disease.
Hazard is from percutaneous injury, ingestion, or mucous
membrane exposure.
Some agents with environmental or agricultural impact.
BSL 3: Indigenous or exotic agents with potential for aerosol
transmission; disease may have serious or lethal
consequences
BSL 4: Dangerous/exotic agents which pose a high risk of lifethreatening disease, aerosol-transmitted lab infections or
related agents with unknown risk of transmission
Standard Laboratory Practices
To minimize the generation of aerosols
Discharge from pipettes should never be dropped from a
height.
Contents should be allowed to run down the wall of the tube or
bottle or be discharged as close as possible to the fluid or agar level
Gently manipulate lab equipment and specimens, avoid
sudden and unnecessary movements
Use disposable inoculating loops
When a procedure or device creates aerosols within a
closed container, wait a few minutes before opening
Standard Practices
Biosafety Manual
Record of personnel working in the lab
Clearly defined lab practices
Standard and specific protocols for disinfection
and sterilization
Methods to prevent protocol drift
Lab protocol policy
Standard Microbiological Practices
Principal Investigator
responsible for enforcing
access requirements as
approved in Biosafety
Manual
Wash hands
After handling viable materials
After removing gloves
Before leaving the lab
Prohibit eating, drinking,
smoking , and storing food
for human consumption in
the lab
Standard Microbiological Practices
Use mechanical pipettes
Use sharps containers
Standard Laboratory Practices
Broken Glass
DON’T
Touch broken glass with
hands
Do
Use a mechanical device
to pick up the sharp
Tongs
Tweezers
Forceps
Dust pan and brush
Standard Laboratory Practices
Autoclaving
The two common steam-sterilizing
temperatures
121oC (250oF), 15 psi
132oC (270oF), 30 psi
Gravity displacement autoclave
High-speed pre-vacuum sterilizers
Gravity displacement Autoclave
Gravity displacement
Autoclave failure
Closed bag
Overloaded Autoclave
Standard Laboratory Practices
Use plastic as a substitute
for glass
Reduce risk of injuries
Biosafety level 1
Applicable for work involving well-characterized
agents
not known to cause disease in immunocompetent
adult humans
minimal potential hazard to laboratory personnel,
animals, plants and/or the environment.
Examples
Recombinant DNA work with Risk Group 1 standard
E. coli host/vector systems.
Risk Group 1 plant/animal & Human agents
Other Examples:
Bacillus subtilis
Pseudomonas fluorescens
Most plant pathogens, e.g. Aschochyta spp.
Adenoviruses Types 1-4
Many non-zoonotic endemic animal disease agents.
Exempt organisms under NIH guidelines
BSL-1 facility
BSL-1 facilities
Sink for hand washing
Work surfaces easily cleaned
Bench tops (can use plastic backed absorbent pads
on the surface)
Sturdy furniture
Operable windows fitted with fly screens
Biosafety Level 2
Applicable for work involving agents of moderate
potential hazard to personnel, plants, animals and/or the
environment.
Differs from BSL-1 in that:
Requires specific training in handling pathogenic agents
Supervision by scientists competent in handling infectious
agents
Access limited when work being conducted
Extreme caution with contaminated sharps
Use of biosafety cabinet to reduce risk of splash and spills
Biosafety Level 2
Examples:
Salmonella spp.
Toxoplasma spp.
Hepatitis B virus
Some established human cell
lines, e.g. HEK-293, HeLa
E. coli O157:H7
Antibiotic treatment and immunizations are
available.
Hepatitis B vaccine series
Biosafety Level 2
All BSL-1 Facilities PLUS
Autoclave
Material to be moved in leak proof
containers
Eyewash station
Test weekly
Safety Shower
Test monthly
BSL-2
BSL2
Laboratory door is closed when work is conducted
should be self-closing with locks
Biohazard warnings and updated biosafety manual in lab
Eye wash station is readily available
Vacuum lines equipped with HEPA filters
Biosafety Cabinets
Ventilation: Ideally negative pressure (inward flow of air)
Standard Laboratory Practices
As BSL increases
all Standard Microbiological Practices & PPE from the lower
levels carry forward PLUS
Additional PPE (as dictated by risk assessment)
Lab coat, gown, gloves
required & must be removed before leaving the lab
Eye, face & respiratory PPE per risk assessment in animal
rooms
All Lab personnel provided with immune competence
information and conditions that may predispose them to
infection
All lab personnel responsible for self-identifying to
healthcare provider for counseling and guidance
Safety Equipment
Class II biosafety Cabinet
Must be annually certified
Grinding, blending , mixing, sonic
disruption, tissue harvest, pipetting,
handling liquid stock cultures, and
intranasal animal inoculations
Large volumes and/or high
concentrations of infectious agents
May use centrifuge with sealed rotor
heads or centrifuge safety cups
Rotors or safety cups should be
opened in a biological safety cabinet
Biosafety cabinet
high efficiency particulate air
filtration system
BSC
HEPA filter
Traps
particulate only
Traps particles
down to 0.3um
Chemical
vapors, fumes
and vapors
pass through
BSL
Isolated from other work areas
Removed from high traffic areas
Away from air ducts
Away from laboratory entry doors
Operating in BSC
Read manufacturer’s recommendations for operation of cabinet
Turn BSC on and allow to run for 10-15 minutes
Confirm inward flow
Load BSC with all needed supplies
Enter straight into cabinet, perform work in controlled methodical
manner
At end of work decontaminate all items to be removed from cabinet
Decontaminate interior of cabinet
Allow cabinet to run for 10-15 minutes
BSC operations
Layout of equipment
Clean materials separated from dirty items
Avoid rapid air movement at the face of cabinet
Do NOT allow the front or rear grills to be
obstructed
Aerosol generating activities should be done
towards the rear of cabinet
BSC operations
Not designed for chemical use
May use for non-volatile toxic chemicals or low
level radioactive materials
May use for “minute” amounts of volatile
chemicals
Cabinets must be certified Annually
Place all materials into the cabinet before starting
work
BSC operations
CAUTION!!
Chemicals may damage the HEPA filter
Exposure risk: chemical/infectious agents
Volatile chemicals NOT retained by HEPA filter
Exposes personnel if not exhausted
BSC fans are not spark proof
Chemicals may result in fire /explosion
Open flames, Burners in BSC
Disrupts air flow
Causes excessive heat build-up
May damage HEPA Filter
Presents potential fire or
explosion hazard
May inactivate manufacturer’s
warranties
Centrifuges
Follow Manufacturer’s operating instructions
Check tubes for cracks/chips
Tightly seal all tubes and cups
Ensure that the rotor is locked to spindle and the bucket
seated
Close lid during operation
Allow to come to complete stop before opening
Centrifuge Hazards
Operator error
Mechanical failure
Lab equipment failure
(tubes etc.)
Aerosol generation
Personal injury
Physical damage
BSL-2 special practices
Person with increased risks
Not allowed in lab or animal rooms
Only those advised or hazards and meet entry
requirements are provided access
Medical surveillance program
Determined by risk assessment
Immunizations
Requires written employee informed consent
Baseline serum samples may be required (case by case)
Requires written employee informed consent
Breach in primary
containment
Needle stick
Aerosolizing activities
performed on the open
bench
Splash soaks through lab
coat
agent can pass through intact
skin
Breach in secondary
containment
Biosafety Cabinet not
functioning properly
Loss of power to the
building HVAC system
Door not closed while
working with agent
Improper use of the
Biosafety Cabinet
Open a centrifuged tube
at the open bench that
contains a BSL-2
organism.
Explanation:
Unequal pressure may be
created when centrifuging
Opening the lid is likely to
create aerosols
BSL-2 potentially
aerosolizing work
performed in uncertified
BSC.
Explanation:
An uncertified BSC may not
be operating correctly and
could contaminate the air
space outside the cabinet
Using a wire transfer loop
and a Bunsen burner pick
a Campylobacter colony
and streak for isolation
work in the BSC
Explanation:
Bunsen burners can disrupt
BSC airflow
Transfer liquid cultures
with a transfer pipette to
produce BSL-2 organism
serial dilutions
Work performed at the
open bench.
Explanation:
Pipetting produces
aerosols. This activity
should be done in BSC
Spill decontamination
Alert co-workers
Define/isolate contaminated area
Put on appropriate PPE (personal protective equip.) to include gloves, lab coat and
face shield (if appropriate)
Remove/glass/glass shards with forceps or scoop
Apply absorbent towels to spill – Do NOT apply disinfectant directly to the spill as this
may aerosolize the agent
Apply disinfectant to towel surface
Allow adequate contact time (generally 10-60 minutes)
Remove towels, mop up; clean with alcohol or soap/water or other agent as
appropriate.
Dispose of materials in biohazardous waste
Notify Primary Investigator/Lab Director
Spill in Biosafety cabinet
Small spills
Leave the cabinet running and close the view screen for
about 5 minutes.
This will allow aerosols to settle before starting cleanup.
The drain pan should be flooded with appropriate
disinfectant.
Leave the disinfectant in the pan for required contact
time
longer if the spill involved a high organic load and an organic
load sensitive disinfectant is used (e.g. bleach).
The disinfectant then needs to be drained out and the surfaces
thoroughly cleaned with water to prevent corrosion when a
corrosive disinfectant is used (bleach)
Personal Exposure
Clean exposed surface with soap/water
1 minute
eyewash
15-20 min.
Apply first aid and treat as an emergency
Seek medical attention
Notify supervisor or PI
Contact Environmental Health and Safety
Major spill
Any spill that take more than 30 mins to clean up
Not including the deactivation time
Notify others in the lab
If the spill cannot be handled by lab personnel
evacuate the area
call Environmental Health and Safety.
If the spill occurs after normal working hours call 911.
Stay out of the lab and keep others out until help arrives
General Guidelines for Surface
Decontamination
AGENT
DISINFECTANT
INACTIVATION TIME
Recombinant DNA
10% Bleach
20 minutes
Bacterial Spores
>6% Hydrogen Peroxide
30 minutes
Vegetative Bacteria
10% Bleach
20 minutes
Viruses & Viroids
10% Bleach
20 minutes
Fungi
10% Bleach
20 minutes
Feline Parvovirus
20% Bleach
30 minutes
Free Living
Cryptosporidium
70% Ethanol
10 minutes
Most Parasites
10% bleach
30 minutes
Prions
50% bleach
60 minutes
Biosafety Level 3
Applicable for work with infectious agents
which may cause serious or potentially lethal
disease as a result of exposure by inhalation
route exposure.
BSL3
Exposure potential to pathogens spread by
aerosol
Infection serious, possibly lethal
Examples:
M. tuberculosis
St. Louis encephalitis virus
Coxiella burnetii
West Nile
Hantavirus
SARS
Avian Influenza H5N1
BSL3
BSL-1 and 2 Facilities PLUS:
Separate building or isolated zone
Restricted Access
Double door entry
BSL-3 has Ante room entry or similar design
Directional inward airflow
BSL-3 is negative with respect to corridor
Single-pass air
Exhaust air is not re-circulated to other rooms
10 /12 air changes/hour
BSL3
BSL3
Enclosures for aerosol generating equipment
(Biosafety Cabinets)
Enclosed centrifuges
Room penetrations sealed
Walls, floors and ceilings are water resistant for
easy cleaning
Porous acoustical ceiling tiles are not used
Floor is monolithic, continuous cove molding
extending at least 4 inches up the wall
Safety equipment
BSL-1 and 2 Safety Equipment PLUS:
BSC class II or III to
manipulate infectious
material
BSL3
BSL-1 and 2 Safety Equipment PLUS:
Respiratory protection
may be indicated
Medical surveillance
Training
Quantitative Fit Test
Special Practices
Use bioaerosol
containing equipment
No work on open
bench
All procedures in BSC