Transcript Biosafety Awareness Training For Laboratory Personnel University of Idaho Environmental Health and Safety & Office of Research Assurances0

Biosafety Awareness Training
For Laboratory Personnel
University of Idaho
Environmental Health and Safety
&
Office of Research Assurances
2010
0
An agent of biological origin that has the
capacity to produce deleterious effects on
humans, animals, plants and/or the
environment.
Some biohazard examples: recombinant
DNA, microorganisms, Select Agents &
toxins, prions, virions, viroids, biological
allergens.
1
Laboratory Associated Infections
Laboratory Associated Infections (LAIs)
•
•
•
•
•
Survey data dating back to the late 1970’s
More than 5,000 labs surveyed
4.091 cases cited
159 different agents involved
168 deaths
Top ten list of laboratory associated infections:
1.
2.
3.
4.
5.
Brucella sp.
Q Fever
Hepatitis B
Typhoid
Tularemia
6. Tuberculosis
7. Dermatomycosis
8. Venezuelan Equine Encephalitis
9. Psittacosis
10. Coccidioidomycosis
2
Laboratory- Associated Infections
Causes for lab-associated infections
• Improper Facility Design
1%
• Containment Equipment Failure
9%
 Improper laboratory Practices
90%
 >80% have been implicated to involve aerosols
– Inspired investigations into the behavior of aerosols generated
in the laboratory
– Guidelines for protecting microbiological lab workers were
developed using:
• Historical data
• Understanding risks associated with different routes of
transmission
3
Picture of Aerosol Production
When Pipetting Liquid
4
Preventing Laboratory Associated Infections:
A safe work environment results from a
combination of :
•
Appropriate engineering controls
•
Management/administrative controls
•
Work practices and procedures
•
Medical surveillance program (as determined by
risk assessment)
5
Principles of Biosafety
Biosafety Guidelines reflect combinations of:
 Laboratory Practices and Techniques
 Standard Practices
 Special Practices
 Safety Equipment (Primary Barriers)
 Laboratory Facilities (Secondary Barriers)
6
Principles of Biosafety
Introduction
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
NIH Guidelines for Research Involving Recombinant DNA Molecules,
April 2002:
•
http://www4.od.nih.gov/oba/rac/guidelines/guidelines.html
World Health Organization : Laboratory Biosafety Manual - Third Edition
•
http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf
7
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
8
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
9
Risk Assessment
• Risk assessment is an important responsibility
shared between directors, principal investigators,
institutional biosafety committees, animal care and
use committees, biosafety professionals, etc.
• Risk assessment is used to:
– Identify the hazards associated with a known infectious
or potentially infectious agent or material
– Activities that can result in an exposure
– Likelihood that such an exposure will cause an LAI
– Consequences of such an infection
10
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
• Operational integrity of containment and facility
safeguards
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Risk Assessment
• Hazardous Characteristics of Biological Agents
– Capability to infect and cause disease in a susceptible
host
– Virulence as measured by the severity of disease
– Availability of preventive measures and effective
treatments for the disease
Resources:
BMBL 5th edition
NIH Guidelines
World Health Organization Biosafety Manual
12
Risk Group
Classification
NIH Guidelines for Research
Involving Recombinant DNA
Molecules 2002
World Health Organization Laboratory
Biosafety Manual 3rd ed. 2004
Risk Group 1
Agents that are not associated with
disease in healthy humans
(No or low individual and community risk) A
microorganism that is unlikely to cause human or
animal disease
Risk Group 2
Agents that are associated with human
disease which is rarely serious and for
which 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.
13
Risk Assessment
The BMBL provides a 5 step approach to the risk
assessment process.
• First Step:
• Identify the hazards associated with the agent and do an
initial assessment of the risk
• Capability to infect and cause disease
• Severity of disease
• Availability of preventive measures and effective treatment
• Utilize your resources
• BMBL, NIH, WHO and other references for
information
• Always keep the generation of aerosols in mind
14
Risk Assessment
• Second Step:
– Identify laboratory procedure hazards
• Agent concentration
• Suspension volume
• Equipment and procedures that generate small particulates and
aerosols (cell sorters)
• Sharps
• Procedures involving animals
– Bites, scratches, zoonotic diseases
• Complexity of a laboratory procedure
– Changes in procedures
– New techniques and/or equipment
15
Risk Assessment
• Third Step:
– Make a final determination of the appropriate biosafety
level and select additional precautions indicated by the
risk assessment
• Based on understanding of lab practices, safety equipment, and
facility safe guards
• Additional safe guards may be warranted based nature of
protocol
• A risk assessment indicate a need to alter the recommended
facility safe guards in some way. This will require careful
review by both safety and facility personnel
• Recognize that laboratory personnel will differ in their
susceptibility to disease
– Pre-existing conditions, medications, compromised immunity,
pregnancy, breast-feeding, etc.
– Medical Surveillance
16
Risk Assessment
• Fourth Step:
– Evaluate proficiencies of staff regarding safe practices
and the integrity of safety equipment
• Protection of laboratory workers, other staff and the public
depends on the laboratory workers themselves
• Laboratory director or principal investigator should ensure that
personnel have acquired the technical proficiency to handle the
agents and the equipment safely
• Laboratory director or principal investigator should ensure that
the necessary safety equipment is available and operating
properly
– A non-certified biosafety cabinet represent a potentially serious
safety hazard
17
Risk Assessment
• Fifth Step:
– Review the risk assessment with a biosafety
professional, subject matter expert, and the institutional
biosafety committee
• Review of the risk assessment and the selected safeguards by
knowledgeable individuals is always beneficial and often
required by regulatory or funding agencies
• Review of high risk protocols by the IBC should be standard
practice
• Adopting this step voluntarily will promote the use of safe
work practices in the laboratory
18
Risk Assessment – Relationship
to Chain of Infection
19
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.
20
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
21
Biosafety Level 1
Applicable for work involving well-characterized
agents not known to consistently cause disease in
immunocompetent adult humans and present
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
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Biosafety Level 1
• Other Examples:
– Bacillus subtilis
– Naegleria gruberi
– Pseudomonas fluorescens
– Most plant pathogens, e.g. Aschochyta spp.
– Adenoviruses Types 1-4
– Most established (2°) human cell lines
– Most non-zoonotic endemic animal disease
agents.
– Exempt organisms under NIH guidelines
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Biosafety Level 1
Laboratory Facilities
• Wastes are separated,
labeled, and stored
properly
• Work can be done on open
bench top
24
Biosafety Level 1
Laboratory 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
25
Facility Design
• Doors for access control
• Lab easily cleaned and
decontaminated
• Adequate space between benches
and equipment
• Bench tops impervious to water
and resistant to heat, organic
solvents, acids, alkalis, and
chemicals used for
decontamination
• Lab chairs used with agents must
have a nonporous cleanable
26
surface
Facility Design
– Laboratory location
• There are no specific requirements for isolating
the BSL-1 laboratory
– Laboratory ventilation
• Though there is no specific requirement for more
than six air changes per hour, it may be
necessary if there are volatile chemicals in use
• May require a chemical fume hood
• Laboratory should be negative with respect to
the hallway (air moves from outside hallway into
the lab)
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Biosafety Level 1
Standard Microbiological Practices
– Principal Investigator responsible
for enforcing the University of
Idaho access requirements as
outlined in the approved 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
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Biosafety Level 1
Standard Microbiological Practices
– Use mechanical
pipetting devices
– Mouth pipetting is
prohibited
– Policy for the safe
handling and
disposal of sharps
29
Biosafety Level 1
Needles & Sharps Precautions
DON’T
• Break, bend, re-sheath or
reuse syringes
or needles
DO
• Use single-use needles and
syringes whenever possible
• Use sharps containers
30
Biosafety Level 1
Sharps Precautions
• Discard needles, syringes,
and similar devices in a leak
proof and puncture resistant
container
• Non-disposable sharps must
be placed in a hard walled
container for transport to a
processing area for
decontamination preferably
by autoclaving
• Contaminated sharps must be
decontaminated by
appropriate means prior to
disposal
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Biosafety Level 1
Needles & Sharps Precautions
DON’T
• Touch broken glass with
hands
Do
• Use a mechanical device
to pick up the sharp
–
–
–
–
Tongs
Tweezers
Forceps
Dust pan and brush
32
Biosafety Level 1
Needles & Sharps Precautions
DO
•Use plastic as a
substitute for glass
•Whenever possible
adopt improved
engineering and work
practice controls that
reduce the risk of
sharps injuries
33
Biosafety Level 1
Standard Microbiological Practices
– Minimize splashes and aerosols
– Decontaminate work surfaces daily
– Decontaminate wastes by approved methods
– Maintain insect & rodent control program
34
Biosafety Level 1
Standard Microbiological Practices
• Hand washing
• Liquid soap
preferable to bar
soap
• 20 seconds of
vigorous lathering
• Paper towel to dry,
use towel to turn off
faucet
35
Biosafety Level 1
Personal Protective Equipment
• Biosafety cabinets are not
usually required for work with
agents assigned to BSL1
• Lab coats, gowns,
(recommended not required)
• Disposable gloves required,
latex or nitrile (risk
assessment)
• Protective eyewear and/or a
face shield (splash guard)
36
Biosafety Level 1
• Supervision
– Scientist with general training in
microbiology or related science
– Establishes general safety procedures
– Provides protocol specific training to
personnel
• Lab Personnel
– Specific training in lab procedures
– Follow prescribed protocols
37
Biosafety Level 1
Special Practices
•
There are no “special” practices
required for activities conducted at
BSL-1
38
Biosafety Level 2
Applicable for work involving agents of moderate
potential hazard to personnel, plants, animals and the
environment.
Differs from BSL-1 in that:
a. Requires specific training in handling pathogenic
agents & supervision by scientists competent in
handling infectious agents
b. Access limited when work being conducted
c. Extreme caution with contaminated sharps
d. BSC’s & other containment of work with aerosol or
splash potential
39
Biosafety Level 2
Examples:
–
–
–
–
Salmonella spp.
Toxoplasma spp.
Hepatitis B virus
Some established human cell
lines, e.g. HEK-293, HeLa
– E. coli O157
• Antibiotic treatment and immunizations
are available.
– Example the Hepatitis B vaccine series
40
Biosafety Level 2
• BSL-1 Facilities
PLUS:
– Autoclave available
• place waste in autoclave
bag before removing
from BSC
• All materials to be
decontaminated outside
of the immediate lab
must be placed in a
durable, leak-roof
container for transport
– Eyewash station
• Test weekly
– Safety shower
• Test monthly
41
Biosafety Level 2
Laboratory Facilities
• Laboratory door is closed when work
is conducted (should be self-closing
with locks)
• Eye wash station is readily available
• Vacuum lines equipped with HEPA
filters
• Adequate illumination
• Biosafety Cabinets (equivalent
containment equipment)
• Ventilation: Ideally negative pressure
(inward flow of air) [risk assessment]
• Consider locating new facilities away
from public areas
42
Biosafety Level 2
Standard Microbiological Practices & PPE
• As the biosafety level increases, all of the Standard
Microbiological practices & PPE from the lower
levels carry forward PLUS:
• Additional PPE (as dictated by risk assessment)
• Lab coat, gown, tyvek (required & must be removed before leaving the lab
area) [Gloves must also 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
43
Biosafety Level 2 Safety Equipment
•BSL-1 Equipment PLUS:
Properly maintained & annual certified biosafety cabinets (class
II) for work with infectious agents involving:
– Aerosols and splashes
• 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
44
Biosafety Level 2 Safety Equipment
Room air in at A through front grille and
under work surface
•Air is blown via F through the rear air
plenum E to the top of the cabinet
Class II-A Biosafety Cabinets
•Air is divided into two chambers at D
• Thirty percent of the air is exhausted out C
through at HEPA filter into the lab room.
•Seventy percent of the air B is directed
through another HEPA filter down to the
work surface
45
Biosafety Level 2 Special Practices
• Policies and procedures
for entry “Restricted
Access”
• Biohazard warning signs
• Biosafety manual specific
to lab developed &
followed
• Training with annual
updates
46
Biosafety Level 2 Special Practices
 Use leak-proof transport
containers
 Gasket seal lid
 Animals and Plants not
used in the research are
not allowed in the lab
 Equipment must be
decontaminated before
repair maintenance or
removal from the lab 47
Biosafety Level 2 Special Practices
• In general persons at increased risk of acquiring
infection are not allowed in lab or animal rooms
• Only those advised of hazards and meeting entry
requirements are provided access
• Medical surveillance (as determined by risk
assessment)
– Immunizations
• Requires written employee informed consent
– Baseline serum samples may be required (case by
case)
• Requires written employee informed consent
48
Biosafety Level 2
Special Practices
• Principal Investigator responsible for ensuring appropriate and at a
minimum annual Biosafety training for all lab personnel
• Potentially infectious material and wastes are placed in covered
containers that prevent leakage during collection, handling,
processing, storage, transport or shipping.
• Decontaminate work surfaces and equipment with effective
disinfectant on a routine basis.
• Report spills and accidents (Incident log)
• No animals in laboratories (All non-research animals are kept out of
the laboratory
49
Biosafety Level 2 Special
Practices
• Incidents that may result in exposure – Know what
to do:
• What constitutes a biological exposure risk?
• What to do when there has been a potential
exposure?
• How to clean up a biological spill?
• When should you seek assistance with spill clean
up?
50
What Constitutes a Biological
Exposure Risk
• Breach in primary
containment, e.g.
– Needle stick
– Aerosolizing activities
performed on the open
bench
– Splash soaks through
lab coat (agent can
pass through intact
skin)
• Breach in secondary
containment, e.g
– 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
51
Examples of Potential Biological
Releases
• 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
52
Examples of Potential Biological
Releases
• Using a wire transfer
• Transfer liquid
loop and a Bunsen
cultures with a transfer
burner pick a
pipette to produce
Campylobacter colony
BSL-2 organism serial
and streak for isolation
dilutions. Work
work in the BSC
performed at the open
bench.
• Explanation: Bunsen
burners can disrupt
• Explanation: Pipetting
BSC airflow
produces aerosols.
This activity should be
performed in the BSC53
Surface 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
54
Biosafety Cabinet Spill
Decontamination
• If the spill of an infectious agent was enough to create puddles or
liquid in the drain pan then the following procedure should be
followed:
– a. Leave the cabinet running and close the view screen for about 5
minutes. This will allow aerosols to settle before starting cleanup.
– b. 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)
55
Personal Exposure
• Clean exposed surface with soap/water,
(1 minute), eyewash (eyes) 15-20 min.,
or rinse mouth 3x’s with water
• Apply first aid and treat as an emergency
• Notify EH&S – fill out report and other
forms as requested
• If appropriate report to medical clinic for
treatment/counseling
56
Major Spills
• This is a spill of a potentially biohazardous
material that will take more than 30 minutes
to clean up (not including the agent
deactivation period of 10-60 minutes
depending on the biological agent and
disinfectant in use)
Call EH&S for Assistance 885-6524
57
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
Practical Disinfectants for use in Recombinant DNA Research
NE=Not Effective, b=variable results dependent on virus
Reference: NIH Guidelines for working with Recombinant DNA Lab Safety Monograph (Appendix D updated)
Liquid
Disinfectants
Practical Requirements
Use/Dilution
Category
Contact time
minutes
Lipo virus
Broad
Spectrum
Inactivates
Vegetative
Bacteria
Lipoviruses
Nonlipid
Viruses
Important Characteristics
Bacterial
Spores
Quaternary
Ammonia
Compounds
0.1-2.0%
10
NE
+
+
Phenolic
Compounds
1.0-5.0%
10
NE
+
+
b
Chlorine
Compounds
500ppm(a)
10
30
+
+
+
+
Iodophor
251600ppm(a)
10
30
+
+
+
+
Alcohol, Ethyl
70-85%
10
NE
+
+
Alcohol,
isopropyl
70-85%
10
NE
+
Formaldehyde
0.2-8.0%
10
30
Glutaraldehyde
2.0%
10
30
Effective
Shelf life >
1week
Effective
for
Surface
Decontam
ination
+
+
+
+
+
+
+
b
+
+
+
b
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Liquids
For
Discard
+
Summary of Disinfectant Activities
Disinfectant
Disinfection
Level
Bacteria
Lipophil.
Viruses
HydroPhilic
Viruses
M.
tuberculosis
Fungi
Comments
Quaternary
Ammonium
(0.5-1.5%)
low
+
+
-
-
+/
Ineffective against bacterial spores. May be
ineffective against Pseudomonas and other
gram negative bacteria; recommendation
limited to environmental sanitation
Alcohols (ethyl
and isopropyl)
60-85%
Intermed.
+
+
-
+/
+
Ethyl or isopropyl alcohol at 70-80%
concentration is a good general purpose
disinfectant; not effective against bacterial
spores. , high concentrations of organic
matter diminish effectiveness; flammable
Phenolics
(0.4%-5%)
Intermed.
+
+
+/-
+
+
Not sporicidal; phenol penetrates latex
gloves; eye/skin irritant; remains active upon
contact with organic soil; may leave residue
Chlorine (1001,000 ppm)
Intermed.
+
+
+
+/
+
Not generally sporicidal; inactivated by
organic matter; fresh solutions of hypochlorite
(chlorox) should be prepared weekly;
corrosive; irritating to eyes and skin
+
+
+
+/
+/
Recommended for general use. Wescodyne
diluted 1 to 10 is a popular disinfectant for
washing hands. Inactivated by organic matter
+
+
+
+
+
Used to sterilize surgical instruments that can
not be autoclaved; strong odor; use with
adequate ventilation. Not for use on
environmental surfaces. Because it is a
sensitizer and causes asthma it is not
recommended for laboratory use.
Iodophors (301,000 ppm
iodine)
Glutaraldehyde
(2-5%)
Intermed.
high
Biosafety Level 2
Supervision
Supervisor is a competent scientist who understands the risks
associated with the agents being used. Supervisor has
increased responsibilities
• Limits access
• Must make sure personnel are training regarding
the potential hazards in the lab
• Develops biosafety manual specific to lab
activities
Lab Personnel
Aware of potential hazards
Demonstrate proficiency in both standard and special
practices/techniques before working with RG-2 agents
Responsible for self identification to health care provider
61
of conditions that may put them at increased risk
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.
62
Biosafety Level 3
– 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
63
Biosafety Level 3
Laboratory Facilities
• BSL-1 and 2 Facilities PLUS:
– Separate building or isolated zone
• Ag Biotech BSL-3 is isolated from neighboring labs
• 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
64
Biosafety Level 3
Laboratory Facilities
• Double door entry
• Air movement is from the
corridor to the BSL-3
procedure area
• Room exhaust air may or
may not be HEPA filtered
but is not re-circulated
65
Biosafety Level 3
Laboratory Facilities
• BSL-1 and 2 Facilities PLUS:
– 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 should be monolithic, continuous cove
molding extending at least 4 inches up the wall
– No upholstered furniture in the BSL-3
66
Biosafety Level 3
Laboratory Facilities
• BSL-1 and 2 Facilities
PLUS:
– Vacuum lines must be
protected with in line
HEPA filters
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Biosafety Level 3
Standard Microbiological Practices
The same as those for BSL-1 and BSL-2
68
Biosafety Level 3
Safety Equipment
• BSL-1 and 2 Safety
Equipment PLUS:
– BSC class II or III to
manipulate infectious
material
69
Biosafety Level 3
Safety Equipment
• BSL-1 and 2 Safety
Equipment PLUS:
– Respiratory protection
may be indicated
– Medical surveillance
– Training
– Quantitative Fit Test
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Biosafety Level 3
Special Practices
Supervision
• Supervisor is a competent scientist experienced
working with agents
• Establishes criteria for entry
• Restricts access
• Develops policies/procedures
• Trains lab personnel in protocol/agent specific
safety practices
• Develops laboratory safety manual
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Biosafety Level 3
Special Practices
• BSL-2 Special Practices PLUS:
– Work in certified BSC
– Use bioaerosolcontaining equipment
• Centrifuge tubes in containment cups
– Decontaminate spills
promptly using approved methods
– No work with open vessels conducted on the bench
– All procedures involving the manipulation of infectious
material must be conducted in a BSC or other physical
containment devices
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Biosafety Level 3
Special Practices
Lab Personnel
Strictly follow guidelines
Demonstrate proficiency
Receive appropriate training
Report incidents
Participate in medical surveillance if required
(Agent and Protocol dependent)
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BSL-3 Requirements (summary)
Facility design, operational parameters and procedures must be verified prior to operation and reverified & documented annually
•
Appropriate Containment
– Risk assessments@
•
Primary Containment -Practices
– All Waste decontaminated
• Facility must have autoclave
– Decontamination of all reusable lab
clothing, (coats) before laundering
– Security (controlled access)*
– Select Agent (if applicable)*
– Base line serum (as appropriate)
•
Primary containment –Equip. &PPE
– BSC*- Or other physical barriers For
all open manipulations of agents
– PPE –Respiratory protection as required
– Sealed rotors/safety buckets (as needed)
•
Emergencies
– Emergency Response Plans* must be
posted in the lab
•
•
Secondary Containment
– Physical separation
– Negative pressure – Exhausted air not
re-circulated, 10-12 air changes per
hour
– Double access doors
– Enclosures for aerosol generating
equipment
– Room penetrations sealed
– Walls floor and ceiling are water
resistant for easy cleaning
– Vacuum lines protected with HEPA
filters and may require additional
disinfectant traps as per risk
assessment.
– Hands free sinks
– Verify directional (-) air flow
– Under failure conditions no (+)air
flow
Biosafety Manual must be developed that
is specific to the risks of the agent and the
work activities@*
Biosafety Level 3 Lab
Ag Biotech
• Experiments involving the use of organisms
requiring BSL-3 containment must first be
approved by:
– The Biosafety Committee (MUA)
– The BSL-3 Use Committee
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Biosafety Level 3 Lab
Ag Biotech
• Laboratory personnel must complete the following :
• Biosafety Awareness for Laboratory Personnel (EHS)
• Protocol specific training (PI)
• Review of the Biosafety Level-3 Laboratory Procedures
Manual
• Additional training as required by approved protocol
• If the protocol requires respiratory protection and/or the
administration of a vaccine. Then personnel must participate
in medical surveillance
• The Principal Investigator must verify in writing that
his/her lab personnel have completed these requirements.
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Biosafety Level 4
Suitable for work involving dangerous and exotic
agents that pose a high risk of aerosol-transmitted
laboratory infections and life-threatening disease.
PLEASE NOTE:
Organisms requiring BSL-4 containment are NOT
allowed to be possessed at the University of
Idaho!
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Biological Safety Cabinets
78
Biological Safety Cabinets
• Product protection
• Personal protection
• Environmental protection
79
Biological Safety Cabinets
Types
A. Class I
•
•
•
•
•
Inward airflow protects researcher
Typically hard ducted to exiting exhaust
system
Air is drawn through HEPA filter as it enters
the exhaust plenum
Exhausts to the outside protects environment
No protection for product
80
Biological Safety Cabinets
Class II
• Provides worker, product
and environmental
protection
• “Sterile” work area
• Use for work with
aerosol-transmissible
microorganisms
• Use for tissue
culture/virology
81
Biological Safety Cabinets
Class III
• Totally enclosed,
ventilated to the outside
via hard ducting
• Suitable for working with
BSL-3 or BSL-4 agents.
82
Biological Safety Cabinets
HEPA Filter
• High Efficiency Particulate Air filter
• Traps particulates only
• Chemical vapors, fumes and vapors pass
through
• Traps particles down to 0.3um
83
Biological Safety Cabinets
HEPA Filters
• Metal or wood framed
• Continuous sheet of
flat filter medium with
aluminum separators
• Gasket sealed
• Adhesive bond
between filter pack
and frame
84
Biological Safety Cabinets
Operation Location
•
•
•
•
Isolated from other work areas
Removed from high traffic areas
Away from air ducts
Away from laboratory entry doors
85
Biological Safety Cabinets
Operating Procedures
• Read manufacturer’s recommendations for operation of
cabinet
• Load BSC with all needed supplies
• Turn BSC on and allow to run for 10-15 minutes
• Confirm inward flow
• 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
• Shut off
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Biological Safety Cabinets Safe Operation
Class II Biosafety Cabinets
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
87
cabinet
Biological Safety Cabinets
Safe Operation
• Always enter straight into the cabinet – no
sweeping motions
• Place materials well within the cabinet- not
on the front grill
• Place discard pan within cabinet
• Watch for disruptions of laminar flow
• Decontaminate materials before they are
removed from the cabinet
88
Biological Safety Cabinets
Safe Operation
• 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
89
Biological Safety Cabinets
Safe Operation
• 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
– Never use NFPA 4 flammables
90
A Word About Biosafety Cabinets, Open
Flames, Bunsen Burners, etc,………..
• Disrupts air flow
• Causes excessive heat
build-up
• May damage HEPA Filter
• Presents potential fire or
explosion hazard
• May inactivate
manufacturer’s warranties
91
Centrifuges
92
Centrifuges
Operating Procedures
•
•
•
•
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
93
Centrifuge Hazards
• Operator error
• Mechanical failure
• Lab equipment failure
(tubes etc.)
• Aerosol generation
• Personal injury
• Physical damage
94
Centrifuges
Safe Operation
•
•
•
•
•
•
•
•
•
•
Use safety cups
Disinfect regularly and after all spills or breakages
Lubricate O-rings and rotor threads
Do not use rotors that have been dropped
Contact the centrifuge representative for specific
information and follow the 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
95