FDS
Biosafety Training
Shulin Chen Lab
Developed and posted 11/14/08
http://www.bio-safety.wsu.edu/biosafety/
Outline
• Introduction
• Biosafety Levels
 Practices
 Facilities
• Biosafety Cabinet
• Case Study
• Resources
• Regulations
Biosafety Introduction
Practicing Science Safely
Means That Before Any
Activity...
 YOU KNOW the risks
 YOU KNOW the worst
things that could happen
 YOU KNOW what to do if
they should happen
 YOU KNOW AND USE the
prudent practices,
protective facilities, and
protective equipment
needed to mitigate the
risks
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
Appropriate Biosafety Level
Agent Risk Group + Risk Assessment =
Appropriate Biosafety Level
Agent Risk Group Resource:
http://www.absa.org/riskgroups/index.html
Risk Group Examples:
RG-1
E. coli (standard host vector
systems)
Most plant pathogens
e.g., Aschochyta spp.
Adenovirus type 1-4, most
human cell lines
RG-2
Klebsiella, Listeria, E. coli O157,
Human Adenovirus, Candida
spp. Giardia lamblia, Some
human cell lines HEK-293 HeLa
(characterized agents)
RG-3
Mycobacterium tuberculosis, M.
bovis, Coccidioides immitis,
HIV, R. richettsii
A Complete Risk Assessment includes
• Agent
Characterization
(Risk Group RG)
• Personnel Factors
(experience)
• Work Activity
Factors
• Environmental
Factors
• Equipment Factors
• Risk Consequences
• Probability Profile
Risk Assessment
• Risk of Activity – same agent can have
different containment levels:
 Procedures that produce aerosols have
higher risk
 Procedures using needles or other sharps
have higher risk
 Handling blood, serum or tissue samples
may have lower risk
 Purified cultures or cell concentrates may
have higher risk
 Large volumes (>10 L) have higher risk
Protection Should Match the Risk: What is the
Expected Route of Infection?
AGENT / LAI*
PRIMARY HAZARDS
*LAI= Laboratory Acquired Infections
Klebsiella spp.
Direct contact of mucous membranes with contaminated
objects, inhalation of infectious aerosols; accidental parenteral
inoculation; ingestion
LAI’s have been reported
Staphylococcus epidermidis
Direct contact of mucous membranes, accidental parenteral
inoculation.
AGENT
Pathogenicity/Epidemiology
Klebsiella spp.
Pathogenicity:
Staphylococcus epidermidis
Pathogenicity: Generally considered to be part
Frequent cause of nosocomial
urinary and pulmonary infections; wound infections,
secondary infection in lunch of patients with chronic
pulonary disease, enteric pathogenicity,
(enterotoxin).
Epidemiology: Worldwide distribution. 2.3 of
all infection due to Klebsiella spp. Are hospitalacquired: causes 3% of all acute bacterial
pneumonia; common source of nosocomial
outbreaks.
of normal Human flora but has been documented as
a pathogen in numberous cases of bacteremia,
surgial wounds, urinary tract and ophthalmologic
infections especially in immunocompromised hosts.
Epidemiology: World wide distribution.
Associated with nosocomial infections.
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 a Spill Clean
up?
What Constitutes a
Biological Exposure
Risk?
What to do when there is a
breach?
Tell your supervisor and the
Biosafety Manager for WSU. Fill
out an incident report, and all
other paper work as required.
What to do when there is a near
miss?
Fill out an incident report.
• Breach in Primary or
Secondary Containment
• What is Primary
Containment?
• What is Secondary
Containment?
Primary Containment
• Lab practices – standard lab practice,
limited access, biohazard warning sign,
sharps/needle precautions, SOPs,
decontamination, waste.
• Safety equipment – biosafety cabinets
(BSC), sharps containers, sealed rotors.
• Personal protective equipment (PPE) –
lab coat, gloves, goggles, respirators.
Establishing Precautions to Minimize Risk
Primary containment
Secondary Containment - Facilities
• Facility provides containment through
traditional construction BSL-1 & BSL-2
 Best practices HVAC provides negative
pressure in BSL-2 labs. Depending on
risk assessment this may be required.
• Facility provides containment through
special design features BSL-3
 Anterooms
 Double doors
 Alarmed redundant HVAC systems
Possible Biological Release Scenarios
Is This a Release?
1. Open a centrifuged tube
at the open bench that
contains Klebsiella
spp.?
2. Using a wire transfer
loop and a bunsen
burner pick a Klebsiella
colony and streak for
isolation work in the
BSC.
Is This a Release?
3. Transfer liquid cultures
with a transfer pipette to
produce BSL-2
organism serial
dilutions. Work
performed at the open
bench.
Answers to Release Scenarios
1. Open a centrifuge tube of
Klebsiella at open bench
2. Bunsen burner in the BSC
•
•
Centrifugation causes the
pressure inside the
centrifuge tube to be altered
from the ambient pressure.
When opening the lid
aerosols are formed.
Performing this action at
the open bench exposes
you and your coworkers to
potentially infectious
aerosols
Bunsen burners cause air
turbulence in the Biosafety
Cabinet which may lead to
escape of bio-aerosols and
subsequent exposure to
infectious aerosols to the
cabinet worker and others
in the laboratory.
Answers to Release Scenarios
3.
Infectious liquid transfer at
bench
•
Any manipulation of infectious
liquids potentially creates
aerosols. To perform
manipulations of infectious
liquids safely this work should
be done in the Biosafety
Cabinet
Summary
In all three of these case
studies there has been a
potential release of
infectious aerosols.
NOTE: The following slide
shows a picture of
aerosol generation when
expelling a liquid from a
pipette.
Spill Clean up of Biological Agents
Surface Contamination:










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 20
minutes)
Remove towels, mop up; clean with alcohol or
soap/water or other agent as appropriate.
Dispose of materials in biohazardous waste
Notify lab instructor
Spill in a BSC
• 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 10% bleach is used. The disinfectant
then needs to be drained out and the surfaces
thoroughly cleaned with water to prevent corrosion.
Spill Clean up of Biological Agents
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 lab instructor – fill out incident report
and other forms as requested
• If appropriate report to medical clinic for
treatment/counseling
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 911 for assistance
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
+
Know and Use
• prudent practices,
• protective facilities, and
• protective equipment
needed to mitigate risks
Recommended Biosafety Containment & Practices – Infectious Agents
BSL
1
Agents
Not known to
consistently cause
disease in healthy
adults
Practices
Primary Containment
• Wash hands after
•
•
•
•
•
•
handling viable
materials
No smoking, eating,
handling contact lenses,
applying cosmetics
No mouth pipetting
Minimize aerosols&
splashes
Decontaminate work
surfaces daily
Do NOT wear gloves out
of lab
Remove gloves before
touching door handles
phones etc.
Safety Equipment
Primary Barriers
None required
Facilities
(Secondary Barriers)
•Doors
•Sink required
•Work surfaces easily
cleaned & impervious
to water
•Sturdy furniture
Recommended Biosafety Containment & Practices – Infectious Agents
BSL
2
Agents
Associated with
human disease,
hazard = percutaneous injury, ingestion, mucous membrane exposure
Practices
(Primary Containment)
BSL-1 practice plus:
•Limited access
Lab entry and exit
policies.
•Biohazard warning signs
at entrance of lab
•“Sharps” precautions
•Decontaminate stock
cultures prior to disposal
•BSM - SOP
•Report Spills & Accidents
•Leak-proof containers for
transport
•PPE required (as approp.)
•Documented training
Safety Equipment
(Primary Barriers)
Facilities
(Secondary Barriers)
Primary barriers =
BSL-1 plus:
BSC I or II or other
Autoclave available
physical barriers,
Locking Doors
e.g. splash shields,
BSC (as needed)
covered centrifuges
etc. -For all open
L Eyewash readily
manipulations of
available
agents that cause
Negative pressure in
splashes or aerosols
laboratory (ideal)
HEPA filters required
on vacuum lines
Personal Protective
Equipments PPE’s:
laboratory coats;
gloves; face
protection as
needed
Biosafety Cabinet
• What is a Biosafety Cabinet (BSC)?
• Understand Equipment Limitations
• Safe Use of a BSC
 Why & When to Use a BSC
• SOP for BSC
• BSC Maintenance / Certification
 Decontamination
What is a Biosafety Cabinet?
• A BSC is a piece of equipment that is only as
•
good as the users understanding of how to
appropriately use and maintain it
There are different Classes of BSC’s
 Class I – user protection but no product protection
 Class II – product, user and environment protection
• Provides a ~100ft/min protective air barrier
 Class III- Air tight - use with Risk Group 3/4 agents
• Fume hoods and laminar flow clean work
benches are NOT Biosafety Cabinets.
What is a Biosafety Cabinet
Outline
• What is a Biosafety Cabinet (BSC)?
• Understand Equipment Limitations
• Safe Use of a BSC
 Why & When to Use a BSC
• SOP for BSC
• BSC Maintenance / Certification
 Decontamination
Understand Equipment Limitations
• Fume Hoods
• Not for use with infectious
materials or environmentally
dangerous organisms
• Exhaust air not HEPA
filtered; not easily
decontaminated
• Laminar Clean Air
 Horizontal
• Air blows through HEPA at
rear of work surface across
work surface and into face of
user
• NOT for use with lab
animals, potentially
infectious materials, drug
formulations.
 Vertical
• Air blows through HEPA on
top of work surface
downward
• Air may flow under a sash
and into the room. Some
models don’t even have
sashes
• Turbulence can distribute
aerosols into user’s
breathing zone
• Not for use with potentially
infectious materials
 Reverse-flow
• Pull air from front of cabinet
through pre-filter and HEPA
at rear
• Used to reduce user’s
exposure to animal urine,
dander etc. (with PPE)
• Not for work with biohazards
(no containment)
Understand Equipment Limitations
• BSC class I
 Inward airflow
protects worker
 Exhaust to outside
w/wo HEPA filter
 No product
protection
 Not for use with
tissue culture
• BSC class II
 “Sterile” work
surface
 Protects worker,
product and
environment
 For use with RG 2-3
agents
 Restrictions for
flammables and
chemicals depending
on type of class II
BSC
Understand Equipment Limitations
• Class II BSC
 Type A2 – 30% exhausted to the room
• Not recommended for hard ducting or chemical usage
 All Type B cabinets – air flow from the rear grill is
discharged into the exhaust system so activities that
may generate chemical vapors should be conducted
towards the rear of the BSC
 Type B3 – 30% exhausted to outside
• Minute chemical usage allowed
 Type B1 – 70% exhausted to outside
• Minute amount of volatile chemicals allowed
 Type B2 – 100% exhausted to outside
• Small amount of volatile chemicals allowed
• Not a good choice for tissue culture work
Outline
• What is a Biosafety Cabinet (BSC)?
• Understand Equipment Limitations
• Safe Use of a BSC
 Why & When to Use a BSC
• SOP for BSC
• BSC Maintenance / Certification
 Decontamination
• Resources
Safe Use of a Biosafety Cabinet
 Must be isolated from other work areas,
lab entry, high traffic areas and away from
air ducts
 Must be operated properly to minimize
risk (more info later in SOP section)
• Sash must be kept at manufacturers
recommended height while in use with
biological agents.
Safe Use of a Biosafety Cabinet
 Flames are not a constant temperature
and therefore can cause air turbulence
inside a BSC
• Compromise protective air barrier integrity
 Are not spark/fire proof. Gas Bunsen
burners should not be used in BSC’s
• 70% recirculated (Class II type A2)
Uncombusted gas (yellow flame) captured in
plenums. UV light – microscopic holes in
gas tubing. Net result high explosion hazard
Bunsen Burner - BSC Fire
Safe Use of a Biosafety Cabinet
 UV lights
• Cause skin cancer and cataracts. Therefore
eyes and skin should not be exposed.
• Not reliable as primary decontamination




Line of site
Poor penetration
UV light turns on at 80 micro watts /square cm
UV light only kills at 160 micro watts/square cm
• Secondary decontamination
 15 minutes generally sufficient
 More than 15 minutes eats up plastics and glue
in HEPA filter and in long term can destroy
BSC integrity and cause cabinet leak test to fail
Safe Use of a Biosafety Cabinet
• Do’s
 Keep supplies to a minimum
and ~6” from sides
 Discard all infectious materials
inside the BSC (disinfectant)
 Understand how the BSC
works
 Turn the cabinet on for
manufacturer recommended
time before using it
 Inform your supervisor if you
are immuno-compromised
 Wear a mask if you are
coughing or sneezing
• Don’ts
 Rely on the UV light to
decontaminate
 Put anything on the front grill
 Move quickly
 Use sweeping motions
 Keep supplies close to the
sides and back
 Move the sash below or above
the recommended standard
 Use a gas flame in the hood
 Remove material before
disinfecting. When working
with potentially infectious
material
FDS
Safe Use of a Biosafety Cabinet
1.
What happens if the front
sash is up too high?
Safe Use of a Biosafety Cabinet
1. Question: Front sash up too high
Answer: Down flow of clean (HEPA
filtered) air will provide more pressure
than inflow of air through the front
sash and safety is compromised i.e.
agent contamination of the user and
environment is likely.
FDS
2. What Happens If The Front Sash
Is Down Too Low?
Safe Use of a Biosafety Cabinet
2. Question: Front sash down too low.
Answer: In flow of air through front sash
is stronger than down flow of air. Net
result is compromised product
integrity, i.e. outside air flows into the
cabinet providing a source of potential
contamination.
FDS
3. What Happens If Objects Are
Placed On The Front Grill Of The
BSC?
Safe Use of a Biosafety Cabinet
3. Question: Effect of objects placed on
the front grill.
Answer: The front grill is one of the
dirtiest areas of the entire lab so
placing items on this grill is not a good
idea. Additionally this interferes with
air balance and causes too much down
flow of air which creates safety issues,
i.e. release of agent outside of the
cabinet.
FDS
4. What Happens When Your Arm
Moves Over The Work Inside The
BSC?
Safe Use of a Biosafety Cabinet
4. Question: Arm moves over work area.
Answer: Down flow air moves
contaminants on your arm/lab coat
down onto the work surface
compromising product sterility.
Note: If you are right handed the waste
container should be on the right side of
the BSC.
FDS
5. What Happens When People
Walk Behind The BSC?
Safe Use of a Biosafety Cabinet
5. Question: Person walks behind the
Biosafety Cabinet.
Answer: Protective air current is ~100
feet / minute = a little over one mile an
hour. People walk at 2-3 miles per
hour and this can disrupt the
protective air current of the biosafety
cabinet.
Outline
• What is a Biosafety Cabinet (BSC)?
• Understand Equipment Limitations
• Safe Use of a BSC
 When to Use a BSC
• SOP for BSC
• BSC Maintenance / Certification
 Decontamination
• Resources
When to Use a BSC
• Product Protection
 Tissue Culture
• User /Environmental Protection
 Aerosolizing activities with Risk Group 2
agents.
Aerosol Producing Activities
• Sonication, blending, mixing, vortexing
• Almost any liquid manipulation
• Centrifuging
• Pouring
• Pipetting
• Opening containers at non-ambient
pressures, (e.g. fermenters, freezer vial)
• Loading syringes and injections
Aerosol Producing Activities
• Intranasal inoculation of animals
• Changing animal bedding
• Harvesting tissue, eggs
• Tissue grinder/homogenizers
• Lasers
• Cell sorters
• Necropsy
(cont.)
Aerosol Control Procedures
• Biosafety Cabinet (BSC) properly
•
•
•
•
•
maintained, used and certified
No blow-out of pipettes
Sidewall contact when delivering liquids
Use of aerosol-minimizing tips
Secondary enclosure for sonicators, etc.
Capped centrifuge tubes, sealed cups,
carriers and rotors
Outline
• What is a Biosafety Cabinet (BSC)?
• Understand Equipment Limitations
• Safe Use of a BSC
 Why Use a BSC
• SOP for BSC
• BSC Maintenance / Certification
 Decontamination
• Resources
Why Use a BSC? – User Protection
Account for 50% of LAI’s (Lab Acquired Infections)
1. Inhalation exposure to infectious aerosols
Account for 20% of LAI’s
1. Parenteral Inoculation (to include animal bites and
scratches)
2. Spills, splashes, skin and mucous membrane
exposure
3. Ingestion
Outline
• What is a Biosafety Cabinet (BSC)?
• Understand Equipment Limitations
• Safe Use of a BSC
 Why & When to Use a BSC
• SOP for BSC
• BSC Maintenance / Certification
 Decontamination
• Resources
BSC
Standard Operating Procedure
Start up Procedure
• Pre-plan to minimize
supplies and movements
in and out of the cabinet
• Turn on for manufacturer’s
recommended time prior
to use
• Ensure that back grill
paper catch is clear
Start up Procedure
• Verify air flow
• Verify drain valve is closed
• Disinfect cabinet surfaces
• Place all materials inside
the cabinet (disinfect as
appropriate)
• Verify view screen height
is appropriate
BSC
Standard Operating Procedure
Work in the Cabinet
• Wash hands and arms
• Do NOT cover the front or
back grill with anything
• Use slow movements
• Avoid door and people
movement beside cabinet
• Avoid open flames
Work in the Cabinet
• Place pipettes exposed to
infectious materials in
disinfectant inside the
cabinet before removing
• Safe work area is 6 inches
from sides of cabinet
• Enclose all potentially
contaminated material &
disinfect surfaces before
removing from BSC
• Disinfect work surfaces
Standard Operation Procedure - BSC
• Develop from Manufacturer’s guidelines
• Baker SOP available in BSL-2 Biosafety
Manual Template (supplemental
resource D)
 http://www.bio-safety.wsu.edu/forms.asp
Outline
• What is a Biosafety Cabinet (BSC)?
• Understand Equipment Limitations
• Safe Use of a BSC
 Why & When to Use a BSC
• SOP for BSC
• BSC Certification
• Resources
BSC Certification
Certification
• Must be certified every
year / after installation /
and after the BSC is
moved
• NSF certifier to provide
certification to NSF
standards requires
 2 HP compressor
 *Pump designed for aerosol
challenge
Certification
• Check air-flows
 100-110 ft/min in-flow (A2)
 75 ft/min in-flow (A1)
 65-75 ft/min down-flow
• Check HEPA filter
 Particle generator* – 18-20
lbs pressure to produce 0.3
micron particle size (99.9%
efficiency test)
Outline
• What is a Biosafety Cabinet (BSC)?
• Understand Equipment Limitations
• Safe Use of a BSC
 Why & When to Use a BSC
• SOP for BSC
• BSC Maintenance
• Resources
BSC Maintenance
Maintenance
Maintenance
•
•
•
•
Keep the cabinet clean
If cabinet is off keep front
sash down to minimize dust
Clean up spills that get
under the cabinet
immediately http://www.bio-
•
•
safety.wsu.edu/biosafety/
•
•
•
Disinfect before & after use
Bleach pits stainless steel
Monitor minihelic gauge
•
Don’t store materials in the
hood
Verify that the front grill/back
grill and paper catch are
unobstructed
Once a month disinfect the
bottom of the hood and then
drain
>10-12 year old BSC’s - stop
stocking parts
Outline
• What is a Biosafety Cabinet (BSC)?
• Understand Equipment Limitations
• Safe Use of a BSC
 Why & When to Use a BSC
• SOP for BSC
• Decontamination
• Resources
Decontamination
• Microorganisms can be grouped in terms of
resistance to disinfectants.
Highly Susceptible
Susceptible
Resistant
Highly Resistant
Extremely Resistant
Microbial Chemical Resistance
Highly Susceptible
Mycoplasmas
Susceptible
Gram +&- bacteria
Fungal Spores
Resistant
Non-Enveloped Viruses
Mycobacteria
Highly Resistant
Bacterial Endospores
Protozoal Oocytes
Extremely Resistant
Prions
Effectiveness of Chemical
Disinfectants
• Effectiveness is influenced by:
 Composition of chemical disinfectants
 Concentration of microorganisms and
chemical disinfectants
 Contact time with the Disinfectant
 Presence of organic matter
 Presence of interfering substances
 Temperature at which they are used
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
Case Study – Putting it all Together
• Work with M. bovis.
 rDNA
 M. bovis culture
• Agent Risk Group
http://www.absa.org/riskgroups/bacteriasearch.php?genus=Mycobacterium
• Risk Assessment
• Appropriate BSL
• Special Considerations
Practicing Science Safely
Means That Before Any
Activity...
 YOU KNOW the risks
• MSDS
 YOU KNOW the worst
things that could happen
• Sick/Die
 YOU KNOW AND USE the
prudent practices,
protective facilities, and
protective equipment
needed to mitigate the
risks
• Risk Assessment
 YOU KNOW what to do
• Follow Biosafety Manual
A Complete Risk Assessment includes
• Agent
Characterization
(Risk Group RG)
• Personnel Factors
(experience)
• Work Activity
Factors
• Environmental
Factors
• Equipment Factors
• Risk Consequences
• Probability Profile
A- Agent Characterization
• Pathogenicity of material – disease incidence and severity
• Routes of Transmission – parenteral, airborne or ingestion
• Agent Stability – ease of decontamination
• Infectious Dose – LD50
• Concentration – infectious material/volume & working volume
• Origin of material - Wild Type, exotic, primary cells
• Availability of effective prophylaxis – Hepatitis B vaccine
• Medical surveillance – effectiveness & availability of treatment
A- Agent Characterization
• Pathogenicity of material – 4th most common LAI, MDR strain
•
•
•
•
•
•
•
used increases the risk.
Routes of Transmission – parenteral, airborne & ingestion
Agent Stability – Resistant to many disinfectants
Infectious Dose – 10 bacilli by inhalation
Concentration – cultures grown on plates
Origin of material - Wild Type
Availability of effective prophylaxis – INH (Isoniazid)
available, ineffective for most MDR strains
Medical surveillance – treatment of MDR Mycobacterium
problematic
B - Personnel Factors
• Biosafety training
• SOP training
• Health Assessment (immunosuppressed)
• Experience with the Agent (B, I, A)
•
•
•
•
•
 Beginner, Intermediate, Advanced
MSDS read
Experience with the procedures (B,I,A)
Use of PPE training
Job hazard analysis undertaken
Allergies (animal, environmental)
C- Work Activity Factors
• Aerosol generating
potential
• Potential for selfinoculation (needle stick,
lesion)
• Sample origin and
concentration
• Volume of pathogen used
• Animal use (types,
potential viral shedding,
bites and scratches)
• Replication competency
• Recombinants
• Cell line characteristics
• Toxin production (y/n, MSDS))
• Modification of pathogen (y/n, result
/ implication)
• Vector use (y/n, describe)
• Inventory Records
• Contingency plan (exposure,
accidental releases / spills)
• Techniques – cryogenics, cytometry
• Disinfectant used as directed
D – Environmental Factors
•
•
•
•
•
•
•
Level of containment available
Degree of monitoring of containment factors
Impact of external activities
Biosecurity (access and inventory control)
Availability and status of emergency support
Housekeeping and Trades Personnel
Access by public
E – Equipment Factors
•
•
•
•
•
•
Equipment Maintenance (frequency, status)
Manual
Reservoirs empty &disinfected
Standard Operating Procedures
Location of use
Ventilation Consideration
F - Rating of Risk Consequences
•
Rating of Risk Consequences
Class
Rating
Consequences
4
Catastrophic
People:
Environment:
Business:
fatalities, evacuation outside site area
irreversible, long-term damage outside site area
total loss:
> $2 million
Interruption: > 2 months
image:
severely damaged, > 1 week, national
3
Critical
People:
Environment:
Business:
serious injuries, effects outside site area
reversible, short-term damage outside site area
total loss:
> $100,000 - $2 million
Interruption: > 2 – 8 weeks
image:
damaged, > 1 week, regional
2
Marginal
People:
Environment:
Business:
minor injuries, annoyance outside site area
only site area effected
total loss:
> $5,000 - $100,000
Interruption: > 1 – 2 weeks
Image:
< 1 week, local
1
Negligible
People:
Environment:
Business:
no effects
only building effected
total loss:
< $ 10,000
Interruption: > 1 week
Image:
no effects
F - Rating of Risk Probability
Table 2 - Rating of Probability
Class
Rating
Probability
Definition
A
Frequent
More than once a year
Likely to occur repeatedly in life cycle system
B
Moderate
Once per year
Likely to occur several times in life cycle system
C
Occasional
Once in 5 years
Likely to occur sometime in life cycle system
D
Rare
Once in 25 years (e.g. once in the life cycle of the
system)
Not likely to occur in system life cycle, but possible
E
Unlikely
Once in 100 years
Will occur once in a lifetime of a site
F
Very Unlikely
Once in 1,000 years
Almost impossible to occur
G -Probability Profile
Risk Profile/Probability
Consequences
Probability
Frequent
A
Moderate
B
Occasional
C
Rare
D
Unlikely
E
Very Unlikely
F
Negligible
1
Marginal
2
Critical
3
Catastrophic
4
Resources - People
•
WSU – Biosafety Pullman and offsite campuses
 Lorraine McConnell 509-335-4462
lorrmcc@wsu.edu
•
WSU- EH&S Pullman
 Mike Kluzik
509-335-9553
mkluzik@wsu.edu
Resources
•
•
Biosafety in Microbiological and Biomedical
Laboratories:
http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.
htm
American Biosafety Association:
http://www.absa.org/XriskgroupsX/index.html
•
Canadian Biological MSDS’s: http://www.phacaspc.gc.ca/msds-ftss/index.html
•
WSU Biosafety Web Page: http://www.biosafety.wsu.edu/biosafety
Web Resources - BSC
• CDC / NIH Guidelines on Biosafety Cabinets:
 http://www.ors.od.nih.gov/ds/pubs/bsc/contents.html
• AIHA – BSC Safety Information:
 http://www2.umdnj.edu/eohssweb/aiha/accidents/BSC.ht
m#Biosafety%20Cabinet
• CDC / NIH 2nd ed. “Primary Containment for
Biohazards”:
 file:///Z:/Biosafety%20Cabinets/CDCPrimary%20Containment%20for%20Biohazards%20(BS
C's).htm
• Ohasis – Office of Health & Safety Primary
Containment for Biohazards Section V.
 file:///Z:/Biosafety%20Cabinets/Primary%20Containment
%20for%20Biohazards.htm
Web Resources - BSC
• CDC – Laboratory Biosafety Guidelines 3rd edition:
 http://209.85.173.104/search?q=cache:h373QUWmYfEJ:
www.phac-aspc.gc.ca/publicat/lbg-ldmbl04/pdf/lbg_2004_e.pdf+CDC+2007+3rd+edition+of+Selec
tion+Installation+and+Use+of+BSC&hl=en&ct=clnk&cd=
5&gl=us&client=firefox-a
• ABSA Position Paper on the Use of Ultraviolet Lights
in Biological Safety Cabinets:
 http://209.85.173.104/search?q=cache:tbuGD9FOadAJ:w
ww.ehs.umass.edu/ABSA%2520UV%2520light%2520pap
er.pdf+position+paper+on+the+use+of+ultraviolet+lights
+in+biological+safety+cabinets&hl=en&ct=clnk&cd=1&g
l=us&client=firefox-a
Web Resources - BSC
• BMBL Biosafety Cabinet Appendix A (4th ed):
 http://www.cdc.gov/od/ohs/biosfty/bmbl4/b4aa.htm
• The Baker Company
 http://www.bakerco.com/resources/intro.php
• NUAIRE How to select a BSC
 http://www.nuaire.com/products/biological_safety_cabin
ets/select_a_bio_cabinet.htm
• NSF Accreditation Standards
 http://www.nsf.org/business/biosafety_accreditation/ind
ex.asp?program=BiosafetyCabCert
Regulations
• OSHA Bloodborne Pathogen Standard:
•
•
•
•
•
http://www.osha.gov/SLTC/bloodbornepathogens/ind
ex.html
Centers for Disease Control and Prevention (CDC):
http://www.cdc.gov/od/sap/
NIH Recombinant DNA Guidelines:
http://www4.od.nih.gov/oba/rac/guidelines/guidelines
.html
CDC Interstate Shipment of Etiological Agents:
http://www.cdc.gov/od/ohs/biosfty/shipregs.htm
CDC Etiologic Agent Import Permit Program:
http://www.cdc.gov/od/eaipp/
APHIS – USDA: http://www.aphis.usda.gov/vs/ncie/
THANK YOU