BSL-3 Training & Work
Practices
EMD545b
Lecture #12
You Are Here!
BSL-4
BSL-3
BSL-1 & BSL-2
BSL-3
Training
Objectives
Promote Awareness of:
– BSL-3 Program
– BSL-3 Facility Design & Operation
– Aerosol containment
– BSL-3 Work Practices & PPE
– BSL-3 Emergency Response Procedures
Minimize/prevent lab-acquired
infections
Biosafety Level 3
“Laboratory personnel have specific
training in handling pathogenic and
potentially lethal agents, and are
supervised by competent scientists who
are experienced in working with these
agents.”
CDC/NIH BMBL, 4th Ed. 1999
Personnel Training
Classroom
– Biosafety, Bloodborne Pathogens, Lab
Chemical Safety, BSL-3 Training,
Shipping/Transport
Tours
– Orientation covering entry, facility equipment
and use, exit
– Review of monitors, alarms and contingency
plans
Personnel Training
Prior work experience
– 2 months (320 hours) with BSL-2 pathogens
– 15 hours (15 supervised visits) at BSL-3
– Relevant education
Accept training from other institutions,
but…
– Require onsite orientation &
– Successful completion of observation
BSL-3 Work Practice
Observation
1 – 2 Reviewers from BSL-3 Subcommittee
Monitor:
– Entry/exit
– Compliance with PI’s SOP
– Aerosol containment
– Safe handling of sharps (animal BSL-3)
– Awareness of emergency procedures
– Comfortable? Safe? Proficient?
BSL-3 Work Practice
Approved?
Observation
Approved?
– Issue certificate to PI, Researcher, Committee, File
– Grant independent access
– Cleared to sponsor/train new researchers
Not Approved?
– Letter to PI/Researcher noting issues
– Requirements (more practice time)
– Retest date?
Multiple failed observations?
BSL-3 PPE
PERSONAL PROTECTIVE
EQUIPMENT
Protect: skin
clothing
mucous membranes
respiratory system
Use: gloves (double, kevlar)
lab coats, solid-front gowns
sleeve covers
full face protection
respiratory protection
PERSONAL PROTECTIVE
EQUIPMENT
Disposable
Decontamination
Dedicated to area
Donning/Doffing
– Compromised (wet/contaminated/torn)
– Respiratory Protection Program
Gloves
Donning
– check before use
– size, fit, taping wrists
Doffing
– remove aseptically
– change frequently
Double gloving
– remove outer pair in
contaminated field
Dedicate to work
area
Respiratory Protection
Disposable N-95 mask
and N-99/N-100
respirator
Powered-Air-Purifying
respirator
Annual training, medical
exam, fit testing
Personal Protective
Equipment
“A man in armor is his armor’s
slave.”
Robert Browning
For work in a Biosafety Cabinet
solid-front or wrap-
around gown
gloves (double
gloving)
face protection (face
shield or mask and
safety glasses)
Use of a HEPA-filtered PAPR for additional
protection during high-risk research procedures
For work outside a Biosafety
cabinet
solid-front or wrap-
around gown or jump
suit
gloves (double gloving)
face protection (face
shield or mask and
safety glasses)
Respiratory protection
if aerosols are
generated
Personal Protective
Equipment (PPE)
Maintain supply of PPE
Don’t reuse potentially contaminated
PPE
Place disposal PPE into Biological waste
Autoclave reusable PPE
Don’t wear PPE outside the laboratory
BL3 Practices
“All procedures involving the
manipulation of infectious materials
are conducted within biological safety
cabinets or other physical
containment devices . . .”
CDC/NIH BMBL, 5th Ed., 2007
Preparation Procedures
Entry Procedures
Verify airflow
Enter ante room
Close outer door
Sign log book
Don PPE
Supplies
Enter laboratory
Perform all work within the biosafety cabinet
Effective Use of Biosafety
Start up
Cabinet
Wipe down
Loading materials and equipment
Work techniques
– Waste collection
– Load/unload centrifuge rotors/buckets
Final purging and wipe down
Shut down
Protect Vacuum System From Contamination
Install hydrophobic or HEPA filter between
the vacuum source and the overflow flask
Proper Use of Centrifuges
utilize aerosol containment devices
follow preventive maintenance procedures
– log book
– O-rings
– tubes
– wipe down
Proper Use of Centrifuges
load and unload
rotors and
buckets
in
the biosafety
cabinet
Microfuge
Avoid unless unit includes aerosol-proof
containment barrier
Use microfuge tubes with O-rings; avoid
snap cap tubes which often leak
Contact OEHS prior to placing microfuge
within the biosafety cabinet - high RPM’s
may overcome containment
Avoid needles and other sharps. Use safe
sharps devices and extreme caution if sharps
must be used.
Place all reusable sharps in a labeled decon receptacle;
soak for at least 10 minutes prior to removal
If sharps are required, use forceps or clamps
to keep your hands away from the blade
Transport of BL3 Materials
(between labs)
leak-proof non-breakable primary
container
place primary into similar secondary
container
outer container labeled
–biohazard warning label
–name of agent
Transport of BL3 Materials
(between labs)
Terminating BL3 Work
Waste Disposal/Decontamination
Disinfection of work surfaces
Disposal/Decontamination of PPE
Handwashing
Exit
Wipe all items with disinfectant prior to
removal from the biosafety cabinet
Collect pipettes in the biosafety cabinet. Soak in
disinfectant or seal in biohazard waste bag before
disposal.
Collect non-sharp biowaste in biohazard bags
within the biosafety cabinet
Pipette liquids carefully to avoid generating
aerosols. Allow liquids to gently flow down side
of flasks and tubes.
Decontaminate all liquid waste before removal
from the biosafety cabinet; final concentration of
10% bleach for 30 minutes prior to disposal in
sanitary sewer (lab sink).
Rank Order of Resistance to
Disinfection
Prions (MOST DIFFICULT TO INACTIVATE)
Protozoan cysts
Bacterial spores
Non-enveloped (hydrophilic) viruses
Mycobacterium
Fungal spores, fungi
Vegetative bacteria
Enveloped viruses (lipophillic)-(EASY to DESTROY)
Promptly disinfect spills within the biosafety
cabinet - cover area with paper towel(s) and
spray disinfectant through towel.
Collect all biological waste within the
biosafety cabinet
Add water to dry biohazard waste prior to
sealing the bag to promote generation of steam
inside the bag during the autoclave run (10 ml
for small/250 ml large)
Wipe exterior of sealed biowaste bag before
removal
Decontaminate rear and side walls after use
Decontaminate inside front view screen after each
use
Decontaminate work surface, front & rear
grilles after each use. Periodically clean
below the work surface.
Discard outer gloves w/in biosafety cabinet, avoid
touching outer portion of glove
Remove gown;
avoid touching
exterior if possible
Place disposable
PPE in
biomedical waste
Remove
face
protection
last
Wash hands
after
removing
PPE
Autoclave located
within BSL-3
Laboratory
All waste from BSL-3
lab must be
autoclaved prior to
disposal
Label autoclaved BSL-3
waste as “Pathological
Waste” for incineration
Verification of Decontamination
Autoclave tape
– time/temperature/date records on chart
Chemical indicators (melt pellets)
– fuse if temperature reached for at least 1
second
Biological Spore indicators
– Bacillus stearothermophillus
– Bacillus subtilis var. niger
Manifest for biomedical waste (incineration)
