Controls in Biosafety II:
Work Practices
and Personal Protective
Equipment
EMD 545b
Lecture #6
Biosafety Work Practices
 Foundation matrix
 Routes of Exposure on Y axis
 7 Basic biosafety work practices on X axis
 Which practices block each route of
exposure?
 Majority of biosafety work practices stem
from the core 7 practices
Routes of Exposure
 Ingestion
 Inhalation
 Mucous membranes (eyes, nose, mouth)
 Percutaneous
Biosafety Work Practices
 No mouth pipetting
 No eating, drinking, smoking, food storage
 Wear personal protective equipment
 Work carefully to avoid creation of aerosols
 Eliminate/avoid the use of sharps
 Decontaminate work surfaces/spills
 Wash hands after removing PPE, skin
contact (keep hands away from face)
Biosafety Work Practices
 Administrative
– establish safety policies (written SOP)
– restrict access to lab to those involved in work
– post entry requirements (immunizations, PPE)
– labels on equipment storing biohazards, door
sign with agent and contact information
– ensure training for all who work with hazards
– advise all workers of any special hazards
– workers must demonstrate proficiency
Biosafety Work Practices
 Transport/Shipping
– use two leak proof labeled containers for oncampus shipment (include contact info)
– avoid the use of glass primary or secondary
containers whenever feasible
– Use UN-Approved infectious substance
shippers for public roadways, air, other
methods of conveyance
– All shippers/transporters trained biennially
Biosafety Work Practices
 Contain other aerosol generating procedures
– any procedure that imparts energy to a
microbial culture, includes:
•
•
•
•
•
•
homogenizing, blending, shaking, grinding
vortexing, mixing, pipetting, spills
opening tubes which are under pressure (vacutainer)
removing syringe from vial, expelling air
animal bedding, cage dust, animal hair, necropsy
droplets from pipets to bench, liquid to liquid (from
height), forcibly expelling liquid from pipette
Bioafety Work Practices
 Personnel
– ensure reporting of all near misses or incidents
– report immunosuppression/compromised skin
– evaluate new procedures before initiation
– never initiate work without authorization
– address all medical requirements before starting
– know signs/symptoms of disease, report them
– always work carefully, avoid short cuts, follow
written SOP’s
– keep pets, other unrelated items out of lab
Foundational Biosafety Practices
 Hand washing
 No mouth pipetting
 No eating or drinking in lab
 Minimize aerosol
generation
 Decontaminate work
surfaces
 Safe sharps handling
 Wear applicable PPE
Foundational Biosafety Practice
#1
Confine Aerosols
Aerosols
– Procedures that impart energy into a microbial
suspension are a potential source of aerosol
(Chatigny, 1974)
– Many common lab procedures and accidents have
capability of releasing aerosols
– homogenization, sonication, blending, mixing, grinding,
shaking, vortexing, spills, opening vials, pipetting, animals
excreting agent, opening vials under pressure, etc.
Confine Aerosols!
Promote Awareness of:
– Aerosol containment
– Work Practices & PPE
– Emergency Response
Procedures
 Minimize/prevent lab-
acquired infections
Aerosols are generated in
many procedures
Viable Particles Recovered from Air
(Chatigny, 1974)
 Procedure
–
–
–
–
–
–
–
sonic oscillator
mixing w/ pipette
overflow from mixer
opening lyophilized vial
top removed after blending
dropping flask of culture
dropping lyophilized
culture
 # Particles/ft3 of air
–
–
–
–
–
–
–
6
7
9
135
1500
1551
4839
Aerosol spread from open flame
Correct Use of Biosafety Cabinets
 Purge air before and
after use
 Load supplies prior to
work
 Wipe items with
disinfectant before
loading (or removing)
 Clean up spills
promptly
Biosafety Work Practices
 Effective Use of the Biosafety Cabinet
– use to contain aerosols (pipetting, other work)
– decontaminate interior before/after use
– work at least 4” inside front grille
– minimize movement of hands out of cabinet
– keep traffic behind operator to a minimum
– avoid the use of an open flame inside cabinet
Centrifuge Containment
 Load/unload sealed
buckets in a
biosafety cabinet
 Wait 2 to 5 minutes
after run to allow
aerosols to settle
 Decontaminate
centrifuge and
buckets after each
use
Biosafety Work Practices
 Centrifugation Precautions
– use secondary containment (decon after use)
– check tubes for cracks or deformities before
use
– always use sealed primary tubes
– don’t overfill primary containers
– balance loads to avoid damage to centrifuge
– unit should have solid lid and interlock
Confinement at Source
Incident Response:
Exposures and Spills
Emergency
Response
 Aerosol exposure
– BL2
• leave lab upon release
• Wash wounds, flush face/eyes
if exposed, wash hands
• post sign, keep area vacated for
20 - 30 minutes to allow aerosols
to settle
• decontaminate personnel
clothing, skin
• notify lab supervisor, biosafety,
and Health Services if necessary
Foundational Biosafety Practice
#2
Hand Washing
Hand Washing
 Hands must be washed
after removing:
– gloves
– other PPE
– and whenever gloves
are damaged or torn
Foundational Biosafety Practice
#3
No Mouth Pipetting
Careful Pipetting Techniques
 Never blow out last
drop in pipette
 Use pipette aids with
filters
 Horizontal pipette
collection tubs
 Never mix by suction
+ expulsion
Minimize Aerosols
 Careful pipetting
practices
 Avoid drops onto hard
surfaces
 Wipe up spills
promptly with
appropriate
disinfectant
Careful Pipetting Techniques
 Discharge liquid down side of container
 Deliver as close as possible to contents
 Work over plastic-backed absorbent matting
Avoid Splatter
1
Incorrect
3
Correct
2
Incorrect
Minimize Aerosols
 Use capped tubes
when mixing,
blending, or vortexing
 Pour liquids carefully
 Avoid bubbles
Foundational Biosafety Practice
#4
Eliminate or Work Safely with Sharps
Use Extreme Care with Sharps
 Percutaneous exposure risk
– Employ safe work practices
– Utilize safe sharp devices
 Aerosol risk
– Use biosafety cabinet for
removal of air from needle
 Sharps also includes scalpels,
blades and wires.
Safe Sharps Devices
 Only use sharps if
absolutely required as
part of a process.
 Ongoing evaluation of
new safety devices
Safe Use of Sharps
 Keep hands away from
needles.
 Use mechanical methods
for needle removal.
 Never bend, recap or
manipulate sharps by
hand.
Safe Use of Sharps
Disposal/Reprocessing
 Dispose of entire unit
into sharps container.
 Collect reusable sharps
in labeled, leak proof
container.
Infectious Waste Disposal
 All cultures and stocks
are decontaminated
before disposal.
 Wastes must be in
closed containers for
transport out of lab.
 Infectious waste
containers must be
labeled.
Infectious Waste Disposal
 Written SOP’s for:
– Waste management
– Regulated sharps
– Other sharps (tips,
pipettes, glass
items, etc.)
– Liquid waste
Foundational Biosafety Practice
#5
No Eating, Drinking or Smoking
(Avoid touching your eyes, nose, mouth)
Foundational Biosafety Practice
#6
Personal Protective Equipment (PPE)
Personal Protective Equipment
(PPE)
Purpose of PPE:
• Barrier against skin,
mucous membrane or
respiratory exposure to
infectious agents
• To prevent spread of
contamination.
Laboratory Clothing
 Protects the worker
from splash and splatter
during procedures.
 Protects worker’s
clothing from
contamination.
 Can be used to protect
product from
contamination.
Laboratory Clothing: Important
Considerations
 Choice of fabric: must
be appropriate for use
– Fire retardant and
resistant to chemicals
– Water repellant if risk
of splash and splatter.
Can add plastic apron
 Type and use must be
prescribed in SOP or
manual and staff trained
Laboratory Clothing: Important
Considerations
 Staff must be trained in
aseptic removal
procedures-gloves last!
 Reusable clothing must
withstand autoclaving
 Laundry facilities must
be provided. Lab
clothing not to be taken
home.
Removing Gloves
1
Removing Gloves
1
2
Removing Gloves
1
2
3
Removing Gloves
1
4
2
3
Gloves: Important Considerations
 Avoid wetting gloves (disinfectants
enhance permeation)
 Change gloves as soon as feasible after
contamination
 Use utility gloves or
double glove for spills
 Examine utility gloves
after washing for
integrity
Personal Protective Equipment
(PPE)
Limitations of PPE:
• Does not eliminate the
hazard.
• Integrity wanes with
use (change gloves
frequently)
• Not all gloves created
equal - select best
glove for the task.
Gloves: Important Considerations
 Latex allergies: Alternatives must be provided
 Leather, mesh and kevlar used with animal
handling (cut, scratch or slice protection)
 Consider chemical hazards
 All gloves not created equal
Eye and Face Protection
Eye and face protection
appropriate for the task:
Safety glasses - must
have side protectors
Goggles
Face shield and
goggles if risk of
splash and splatter
Respirators
 Air Purifying Respirator
(APR)
 Uses a filter medium to
remove contaminant
– Reduces aerosols
– Non-powered - masks
– Powered - PAPR
 Assigned Protection Factor
– APF = 10 - 50 (NIOSH)
for Air Purifying or
Powered Air Purifying
Respirators
 Air supplying
– Air supplied from safe area
– Supplied Air Respirator
(SAR)
– Air line and + pressure
– Self Contained Breathing
Apparatus (SCBA)
 Protection Factor!
– APF 10,000 (pressure
demand SCBA)
Other Considerations
 Cover exposed skin, dress sensibly
 Avoid shorts, sandals in the lab
 Use shoes with non-slip
soles
 Shoe covers for spills or
contamination control
 Rubber boots may be
in wet areas
needed
Foundational Biosafety Practice
#7
Decontamination/Disinfection
Disinfecting & Cleaning
Biosafety Work Practices
 Disinfection
– select appropriate combination of chemical,
concentration, and contact time
– keep working solutions of disinfectant at bench
– use work surface covers (replace frequently)
– promptly clean/decontaminate work surfaces &
equipment, biohazard waste (and spills)
– spot treat reusable protective clothing or
launder (never bring home for cleaning)
3 C’s of Disinfection
 Chemical
– Select disinfectant effective against target
organism
 Concentration
– Prepare the “referenced” solution
 Contact time
– Disinfectants don’t work instantaneously
– EPA uses a 10 minute contact time for testing
Disinfectants
Disinfectants will destroy or inactivate specific
viruses, bacteria, and pathogenic fungi, but not
necessarily their spores.
 Chlorine (household) bleach
– 1% dilution for work surfaces
– 10% for spills or porous surfaces
Disinfectants
 Alcohol
– ethyl alcohol or isopropyl alcohol diluted to
70-85% in water for surface decontamination
 Other
– classified by EPA as tuberculocidal and
virucidal
– follow manufacturer’s written instructions for
use
Decontamination
 Encompasses:
– antisepsis, disinfection, decontamination, and
sterilization
– Antisepsis
• chemical applied to living tissue that will control or
arrest the growth of a microorganism
– Decontamination
• disinfection or sterilization of contaminated
materials
Decontamination
 Disinfection
– To free from infection, reduction of
contaminant load to a safe level, does not imply
total destruction of all microorganisms. Will
not always destroy spores.
 Sterilization
– The destruction of all forms of microbial life.
Difficult to achieve (usually referred to a very
low chance that a microbe survived).
Spill Clean-Up Kit
 disinfectant
 paper towels
 gloves
 face protection
 biohazard bags
 forceps
 dust pan & brush
Personal Protective Equipment
Biohazards
Personal Protective Equipment (PPE)
 Protection of:
 Use:
 Skin
 Gloves (double,
 Clothing
 Mucous membranes
 Respiratory system
kevlar)
 lab coats, solid-front
gowns, sleeve covers
 Full-face protection
 Respirator
OSHA Standard 1910.1032
 General requirements for PPE, employers
must:
– conduct hazard assessment, select equipment
– inform employees of hazards and equipment
selected for protection
– provide proper sizes are available
– train employees
• what PPE, when to wear, how to put on, wear and
take off, limitations, care, maintenance, disposal
Gloves
 Latex (allergy)
 Nitrile
 vinyl, PVC
 Polyvinyl Alcohol
 Neoprene
 Silver Shield
 Kevlar
 Gauntlet
 Temperature resistant
– autoclaves, cryogens
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
Lab Coats/Gowns
 Lab coat
 Back-fastening gown, scrubs
 Aprons, jump suits (w/ hood, booties)
 Level A, B, C, D suits
 Knit grip cuff vs. open cuff, sleeve covers
 Disposable/Reusable
– Treat onsite, use laundry service, no home use
– Hook in lab, remove prior to leaving
For work in a Biosafety Cabinet
 solid-front or
wrap-around
gown
 gloves (double
gloving)
 face protection
(face shield or
mask and safety
glasses)
Face Protection
 Safety glasses
 Goggles
 Face shield
 Surgical
mask/shield
 Surgical
mask/goggles
 Impact resistant
face shield
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
Respiratory Protection
 Disposable N-95 mask and N-
99/N-100 respirator
 Powered-Air-Purifying
respirator
 Annual training, medical
exam, fit testing
Respiratory Protection
 Disposable Respirators (95%, 99%, 100%
efficiencies)
– N - not resistant to oil
– P - partially resistant to oil
– R - resistant to oil
 Medical questionnaire
 Annual training
 Qualitative & quantitative fit testing