Controls in Biosafety II: Work Practices and Personal Protective Equipment

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Controls in Biosafety IIWork Practices and
Personal Protective Equipment

• EMD 545b
• Lecture 6

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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

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Routes of Exposure

• Ingestion
• Inhalation
• Mucous membranes (eyes, nose, mouth)
• Percutaneous

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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)

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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

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Biosafety Work Practices

• Transport/Shipping
• use two leak proof labeled containers for
  on-campus 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

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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

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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 SOPs
• keep pets, other unrelated items out of lab

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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

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Foundational Biosafety Practice 1

• Confine Aerosols

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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.

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Confine Aerosols!

• Promote Awareness of
• Aerosol containment
• Work Practices PPE
• Emergency Response Procedures
• Minimize/prevent lab-acquired infections

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Aerosols are generated in many procedures
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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

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Aerosol spread from open flame
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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

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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

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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

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Biosafety Work Practices

• Centrifugation Precautions
• use secondary containment (decon after use)
• check tubes for cracks or deformities before use
• always use sealed primary tubes
• dont overfill primary containers
• balance loads to avoid damage to centrifuge
• unit should have solid lid and interlock

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Confinement at Source
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Incident Response Exposures and Spills
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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

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Foundational Biosafety Practice 2

• Hand Washing

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Hand Washing

• Hands must be washed after removing
• gloves
• other PPE
• and whenever gloves are damaged or torn

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Foundational Biosafety Practice 3

• No Mouth Pipetting

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Careful Pipetting Techniques

• Never blow out last drop in pipette
• Use pipette aids with filters
• Horizontal pipette collection tubs
• Never mix by suction expulsion

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Minimize Aerosols

• Careful pipetting practices
• Avoid drops onto hard surfaces
• Wipe up spills promptly with appropriate
  disinfectant

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Careful Pipetting Techniques

• Discharge liquid down side of container
• Deliver as close as possible to contents
• Work over plastic-backed absorbent matting

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Avoid Splatter
3 Correct
1 Incorrect
2 Incorrect
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Minimize Aerosols

• Use capped tubes when mixing, blending, or
  vortexing
• Pour liquids carefully
• Avoid bubbles

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Foundational Biosafety Practice 4

• Eliminate or Work Safely with Sharps

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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.

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Safe Sharps Devices

• Only use sharps if absolutely required as part of
  a process.
• Ongoing evaluation of new safety devices

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Safe Use of Sharps

• Keep hands away from needles.
• Use mechanical methods for needle removal.
• Never bend, recap or manipulate sharps by hand.

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Safe Use of SharpsDisposal/Reprocessing

• Dispose of entire unit into sharps container.
• Collect reusable sharps in labeled, leak proof
  container.

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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.

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Infectious Waste Disposal

• Written SOPs for
• Waste management
• Regulated sharps
• Other sharps (tips, pipettes, glass items, etc.)
• Liquid waste

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Foundational Biosafety Practice 5

• No Eating, Drinking or Smoking
• (Avoid touching your eyes, nose, mouth)

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Foundational Biosafety Practice 6

• Personal Protective Equipment (PPE)

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Personal Protective Equipment (PPE)

• Purpose of PPE
• Barrier against skin, mucous membrane or
  respiratory exposure to infectious agents
• To prevent spread of contamination.

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Laboratory Clothing

• Protects the worker from splash and splatter
  during procedures.
• Protects workers clothing from contamination.
• Can be used to protect product from contamination.

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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

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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.

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Removing Gloves
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Removing Gloves
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Removing Gloves
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Removing Gloves
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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

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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.

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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

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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

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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

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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)

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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 needed in
  wet areas

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Foundational Biosafety Practice 7

• Decontamination/Disinfection

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Disinfecting Cleaning
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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)

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3 Cs of Disinfection

• Chemical
• Select disinfectant effective against target
  organism
• Concentration
• Prepare the referenced solution
• Contact time
• Disinfectants dont work instantaneously
• EPA uses a 10 minute contact time for testing

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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

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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 manufacturers written instructions for use

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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

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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).

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Spill Clean-Up Kit

• disinfectant
• paper towels
• gloves
• face protection
• biohazard bags
• forceps
• dust pan brush

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Personal Protective EquipmentBiohazards
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Personal Protective Equipment (PPE)

• Use
• Gloves (double, kevlar)
• lab coats, solid-front gowns, sleeve covers
• Full-face protection
• Respirator

• Protection of
• Skin
• Clothing
• Mucous membranes
• Respiratory system

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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

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Gloves

• Latex (allergy)
• Nitrile
• vinyl, PVC
• Polyvinyl Alcohol
• Neoprene
• Silver Shield
• Kevlar
• Gauntlet
• Temperature resistant
• autoclaves, cryogens

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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

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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

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For work in a Biosafety Cabinet

• solid-front or wrap-around gown
• gloves (double gloving)
• face protection (face shield or mask and safety
  glasses)

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Face Protection

• Safety glasses
• Goggles
• Face shield
• Surgical mask/shield
• Surgical mask/goggles
• Impact resistant face shield

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Use of a HEPA-filtered PAPR for additional
protection during high-risk research procedures
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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

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Respiratory Protection

• Disposable N-95 mask and N-99/N-100 respirator
• Powered-Air-Purifying respirator
• Annual training, medical exam, fit testing

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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