Biosafety Training

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Biosafety Training

• University of Ottawa
• Office of Risk Management
• ?Human Resources - Occupational Health
  Disability Leave

v0501
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Biosafety Outline

• Introduction
• Laboratory Associated Infections
• Blood-borne Pathogens
• Classification of Biohazards
• Infection/Biohazard Control
• Spill Response
• Biomedical Waste
• Regulations

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What is Biosafety?

• Measures employed when handling biohazardous
  materials to avoid infecting oneself, others or
  the environment.
• Achieved through
• Engineering Controls
• Administrative Controls
• Practices and Procedures
• Personal Protective Equipment

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What is a Biohazard?
A potential hazard to humans, animals or the
environment caused by a biological organism, or
by material produced by such an organism

• Examples
• Viruses, bacteria, fungi, and parasites and their
  toxins.
• Blood and body fluids, as well as tissues from
  humans and animals.
• Transformed cell lines and certain types of
  nucleic acids .

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Whos Responsible, who are the Stakeholders?

• EXTERNALLY
• Health Canada
• Canadian Food Inspection Agency
• Transport Canada
• Ontario Ministry of Labour
• Emergency Response Personnel
• Suppliers Contractors
• Community

• INTERNALLY
• Vice-President (Research)
• Committees
• University Services (ORM, HR, PRS, PS)
• Deans, Chairs, Principal Investigators,
  Employees, Students
• Manager of Biological Containment Suite

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Key Services

• Office of Risk Management
• Training
• Interface with Regulatory Bodies
• Biosafety Program

• certifications
• training
• procedures
• inspections
• contingency planning
• accident/incident follow-up

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Key Services

• HR (Occupational Health, Disability and Leave)
• Medical surveillance
• Immunizations
• Medical Follow-up
• Interface with Workplace Safety and Insurance
  Board

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Why are we concerned about biohazardous materials?

• Potential for acquiring a laboratory-associated
  infection (LAI)
• Contamination of the environment
• Contamination of research
• Public perception

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Laboratory Associated Infections
Susceptible Host
Infection Source

• Immune system
• Vaccination status
• Age

• Cultures and stocks
• Research animals
• Specimens
• Items contaminated with above

Route of Transmission

• Percutaneous inoculation
• Inhalation of aerosols
• Contact of mucous membranes
• Ingestion

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LAIs

• Only 20 causative or defined event
• 80 of which are caused by human error
• 20 are caused by equipment failure
• Top 4 accidents resulting in infection
• Spillages splashes
• Needle and syringe
• Sharp object, broken glass
• Bite or scratch from animals or ectoparasites

http//www.weizmann.ac.il/safety/bio2.html
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LAIs
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Bloodborne Pathogens (BBP)

• Sources
• Blood
• Semen
• Vaginal Secretions
• Body Fluids
• Cerebrospinal
• Amniotic
• Synovial
• Tissue Cultures
• Organ Cultures
• Infected Experimental Animals

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Risk of Exposure

• Pathogen involved
• Type of body fluid
• Route of exposure
• Duration of exposure
• Volume of blood involved in exposure
• Concentration of virus at time of exposure
• PPE worn

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Specific Examples of Bloodborne Pathogens

• Hepatitis B
• Hepatitis C
• HIV

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Issues to Consider

• Symptoms
• Mode of transmission
• Incubation period
• Survival outside host
• Communicability
• Immunization
• Prophylaxis / Treatment

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If An Exposure Occurs (or the possibility of
exposure)

• Initiate first aid
• Notify your supervisor / designated person
• Report to hospital emergency department or
  Universitys Health Services
• Report incident to OHDL
  
• Occupational Health, Disability and Leave Office
  telephone ext. 1472 http//www.uottawa.ca/service
  s/hr/frames.html

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Universal Precautions

• Minimum standard of practice for preventing the
  transmission of BBP
• Includes - Education
• - Hand washing
• - Wearing protective barriers
• - Use safe work practices

If samples cannot be guaranteed non-infective
treat as infectious!
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Classification of Biohazards

• Conventional Agents
• Unconventional Agents
• Recombinant DNA
• Tissue Culture
• Animal Work
• Anatomical Specimens

Class D, division 3 of WHMIS (Poisonous and
Infectious Material - Biohazardous Infectious
Material)
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Classification of Biohazards
_

• As the level ? so does
• the risk of the organism to humans, animals,
  plants and/or the environment
• the procedural and facility requirements
• the level of containment required
• the degree of protection for personnel, the
  environment and the community.

BSL 4
BSL 3
BSL 2
BSL 1
_
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Conventional Agents
Unlikely to cause disease in healthy workers or
animals
Rarely cause serious human or animal disease
May cause serious disease
Likely to cause very serious disease
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Unconventional Pathogens

• TSE prion diseases lethal transmissible
  neurodegenerative conditions
• Creutzfeld-Jakob disease, Variant C-J Disease,
  Mad Cow Disease, Scrapie, Chronic Wasting
  Disease.
• Resistant to destruction by procedures that
  normally inactivate viruses.
• Contact ORM to assess requirements (containment,
  procedures, waste disposal, etc.)

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Recombinant DNA
Genetic Engineering in vitro incorporation of
genetic material from one cell into another

• Canada Level of risk depends on source of DNA,
  vector and host.
• The Biosafety Committee will assist the
  investigator in this determination.

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Tissue Culture

• Have the potential to contain pathogenic
  organisms
• In general

Human non-human primate, and mycoplasma-containi
ng cell lines
Level 2
Level 1
Others
A detailed risk assessment should be undertaken
when using a new cell line.
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Animal Work

• Animals can harbour infectious organisms
  (naturally or introduced)
• Level dependent on type of work being conducted.
• Special Animal Care training is required for all
  personnel working with animals.
• All work involving animal use must receive prior
  approval from the Animal Care Committee

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Anatomical Specimens

• All specimens should be considered infectious due
  to potential presence of infectious agents
• Important to consider the type of specimen
• blood, organs, tissues
• Spinal sample, brain tissue
• From infectious patient
• In general Level 2 but it depends on the nature
  of the work.

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Infection/Biohazard Control

• Engineering Controls
• Administrative Controls
• Practices and Procedures
• Personal Protective Equipment

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Engineering Controls

• Technology based, reduce or eliminate exposure to
  hazards by changes at the source of the hazard.
• Containment
• Primary vs Secondary
• Containment levels

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Primary Containment

• First line of defence.
• Ensures protection of personnel and immediate
  environment from exposure to the infectious
  agent.
• Protective envelope that encapsulates the
  infectious agent or animal.
• Petrie dish, vial, stoppered bottle.
• Biological safety cabinets, glove boxes and
  animal caging equipment.
• Effectiveness of control is based on the
  integrity of the containment.

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Secondary Containment

• Protects the environment external to the
  laboratory from exposure.
• Includes facility design and operational
  practices.

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Biosafety Containment Levels

• Containment Levels similar to Risk Levels.
• Biohazards Committee will evaluate the research
  proposals to ensure adequate containment .
• Level 1
• Level 2
• Level 3
• Level 4

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Level 1

• Basic laboratory
• Requires no special design features
• Biosafety cabinets are not required and work may
  be performed on the open bench.

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Level 2

• Clinical, diagnostic, research and teaching
  facilities with level 2 agents.
• Requires a class I or class II biological safety
  cabinet if any potential for aerosol or splash
  exists.
• An emergency plan for handling spills must be
  developed.
• Access should be controlled.

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Level 3

• Specialized design and construction
• primary barriers to protect the individual
• secondary barriers to protect the environment
• All staff must undergo special training on the
  agents being used, PPE, equipment, waste
  management as well as practices and procedures
  above and beyond the scope of this course.

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Level 4

• Only one level 4 facility in Canada (Canadian
  Centre for Human and Animal Health in Winnipeg,
  Man.)
• Design specifications are extremely stringent,
  worker is completely isolated from infectious
  material.

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Biological Safety Cabinets

• Effective means of physical containment for
  biological agents, especially when aerosols are
  generated.
• HEPA filters remove particles (min 0.3 microns)
  with 99.97 efficiency.
• There are 3 main classes of cabinets (I, II,
  III) which provide various levels of protection.

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Biological Safety Cabinets
VS

• Laminar flow hoods
• NOT biological safety cabinets
• Vertical or horizontal laminar flow
• HEPA filtered air (intake)
• product protection only

• Biological Safety Cabinet
• HEPA filtered laminar air flow and
• exhaust
• personnel, environment often
• product protection

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Working safely in a BSC

• Before using the cabinet
• Ensure BSC is certified
• Turn off UV lamp turn on fluorescent lamp
• Disinfect work surfaces with appropriate
  disinfectant
• Place essential items inside cabinet
• Allow the blower to run for 5-10 min before work

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Working safely in a BSC

• During use
• Ensure material and equipment is placed near the
  back of the hood, especially aerosol-generating
  equipment. Do not block any vents.
• Use techniques that reduce splatter and aerosols.
• General work flow should be from clean to
  contaminated areas.
• Minimize movement so as not to impede air flow.
• Open flame in BSCs is controversial.

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Working safely in a BSC
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Working safely in a BSC

• After completion of work
• Leave blower on at least 5 minutes to purge
  cabinet
• Remove and decontaminate equipment and materials
• Disinfect cabinet surfaces
• Turn off blower and fluorescent lamp, turn on UV
  lamp

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Working safely in a BSC

• Maintenance
• Twice daily - Work surfaces wiped down
• Weekly - UV lamp should be wiped clean
• Monthly - All vertical surfaces wiped down
• Annually - UV lamp intensity verified.
• - Decontamination with formaldehyde gas (ORM)
• - Certification (ORM)

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Administrative Controls
Program based, information and methods to
minimize risk of exposure.

• Risk assessment
• Medical Surveillance
• Training/Education
• Resources
• Inspections
• Signs Labeling

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Administrative Controls

• Risk Assessment
• Will determine type of containment, procedures,
  and safety equipment required
• Responsibility of users, additional assistance is
  available from ORM
• Consider areas such as experimental design,
  procedures to be employed and personal
  experience/knowledge, etc.

http//www.hc-sc.gc.ca/pphb-dgspsp/ols-bsl/lbg-ldm
bl/pdf/lbg-3e-draft.pdf
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Administrative Controls
Risk Assessment Know your Agent

• Know and understand the various characteristics
  of the agent(s) you are working with.
• This information is available from
• MSDSs
• Suppliers or manufacturers
• Example

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Administrative Controls

• Medical Surveillance
• Training Education
• Lab specific policies and procedures
• Biosafety training
• Resources
• ORM web site, Biosafety page
• Faculty web sites
• Biosafety Manual
• Training Videos

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Administrative Controls

• Inspections
• Routine self-inspections
• Biosafety Inspection Checklist available on-line
• In addition, ORM, EHSOs and OHS will inspect
  labs to ensure compliance with regulations/
  guidelines and provide feedback.

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Administrative Controls

• Signs Labeling
• Biohazard warning signs must be posted on doors
  to rooms where biohazardous materials are used.
• Biohazard labels should be placed on containers,
  equipment and storage units used with biological
  agents.

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Practices and Procedures

• General Safety Guidelines
• Good Microbiological Practice
• Handwashing
• Specific Procedures
• Centrifuges
• Needles Syringes and other sharps
• Pipettes
• Blenders, Grinders, Sonicators Lyophilizers
• Inoculation Loops
• Cryostats

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General Laboratory Safety Guidelines

• Mostly common sense, but you must understand the
  hazards you face in the laboratory and be
  adequately trained to deal with them.
• Basic must knows for all labs.
• Examples?

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Good Microbiological Practice (GMP)

• Basic code of practice that should be applied to
  all types of work involving microorganisms
• Objectives
• prevent contamination of laboratory workers and
  the environment
• prevent contamination of the experiment/samples
• Application of aseptic technique, minimization of
  aerosols, contamination control, personal
  protection, emergency response

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Handwashing

• One of the single effective means of preventing
  infections
• IF done properly and frequently
• When to wash?

• Before starting any manipulations
• Before leaving the lab
• When hands are obviously soiled
• Before and after completing any task in a BSC
• Every time gloves are removed
• Before contact with ones face or mouth
• At the end of the day

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Safe use of Centrifuges

• Before use
• Stress lines? Overfilled? Balanced?
• Caps or stoppers properly in place?
• Run conditions achieved?
• Use sealable buckets (safety cups) or sealed
  rotors
• After run
• Centrifuge completely stopped?
• Spills or leaks?
• Allow aerosols to settle (30 min) or open in a
  BSC.

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Needles and Syringes

• Avoid use whenever possible
• Use a BSC for all operations with infectious
  material
• Fill syringes carefully
• Shield needles when withdrawing from stoppers

• Do not bend, shear or recap needles.
• Dispose of all used needles/syringes in yellow
  sharps containers

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Pipettes

• Mouth pipetting is prohibited.
• All biohazardous materials should be pipetted in
  BSCs.
• Never force fluids out, use to deliver
  pipettes.
• To avoid splashes, allow discharge to run down
  dispense the receiving container wall.
• Never mix material by suction and expulsion.
• Reusable pipettes should be placed horizontally
  in a disinfectant filled pan.

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Blenders, Grinders, Sonicators and Lyophilizers

• Operate in a BSC whenever possible. Allow
  aerosols to settle for 5 minutes before opening.
• Safety Blender
• Do not use glass blender jars
• Decontaminate immediately after use
• Lyophilizers
• Use glassware designed for vacuum work, ensure
  there is no damage before using.
• All surfaces should be disinfected after use
• Use vapour traps whenever possible.

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Inoculation Loops

• Sterilization in an open flame may create
  aerosols which may contain viable microorganisms.
• Shorter handles minimize vibrations.
• Disposable plastic loops are good alternatives.

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Cryostats

• Wear gloves during preparation of frozen sections
  and heavy gloves when accessing the cryostat.
• Decontaminate frequently (70 Ethanol or
  specific to agent)
• Guards and wheel locks!

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Personal Protective Equipment

• PPE can become an important line of defence (last
  line of defense).
• Responsibility of both the user and the
  supervisor to ensure that PPE is worn

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PPE

• Criteria for consideration
• Routes of exposure that need to be blocked
• Degree of protection offered
• Ease of use
• Only effective if correctly selected, fitted,
  used and cared for, and the individual is trained
• Ensure PPE is removed before leaving the lab.

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PPE

• Footwear
• Closed toed shoes should always be worn. Booties
  are worn in some higher containment labs and
  animal facilities.
• Lab Coats/Gowns
• Long-sleeved, knee length with snaps
• Elastic cuffs
• Back-closing gowns
• Periodic cleaning required

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PPE

• Gloves
• Latex, nitrile vinyl for work with biological
  agents
• Exam gloves should not be reused, change
  frequently. Utility gloves can be disinfected and
  reused if they show no sign of degradation.
• Consider tensile characteristics, length of cuff
• Double gloving
• ORM can provide assistance finding an alternative
  for people with allergies.
• Gloves are not to be worn in public places

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PPE

• Eye Face Production
• Goggles, safety glasses to protect the eyes
• Full face shield to protect facial skin.
• Respirators
• Only personnel who have been fit-tested and
  trained should wear respirators.

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Spills

• Spill response will vary depending on
• What was spilled?
• How much was spilled?
• Where was the spill?
• What is the potential for release to the
  environment?
• Spills should be cleaned up immediately (unless
  an aerosol was generated), to ensure proper
  decontamination.
• Ensure appropriate PPE is worn and clean-up
  equipment is readily available.

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Spills General Clean-up

• Cover spill area with absorbent material
• Soak the spill area with an appropriate
  disinfectant (i.e. 10 bleach)
• Pour disinfectant from the outside surface of the
  absorbent material towards the inside
• Ensure any broken glass is picked up (with
  forceps!) and placed in a sharps container
• Leave on for 20 to 30 minutes
• Wipe up with absorbent material
• Waste should be disposed in appropriate biohazard
  bags and where possible autoclaved

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Spills Special Cases

• Within a Centrifuge
• Within a BSC
• Open Areas (lab, during transport)
• The spill response plan template is available at
  http//www.uottawa.ca/services/ehss/SPILLRESPONSEP
  LAN.pdf

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Spills

• All users of biological materials should be
  familiar with the spill clean-up procedures.
• All spills are to be reported ASAP to the lab
  supervisor and ORM.
• Additional assistance is available from
• ORM x 5892
• Your departmental safety officer
• ERT x 5411 (through Protection)

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Decontamination, Disinfection and Sterilization

• Decontamination Free of contamination, the
  destruction of microorganisms to a lower level
  such that it removes danger of infection to
  individuals.
• Sterilization The complete destruction of all
  viable microorganisms.
• Disinfection Use of agents (physical or
  chemical) to destroy harmful organisms on
  inanimate objects (not necessarily all organisms)

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Decontamination Physical

• Heat
• Autoclaving (most practical and recommended)
• Incineration (for disposal of sharps and tissues)
• Irradiation
• UV light (wavelength of 253 nm is germicidal)
• Gamma (disrupts DNA and RNA)
• Filtration
• HEPA (biological safety cabinets, ventilation)

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Autoclaves

• Items that CAN be autoclaved
• Cultures and stocks of infectious material
• Culture dishes and related devices
• Discarded live and attenuated vaccines
• Contaminated solid items (petrie dishes,
  eppendorf tips, pipettes, gloves, paper towels)
• Items that CAN NOT be autoclaved
• Chemical, chemotherapeutic or radioactive waste
• Bleach
• Certain kinds of plastics
• Sharps (not at the University of Ottawa)

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Autoclaves

• Preparation of waste
• Use only approved autoclave bags.
• Do not overfill autoclave bags
• Separate material for re-use from that which will
  be disposed and dry from liquid material .
• If outside of bag is contaminated, double bag.
• All flasks containing biological material should
  be capped with aluminum foil.
• Ensure items are labeled with contact
  information.

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Safe Use of Autoclaves

• Many autoclaves are now run by dedicated staff,
  however, if you are operating an autoclave
• Learn how to use!
• Ensure PPE is worn
• Recognize acceptable material and packaging
• Proper loading and unloading

All users/operators must fill out the Autoclave
User Questionnaire and receive training!
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Decontamination Chemical

• Generally for disinfection rather than
  sterilization
• Choice depends on
• Type of material to be disinfected
• Organic load
• Chemical characteristics
• Most common are chlorine compounds and alcohols
  (broad range)

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Disinfection What to use for my organism?

• Viruses
• Enveloped (HIV, Herpes)
• 2 domestic bleach
• 75 Ethanol
• Quaternary ammonia
• 6 formulated Hydrogen peroxide
• Non enveloped (Hepatitis, Adenovirus)
• 10 domestic bleach
• 6 formulated Hydrogen peroxide
• Gluteraldehyde
• Formaldehyde

• Bacteria
• Vegetative bacteria (E.coli, Staph)
• 2 domestic bleach
• 75 Ethanol
• Quaternary ammonia
• 6 formulated Hydrogen peroxide
• Mycobacteria and fungi
• 10 domestic bleach
• 75 Ethanol
• Phenolic compounds
• 6 formulated Hydrogen peroxide
• Spore forming bacteria (Bacillus)
• 10 domestic bleach
• Gluteraldehyde
• Formaldehyde
• 6 formulated Hydrogen peroxide

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Waste Management

• Biomedical waste
• Discarded biological material from teaching,
  clinical and research laboratories and
  operations. Biomedical waste includes but is not
  limited to
• Animal waste
• Biological laboratory waste
• Human anatomical waste
• Human blood and body fluid waste
• Sharps

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Waste Management

• All biological waste should be decontaminated
  prior to disposal (including level 1 agents).
• Treated waste is no longer considered
  biomedical (i.e. microbiological waste, blood
  and bodily fluid waste) and can be disposed in
  the regular waste stream.
• Any waste that cannot be treated (i.e. sharps,
  carcasses, tissues and body parts) remains
  biomedical waste and must be incinerated off
  site.

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Waste Disposal
Biomedical Waste (untreated)
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Waste Disposal
Biomedical Waste (treated)

In compliance with Sewer use by-laws
With H2O 110
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Special Waste

• EtBr
• Toxins
• Recombinant DNA
• Contact ORM

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Transportation

• Important Considerations
• does material need to be transported at all
• packaging requirements
• means and route of transportation
• regulatory requirements
• Between lab transfers - 4 sided cart, sealed
  primary container, secondary container, low
  traffic route.
• Off Campus transfers consult ORM

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Shipping Receiving

• Transportation of Dangerous Goods Act Class 6.2
  of (Infectious Substances)
• International Air Transport Association
• HC/CFIA restrictions
• Ensure
• Proper classification
• Proper packaging
• Proper labeling
• Proper documentation
• Import/Export Permits

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Purchasing

• Importation permits required by Health Canada or
  CFIA for certain agents
• US restrictions
• Ensure you meet all criteria and have all
  pertinent documentation

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Security

• Controlled access
• physical barriers (structural design,
  departmental design, key/card access, etc.)
• psychological barriers (obvious presence of
  identifiable security personnel, security
  culture, use of monitoring tools)
• monitoring activities (security patrols,
  departmental monitoring, key control program)
• personnel clearance

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Inventory

• What material is presently being used and/or
  stored in the lab
• Location
• Expiry date
• How much, use log
• MSDSs

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The Bottom Line

• If you are not careful and diligent with
  biological agents you risk
• Infecting yourself, others or the environment
• Contaminating your research
• Having Health Canada, Canadian Food Inspection
  Agency, Ministry of the Environment or Transport
  Canada after you

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Conclusions

• Biosafety - ensuring that individuals and the
  environment are not infected
• Biohazards - microorganisms, blood and body
  fluids, tissues and tissue culture
• Everyone within the University community is
  responsible
• With proper knowledge, planning and care, a
  biological exposure is avoidable.

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Biosafety Website Orientation

• http//www.uottawa.ca/services/ehss/biosafety.htm

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