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Biosafety Training. University of Ottawa * Office of Risk Management  Human Resources - Occupational Health Disability & Leave. v0501. Biosafety Outline. Introduction Laboratory Associated Infections Blood-borne Pathogens Classification of Biohazards Infection/Biohazard Control

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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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Who’s Responsible, who are the Stakeholders?

INTERNALLY

  • Vice-President (Research)

  • Committees

  • University Services (ORM, HR, PRS, PS)*

  • Deans, Chairs, Principal Investigators, Employees, Students

  • Manager of Biological Containment Suite

EXTERNALLY

  • Health Canada

  • Canadian Food Inspection Agency

  • Transport Canada

  • Ontario Ministry of Labour

  • Emergency Response Personnel

  • Suppliers & Contractors

  • Community


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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

  • Percutaneous inoculation

  • Inhalation of aerosols

  • Contact of mucous membranes

  • Ingestion

Infection Source

Susceptible Host

  • Immune system

  • Vaccination status

  • Age

  • Cultures and stocks

  • Research animals

  • Specimens

  • Items contaminated with above

Route of Transmission


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LAI’s

  • 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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LAI’s


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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 University’s Health Services

  • Report incident to OHDL

    Occupational Health, Disability and Leave Office telephone ext. 1472 http://www.uottawa.ca/services/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

  • Canada: Level of risk depends on source of DNA, vector and host.

  • The Biosafety Committee will assist the investigator in this determination.

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


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Tissue Culture

  • Have the potential to contain pathogenic organisms

  • In general;

Human & non-human primate, and mycoplasma-containing 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 BSC’s 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-ldmbl/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;

    • MSDS’s

    • 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 OH&S 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?

b

i

o

s

a

f

e

t

y


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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 one’s 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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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

Needles and Syringes


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Pipettes

  • Mouth pipetting is prohibited.

  • All biohazardous materials should be pipetted in BSC’s.

  • 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/SPILLRESPONSEPLAN.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?

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*

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


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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 1:10


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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

    • MSDS’s


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The Bottom Line

  • If you are notcareful 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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