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Introduction to Biosafety
Environmental Health and Safety
Objectives
Have a good working knowledge of risk groups and
containment levels
Recognize the risks associated with biohazards in the
laboratory
Use safe practices when handling biohazards
Understand the strengths and limitations of autoclaves
and disinfectants
Know how to respond to accidental exposures and spills
of biohazardous material
www.mcgill.ca/ehs/laboratory/biosafety/manual
Regulations & Guidelines
Public Health Agency of Canada (PHAC) Guidelines
Canadian Food Inspection Agency (CFIA) Standards
Environment Canada – New Substances Notification
Transport Canada – Transport of Dangerous Goods
Workplace Hazardous Materials Information System
(WHMIS)
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RegulationsP
H
A
C
Human Pathogens
C
F
I
A
Animal Pathogens
Canadian Biosafety Standards and Guidelines
Prions
C
F
I
A
CFIA Standards
Containment Standards for Facilities Handling Plant Pests
Containment Standards for Facilities Handling Aquatic Animal Pathogens (2010)
Environment Canada
New Substances Notification Regulations
(Organisms)
Reporting mandatory for manufactured/imported microorganisms if: Not on Domestic Substances List
Research & development exemption unless: Introduced into experimental field
study without containment
CLASS AND SYMBOL CHARACTERISTICS PRECAUTIONS
Class D Poisonous and
Infectious Material
Microbiological agents (e.g.,
bacteria, viruses, fungi and
their toxins) that may cause
illness or death
Wear the recommended protective
equipment and clothing
Work with these materials in
designated areas
Disinfect area after handling
Wash hands after handling
Division 3:
Biohazardous
Infectious
Materials
WHMIS (Workplace Hazardous
Materials Information System)
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Where to find MSDS for infectious substances?
http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/index-eng.php
http://www.inspection.gc.ca/english/anima/disemala/disemalae.shtml
Transport of Dangerous GoodsDivision 6.2 Biological Substances
Transport Canada -TDG RegulationsDivision 6.2 Infectious Substances
Packaging and labeling of biological
agents or toxins transported to
another entity
Mandatory training for all who
transport (shipping and/or receiving)
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Definition: Biohazards Biohazards
Bacteria
Viruses
Fungi
Parasites
Plants and algae
Toxins of biologicalorigin
Biohazards
Prions
Human tissues, blood and body fluids
Animals, animal tissues and carcasses
Cell lines
Nucleic acids
Genetically modified strains & organisms
Salmonella typhi
TetrodotoxinMould spores
Formaldehyde
Ethidium bromide
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Consequences of Lapses
Personnel exposure
Contamination of research
Environmental release
Negative public perception
You or a colleague could be exposed...
Unvaccinated
student
Not handled by
victim
Genetically
modified strain
Stored in freezer,
not used for 5
years
You or a colleague could be exposed...continued
27 students, 1
teacher infected
Sheep anatomy
experiment
Lab disorganized,
unsanitary
Dean, party
secretary relieved
of duties
You or a colleague could be exposed...
Associate
Professor
Attenuated strain
Cause of death:
hemochromatosis
-induced iron
overload
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...your research could be exposed...
Cell culture contamination with mycoplasma
…as well as the environment...
>600 animals
culled
Loss of £3.5
millions per day
Lasted >20 days
Laboratory-Acquired Infections
(LAI)
LAI: Routes of Transmission
Direct contact (e.g. splash)
Inhalation
Ingestion
Parenteral inoculation
Indirect contact
Vectors: animal/insect bites
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LAI: Routes of Transmission
Inhalation - aerosols
Infectious airborne particles
Large droplets (100um) settle
Small droplets respirable
5-10 microns: upper
respiratory tract
<5 microns: lungs
LAI: Routes of Transmission
Inhalation - aerosols
Some aerosol generating procedures:
homogenizing, blending, grinding
vortexing, mixing, pipetting
centrifugation
opening snap-cap tubes
animal cage changing, necropsy
streaking agar plates
spills
LAI: U.S. and Canada 2000 - 2009
Under-reported (81 cases)
34 infections confirmed
3 deaths
Most common causes:
Inoculation (10)
Skin & mucous membrane contamination (3)
Unknown (21)
Public Health Agency of Canada 2009
What to do after accidental exposure?
1. Ask for help
2. Serious injury: dial
3. Needlesticks and cuts: wash with soap and
water
4. Splashes: flush eyes, mouth, nose
911 and Security
Downtown: 514-398-3000Macdonald Campus: 514-398-7777MNI: 55-555
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What to do after accidental exposure?
5. If required, seek medical attention:
Student Health Services
Downtown
M-F 9:00 – 16:00 Brown Bldg, Rm. 3300
514-398-6017
Macdonald Campus
M/W/Th 9:00 -15:30 Centennial Centre, Rm. CC1-124
T/F 13:00-15:30
514-398-7565
Nearest hospital or clinic
What to do after accidental exposure?
6. Notify supervisor
7. Complete Accident, Incident & Occupational
Disease Report Page 1: ‘victim’
Page 2: supervisor/safety representative
Submit to EHS (MNI Rm 778 if working at MNI)
8. Monitor for symptoms - most pathogens
have an incubation period
Accidental ExposureBloodborne pathogens
www.mcgill.ca/ehs/laboratory/ohs/bloodborne-pathogens
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Risk Groups & Containment Levels Risk Groups
Categorization of relative hazards of infective
organisms (4 levels)
Risk classification:
Pathogenicity
Infectious dose
Mode of transmission
Host range
Effective prevention, e.g. vaccine
Effective treatment, e.g. antibiotic, antiviral PEP
Risk Groups
Risk Group 1
• Low individual risk
• Low community risk
• Unlikely to infect healthy humans & animals
Standard molecular biology E. coli K12, Lactobacillus spp., Bacillus subtilis,
Micrococcus spp., Aerococcus spp.
Risk Groups
Risk Group 2
• Moderate individual risk
• Low community risk
• Treatment/prevention available
Listeria spp., Salmonella spp., Leishmania spp., Toxoplasma spp., Ascaris
spp., enteropathogenic E. coli, cell lines
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Risk Groups
Risk Group 3
• High individual risk
• Low community risk
• Cause serious disease, treatment/prevention usually available
Hantavirus, Bacillus anthracis, Yersinia pestis, Histoplasma capsulatum,
cultures of Hepatitis B or HIV
Risk Groups
Risk Group 4
• High individual risk
• High community risk
• Cause serious or lethal disease by casual contact
• Treatment/prevention notusually available
Marburg virus, Ebola virus, Herpesvirus simiae, Crimean-Congo hemorrhagic
fever
Saccharomyces cerevisiae
Clostridium difficile
Bovine Spongiform
Encephalopathy
Containment Levels
Minimum requirements for safe handling (4 levels) Operational practices
Safe work practices
Engineering, technical, physical: Location & access
Surface finishes & casework HVAC
Containment perimeter (windows, autoclave location, etc.)
Services (water, drains, gas, electricity, equipment)
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Risk Groups vs Containment levels
Risk groups agent-specific
Containment levels based on:
Risk groups
Potential for aerosol generation
Quantity
Concentration
In vitro vs in vivo
RG and CL not always the same
Containment Level 1
Containment Level 1
Physical requirements:
Basic design features
Operational requirements:
Work may be done on open bench top
BSC not required, may be used for sterility
Good microbiological practices (good hygiene)
Containment Level 2
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Containment Level 2
Additional physical requirements, e.g.
Limited access, signage, lockable doors
Resistant, non-absorptive surfaces (for disinfection)
Containment of aerosols, e.g.:
BSC
Centrifuges with sealed rotors or safety cups
Minimize environmental contamination:
Handwashing sinks
Decontamination facilities (autoclaves)
Containment Level 2+
Level 2 physical, Level 3 operational
Mandatory Standard Operational Procedure (SOP)
Entry, exit protocols
Personal protective equipment
Experimental procedures
Effective use of BSC
Safe use of lab equipment
Emergency procedures
Decontamination and waste disposal
Specific training required, must be documented
Containment Levels 3 and 4
Level 3
Respiratory protection
HEPA filtration of lab exhaust
Strictly controlled access
Level 4
Isolated facility with sealed perimeter
Positive pressure suits or Class 3 BSC
Effluent sterilization system
Risk Assessment
Biohazards
Contamination of research
Contamination of the
environment
Public perception
Laboratory associated infection
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Human Blood and Body Fluids
May contain bloodborne pathogens e.g.
HBV, HCV, Treponema (Syphillis), HIV
Use universal precautions
HBV vaccination
Cell Cultures
Risks based on:
Source of tissue
Primary vs. characterized cell line
ATCC screens for bacteria, fungi,
mycoplasma only
Presence of pathogens: Naturally
Via contamination, recombination or
transfection
Cell Lines
May contain viral DNA sequences:
HEK 293: Adenovirus-5
COS-1, COS-7, SVEC4-10: Simian virus-40
Daudi: Epstein-Barr virus
HS-5: Human papillomavirus-16
HeLa: Human papillomavirus
RWPE-1: Human papillomavirus
Cell Cultures
Contaminants can affect:
Growth of cells
Cell metabolism
Cell morphology and swelling
Genome structure
Recombination
Yield of virus production
…
Pauwels et al. 2007
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Recombinant Organisms
Case-by-case assessment based on
Risks associated with recipient & donor
Function of gene expressed in recombinant
Host range alteration
Replication capacity of recombinant
Capability to revert to wild type
Viability of host-vector system outside lab
Viral Vectors
Principles of Biosafety, PCIH 2004
Risk Group Virus
1Adeno-Associated virus
Baculovirus (insect cells)
2
Adenovirus
Herpesvirus (EBV)
Poxvirus (vaccinia, fowlpox)
2+ or 3Lentivirus (HIV, SIV)
Flavivirus
Animals
Risks based on type of work
being conducted
Animals can harbour infectious
organisms (natural or introduced)
Animal Use Protocols
Occupational Health Program
You are transfecting a containment level 1 cell line
with a containment level 2 vector.
What is the containment level of the transfected
cell line?
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You are culturing small volumes of recombinant
E. coli K-12 expressing constitutively a
mammalian oncogene for injection in mice.
What factors increase the risk of this project?
Definition: Biosecurity
Prevention of theft, misuse, intentional release
Specific for facility
Includes:
physical protection (e.g. card access, locks)
personnel suitability/reliability
pathogen accountability (e.g. inventory & tracking)
incident reporting, emergency response
Definition: Biosafety
Measures employed when handling
biohazardous materials to avoid infecting
oneself, others or the environment
Engineering controls
Reduce or eliminate
hazards at source
First and best strategy
Administrative controls
Engineering Controls
Handwashing facilities
Autoclave for waste decontamination
Cleanable surfaces
Adequate lighting
Biological safety cabinets
Examples:
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Engineering Controls
Filters for flasks, pipette tips & vacuum lines
Plasticware substituted for glassware
Gasketed blenders, homogenizers
Sealed centrifuge rotors, safety cups
Microincinerators………
Examples:
Administrative Controls
Limited access
Safety manual available and use encouraged
Insect/rodent control
Record-keeping
Medical surveillance
Training
Spill response plan
Inventory
Examples:
Administrative Controls
Inventory
Description (cell lines, organisms, vectors, etc.)
Supplier name, if applicable
Quantity and volume
Location
Date in storage
Date out of storage
Initials
Occupational illnesses in the US
BioWarfare Program (1943-1969)
Era YearsCase by million man
hours of lab work
Pre-BSC 1943-50 35
BSC 1951-59 9.1
Vaccines & BSC’s 1960-69 2
Wedum AG. J Amer Biol Safty Assn 1996;1:7-25
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Standard Microbiological Practices
Eating, drinking, applying cosmetics,
inserting and removing contact
lenses, storing food and utensils not
permitted
Minimize splashes and aerosols
Mouth pipetting prohibited
Standard Microbiological Practices
Cover open wounds, cuts, scratches with
waterproof dressing
Work on plastic-backed absorbent mat
Transport biohazards in leak-proof containers
Use 4-sided cart
Decontaminate work surfaces daily
Decontaminate waste
Standard Microbiological Practices
Handwashing
Effective in preventing infection
When to wash?
Before starting any manipulations
When hands are obviously soiled
Whenever gloves are removed
Whenever leaving the lab
Use blunt-end needles if possible
Do not bend, shear, recap
needles
Discard immediately after use
Separate contaminated and non-
contaminated
Use puncture-proof waste
containers
Standard Microbiological Practices
Safe use of sharps
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CFU recovered from operator’s gloves
Run Before pouringAfter pouring into
centrifuge tubes
1 0 48 000
2 0 48 000
3 0 7900
4 0 8900
Average 0 28 000
Suspension poured contained 109/ml. Flavobacterium (Burnett et al. 2005)
Standard Microbiological Practices
Safe pipetting
Use mechanical pipetting devices
Avoid mixing by vigorous suction & expulsion
Discharge liquid down side of container
Deliver as close as possible to contents
Never blow out last drop
Personal Protective Equipment
No shorts, open shoes, etc.
Use personal protective equipment
Fastened lab coat
Gloves
Eye protection
Respirators only if fit-tested
Contact EHS
McGill PPE Policy
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Sterilization & Disinfection Definitions
Sterilization
Destruction of all microorganisms, including spores
Disinfection
Destruction of microorganisms – may not kill spores
Decontamination
Destruction or reduction of pathogens to safe level
Microbial Resistance
Prions
Bacterial spores, protozoan cysts (Bacillus subtilis, Clostridium sporogenes)
Mycobacteria(Tubercle bacterium)
Non-lipid or small viruses (non-enveloped)(Poliovirus, Coxsackievirus, Rhinovirus)
Fungi(Trichophyton, Cryptococcus, Candida, Aspergillus)
Vegetative bacteria(P. aeruginosa, S. aureus, S. choleraesuis)
Lipid or medium-size viruses (enveloped)(Herpes simplex, HBV, HIV, CMV)
Sterilization & DisinfectionPhysical Methods
Heat: dry, moist
UV light
Infrared radiation (small metal & glass items)
Microwaves (biowaste, liquids, nonmetallics)
Filtration (heat- or chemical- sensitive liquids)
Gamma irradiation (chemical-, heat-, pressure-
sensitive items)
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Alcohols (70-85%)
Aldehydes (glutaraldehyde, formaldehyde)
Phenolic compounds (HG7)
Halogens (iodine, chlorine)
Quaternary ammonium compounds (“Quats”)
Oxidizing agents (hydrogen peroxide, ethylene oxide gas)
Sterilization & DisinfectionChemical Agents
Number and nature of microbes
Type of item to be treated
Purpose (disinfection vs sterilization)
Interactions with other chemicals
Amount of soil/organic matter
Selection CriteriaChemical Agents
Required contact time
Toxicity to cultures, humans, etc
pH, temperature, hardness of dilution water
Cost
Selection CriteriaChemical Agents
Organic Load
DisinfectantLog reduction of Listeria monocytogenes
TSB* Serum Milk
70% Ethanol 3 1 1
Sodium hypochlorite 5 1 1
Glutaraldehyde 3 3 1
Gauggel et al. 2004
Ex: Blood, sputum, milk, bedding, feed, manure
Proteins physically protect and stabilize many
microorganisms
*TSB: trypticase soy broth (soybean casein)
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Contact Time
Extending contact time may increase
effectiveness
DisinfectantLog reduction of Mycobacteria spp.
1 min 10 min 20 min
70% Ethanol 1 4 5
Sodium hypochlorite 5 5 5
Glutaraldehyde 1 2.5 5
Gauggel et al. 2004
www.mcgill.ca/ehs/biosafety/manual/
Autoclaves Autoclaves
Hazards:
Steam
Heat
High chamber pressure
Potential exposure to
biohazards
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Autoclaves
Mode of action: coagulation of
proteins
Effective conditions: Gravity displacement:121oC/15 psi, 15-90
mins
Pre-vacuum: 132oC/27 psi for 4-20 mins
Steam must contact material
For heat and moisture-stable material
Autoclaves
Gravity: Longer sterilization time, lower temp and pressure
than pre-vac
For dry materials (tubes, tips, pipettes, etc.) and
liquids
Pre-vacuum
Creates vacuum to pull steam into chamber, removes
air pockets
Shorter cycle, higher temp and pressure than gravity
Not all items can withstand temp or pressure
Autoclave
Do not autoclave:
Chemicals (solvents,
corrosives)
Radioactive materials
Bleach and other
chlorinated products
Plastic and glass not rated
for autoclave use
Primary & Secondary Containers
Use only approved
autoclave bags
Use secondary
containers to contain
potential spillage
Ensure that they are
autoclavable
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Primary & Secondary Containers
Best:
Polypropylene (PP) and polycarbonate (PC)
Borosilicate glass (Pyrex)
Stainless steel
Poor:
Polystyrene (PS), polyethylene (PE) and high
density polyethylene plastics (HDPE)
Regular glassware.
Solids:
pack for steam penetration, do not close bags/containers tightly
Do not overpack autoclave bags (max 75%)
Liquids: Loosen screw caps or use self-venting
caps to avoid rupture
Do not fill more than 2/3 full
Cap open containers loosely with foil
Packing & Loading
Clean drain screen
Avoid crowding or stacking
Items must not touch top or sides
Do not place items on autoclave floor
- use racks or secondary trays
Know which cycle to use (e.g. liquid
vs dry)
Packing & Loading
Wear insulated gloves or mitts
Ensure chamber pressure gauge
reads 0 psi
“Crack” open door, wait 5-10 mins
Liquids
Wear rubber apron in addition
Beware of bubbling (explosion hazard)
Remove load, allow to cool before
handling
Unloading
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Temperature Sensitive Tape
Indicates autoclaving temperature
reached
Markings appear at ~121°C
Markings do not prove successful
sterilization of contents
Biological Indicators
Use biological indicators, e.g.
Geobacillus stearothermophilus,
regularly (weekly) to prove that
effective cycle in use
Indicator spores killed after 15
mins at 121oC
Contact EHS for more information Virginia Tech Environmental
Health & Safety 2009
Spill Kit
Disposable protective clothing
Absorbent paper
Autoclavable container or bags
(if using autoclave)
Appropriate disinfectant (fresh)
Autoclavable (or disposable)
forceps, dustpan
Spill Response
Don’t panic
Remove contaminated clothing
Assess degree of contamination
& action needed
Allow aerosols to settle
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Spill Response
Don protective clothing
Cover spill with disinfectant-
soaked towel
Gently pour disinfectant
around perimeter
Work disinfectant toward
centre
Let sit 30 minutes
Spill Response
Wipe down walls, equipment,
etc.
Pick up broken sharps with
forceps, place in sharps
container
Pick up (forceps, dustpan)
absorbent materials and dispose
appropriately
Spill in BSC
Leave BSC running
Follow spill response procedure
If material has gone through grill:
Pour disinfectant through grill
Allow sufficient contact time
Open drain valve, collect liquid for
disposal
Video
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Biohazards ApplicationsMcGill Biohazards Policy*
``Prior to beginning work with
biohazardous materials, the responsible
person must complete and submit an
Application to Use Biohazardous
Materials to Environmental Health &
Safety for review and approval.``
*University Laboratory Safety Committee September 24, 2007
Biohazards Applications
Completed by PI/lab supervisor prior to
starting project
Required for research, teaching, diagnostic
activities using biohazards
Required for all activities, including Level 1
Submit to EHS for approval (~1 week)
Biohazards ApplicationsGeneral requirements for Master’s and
Doctoral theses*
“Research involving..........microorganisms, living cells, other biohazards .......... must have had the appropriate compliance certification. Copies of any certificates of compliance must be provided to the Thesis Office at the time of submission”
*Graduate and Postdoctoral Studies: http://www.mcgill.ca/gps/thesis/guidelines/general-requirements
Environmental Health & Safety
Contacts
Telephone: 514-398-4563
Fax: 514-398-8047
Email: [email protected]
Website: www.mcgill.ca/ehs