Download Biosafety At the University of Ottawa

BIOSAFETY
TRAINING
Tina Preseau*
& Rita ToussaintArchambault
*Office of Risk Management
Human Resources - Occupational Health
Disability & Leave
COURSE OUTLINE








Introduction
Laboratory Associated Infections
Blood-borne Pathogens
Classification of Biohazards
Infection/Biohazard Control
BIOSAFETY
Spill Response
Biomedical Waste
Regulations
INTRODUCTION
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 product.
Blood and body fluids, as well as tissues from humans and
animals.
Transformed cell lines and certain types of nucleic acids .
WHAT IS BIOSAFETY?
 Measures employed when handling biohazardous
materials to avoid infecting oneself, others or the
environment.
 Achieved through;
 Administrative Controls
 Engineering Controls
 Personal Protective Equipment
 Practices and Procedures
WHAT IS BIOSECURITY?
 Measures employed to protect biohazardous
materials, or critical relevant information, against
theft or diversion by those who intend to pursue
intentional misuse.
 Achieved through;
 Physical barriers
 Psychological barriers
 Monitoring Activities
 Personnel Clearance
WHO ARE THE STAKEHOLDERS?
INTERNALLY
EXTERNALLY
 Vice-President (Research)

 Committees
 University Services (ORM, HR, 
PRS, PS)

 Deans, Chairs, Principal
Investigators, Employees,

Students

 Manager of Biological

Containment Suite
Public Health Agency of
Canada
Canadian Food Inspection
Agency
Environment Canada
Transport Canada
Ontario Ministry of Labour
Emergency Response
Personnel
 Suppliers & Contractors
 Community
KEY SERVICES
 Office of Risk Management
 Training
 Interface with Regulatory Bodies
 Biosafety Program
certifications
training
procedures
inspections
contingency planning
accident/incident follow-up
KEY SERVICES
 HR (Occupational Health, Disability and Leave)
 Medical surveillance
 Immunizations
 Medical Follow-up
 Interface with Workplace Safety and Insurance Board
WHY ARE WE CONCERNED?
 Potential for acquiring a laboratory-associated
infection (LAI)
 Contamination of the environment
 Contamination of research
 Public perception
LABORATORY ASSOCIATED
INFECTIONS
LABORATORY ASSOCIATED INFECTIONS
Infection Source




Susceptible Host



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
LAIS

Only 20% causative or defined event



80% of which are caused by human factors
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
LAIS
WHO
WHAT
WHERE
WHEN
HOW
Researcher
SARS
Taiwan
December 2003
Microbiologist
West Nile Virus
United States
August 2002
Laceration
Laboratory
Worker
Meningococcal
Disease
United States
2000
Aerosol?
Laboratory
worker
Vaccinia virus
United States
2004
Many ways
BLOOD-BORNE PATHOGENS
BLOODBORNE PATHOGENS (BBP)
 Sources




Blood
Semen
Vaginal Secretions
Other Bodily Fluids
Cerebrospinal
Amniotic
Synovial
 Tissue Cultures
 Organ Cultures
 Infected Experimental Animals
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
SPECIFIC EXAMPLES OF BBPS
Hepatitis B
Hepatitis C
HIV
ISSUES TO CONSIDER







Symptoms
Mode of transmission
Incubation period
Survival outside host
Communicability
Immunization
Prophylaxis / Treatment
IF AN EXPOSURE OCCURS




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.rh.uottawa.ca/00_main/index_f.asp
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 noninfective …… treat as infectious!
BIOHAZARD
CLASSIFICATION
BIOHAZARD CLASSIFICATION






Conventional Agents
Recombinant DNA
Tissue Culture
Animal Work
Anatomical Specimens
Unconventional Agents
Class D, division 3 of WHMIS
(Poisonous and Infectious Material - Biohazardous
Infectious Material)
BIOHAZARD CLASSIFICATION
 Organisms are categorized into a group base on the
particular characteristics of each organism, such as
 Pathogenicity





Infectious dose
Mode of transmission
Host Range
Availability of effective preventive measures
Availability of effective treatment
BIOHAZARD CLASSIFICATION
 Organisms are categorized base on the measures
required for handling each organism safely in a
laboratory setting, such as
 Engineering Requirements
 Operational Requirements
 Technical Requirements
 Physical Requirements
CONVENTIONAL AGENTS
Risk
Group
Individual
Risk
Community
Risk
Containment
Level
1
Low
Low
Level 1
E.coli, B. subtilis, S. aureus,
Trichoderma reesei
Level 2
Streptococcus & Salmonella
spp, Measles, Adenoviruses,
Hepatitis A, B & C, Influenza
Level 3
Bacillus anthracis,
Mycobacterium tuberculosis,
HIV, Yellow fever virus
Level 4
Lassa virus, Ebola virus,
Marburg virus
Unlikely to cause disease
in healthy workers or
animals
2
Moderate
Limited
Rarely cause serious
human or animal disease
3
High
Low
May cause serious disease
4
High
High
Likely to cause very
serious disease
Examples
RECOMBINANT DNA
Genetic Engineering = in vitro incorporation of genetic
material from one cell into another
 In Canada the level of risk depends on source of
DNA, vector and host.
The Office of Risk Management will assist the
investigator in this determination.
TISSUE CULTURE
 Have the potential to contain pathogenic organisms
 In general;
Human & non-human primate, and mycoplasma-containing cell
lines
Level 2
Others
Level 1
A detailed risk assessment should be
undertaken when using a new cell line.
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
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.
UNCONVENTIONAL PATHOGENS
 TSE
prion
diseases;
lethal
neurodegenerative conditions
transmissible
 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.)
WHERE CAN WE FIND THESE?
Biology
Earth
Sciences
Bio-Engineering
University
Engineering
Human
Kinetics
Civil
Chemical
Chemistry
Medicine/
Nursing
INFECTION/BIOHAZARD
CONTROL
INFECTION/BIOHAZARD CONTROL




Administrative Controls
Engineering Controls
Personal Protective Equipment
Practices and Procedures
ADMINISTRATIVE CONTROLS
 Program based, information and methods to
minimize risk of exposure:






Risk assessment
Medical Surveillance
Training/Education
Resources
Inspections
Signs & Labeling
ADMINISTRATIVE CONTROLS
Risk Assessment
 Will determine type of containment, procedures, and safety
equipment required
 Responsibility of users, additional assistance is available from
the ORM
 Consider areas such as; experimental design, procedures to be
employed and personal experience/knowledge, etc.
 Know and understand the various characteristics of the agent(s)
you are working with. (Material Safety Data Sheets and
suppliers or manufacturers)
ADMINISTRATIVE CONTROLS
Medical Surveillance
Training & Education
 WHMIS
 Lab specific policies and procedures
 Biosafety training
Resources




ORM web site, Biosafety page
Faculty web sites
Biosafety Manual
Training Videos
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.
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.
ENGINEERING CONTROLS
 Technology based, reduce or eliminate exposure to
hazards by changes at the source of the hazard.
 Containment:
 Types: Primary and Secondary
 Containment levels
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, etc.
Effectiveness of control is based on the
integrity of the containment.
SECONDARY CONTAINMENT
 Protects the environment external to the laboratory
from exposure
 Includes facility design and operational practices
CONTAINMENT LEVEL 1
 Basic laboratory
 Requires no special design features
 Biosafety cabinets are not required and work may be
performed on the open bench.
CONTAINMENT 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.
CONTAINMENT 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.
CONTAINMENT 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.
BIOLOGICAL SAFETY CABINETS
 Effective means of primary 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.
BIOLOGICAL SAFETY CABINETS
Biological Safety Cabinet
Laminar flow hoods
HEPA filtered laminar air flow
Exhaust
Personnel,
environment
&
product protection
Vertical or horizontal laminar
flow
HEPA filtered air (intake only)
Product protection only
VS
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
WORKING SAFELY IN A BSC
While using the cabinet:
 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
WORKING SAFELY IN A BSC
WORKING SAFELY IN A BSC
After using the cabinet:




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
WORKING SAFELY IN A BSC
Maintenance:




Before and after each use - 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)
PERSONAL PROTECTIVE EQUIPMENT
(PPE)
 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
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
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
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.
PPE
Eye & Face Protection
 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.
PRACTICES AND PROCEDURES





General Safety Guidelines
Good Microbiological Practice
Handwashing
Suspicious Packages
Specific Procedures






Centrifuges
Needles & Syringes and other sharps
Pipettes
Blenders, Grinders, Sonicators & Lyophilizers
Inoculation Loops
Cryostats
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 be known for all labs.
b i o s a f e t y
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
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
SUSPICIOUS PACKAGES
Unopened





Do not open and do not shake
Place in secondary container or cover
Inform others of the situation
Clear the room and section off the area
All individuals who may have come into contact with the
material must wash their hands
 Call Protection Services and wait for their arrival
 List all the individuals present in the room or area when the
package arrived. Give this list to Protection Services for followup
SUSPICIOUS PACKAGES
Opened
 Contents Intact





Do not manipulate contents further
Cover the package
Inform others of the situation
Clear the room and section off the area
All individuals who may have come into contact with the
material must wash their hands
 Call Protection Services and wait for their arrival
 List all the individuals present in the room or area when the
package arrived. Give this list to Protection Services for followup
SUSPICIOUS PACKAGES
 Contents not intact (spilled)




Do not try to clean up the spill
Gently cover the spill
Inform others of the situation
All individuals who may have come into contact with the
material must wash their hands
 Call Protection Services
 Remove heavily contaminated clothing (place in bag) and
shower using soap and water
 List all the individuals present in the room or area when the
package arrived. Give this list to Protection Services for followup
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.
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
PIPETTES
 Mouth pipetting is prohibited.
 Never force fluids out.
 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.
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
INOCULATION LOOPS
 Sterilization in an open flame may create aerosols
which may contain viable microorganisms.
 Use a shielded electric incinerator
 Shorter handles minimize vibrations
 Disposable plastic loops are good alternatives
CRYOSTATS
 Wear gloves during preparation of frozen sections
and heavy gloves when accessing the cryostat.
 Decontaminate frequently (100 or 70% Ethanol)
SPILL RESPONSE
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.
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 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
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.p
df)
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 Services)
BIOMEDICAL WASTE
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)
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)
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)
AUTOCLAVES
Items that CAN NOT be autoclaved:





chemicals (flammables, oxidizers, phenols, acids, alkalides)
chemotherapeutic or radioactive waste
Bleach (or other chlorinated products)
certain kinds of plastics
sharps (not at the University of Ottawa)
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
SAFE USE OF AUTOCLAVES
 Many autoclaves are now run by dedicated staff,
however, if you are operating an autoclave;




Learn how to use it!
Ensure PPE is worn
Recognize acceptable material and packaging
Proper loading and unloading
 All users/operators must take the autoclave training
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)
WHAT TO USE FOR MY AGENT?
Vegetative bacteria (E.coli,
Viruses




Enveloped (HIV, Herpes)
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




2% domestic bleach
75% Ethanol
Quaternary ammonia
6% formulated Hydrogen peroxide*
Non enveloped (Hepatitis,




Adenovirus)
10% domestic bleach
6% formulated Hydrogen peroxide*
Gluteraldehyde
Formaldehyde
WASTE MANAGEMENT
Discarded biological material from teaching, clinical
and research laboratories and operations is
biomedical waste. Biomedical waste includes but is
not limited to;





Animal waste
Biological laboratory waste
Human anatomical waste
Human blood and body fluid waste
Sharps
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.
WASTE DISPOSAL
Biomedical Waste (untreated)
WASTE DISPOSAL
Biomedical Waste (treated)
compliance with
* InSewer
use by-laws
With H2O 1:10
SPECIAL WASTE




EtBr
Toxins
Recombinant DNA
Contact ORM
REGULATIONS
KEY REGULATED ACTIVITIES
 Purchasing & Receiving of Biological Agents
 PHAC, CFIA, Environment Canada
 Inventory Records
 Transportation/Transfer
 Transport Canada- TDG
 All Agencies (provincial and federal) emphasize and
expect Biosecurity
PURCHASING
 Importation permits required by Public Health
Agency Canada or CFIA for certain agents
 US restrictions
 Ensure you meet all criteria and have all pertinent
documentation
INVENTORY
 What material is presently being used and/or stored




Location
Expiry date
Use log book for remaining amount
MSDS’s
 Mandatory
SHIPPING AND RECEIVING
 Transportation of Dangerous Goods Act: Class 6.2 of
(Infectious Substances)
 PHAC/CFIA restrictions
 Ensure;





Proper classification
Proper packaging
Proper labeling
Proper documentation
Import/Export Permits
TRANSPORTATION OF DANGEROUS
GOODS








Pre-approved
Authorized Individuals
Lead time (International Regulations….)
Appropriate Scheduling (Holidays, Weekends)
Transportation within the building
Between lab to lab
Colleague to Colleague
Between Institutions
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
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 Public Health Agency of Canada, Canadian Food
Inspection Agency, Ministry of the Environment or
Transport Canada after you
CONCLUSIONS





Biohazards - microorganisms, blood and body
fluids, tissues and tissue culture
Biosafety - ensuring that individuals and the
environment are not infected
Biosecurity - used in the context of protecting
dangerous pathogens and toxins against intentional
removal
Everyone within the University community is
responsible
With proper knowledge, planning and care, a
biological exposure is avoidable.
BIOSAFETY WEBSITE
http://www.uottawa.ca/services/ehss/biosafety.htm