Biosafety Training
Tina Preseau* & Lise Griffith
*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 Spill Response Biomedical Waste Regulations
BIOSAFETY
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 Vice-President (Research) Committees University Services (ORM,
HR, PRS, PS) Deans, Chairs, Principal
Investigators, Employees, Students
Manager of Biological Containment Suite
EXTERNALLY 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
Route of Transmission
Susceptible Host
Infection Source
Percutaneous inoculation Inhalation of aerosols Contact of mucous
membranes Ingestion
Cultures and stocks Research animals Specimens Items contaminated
with above
Immune system Vaccination status Age
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
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
CerebrospinalAmnioticSynovial
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 non-infective …… 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
Risk Grou
p
Individual Risk
Community Risk
Containment
Level
Examples
1 Low Low Level 1 E.coli, B. subtilis, S. aureus, Trichoderma reesei
2 Moderate
Limited Level 2 Streptococcus & Salmonella spp, Measles, Adenoviruses, Hepatitis A, B & C, Influenza
3 High Low Level 3 Bacillus anthracis, Mycobacterium tuberculosis, HIV, Yellow fever virus
4 High High Level 4 Lassa virus, Ebola virus, Marburg virus
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
Conventional Agents
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 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.)
Where can we find these?
Earth Sciences
Earth Sciences
EngineeringCivil
Chemical
EngineeringCivil
Chemical
ChemistryChemistry Medicine/ Nursing
Medicine/ Nursing
HumanKinetics
HumanKinetics
Bio-EngineeringBio-Engineering
BiologyBiology
UniversityUniversity
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 SurveillanceTraining & 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.
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 3
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
Laminar flow hoodsVertical or horizontal laminar flowHEPA filtered air (intake only)Product protection only
Biological Safety CabinetHEPA filtered laminar air flowExhaust Personnel, environment & product protection
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
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)
Working safely in a BSC
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 follow-up
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 follow-up
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 follow-up
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, 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.
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.pdf)
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 fill out the Autoclave User Questionnaire
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)
Disinfection: What to use for my agent?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
VirusesEnveloped (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
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)
With H2O 1:10
In compliance with Sewer use by-laws*
Special Waste
EtBr Toxins Recombinant DNA Contact ORM
regulations
Key Regulated Activities
Purchasing & Receiving of Biological AgentsPHAC, CFIA, Environment CanadaInventory Records
Transportation/TransferTransport 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 & 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
TDG
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