BIOSAFETY TRAINING Pierre Laflamme May 16, 2012 Uottawa2k9

BIOSAFETY TRAINING Pierre Laflamme May 16, 2012 Uottawa2k9
Human Resources - Occupational Health Disability & Leave Pierre Laflamme May 16, 2012 Office of Risk Management, Environmental Health and Safety

COURSE OUTLINE BIOSAFETY Introduction Laboratory Associated Infections
Blood-borne Pathogens Classification of Biohazards Infection/Biohazard Control Spill Response Biomedical Waste Regulations BIOSAFETY -Double Click

INTRODUCTION

What is a BIOHAZARD? Any organism or its toxin that is known to cause disease in humans or animals or that is a potential hazard to humans, animals or the environment. Examples: Microorganisms such as viruses, bacteria, fungi, and parasites and their toxins. Blood, body fluids and tissues from humans and animals. Transformed cell lines

What is BIOSAFETY? The combination of measures employed when handling biohazardous materials to: Protect personnel from exposure to infectious agents Prevent environmental contamination Provide an environment for high quality research while maintaining a safe work place Comply with applicable federal, provincial and municipal requirements The combination of measures employed when handling biohazardous materials to: Protect personnel from exposure to infectious agents Prevent environmental contamination Provide an environment for high quality research while maintaining a safe work place Comply with applicable federal, provincial and municipal requirements

How is BIOSAFETY achieved?
Administrative controls Training, Inspections, Permits and Certificates Engineering Controls Biological Safety Cabinets, Ventilation Personal Protective Equipment Practices and Procedures Medical Surveillance Immunization when necessary How is biosafety achieved? Through Administrative controls: -training -Inspections -Issuing permits and certificates Engineering controls: - Isolation or enclosure of an operation - Biological Safety Cabinets - Ventilation - Substitution for a less toxic material Peronal Protective Equipment -Gloves, goggles, lab coats, face shields Practices & Procedures: -Good practices -SOP -Guidelines -policies - Medical Surveillance And Immunization when necessary

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. Measures employed to protect biohazardous materials, or critical relevant information, against theft or diversion by those who intend to pursue intentional misuse.

How is BIOSECURITY achieved?
Physical barriers Buildings, doors, locks, key card access Psychological barriers Security personnel, cameras Monitoring Activities Patrols, monitoring by support staff Personnel Clearance Access to authorized personnel only physical barriers - structural design to increase the level of security to reflect the transition from general public to laboratory zones - departmental design to control traffic flow patterns - control access: locks, key card access, self-locking doors psychological barriers - obvious presence of identifiable security personnel - obvious presence of security culture - use of cameras, mirrors, mirrored domes (90o,180o, 360o) and other monitoring tools Monitoring activities - patrols of facilities by security personnel - support staff monitoring of departments (secretariat staff located at entrance of departments to monitor access, all staff questioning strangers) - key control programs) Personnel clearance - identifying and restricting access to only authorized personnel personnel clearance identifying and restricting access to only authorized personnel Growing concern about possible use of biologicals as weapons. Theft of any item Be aware of what is going on in your lab Report any strange persons, missing material.

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 Committees (Biosafety, Health, Safety & the Environment) Emergency response personnel (i.e. fire department) Suppliers & Contractors (ATCC, Waste carrier, Biosafety Cabinet certification/repairs) Community (OHSC, general public)

University KEY SERVICES
Office of Risk Management, Environmental Health and Safety Certificates and Permits Training Procedures (Waste disposal) Risk Identification (Inspections) Emergency plans Accident/Incident follow-up Certification (Biohazardous Materials Use) Permits (Importation permits) Training (EHSS & P.I.) Procedures (sharps, gloves, waste, etc.,) Inspections (Risk identification) Emergency Response / Contingency Planning Accident /Incident follow-up Add brief note about role of PRS and PS.

University KEY SERVICES
HR (Occupational Health, Disability and Leave) Medical surveillance Immunizations Medical Follow-up Interface with Workplace Safety and Insurance Board Certification (Biohazardous Materials Use) Permits (Importation permits) Training (EHSS & P.I.) Procedures (sharps, gloves, waste, etc.,) Inspections (Risk identification) Emergency Response / Contingency Planning Accident /Incident follow-up Add brief note about role of PRS and PS.

WHY ARE WE CONCERNED? Potential for acquiring a laboratory-associated infection (LAI) Contamination of the environment Contamination of research

LABORATORY ASSOCIATED INFECTIONS

LABORATORY ASSOCIATED INFECTIONS
Infection Source Microorganisms Cells and tissues Blood and body fluids Any items contaminated with the above Susceptible Host Route of Transmission Immune system Vaccination status Age Percutaneous inoculation (needles and bites) Inhalation of aerosols Contact of mucous membranes Ingestion

LAIs Only 20% of LAIs are related to a causative or defined event
80% are caused by human errors 20% are caused by equipment failure Types of accidents causing LAIs Spills and sprays Needles Sharp objects and broken glass Bites or scratches from animals Studies have shownn that around 80% of laboratory acquired infections are due to unknown or unrecognized causes. Therefore, only 20% of LAIs are related to a causative or defined event. From these, around 80% were caused by human errors while about 20% were caused by equipment failure. Accidents may occur when experiments are performed to fast, when steps in experimental protocols are switched, forgotten or the timing is wrong, when there is a mistake in the quantities used, or when a combination of the above, in addition to other factors, occurs. Previously , oral aspiration through pipettes was the prime cause of laboratory-acquired infections. Now the top 4 accidents causing LAIs Spills and sprays Needles Sharp objects and broken glass Bites or scratches from animals

BLOOD-BORNE PATHOGENS Human Resources Occupational Health Disability & Leave

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

FACTS ABOUT SOME BBPs Pathogen Hepatitis B Hepatitis C HIV
Pathogenicity 2 major forms: asymptomatic symptomatic non-specific symptoms acute infection: non-specific “flu-like”, “mono-like” symptoms Mode of transmission percutaneous/ permucosal exposure to body fluids, organs indirect contact with contaminated lab items (e.g. needles, syringes) Percutaneous exposure to contaminated blood (102 – 103 infectious particles/ mL) intravascular inoculation (e.g. transfusion) of contaminated blood products direct exposure of virus to mucosa (oral, rectal, vaginal) Incubation period usually: days average: days 2 weeks - 6 months most commonly 7 – 10 weeks chronic infection may persist up to 20y before onset of cirrhosis variable generally months between time of infection to development of detectable Ab’s time from HIV infection to diagnosis of AIDS ranges from < 1y to 15y or more Survival outside host Survives in dried blood for long periods (weeks) stable on environmental services for at least 7 days at 25 °C not known similar to hep B (survives in dried blood for long periods…weeks) viable in blood in RT for 42d Cell-free HIV dried on glass coverslips in 10% serum can survive forlonger than 7d, depending on initial titre Laboratory-acquired infections (LAIs) MOST FREQUENTLY occurring LAI lab workers incident rate: 7X > general population health care workers handling blood at higher risk to infection low (e.g. as of 2001, total of 57 cases of documented occupationally acquired HIV among US health care workers [Source:

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, ext

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

Conventional Agents – Risk Groups 1 to 4 Recombinant DNA Tissue Culture Animal Work Anatomical Specimens Unconventional Agents Class D, division 3 of WHMIS (Poisonous and Infectious Material - Biohazardous Infectious Material)

Conventional agents are categorized into risk groups based on their particular characteristics such as: Pathogenicity (Infectivity of the agent - disease, severity, mortality) Infectious dose Mode of transmission (Airborne, Ingestion, Parenteral) Host Range (Animal or human pathogen Availability of effective preventive measures (PPE) Availability of effective treatment Pathogenicity: How infectious is the agent (disease evidence, severity, mortality) i.e. S. aureus versus Ebola Infectious dose: Can vary from one to hundreds of thousands of units. One’s immune system is directly related to the susceptibility to disease. Interaction between microorganisms and host = challenge Mode of transmission: Airborne, ingestion, parenteral Consider aerosols when mode of transmission is unknown. Host Range: Animal pathogen Human pathogen Availability of effective preventative measures: PPE, SOP, guidelines, training Availability of effective treatment: Effective prophylaxis, medical surveillance

Conventional agents are categorized based on the measures required for handling each organism safely in a laboratory setting, such as: Operational Requirements (Protocols, Biological safety cabinets, Lab safety practices) Engineering Requirements (Maintenance, certification, repairs) Physical Requirements (PPE) Engineering requirements: Maintenance, repairs, certification Operational requirements: Protocols, SOP, basic (general) lab safety practices, safety cabinets…. Technical Requirements: Physical requirements:

CONVENTIONAL AGENTS 1 Low Level 1 2 Moderate Limited Level 2 3 High
Risk Group Individual Risk Community Risk Containment Level Examples 1 Low Level 1 Escherichia Coli 2 Moderate Limited Level 2 Bacteria: Streptococcus and Salmonella Viruses: Adenovirus, Hepatitis A, B & C, Influenza 3 High Level 3 Bacteria: Bacillus anthracis and, Mycobacterium tuberculosis Virus: HIV 4 Level 4 Viruses: Ebola virus and Lassa virus Unlikely to cause disease in healthy workers or animals Rarely cause serious human or animal disease May cause serious disease 1 - Unlikely to cause disease in healthy workers or animals. 2 – Rarely cause serious human or animal disease 3 – May cause serious disease 4 – Likely to cause very serious disease Likely to cause very serious disease

RECOMBINANT DNA Recombinant DNA technology or genetic engineering:
in vitro incorporation of genetic material from one cell into another or from one organism to another In Canada the level of risk depends on the source of DNA being transferred the vector the host The Office of Risk Management will assist the investigator in this determination. each case needs to be assessed on its own merits. In general, if none of the components of the genetic manipulation presents any known hazard, and none can be reasonably foreseen to result from their combination, then no biohazard restrictions are needed. However, if one of the components is potentially hazardous, a risk level appropriate for the known hazard is assigned and modified as required.

TISSUE CULTURE Mammalian cell lines have to be considered infectious as they may contain infectious agents Untransformed mammalian cell lines - Risk Group 1 MCF-7 (Human breast carcinoma cell line) NIH 3T3 (Mouse fibroblast cell line) Transformed mammalian cell lines – Risk Group 2 HeLa (Human - contains papovavirus) All mammalian cell lines should be handled in a Level 2 Containment. Cell lines may contain pathogens (naturally or through contamination by adventitious agents, transformation or recombination). Can be contaminated with bacteria, fungi, mycoplasma and prions.

ANIMAL WORK Animals can harbour infectious agents (naturally or introduced) which can be transmitted to humans Scratches, bites, aerosols (needles and litter changes), body fluids and excrements 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 Viruses can easily replicate in this environment, Animals at increased risk of exposure  humans are also at increased risk Scratches Bites Aerosol generation (syringes, litter changes) Faecal matter Other exudates and bodily fluids

ANATOMICAL SPECIMENS All specimens should be considered infectious due to potential presence of infectious agents It’s 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. Blood, organs, tissues – BBP’s Spinal sample, brain tissue - prions

UNCONVENTIONAL PATHOGENS
Includes unconventional agents, slow viruses and prions causing progressive neurological diseases Creutzfeld-Jakob disease in humans, Mad Cow Disease, Scrapie in sheeps and goats Resistant to destruction by chemical and physical procedures that normally inactivate viruses Precautions: Handle tissues as Risk Group 2 or higher Handle formalin-fixed tissues and paraffin-embedded blocks as if still infectious Follow up-to-date disinfection protocols. Transmissible Spongiform Encephalopathy rare diseases widely believed to be caused by unconventional, virus-like infective agents composed largely or entirely of a misshapen host protein. Biosafety level assigned should be established using a risk assessment that accounts for the nature and host range of the agent, as well as the nature of the procedures and concentration and quantity of agent. Precautions Handle tissue as Risk Group 2 or higher Handle formalin-fixed tissues and paraffin-embedded blocks as if still infectious Follow up-to-date disinfection protocols. Bovine Spongiform Encephalopathy Scrapie in sheep and goats. CWD in deer and elk

WHERE ARE BIOHAZARDS FOUND?

INFECTION/BIOHAZARD CONTROL

INFECTION/BIOHAZARD CONTROL
Administrative Controls Engineering Controls Personal Protective Equipment Practices and Procedures Engineering Controls – change at source of hazard, don’t rely on skill or vigilance. Equipment such as BSC, clean benches, ventilation. Administrative Controls – Behaviours or actions that are dictated by regulation or policy. Risk assessment, Biosafety Manual, signs and labels, inspections, training/education, medical surveillance.

INFECTION/BIOHAZARD CONTROL 1. ADMINISTRATIVE CONTROLS

ADMINISTRATIVE CONTROLS
Administrative procedures to minimize the risk of exposure: Risk assessment Training/Education Resources Inspections Permits and Certificates Medical Surveillance Signage The information and resources are provided to you but the onus is on you to use them properly in order to mitigate risk.

Risk Assessment Will determine for each biohazard: Risk group Containment level Operational practices Safety measures Responsibility of users Know and understand the various characteristics of the agent(s) you are working with. (Material Safety Data Sheets and suppliers or manufacturers information sheets) Experimental design – actively growing viral stock, aerosol production, manipulations planned, concentration of agent to be used, quality control of sample (established cell culture vs unknown) Routine procedures vs new protocol

Medical Surveillance Training & Education WHMIS Lab specific policies and procedures Biosafety training, Laboratory safety training Resources ORM web site, Biosafety page Faculty web sites Biosafety Manual Training Videos National and International Biosafety Guidelines National/International guidelines University Policies and Procedures Specific biosafety topics, fundamental issues MSDS’s

Inspections Routine self-inspections Biosafety Inspection Checklist available on-line In addition, ORM and Health, Safety and Risk Officers will inspect labs to ensure compliance with regulations/ guidelines and provide feedback. Risk – contaminated specimens – poor science – LAI’s

Signs & Labeling Biohazard warning signs must be posted on doors to rooms where biohazardous materials are used and/or stored. Biohazard labels should be placed on containers, equipment and storage units used with biological agents. Hazard awareness, one of first questions on the questionnaire is whether or not the lab is informing others of the hazards in that lab. Incubators, freezers, fridges, waste containers

INFECTION/BIOHAZARD CONTROL 2. ENGINEERING CONTROLS

ENGINEERING CONTROLS Technology based
Reduce or eliminate exposure to hazards Containment: Types: Primary and Secondary Levels: 1, 2, 3 and 4 For effective containment, users must be aware of the potential hazards be trained Handle the material safely by adhering to standard microbiological practices and techniques doesn’t rely on skill or vigilance For effective containment, people must be aware of potential hazards, be trained and adhere to established practices and techniques.

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. Petri dish, vial Biological safety cabinets animal caging equipment *Ensure it works!

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 – safe methods for managing infectious agents in the laboratory environment where they are being handled and maintained.

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 with
primary barriers to protect the individual secondary barriers to protect the environment Requires type II or type III biosafety cabinets All staff must undergo specific training on the agents used, PPE, equipment, waste management as well as practices and procedures. Specialized design and construction with: primary barriers to protect the individual secondary barriers to protect the environment Must undergo annual performance, testing and verification. Requires type II or type III biosafety cabinets. Also, all centrifugation must be performed in closed trunnion cup centrifuges All staff must undergo specific training on the agents used above the general hazard information, PPE, equipment, waste management as well as practices and procedures. PPE is very specific, including solid front clothing and dedicated footwear, for use only within the facility Written protocols must be provided and posted A medical surveillance program must be in effect A reporting system for accidents must be in place -

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 The worker is completely isolated from infectious material. The highest level of containment available where all manipulations pose a high risk of exposure and infection There is only one level 4 facility in Canada (Canadian Centre for Human and Animal Health in Winnipeg, Man.) Design specifications are extremely stringent The worker is completely isolated from infectious material. Entry and exits are through airlocks Showers are mandatory All work is performed in class III cabinets or class I or II cabinets with a positive pressure suit.

BIOLOGICAL SAFETY CABINETS
Primary containment Minimize contact between operator and the infectious agent by the use of directional airflows There are 3 main classes of cabinets (I, II, III) which provide various levels of protection. Class II and III BSC contain HEPA filters which remove particles (min 0.3 microns) from supply and exhaust air with 99.97% efficiency . BSC should be located away from doors and high traffic areas Higher classed cabinets provide more protection. Level 1 provides personnel and environment protection, Level 2 provides personnel, environment as well as product protection. There are 4 types of level 2 cabinets, each with different air circulation configurations. Level 3 cabinets provide the most protection, the user works through attached gauntlets and there is a pass through, they are used for BSL level 3 & 4 agents. HEPA= High Efficiency Particulate Air

BIOLOGICAL SAFETY CABINETS
Laminar air flow and HEPA filtered exhaust air Personnel and environment protection HEPA filtered supply air & product protection with Class II + III To be used with biohazards Laminar Flow Hoods or Clean Air Benches Vertical or horizontal laminar flow HEPA filtered supply air only Provide product protection only Not to be used with biohazards Laminar flow clean bench can be used for certain clean activities but not with cell culture material, drug formulations or manipulating infectious material. Horizontal or Vertical. Often used in hospitals for preparation of IV drugs. And, different than fume hoods which are designed to protect personnel by removing chemical fumes and aerosols away from the work area. - BSC’s provide protection for various combinations of product, personnel and environment. VS

WORKING SAFELY IN A BSC Step 1 Before using the cabinet:
Turn off UV lamp; turn on fluorescent lamp Ensure BSC is certified Disinfect work surfaces with appropriate disinfectant Place essential items inside cabinet Allow the blower to run for 5-10 min before work Certification sticker, with date of last certification, should be located on the front of the BSC. BSCs must be certified every year. When the window is closed: The motor will keep working, electrostatic charge (make sure your BSC is designed for that!!!) You keep the working volume sterile You save the HEPA filter life The cabinet is quieter UV lamp can be used safely Efficiency of the UV lamp depends on Age of the tube Exposition time Distance between the UV lamp and the microorganisms Proper regular decontamination including, window and side panels

WORKING SAFELY IN A BSC Step 2 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 Prepare everything you need ahead of time so you are not moving in and out of the hood. Rapid and excessive movements in the hood affect air flow. Open flame is not recommended unless there is absolutely no other method to perform what you want (i.e. can micro-burners be used). Contact the ORM before deciding to use open flame.

WORKING SAFELY IN A BSC Step 3 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 15 min.

WORKING SAFELY IN A BSC Maintenance:
Before and after each use - Wipe down work surfaces Weekly - Clean UV lamp Monthly - Wipe down all vertical surfaces Annually - VerifyUV lamp intensity Decontamination with formaldehyde gas (by ORM) - Certification (by ORM) * UV light is only effective as long as the light is well maintained. Dark spots signify a loss of effectiveness.

INFECTION/BIOHAZARD CONTROL 3. PERSONAL PROTECTIVE EQUIPMENT

PERSONAL PROTECTIVE EQUIPMENT
PPE is an important line of defence Responsibility of both the user and the supervisor to ensure that PPE is worn PPE is specific to each containment level Examples of PPE? Benefits; possible prevention of exposure, potential minimization of risk that exposure can occur, compliments existing controls to enhance personal protection. PPE protects only the individual wearing it

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 the individual is well trained Ensure PPE is removed before leaving the lab PPE is often specific for each containment level

Closed toe and heel shoes only. No sandals!
PPE Footwear Closed toe and heel shoes only. No sandals! Shoe coverings are worn in some higher containment labs and animal facilities. Closed toed shoes protect against spills and injuries from dropped sharps. Elastic cuffs help prevent spills and contamination

PPE Lab Coats/Gowns Protect street clothing from spills
Offer additional body protection Long-sleeved, knee length with snaps Elastic cuffs Back-closing gowns Periodic cleaning required Closed toed shoes protect against spills and injuries from dropped sharps. Elastic cuffs help prevent spills and contamination

PPE Gloves Offer protection against a variety of hazards (heat, cold, chemical agents, biological agents, radioisotopes…) Latex, nitrile, rubber & vinyl for work with biological agents. Gloves should not be reused and should be changed frequently. Utility gloves can be disinfected and reused if they show no sign of degradation.

PPE Eye & Face Protection Goggles, safety glasses to protect the eyes
Full face shield to protect facial skin Offer protection against chemical and biological splashes No contact lenses Respirators Only personnel who have been fit-tested and trained should wear respirators. Worn in atmospheres that pose an infectious or toxic hazard

INFECTION/BIOHAZARD CONTROL 4. PRACTICES AND PROCEDURES

PRACTICES AND PROCEDURES
General Safety Guidelines Good Microbiological Practice Handwashing Receipt of Packages Opening Packages Specific Procedures Centrifuges Needles & Syringes and other sharps Pipettes Blenders, Grinders, Sonicators & Lyophilizers Inoculation Loops Cryostats

GENERAL LABORATORY SAFETY GUIDELINES
Understand the hazards you face in the laboratory Be adequately trained Appropriate PPE must be worn The lab should be kept clean and in order Long hair must be tied back Work surfaces must be cleaned and decontaminated daily The use of needles should be limited Lab doors have to be closed Access to the lab has to be restricted Protect yourself, others and the environment and indirectly the product. Provide at least 5 examples. b i o s a f e t y

1. GOOD MICROBIOLOGICAL PRACTICE (GMP)
Mitigates the risk of: 1. Personnel exposure 2. Contamination a) sample b) environment What are we talking about ?

Universal Precautions: More knowledge about the organism being used = easier to take the necessary precautions Appropriate PPE greatly minimizes risk of exposure Engineered controls (BSC’s) prevent release of aerosols outside cabinet (and helps protect user!) Frequent hand washing = avoid infections The more knowledgeable one is about the organism being used, the easier it is to take the necessary precautions Washing hands prior to and following experiments/manipulations of organisms and at any time contamination is suspected Employ universal precautions when using blood-borne pathogens Disinfection of: work surfaces with a suitable disinfectant before and after experiments/ manipulations of organisms, and materials that have come in contact with your organism Clean spills immediately according to established protocols and disinfect the area thoroughly Keep bench top uncluttered Minimize traffic and unnecessary movements around the work area (movement can stir up air currents which can carry contaminants into the work area). All work with infectious material should be carried out in a specific area, and the material should not be carried throughout or out of the lab unless in a closed or capped container Minimize aerosol generation; if unavoidable, activities should be carried out in a biological safety cabinet Use proper aseptic technique for the transfer and handling or microorganisms and instruments Keep sterile and non-sterile objects separate Minimize exposure to outside air (i.e. keep lids off sterile containers for as little time as possible) Avoid contact with non-sterile surfaces and items (i.e. never place lids/caps onto a work surface, hold lids in an opening-down position), and Hold open containers at an angle whenever possible to prevent contaminants from falling in) Identification and proper disposal of different types of waste (glass & plastic, biomedical, chemical, or radioactive waste)

Prepare yourself for the work: Know what you will be doing Structure the work in a logical fashion (work flow) Prepare the work area Ensure all material that needs to be in the BSC is sterile before placing it there Ensure waste containers are at hand’s reach and are not overflowing and likely to collapse/ fall over Use aseptic technique Consult web [ for SOPs & techniques Properly trained to use equipment accordingly and when in doubt…ASK! Clean up and decontaminate The more knowledgeable one is about the organism being used, the easier it is to take the necessary precautions Washing hands prior to and following experiments/manipulations of organisms and at any time contamination is suspected Employ universal precautions when using blood-borne pathogens Disinfection of: work surfaces with a suitable disinfectant before and after experiments/ manipulations of organisms, and materials that have come in contact with your organism Clean spills immediately according to established protocols and disinfect the area thoroughly Keep bench top uncluttered Minimize traffic and unnecessary movements around the work area (movement can stir up air currents which can carry contaminants into the work area). All work with infectious material should be carried out in a specific area, and the material should not be carried throughout or out of the lab unless in a closed or capped container Minimize aerosol generation; if unavoidable, activities should be carried out in a biological safety cabinet Use proper aseptic technique for the transfer and handling or microorganisms and instruments Keep sterile and non-sterile objects separate Minimize exposure to outside air (i.e. keep lids off sterile containers for as little time as possible) Avoid contact with non-sterile surfaces and items (i.e. never place lids/caps onto a work surface, hold lids in an opening-down position), and Hold open containers at an angle whenever possible to prevent contaminants from falling in) Identification and proper disposal of different types of waste (glass & plastic, biomedical, chemical, or radioactive waste)

Disinfect work surfaces with suitable disinfectant before and after Clean spills immediately and disinfect area thoroughly Keep bench top uncluttered Minimize traffic and unnecessary movements around work area all work with infectious material should be carried out in a specific area Material should not be carried throughout, or out of lab, unless in a closed or capped container Minimize aerosol generation; if unavoidable, carry out activities in a BSC Disinfection of: work surfaces with a suitable disinfectant before and after experiments/ manipulations of organisms, and materials that have come in contact with your organism Clean spills immediately according to established protocols and disinfect the area thoroughly Keep bench top uncluttered Minimize traffic and unnecessary movements around the work area (movement can stir up air currents which can carry contaminants into the work area). All work with infectious material should be carried out in a specific area, and the material should not be carried throughout or out of the lab unless in a closed or capped container Minimize aerosol generation; if unavoidable, activities should be carried out in a biological safety cabinet Use proper aseptic technique for the transfer and handling or microorganisms and instruments Keep sterile and non-sterile objects separate Minimize exposure to outside air (i.e. keep lids off sterile containers for as little time as possible) Avoid contact with non-sterile surfaces and items (i.e. never place lids/caps onto a work surface, hold lids in an opening-down position), and Hold open containers at an angle whenever possible to prevent contaminants from falling in) Identification and proper disposal of different types of waste (glass & plastic, biomedical, chemical, or radioactive waste)

Keep sterile and non-sterile objects separate Minimize exposure to outside air Avoid contact with non-sterile surfaces and items Hold open containers at an angle whenever possible Identify and properly dispose of different types of waste Keep sterile and non-sterile objects separate Minimize exposure to outside air (i.e. keep lids off sterile containers for as little time as possible) Avoid contact with non-sterile surfaces and items (i.e. never place lids/caps onto a work surface, hold lids in an opening-down position), and Hold open containers at an angle whenever possible to prevent contaminants from falling in) Identification and proper disposal of different types of waste (glass & plastic, biomedical, chemical, or radioactive waste)

HANDWASHING One of the single effective means of preventing infections if done properly and frequently When to wash hands? 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 Liquid dispensers should be used rather than bars Level 1 lab - a non antiseptic soap can be used Level 2 lab - requires antiseptic hand washing solutions

RECEIPT OF PACKAGES At Shipping & Receiving
Verify shipment is yours, and expected. Inspect the integrity of container. If damage and breakage possible, transfer the package into a secondary container lined with absorbent paper (absorbent side up) Transfer to a cart with 4 sides for transfer to lab. Decontaminate all the areas in S&R where the package came into contact with. All individuals who may have come into contact with the material must wash their hands REMEMBER AT THIS POINT YOU DO NOT KNOW IF THE SAMPLE HAS BEEN BE BREACHED !

OPENING PACKAGES – IN LAB
Scenario 1: Package appears damaged. Transfer the sample to a biological cabinet and open and inspect each layer of packaging confronted with for signs which would indicate the sample integrity. If damaged, inform your supervisor and ORM (x. 3153) Dispose of sample in the appropriate manner Package must be sterilized or sent for incineration.

OPENING PACKAGES – IN LAB
Scenario 2: Package is intact. Open package in the containment level required by the sample Add sample to inventory Read and file MSDS or supplier information sheet Deface all markings on the package prior to disposal

SAFE USE OF CENTRIFUGES
Before use Check centrifuge tubes for cracks Avoid Overfilling Place caps or stoppers properly Balance loads Use sealed buckets (safety cups) or sealed rotors Before leaving: ensure centrifuge achieves run conditions After run Centrifuge has to be completely stopped before opening the lid Check for spills or leaks before removing samples. Clean spills Allow aerosols to settle (30 min) or open in a BSC Check logs to ensure centrifuge is consistently achieving desired conditions

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 Use safe needle devices if possible; locking units.

PIPETTES Mouth pipetting is prohibited. Never force fluids out.
To avoid splashes, discharge the liquid down 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. Decontaminate after use Blender Do not use glass blender jars Use safety blenders which can be autoclave Lyophilizers (used for dehydration process) Use glassware designed for vacuum work, ensure there is no damage before using Use vapour traps whenever possible Safety blender – designed to prevent leakage from the bottom of the blender jar and to withstand sterilization by autoclaving. They also provide a cooling jacket to avoid biological inactivation. Sonicator - Surface decontaminate the probe.

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

CRYOSTATS Wear gloves during preparation of frozen sections and heavy gloves when accessing the cryostat. Decontaminate frequently (70% Ethanol) Freezing tissue does not necessarily inactivate infectious agents.

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. What was spilled? (What are the physical characteristics and potential hazards of that particular organism?) How much was spilled? (What is the volume and concentration of the organism?) Where was the spill? (In a BSC, in the lab, outside the lab, in a centrifuge?) What is the potential for release to the environment? (Were aerosols or droplets generated?)

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 Leave on for 20 to 30 minutes Pick up any broken glass (with forceps!) and place in a sharps container Wipe up with absorbent material Waste should be disposed in appropriate biohazardous waste container Applies to blood as well

SPILLS-SPECIAL CASES Within a Centrifuge Within a BSC
Open Areas (lab, during transport) The spill response plan template is available at (

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: 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

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) Incineration is done off-site

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 (petri dishes, eppendorf tips, pipettes, gloves, paper towels)

AUTOCLAVES Items that CANNOT 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) Material containing solvents, volatile or corrosive chemicals (Phenol, trichloroacetic acid, ether, chloroform) Chemotherapeutic agents.

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 Autoclaving success depends on heat penetrating all material in the bag.

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 Use orange bags.

DISINFECTION: 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) Chemical characteristics (corrosion, toxic, flammable, etc.)

WHAT TO USE FOR MY AGENT? Viruses Vegetative bacteria (E.coli, Staph)
Enveloped (HIV, Herpes) 2% domestic bleach 75% Ethanol Quaternary ammonia 6% formulated Hydrogen peroxide* Non enveloped (Hepatitis, Adenovirus) 10% domestic bleach Gluteraldehyde Formaldehyde Vegetative bacteria (E.coli, Staph) 2% domestic bleach 75% Ethanol Quaternary ammonia 6% formulated Hydrogen peroxide Mycobacteria and fungi 10% domestic bleach Phenolic compounds Spore forming bacteria (Bacillus) Gluteraldehyde Formaldehyde Notice broad range of bleach

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

 LABEL YOUR WASTE 
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 of 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.  LABEL YOUR WASTE   IDENTIFY CONTENTS 

WASTE DISPOSAL Biomedical Waste (untreated)

*in compliance with sewer use by-laws
WASTE DISPOSAL Biomedical Waste (treated) *in compliance with sewer use by-laws with H2O (1:10)

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 (PHAC) or Canadaian Food Inspection Agency (CFIA) for certain agents Material Transfer Agreements (MTAs) between importer & exporter US restrictions Ensure you meet all criteria and have all pertinent documentation Due to restrictions enforced in US, there may be conditions on a contract if an agent is imported from the US.

BIOMATERIAL ACQUISITION/ MTAs
“How soon do you need it?” “You want it when?” In order to facilitate a quick turnaround, provide ORM with: copies of MSDS’s references (hardcopies) as much background information re: product as possible

INVENTORY What material is presently being used and/or stored Location
Expiry date Use log book for remaining amount MSDS’s Mandatory HC and CFIA expect labs to identify their agents and where they are stored to ensure they are secure

SHIPPING AND RECEIVING
Transportation of Dangerous Goods Act: Class 6.2 (Infectious Substances) PHAC/CFIA restrictions Ensure: Proper classification Proper packaging Proper labeling Proper documentation Import/Export Permits Export permits, Import 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 HC/CFIA restriction The transportation of infectious or potentially infectious material whether it be between laboratories within a department/faculty, or between institutions involves a level of risk of unintentional release of the material to the environment. Legal requirements which regulate how material may be transported - TDG (can the supporting experimental apparatus be taken to the samples), does material need to be transported at all packaging requirements (primary and secondary containers, dry ice etc) means and route of transportation (use of cart with guard rails, low traffic area etc.) regulatory requirements (classification, labelling, signing, documenting)

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 Media

BIOSAFETY WEBSITE http://www.uottawa.ca/services/ehss.biosafety.htm
Biohazardous Materials User Registration Biosafety Health Assessment Survey Practical Training Form

BIOSAFETY TRAINING Pierre Laflamme May 16, 2012 Uottawa2k9

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BIOSAFETY TRAINING Pierre Laflamme May 16, 2012 Uottawa2k9

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