4 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 parasitesand their toxins.Blood, body fluids and tissues from humans and animals.Transformed cell lines
5 What is BIOSAFETY?The combination of measures employed when handling biohazardous materials to:Protect personnel from exposure to infectious agentsPrevent environmental contaminationProvide an environment for high quality research while maintaining a safe work placeComply with applicable federal, provincial and municipal requirementsThe combination of measures employed when handling biohazardous materials to:Protect personnel from exposure to infectious agentsPrevent environmental contaminationProvide an environment for high quality research while maintaining a safe work placeComply with applicable federal, provincial and municipal requirements
6 How is BIOSAFETY achieved? Administrative controlsTraining, Inspections, Permits and CertificatesEngineering ControlsBiological Safety Cabinets, VentilationPersonal Protective EquipmentPractices and ProceduresMedical SurveillanceImmunization when necessaryHow is biosafety achieved?ThroughAdministrative controls:-training-Inspections-Issuing permits and certificatesEngineering controls:- Isolation or enclosure of an operation- Biological Safety Cabinets- Ventilation- Substitution for a less toxic materialPeronal Protective Equipment-Gloves, goggles, lab coats, face shieldsPractices & Procedures:-Good practices-SOP-Guidelines-policies- Medical SurveillanceAnd Immunization when necessary
7 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.
8 How is BIOSECURITY achieved? Physical barriersBuildings, doors, locks, key card accessPsychological barriersSecurity personnel, camerasMonitoring ActivitiesPatrols, monitoring by support staffPersonnel ClearanceAccess to authorized personnel onlyphysical 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 doorspsychological barriers- obvious presence of identifiable security personnel - obvious presence of security culture - use of cameras, mirrors, mirrored domes (90o,180o, 360o) and other monitoring toolsMonitoring 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 personnelpersonnel clearanceidentifying and restricting access to only authorized personnelGrowing concern about possible use of biologicals as weapons.Theft of any itemBe aware of what is going on in your labReport any strange persons, missing material.
9 Who are the STAKEHOLDERS? INTERNALLYVice-President (Research)CommitteesUniversity Services (ORM, HR, PRS, PS)Deans, Chairs, Principal InvestigatorsEmployees, StudentsManager of Biological Containment SuiteEXTERNALLYPublic Health Agency of CanadaCanadian Food Inspection AgencyEnvironment CanadaTransport CanadaOntario Ministry of LabourEmergency Response PersonnelSuppliers & ContractorsCommunityCommittees (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)
10 University KEY SERVICES Office of Risk Management, Environmental Health and SafetyCertificates and PermitsTrainingProcedures (Waste disposal)Risk Identification (Inspections)Emergency plansAccident/Incident follow-upCertification (Biohazardous Materials Use)Permits (Importation permits)Training (EHSS & P.I.)Procedures (sharps, gloves, waste, etc.,)Inspections (Risk identification)Emergency Response / Contingency PlanningAccident /Incident follow-upAdd brief note about role of PRS and PS.
11 University KEY SERVICES HR (Occupational Health, Disability and Leave)Medical surveillanceImmunizationsMedical Follow-upInterface with Workplace Safety and Insurance BoardCertification (Biohazardous Materials Use)Permits (Importation permits)Training (EHSS & P.I.)Procedures (sharps, gloves, waste, etc.,)Inspections (Risk identification)Emergency Response / Contingency PlanningAccident /Incident follow-upAdd brief note about role of PRS and PS.
12 WHY ARE WE CONCERNED?Potential for acquiring a laboratory-associated infection (LAI)Contamination of the environmentContamination of research
14 LABORATORY ASSOCIATED INFECTIONS Infection SourceMicroorganismsCells and tissuesBlood and body fluidsAny items contaminated with the aboveSusceptible HostRoute of TransmissionImmune systemVaccination statusAgePercutaneous inoculation (needles and bites)Inhalation of aerosolsContact of mucous membranesIngestion
15 LAIs Only 20% of LAIs are related to a causative or defined event 80% are caused by human errors20% are caused by equipment failureTypes of accidents causing LAIsSpills and spraysNeedlesSharp objects and broken glassBites or scratches from animalsStudies 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 LAIsSpills and spraysNeedlesSharp objects and broken glassBites or scratches from animals
16 BLOOD-BORNE PATHOGENS Human Resources Occupational Health Disability & Leave
18 RISK OF EXPOSURE Pathogen involved Type of body fluid Route of exposureDuration of exposureVolume of blood involved in exposureConcentration of virus at time of exposurePPE worn
19 SPECIFIC EXAMPLES OF BBPS Hepatitis B Hepatitis C HIV
20 FACTS ABOUT SOME BBPs Pathogen Hepatitis B Hepatitis C HIV Pathogenicity2 major forms:asymptomaticsymptomaticnon-specific symptomsacute infection: non-specific “flu-like”, “mono-like” symptomsMode of transmissionpercutaneous/ permucosal exposure to body fluids, organsindirect 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 productsdirect exposure of virus to mucosa (oral, rectal, vaginal)Incubationperiodusually: daysaverage: days2 weeks - 6 monthsmost commonly 7 – 10 weekschronic infection may persist up to 20y before onset of cirrhosisvariablegenerally months between time of infection to development of detectable Ab’stime from HIV infection to diagnosis of AIDS ranges from < 1y to 15y or moreSurvival outside hostSurvives in dried blood for long periods (weeks)stable on environmental services for at least 7 days at 25 °Cnot knownsimilar to hep B (survives in dried blood for long periods…weeks)viable in blood in RT for 42dCell-free HIV dried on glass coverslips in 10% serum can survive forlonger than 7d, depending on initial titreLaboratory-acquired infections (LAIs)MOST FREQUENTLY occurring LAIlab workers incident rate: 7X > general populationhealth care workers handling blood at higher risk to infectionlow (e.g. as of 2001, total of 57 cases of documented occupationally acquired HIV among US health care workers[Source:
21 ISSUES TO CONSIDER Symptoms Mode of transmission Incubation period Survival outside hostCommunicabilityImmunizationProphylaxis / Treatment
22 IF AN EXPOSURE OCCURS Initiate first aid Notify your supervisor / designated personReport to hospital emergency department or University’s Health ServicesReport incident to OHDLOccupational Health, Disability and Leave Office, ext
23 UNIVERSAL PRECAUTIONS Minimum standard of practice for preventing the transmission of BBP includes:EducationHand washingWearing protective barriersUse safe work practicesIf samples cannot be guaranteed non-infective …… treat as infectious!
25 BIOHAZARD CLASSIFICATION Conventional Agents – Risk Groups 1 to 4Recombinant DNATissue CultureAnimal WorkAnatomical SpecimensUnconventional AgentsClass D, division 3 of WHMIS(Poisonous and Infectious Material - Biohazardous Infectious Material)
26 BIOHAZARD CLASSIFICATION Conventional agents are categorized into risk groups based on their particular characteristics such as:Pathogenicity (Infectivity of the agent - disease, severity, mortality)Infectious doseMode of transmission (Airborne, Ingestion, Parenteral)Host Range (Animal or human pathogenAvailability of effective preventive measures (PPE)Availability of effective treatmentPathogenicity:How infectious is the agent (disease evidence, severity, mortality)i.e. S. aureus versus EbolaInfectious 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 = challengeMode of transmission:Airborne, ingestion, parenteralConsider aerosols when mode of transmission is unknown.Host Range:Animal pathogenHuman pathogenAvailability of effective preventative measures: PPE, SOP, guidelines, trainingAvailability of effective treatment:Effective prophylaxis, medical surveillance
27 BIOHAZARD CLASSIFICATION 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, certificationOperational requirements:Protocols, SOP, basic (general) lab safety practices, safety cabinets….Technical Requirements:Physical requirements:
28 CONVENTIONAL AGENTS 1 Low Level 1 2 Moderate Limited Level 2 3 High Risk GroupIndividual RiskCommunity RiskContainmentLevelExamples1LowLevel 1Escherichia Coli2ModerateLimitedLevel 2Bacteria: Streptococcus and SalmonellaViruses: Adenovirus, Hepatitis A, B & C, Influenza3HighLevel 3Bacteria: Bacillus anthracis and, Mycobacterium tuberculosisVirus: HIV4Level 4Viruses: Ebola virus and Lassa virusUnlikely to cause disease in healthy workers or animalsRarely cause serious human or animal diseaseMay cause serious disease1 - Unlikely to cause disease in healthy workers or animals.2 – Rarely cause serious human or animal disease3 – May cause serious disease4 – Likely to cause very serious diseaseLikely to cause very serious disease
29 RECOMBINANT DNA Recombinant DNA technology or genetic engineering: in vitro incorporation of genetic material from one cell into another or from one organism to anotherIn Canada the level of risk depends onthe source of DNA being transferredthe vectorthe hostThe 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.
30 TISSUE CULTUREMammalian cell lines have to be considered infectious as they may contain infectious agentsUntransformed mammalian cell lines - Risk Group 1MCF-7 (Human breast carcinoma cell line)NIH 3T3 (Mouse fibroblast cell line)Transformed mammalian cell lines – Risk Group 2HeLa (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.
31 ANIMAL WORKAnimals can harbour infectious agents (naturally or introduced) which can be transmitted to humansScratches, bites, aerosols (needles and litter changes), body fluids and excrementsLevel 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 CommitteeViruses can easily replicate in this environment,Animals at increased risk of exposure humans are also at increased riskScratchesBitesAerosol generation (syringes, litter changes)Faecal matterOther exudates and bodily fluids
32 ANATOMICAL SPECIMENSAll specimens should be considered infectious due to potential presence of infectious agentsIt’s important to consider the type of specimenblood, organs, tissuesSpinal sample, brain tissueFrom infectious patientIn general Level 2 but it depends on the nature of the work.Blood, organs, tissues – BBP’sSpinal sample, brain tissue - prions
33 UNCONVENTIONAL PATHOGENS Includes unconventional agents, slow viruses and prions causing progressive neurological diseasesCreutzfeld-Jakob disease in humans, Mad Cow Disease, Scrapie in sheeps and goatsResistant to destruction by chemical and physical procedures that normally inactivate virusesPrecautions:Handle tissues as Risk Group 2 or higherHandle formalin-fixed tissues and paraffin-embedded blocks as if still infectiousFollow up-to-date disinfection protocols.Transmissible Spongiform Encephalopathyrare 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.PrecautionsHandle tissue as Risk Group 2 or higherHandle formalin-fixed tissues and paraffin-embedded blocks as if still infectiousFollow up-to-date disinfection protocols.Bovine Spongiform EncephalopathyScrapie in sheep and goats.CWD in deer and elk
36 INFECTION/BIOHAZARD CONTROL Administrative ControlsEngineering ControlsPersonal Protective EquipmentPractices and ProceduresEngineering 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.
37 INFECTION/BIOHAZARD CONTROL 1. ADMINISTRATIVE CONTROLS
38 ADMINISTRATIVE CONTROLS Administrative procedures to minimize the risk of exposure:Risk assessmentTraining/EducationResourcesInspectionsPermits and CertificatesMedical SurveillanceSignageThe information and resources are provided to you but the onus is on you to use them properly in order to mitigate risk.
39 ADMINISTRATIVE CONTROLS Risk AssessmentWill determine for each biohazard:Risk groupContainment levelOperational practicesSafety measuresResponsibility of usersKnow 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
40 ADMINISTRATIVE CONTROLS Medical SurveillanceTraining & EducationWHMISLab specific policies and proceduresBiosafety training, Laboratory safety trainingResourcesORM web site, Biosafety pageFaculty web sitesBiosafety ManualTraining VideosNational and International Biosafety GuidelinesNational/International guidelinesUniversity Policies and ProceduresSpecific biosafety topics, fundamental issuesMSDS’s
41 ADMINISTRATIVE CONTROLS InspectionsRoutine self-inspectionsBiosafety Inspection Checklist available on-lineIn 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
42 ADMINISTRATIVE CONTROLS Signs & LabelingBiohazard 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
43 INFECTION/BIOHAZARD CONTROL 2. ENGINEERING CONTROLS
44 ENGINEERING CONTROLS Technology based Reduce or eliminate exposure to hazardsContainment:Types: Primary and SecondaryLevels: 1, 2, 3 and 4For effective containment, users mustbe aware of the potential hazardsbe trainedHandle the material safely by adhering to standard microbiological practices and techniquesdoesn’t rely on skill or vigilanceFor effective containment, people must be aware of potential hazards, be trained and adhere to established practices and techniques.
45 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, vialBiological safety cabinetsanimal caging equipment*Ensure it works!
46 SECONDARY CONTAINMENT Protects the environment external to the laboratory from exposureIncludes facility design and operational practices
47 CONTAINMENT LEVEL 1 Basic laboratory Requires no special design featuresBiosafety 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.
48 CONTAINMENT LEVEL 2Clinical, 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.
49 CONTAINMENT LEVEL 3 Specialized design and construction with primary barriers to protect the individualsecondary barriers to protect the environmentRequires type II or type III biosafety cabinetsAll 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 individualsecondary barriers to protect the environmentMust undergo annual performance, testing and verification.Requires type II or type III biosafety cabinets. Also, all centrifugation must be performed in closed trunnion cup centrifugesAll 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 facilityWritten protocols must be provided and postedA medical surveillance program must be in effectA reporting system for accidents must be in place-
50 CONTAINMENT LEVEL 4Only one level 4 facility in Canada (Canadian Centre for Human and Animal Health in Winnipeg, Man.)Design specifications are extremely stringentThe worker is completely isolated from infectious material.The highest level of containment available where all manipulations pose a high risk of exposure and infectionThere is only one level 4 facility in Canada (Canadian Centre for Human and Animal Health in Winnipeg, Man.)Design specifications are extremely stringentThe worker is completely isolated from infectious material.Entry and exits are through airlocksShowers are mandatoryAll work is performed in class III cabinets or class I or II cabinets with a positive pressure suit.
51 BIOLOGICAL SAFETY CABINETS Primary containmentMinimize contact between operator and the infectious agent by the use of directional airflowsThere 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 areasHigher 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
52 BIOLOGICAL SAFETY CABINETS Laminar air flow and HEPA filtered exhaust airPersonnel and environment protectionHEPA filtered supply air & product protection with Class II + IIITo be used with biohazardsLaminar Flow Hoods or Clean Air BenchesVertical or horizontal laminar flowHEPA filtered supply air onlyProvide product protection onlyNot to be used with biohazardsLaminar 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
53 WORKING SAFELY IN A BSC Step 1 Before using the cabinet: Turn off UV lamp; turn on fluorescent lampEnsure BSC is certifiedDisinfect work surfaces with appropriate disinfectantPlace essential items inside cabinetAllow the blower to run for 5-10 min before workCertification 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 sterileYou save the HEPA filter lifeThe cabinet is quieterUV lamp can be used safelyEfficiency of the UV lamp depends onAge of the tubeExposition timeDistance between the UV lamp and the microorganismsProper regular decontamination including, window and side panels
54 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 ventsUse techniques that reduce splatter and aerosols.General work flow should be from clean to contaminated areasMinimize movement so as not toimpede air flowPrepare 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.
55 WORKING SAFELY IN A BSC Step 3 After using the cabinet: Leave blower on at least 5 minutes to purge cabinetRemove and decontaminate equipment and materialsDisinfect cabinet surfacesTurn off blower and fluorescent lamp, turn on UV lamp15 min.
56 WORKING SAFELY IN A BSC Maintenance: Before and after each use - Wipe down work surfacesWeekly - Clean UV lampMonthly - Wipe down all vertical surfacesAnnually - 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.
57 INFECTION/BIOHAZARD CONTROL 3. PERSONAL PROTECTIVE EQUIPMENT
58 PERSONAL PROTECTIVE EQUIPMENT PPE is an important line of defenceResponsibility of both the user and the supervisor to ensure that PPE is wornPPE is specific to each containmentlevelExamples 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
59 PPE Criteria for consideration Routes of exposure that need to be blockedDegree of protection offeredEase of useOnly effective ifcorrectly selected, fitted, used and cared forthe individual is well trainedEnsure PPE is removed before leaving the labPPE is often specific for each containment level
60 Closed toe and heel shoes only. No sandals! PPEFootwearClosed 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
61 PPE Lab Coats/Gowns Protect street clothing from spills Offer additional body protectionLong-sleeved, knee length with snapsElastic cuffsBack-closing gownsPeriodic cleaning requiredClosed toed shoes protect against spills and injuries from dropped sharps.Elastic cuffs help prevent spills and contamination
62 PPEGlovesOffer 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.
63 PPE Eye & Face Protection Goggles, safety glasses to protect the eyes Full face shield to protect facial skinOffer protection against chemical and biological splashesNo contact lensesRespiratorsOnly personnel who have been fit-tested and trained should wear respirators.Worn in atmospheres that pose an infectious ortoxic hazard
66 INFECTION/BIOHAZARD CONTROL 4. PRACTICES AND PROCEDURES
67 PRACTICES AND PROCEDURES General Safety GuidelinesGood Microbiological PracticeHandwashingReceipt of PackagesOpening PackagesSpecific ProceduresCentrifugesNeedles & Syringes and other sharpsPipettesBlenders, Grinders, Sonicators & LyophilizersInoculation LoopsCryostats
68 GENERAL LABORATORY SAFETY GUIDELINES Understand the hazards you face in the laboratoryBe adequately trainedAppropriate PPE must be wornThe lab should be kept clean and in orderLong hair must be tied backWork surfaces must be cleaned and decontaminated dailyThe use of needles should be limitedLab doors have to be closedAccess to the lab has to be restrictedProtect yourself, others and the environment and indirectly the product.Provide at least 5 examples.biosafety
69 1. GOOD MICROBIOLOGICAL PRACTICE (GMP) Mitigates the risk of:1. Personnel exposure2. Contaminationa) sampleb) environmentWhat are we talking about ?
70 2. GOOD MICROBIOLOGICAL PRACTICE (GMP) Universal Precautions:More knowledge about the organism being used = easier to take the necessary precautionsAppropriate PPE greatly minimizes risk of exposureEngineered controls (BSC’s) prevent release of aerosols outside cabinet (and helps protect user!)Frequent hand washing = avoid infectionsThe more knowledgeable one is about the organism being used, the easier it is to take the necessary precautionsWashing hands prior to and following experiments/manipulations of organisms and at any time contamination is suspectedEmploy universal precautions when using blood-borne pathogensDisinfection of:work surfaces with a suitable disinfectant before and after experiments/ manipulations of organisms, andmaterials that have come in contact with your organismClean spills immediately according to established protocols and disinfect the area thoroughlyKeep bench top unclutteredMinimize 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 containerMinimize aerosol generation; if unavoidable, activities should be carried out in a biological safety cabinetUse proper aseptic technique for the transfer and handling or microorganisms and instrumentsKeep sterile and non-sterile objects separateMinimize 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), andHold 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)
71 3. GOOD MICROBIOLOGICAL PRACTICE (GMP) Prepare yourself for the work:Know what you will be doingStructure the work in a logical fashion (work flow)Prepare the work areaEnsure all material that needs to be in the BSC is sterile before placing it thereEnsure waste containers are at hand’s reach and are not overflowing and likely to collapse/ fall overUse aseptic techniqueConsult web [http://www.protocol-online.org] for SOPs & techniquesProperly trained to use equipment accordingly and when in doubt…ASK!Clean up and decontaminateThe more knowledgeable one is about the organism being used, the easier it is to take the necessary precautionsWashing hands prior to and following experiments/manipulations of organisms and at any time contamination is suspectedEmploy universal precautions when using blood-borne pathogensDisinfection of:work surfaces with a suitable disinfectant before and after experiments/ manipulations of organisms, andmaterials that have come in contact with your organismClean spills immediately according to established protocols and disinfect the area thoroughlyKeep bench top unclutteredMinimize 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 containerMinimize aerosol generation; if unavoidable, activities should be carried out in a biological safety cabinetUse proper aseptic technique for the transfer and handling or microorganisms and instrumentsKeep sterile and non-sterile objects separateMinimize 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), andHold 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)
72 4. GOOD MICROBIOLOGICAL PRACTICE (GMP) Disinfect work surfaces with suitable disinfectant before and afterClean spills immediately and disinfect area thoroughlyKeep bench top unclutteredMinimize traffic and unnecessary movements around work areaall work with infectious material should be carried out in a specific areaMaterial should not be carried throughout, or out of lab, unless in a closed or capped containerMinimize aerosol generation; if unavoidable, carry out activities in a BSCDisinfection of:work surfaces with a suitable disinfectant before and after experiments/ manipulations of organisms, andmaterials that have come in contact with your organismClean spills immediately according to established protocols and disinfect the area thoroughlyKeep bench top unclutteredMinimize 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 containerMinimize aerosol generation; if unavoidable, activities should be carried out in a biological safety cabinetUse proper aseptic technique for the transfer and handling or microorganisms and instrumentsKeep sterile and non-sterile objects separateMinimize 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), andHold 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)
73 5. GOOD MICROBIOLOGICAL PRACTICE (GMP) Keep sterile and non-sterile objects separateMinimize exposure to outside airAvoid contact with non-sterile surfaces and itemsHold open containers at an angle whenever possibleIdentify and properly dispose of different types of wasteKeep sterile and non-sterile objects separateMinimize 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), andHold 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)
74 HANDWASHINGOne of the single effective means of preventing infections if done properly and frequentlyWhen to wash hands?Before starting any manipulationsBefore leaving the labWhen hands are obviously soiledBefore and after completing any task in a BSCEvery time gloves are removedBefore contact with one’s face or mouthAt the end of the dayLiquid dispensers should be used rather than barsLevel 1 lab - a non antiseptic soap can be usedLevel 2 lab - requires antiseptic hand washing solutions
75 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 handsREMEMBER AT THIS POINT YOU DO NOT KNOW IF THE SAMPLE HAS BEEN BE BREACHED !
76 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 mannerPackage must be sterilized or sent for incineration.
77 OPENING PACKAGES – IN LAB Scenario 2: Package is intact.Open package in the containment level required by the sampleAdd sample to inventoryRead and file MSDS or supplier information sheetDeface all markings on the package prior to disposal
78 SAFE USE OF CENTRIFUGES Before useCheck centrifuge tubes for cracksAvoid OverfillingPlace caps or stoppers properlyBalance loadsUse sealed buckets (safety cups) or sealed rotorsBefore leaving: ensure centrifuge achieves run conditionsAfter runCentrifuge has to be completely stopped before opening the lidCheck for spills or leaks before removing samples. Clean spillsAllow aerosols to settle (30 min) or open in a BSCCheck logs to ensure centrifuge is consistently achieving desired conditions
79 NEEDLES AND SYRINGES Avoid use whenever possible Use a BSC for all operations with infectious materialFill syringes carefullyShield needles when withdrawing from stoppersDo not bend, shear or recap needles.Dispose of all used needles/syringes in yellow sharps containersUse safe needle devices if possible; locking units.
80 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.
81 BLENDERS, GRINDERS, SONICATORS, AND LYOPHILIZERS Operate in a BSC whenever possible. Allow aerosols to settle for 5 minutes before opening.Decontaminate after useBlenderDo not use glass blender jarsUse safety blenders which can be autoclaveLyophilizers (used for dehydration process)Use glassware designed for vacuum work, ensure there is no damage before usingUse vapour traps whenever possibleSafety 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.
82 INOCULATION LOOPSSterilization in an open flame may create aerosols which may contain viable microorganisms.Shorter handles minimize vibrationsDisposable plastic loops are good alternatives
83 CRYOSTATSWear 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.
85 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?)
86 SPILLS-GENERAL CLEAN-UP Cover spill area with absorbent materialSoak the spill area with an appropriate disinfectant (i.e. 10% bleach)Pour disinfectant from the outside of the absorbent material towards the insideLeave on for 20 to 30 minutesPick up any broken glass (with forceps!) and place in a sharps containerWipe up with absorbent materialWaste should be disposed in appropriate biohazardous waste containerApplies to blood as well
87 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/docs/SPILLRESPONSEPLAN.pdf)
88 SPILLSAll 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 5892Your departmental safety officerERT x 5411 (through Protection Services)
90 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
91 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)FiltrationHEPA (biological safety cabinets, ventilation)Incineration is done off-site
92 AUTOCLAVES Items that CAN be autoclaved: Cultures and stocks of infectious materialCulture dishes and related devicesDiscarded live and attenuated vaccinesContaminated solid items (petri dishes, eppendorf tips, pipettes, gloves, paper towels)
93 AUTOCLAVES Items that CANNOT be autoclaved: chemicals (flammables, oxidizers, phenols, acids, alkalides)chemotherapeutic or radioactive wastebleach (or other chlorinated products)certain kinds of plasticsSharps (not at the University of Ottawa)Material containing solvents, volatile or corrosive chemicals (Phenol, trichloroacetic acid, ether, chloroform)Chemotherapeutic agents.
94 AUTOCLAVES Preparation of waste: Use only approved autoclave bags Do not overfill autoclave bagsSeparate material for re-use from that which will be disposed, and dry from liquid materialIf outside of bag is contaminated, double bagAll flasks containing biological material should be capped with aluminum foilEnsure items are labeled with contact informationAutoclaving success depends on heat penetrating all material in the bag.
95 SAFE USE OF AUTOCLAVESMany autoclaves are now run by dedicated staff, however, if you are operating an autoclave:Learn how to use it!Ensure PPE is wornRecognize acceptable material and packagingProper loading and unloadingAll users/operators must take the autoclave trainingUse orange bags.
96 DISINFECTION: CHEMICAL Generally for disinfection rather than sterilizationChoice depends on:Type of material to be disinfectedOrganic loadChemical characteristicsMost common are chlorine compounds and alcohols (broad range)Chemical characteristics (corrosion, toxic, flammable, etc.)
97 WHAT TO USE FOR MY AGENT? Viruses Vegetative bacteria (E.coli, Staph) Enveloped (HIV, Herpes)2% domestic bleach75% EthanolQuaternary ammonia6% formulated Hydrogen peroxide*Non enveloped (Hepatitis, Adenovirus)10% domestic bleachGluteraldehydeFormaldehydeVegetative bacteria (E.coli, Staph)2% domestic bleach75% EthanolQuaternary ammonia6% formulated Hydrogen peroxideMycobacteria and fungi10% domestic bleachPhenolic compoundsSpore forming bacteria (Bacillus)GluteraldehydeFormaldehydeNotice broad range of bleach
98 WASTE MANAGEMENT Discarded biological material from teaching, clinical and research laboratories and operations is biomedicalwaste.Biomedical waste includes but is not limited to:Animal wasteBiological laboratory wasteHuman anatomical wasteHuman blood and body fluid wasteSharps
99 LABEL YOUR WASTE WASTE MANAGEMENTAll 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
103 KEY REGULATED ACTIVITIES Purchasing & Receiving of Biological AgentsPHAC, CFIA, Environment CanadaInventory RecordsTransportation/TransferTransport Canada- TDGAll Agencies (provincial and federal) emphasize and expect Biosecurity
104 PURCHASINGImportation permits required by Public Health Agency Canada (PHAC) or Canadaian Food Inspection Agency (CFIA) for certain agentsMaterial Transfer Agreements (MTAs) between importer & exporterUS restrictionsEnsure you meet all criteria and have all pertinent documentationDue to restrictions enforced in US, there may be conditions on a contract if an agent is imported from the US.
105 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’sreferences (hardcopies)as much background information re: product as possible
106 INVENTORY What material is presently being used and/or stored Location Expiry dateUse log book for remaining amountMSDS’sMandatoryHC and CFIA expect labs to identify their agents and where they are stored to ensure they are secure
107 SHIPPING AND RECEIVING Transportation of Dangerous Goods Act: Class 6.2 (Infectious Substances)PHAC/CFIA restrictionsEnsure:Proper classificationProper packagingProper labelingProper documentationImport/Export PermitsExport permits, Import permits
108 TRANSPORTATION OF DANGEROUS GOODS Pre-approvedAuthorized IndividualsLead time (International Regulations….)Appropriate Scheduling (Holidays, Weekends)Transportation within the buildingBetween lab to labColleague to ColleagueBetween Institutions
109 TRANSPORTATION Important Considerations: does material need to be transported at allpackaging requirementsmeans and route of transportationregulatory requirementsBetween lab transfers - 4 sided cart, sealed primary container, secondary container, low traffic route.Off Campus transfers – consult ORMHC/CFIA restrictionThe 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 allpackaging 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)
110 THE BOTTOM LINEIf you are not careful and diligent with biological agents you risk:Infecting yourself, others or the environmentContaminating your researchHaving Public Health Agency of Canada, Canadian Food Inspection Agency, Ministry of the Environment or Transport Canada after youMedia
111 BIOSAFETY WEBSITE http://www.uottawa.ca/services/ehss.biosafety.htm Biohazardous MaterialsUser RegistrationBiosafety Health Assessment SurveyPractical Training Form