3 Information for undergrad courses You will be contacted only if you have failed your quiz, <75%. Your instructional assistant will be contacted at the same time.You will not be permitted to attend the biological labs of your course until you pass.
4 Biosafety in Canada: Standards Public Health Agency of Canada (PHAC) through the “Laboratory Biosafety Guidelines” Canadian Food Inspection Agency (CFIA) has “Veterinary Standards for Animal Facilities” Human Pathogens and Toxins Act C-11 Ministry of Environment Canada (MOE) for waste disposal
5 PHAC has “Laboratory Biosafety Guidelines” Sets the standards for work with biological agents such as bacteria, viruses, fungi parasites, prions and hazardous cell lines.The health risk associated with an organism will determine the protocols which must be followed to work with it.
6 Level of Hazard: BSL 1,2,3,4Internationally and in Canada, there are four levels of biosafety, each associated with a type of lab and specific equipment. The personal protection required increases with the level of hazard. Labs, techniques and methods required increased safety with the level of hazard.
7 Risk evaluation to determine level of use Risk evaluation of an agent is part of the responsibility of the supervisor for the project and is reviewed by the University Presidential Biosafety Advisory Committee.All individuals working with an agent must comply with the level of risk.
8 Factors affecting risk agent in use eg. specific bacteriumpathogenicity eg. degree of diseaseinfectious dose eg. # organisms to cause diseasemode of transmission eg. directly by blood or indirectly through the air
9 Factors affecting risk techniques in usehost range eg. does it infect animals as well as humanseffective treatment or prevention eg. is there a vaccine?Potential for aerosol creation eg. sonication, vortexing or homogenization
10 Factors affecting risk Volume of material eg > 10 litersConcentration of materialStability in the environment eg. will it survive on a benchtop?Use of re-combinant DNA eg. oncogenicity, replication ability, host range, ability to revert to wild type
11 Biosafety Level 1 (BSL 1) Low individual risk Low community risk unlikely to cause disease in healthy workers or animalstreatment / prevention is availablecan work on the open bench, examples include: e.coli, normal cell culture (non-viral, non-cancerous)
12 Biosafety Level 2 (BSL 2) Moderate individual risk Limited community riskcauses human or animal diseaseunder appropriate use, is unlikely to cause death to healthy laboratory workers or animalstreatment / prevention is availablework to be completed in a Class II biological safety cabinet (BSC)examples include: adenovirus, Helicobacter pylori, 293 cells, COS cells, retrovirus
13 Biosafety Level 3 (BSL 3) High individual risk Moderate community risk usually causes serious human or animal disease including death. Usually spreads by direct contact of blood or body fluids from one individual to anothergenerally, no cure / prevention availablemust work in a Biosafety Level 3 facility (McMaster)examples include: HIV, Mtb, West Nile Virus
14 Biosafety Level 4 (BSL 4) High individual risk High community risk human disease including death and may be readily transmitted through casual contactno treatment / prevention availablemust work in a Biosafety Level 4 facility (Winnipeg)examples include: Ebola, Marburg virus
15 Which level of organisms are being used in your lab ? Ask to see the biological equivalent of an “MSDS” for the agents you will be using.Or look up the information on the Internet site for the supplier of the product.Or reference a large database such as ATCC orPHAC
16 Basic Safety Practices - BSL 1 These requirements must be followed in all labs working with biological agents.Know and understand them.Know the emergency number; hospital – 5555, campus – 88Ensure that a working alone policy has been developedLab coat – used specifically for tissue cultureSuitable footwear – closed toed, but not necessarily closed heeled
17 Basic Safety Practices Longer hair is to be restrained so that it cannot come into contact with hands, specimens, containers or equipment.Oral pipetting of any substance is prohibited in any lab, use only mechanical fluid transfer devices.
18 Basic Safety Practices No eating or storage of food or drink in the labs…dispose of any food wrapping or containers BEFORE you enter the lab, including water bottles and gum. Keep personal items out of the working area of the lab.
19 Basic Safety Practices It is wise to report any increased susceptibility to infection to your supervisor or medical advisor.Ask the lab supervisor if you are unclear about any hazard or process.Dispose of all biological waste in the designated container only.
20 Waste disposalBiological waste is to be disposed of into the yellow or red bags in the biohazard labeled cardboard boxesSharps (scalpel blades, razor blades, needles on syringes) must be disposed into a sharps container and then disposed as biological waste when ¾ full
21 Basic Safety Practices Open wounds, cuts, scratches and grazes should be covered with waterproof dressings BEFORE working with any biological material. Limit use of sharps and glass. DO NOT RECAP NEEDLES
22 Basic Safety Practices Minimize aerosol creationLabel all biological materialWhere possible and appropriate, protect yourself by immunization esp Hep B when working with blood or body fluids.
23 Personal protection for BSL 1 Protective laboratory clothing must be worn in the lab but not worn out of the lab- closed toed footwearwear lab coats at all timesgoggles – if using contact lenses- if required by the protocol
24 Personal protection for BSL 1 Gloves…latex for water based materials…nitrile or vinyl for organicsChange gloves when contaminated.Wash your hands frequently, particularly when leaving the laboratory.
25 EmergenciesStaff must understand all hazards with which they work, including emergency response.- read and know the emergency and spill response protocol posted in your lab- report any spills or injuries to your supervisorknow the location of spill kits and first aid kitsknow the location of eyewash and shower
26 Trainingpersonnel must receive training on biohazards and safe handling protocols- pass for biotraining is 75%- local lab training to be signed by trainee and trainer- retraining program includes annual update
27 Physical lab requirements for BSL 1 Physical requirements are a functional and well designed labDoors to the laboratory must not be left open
28 Working requirements for BSL 1 Basic microbiology lab practices, keep the lab organized and cleanOpen bench is suitableRoutine decontamination techniques to maintain sterility - contamination of cultures is a risk in an unorganized lab
29 Supervisor responsibilities for BSL 1 Access to the laboratory is restricted to authorized personnelie. those who have been advised of the potential hazards, trained and approved by the supervisor
30 Supervisor responsibilities for BSL 1 Post the lab with a biohazard sign indicating the biosafety Level and organism(s) in use Contaminated materials or equipment leaving the lab must be appropriately decontaminated first
31 Supervisor responsibilities for BSL 1 All materials must be contained so that they will not be released to the environment. Disinfectants effective against the material in use, must be available at all times. Have available leak proof containers for transporting materials between labs.
32 Supervisor responsibilities for BSL 1 Maintain an effective insect and rodent control program. Investigate spills and accidents, keep records, use results in future training.
33 Supervisor responsibilities for BSL 1 Biological safety information, such as a manual, must be available- with procedures for the lab- it must be read and followed by all- it must be updated regularly
34 Supervisor responsibilities for BSL 1 Provide MSDSs or equivalent.Ensure compliance within lab.Any use of animals must comply with the CCAC and any training requirements of the CAF and follow the SOP.
35 Additional biosafety lab information 1) Bunsen burners 2) Ultraviolet light 3) Causes of contamination 4) airflow diagram of a biological cabinet 5) airflow diagram of a fume hood 6) airflow diagram of a clean air bench
36 Bunsen Burners Substitute: Do not use combustibles or flammables in a biological cabinet – 70% recirculated will allow buildup of leaking gasesUpward hot air flow disrupts downward laminar flowMay create holes in the HEPA filtersSplashes may occur (when using loops) and could cause contaminationSubstitute:Micro incineratorAlcohol burnerIf the flame was to extinguish there would be a continuous supply of gas into the cabinet – which is 70 percent recirculated – could cause an explosion
37 Ultra Violet Light UV lights are not recommended: they provide a false sense of security - not giving off UV light (just prior to the end life of the bulb)only disinfects the surface – could contact a layer of media protein covering the pathogencasts a shadow, leaving areas that remain unexposed to the UV wavelengthscould cause burns / damage eyes – turn off when working in the roomUV lights are not recommended, provide only surface disinfection, Require frequent testing - appear to be functioning but are not giving off germicidal UV light, can cause burnsIf UV lights are used be sure the lab has no people in it, close the sash before turning the light on (turn the motor blower off when you close the sash).Equipment will also cast a shadow – show diagram of light / shadow resulting in areas that remain unsterilized
38 Causes of Contamination 1) YOU!hands not clean, bacteria/spore under your nailsgloves – not sterile from the boxdirty lab coatslab coat sleeves should be tapedplace all materials (cleaned) required into hood BEFORE work2) IncubatorAvoid dip in line from CO2 tankClean humidity source regularlyClean all surfaces after spill, including holes on traysIf at ground level – tape bench paper onto floor
39 Class II Biological Safety Cabinet use with BSL 2 agents yes - personnel protectionyes - product protectionyes - environmental protectionDesigned to contain biological hazards, Inward airflow for personnel protection, HEPA filtered exhaust air for environmental protection, Supply air HEPA filter for product protection, Open front with inward airflow for personnel protection, Downward HEPA filtered laminar airflow for product protection, HEPA filtered exhaust air for environmental protection, Exhaust approximately 30% of total air handled and recirculate 70% in cabinet
40 Chemical Fume Hood do not use with biological agents yes - personnel protectionno - product protectionno - environmental protectionOffers only personal protection, always exhausts air to the outside, does not offer protection to the product or the environment, as there is no filtration of intake and exhaust air, draws contaminants in the laboratory air directly over the product being worked on
41 Clean Air Bench use only with BSL 1 no - personnel protectionyes - product protectionno - environmental protectionProvide product protection only, Product protection is provided by creating a unidirectional airflow generated through a HEPA filter, discharge air goes directly into workroom
42 Information for McMaster staff/students Do not ship any samples, or take samples with you on a trip. Check with Mike McGuire to ensure it does not require Transportation of Dangerous Goods complianceFile a Materials Transfer Agreement with Bertha Monrose before you share material with another institution
43 Information for McMaster staff/students Do not transform your own body cells. The immune protection you would normally have has now been bypassedObtain a biosafety approval number from the Biosafety office, HSC 1J11 ext before importing material or working with biological material
44 Information for McMaster staff/students Cell lines can be BSL 1 or BSL 2. Check with your supplier or your supervisor.If you are isolating primary cell lines from ill humans or animals, they may be BSL 2 until they are well characterized. Check with the Biosafety office first.
45 Use of a biological cabinet Some labs may chose to use Class II biological cabinets to maintain sterility for their BSL 1 agents.The following slides cover information for the use of biological cabinets.
46 Before you beginCheck to ensure you are using the right cabinet for your work.Check to see it has been certified in the last 12 months.Ensure the UV light is off anytime anyone is in the room.To start, let the cabinet run for 3 minutes to purge air.
47 Before you beginUse a disinfectant that will kill any organism in the cabinetCheck to see that the window is in the correct positionDo not place pens or paper inside the cabinetNever block the grillesSegregate clean items from dirty ones
48 Before you begin Wear a lab coat and tape the coat at the wrist Pull the gloves over the taped coatSterilize the gloves with a 70% alcohol washAs you place items into the hood, spray them down with alcohol to sterilize
49 Before you begin Place sterile items on the left side Place items from the incubator at center backYou may place a work pad soaked in disinfectant on the center front work areaOn the right side, place a waste bucket for fluids, one for sharps, one for solids
50 Working in the cabinetKeep lab doors closed and keep others out of the roomOnly one person working in the cabinetUse good aseptic techniqueSlow movements directly in or outKeep sterile and non-sterile items separatedDo not use Bunsen burners in cabinet
51 Working in the cabinet Have posted spill protocol nearby No gum, food or drinkSee the two powerpoint presentations on Good Culture Practices and Misidentification and Contamination, to learn more about good practices and why they are important.
52 When you are finishedSpray each item with a disinfectant as you take it out of the cabinetFill the liquid waste to the max fill line to achieve appropriate concentration. Mark on the time when it can be disposedSeal the top of the solid waste bag, spray it, put it into the biohazard cardboard boxClose the top of the sharps container, leave it in the hood
53 When you are finishedSpray all interior surfaces with the disinfectant, remember to spray the plastic window facing you.If possible, leave the cabinet running.If not, let it purge for 3-4 minutes.If there is anything unusual, tell your supervisor and leave a note for the next person who will be using the hood.
54 Clean ----------------------- Dirty Suggested layout for working in a Class II biohoodAnew materialsB materialsfrom incubatorCgarbageD materialsto go back intoincubatorE workingsurfaceF wet materialsolution (bleach)Clean Dirty
55 Contact informationFor additional training videos, biosafety approval, import permits, reporting of spills or lab acquired infections, or general questionsFHSc. Safety OfficeHealth Sciences Center 1J11, extCarol Carte or Karin Cassidy