4.6.2 General Safe Work Precautions 1. All workers should routinely use appropriate barrier precautions to prevent skin and mucous membrane exposure when contact with human blood or other body fluids is anticipated. 2. Eating, drinking, smoking, applying cosmetics or lip balm, and handling contact lenses are prohibited. 3. Gloves shall be worn when touching blood and body fluids, mucous membranes, or non-intact skin, for handling items or surfaces soiled with blood or body fluids, and for performing venipuncture and other vascular access procedures. If a glove is torn or damaged during use, it shall be removed and a new glove used as promptly as safety permits. Disposable gloves shall not be washed or disinfected for reuse. Washing with surfactants may enhance penetration of liquids through undetected holes in the glove. Disinfecting agents may cause deterioration of the glove material. 4. Masks and protective eyewear or face shields shall be worn during procedures that are likely to generate droplets of blood or other body fluids to prevent exposure of mucous membranes of the mouth, nose, and eyes. 5. Long sleeved lab coats completely buttoned or aprons shall be worn during procedures that are likely to generate splashes of blood or other body fluids. Protective clothing should be removed before leaving the area. 6. Hands and other skin surfaces shall be washed immediately and thoroughly if contaminated with blood or other body fluids. Hands shall be washed immediately after gloves are removed since no barrier is 100% effective. 7. Workers shall take precautions to prevent injuries caused by needles, scalpels, and other sharp instruments or devices during procedures, when cleaning used instruments, during disposal of used needles, and when handling sharp instruments after procedures. Needles and syringes should beused only in those situations when there is no alternative. To prevent needlestick injuries, needles should not be recapped, purposely bent or broken by hand, removed from disposable syringes, or otherwise manipulated by hand. After they are used, disposable syringes and needles, scalpel blades, and other sharp items shall be placed in puncture-resistant containers for disposal. The puncture-resistant container shall be located as close to the use area as practical. 8. Workers who have exudative lesions, weeping dermatitis, cuts, open wounds or other breaks in the skin shall either refrain from all direct contact with blood and other body fluids until the condition resolves, or utilize protective barriers to reduce the risk of exposure. 9. Pregnant workers shall be especially familiar with and strictly adhere to precautions to minimize the risk of perinatal transmission of bloodborne pathogens. 4.7 Biosafety Signs and Labels The Public Health Agency of Canada and the Medical Research Council (MRC) require that warning signs and/or symbols be used to inform personnel and visitors of the potential of hazards in the workplace. Specifically, with regard to biohazards, the universal biohazard warning sign must be used to "signify the actual or potential presence of a biohazard and to identify equipment, containers, rooms, materials, experimental animals or combinations thereof, which contain and/or are contaminated with, viable hazardous agents." 1. TRU requires that the universal biohazard symbol be used to designate the presence of agents/substances that are believed to be biohazardous. 2. All laboratories and work areas utilizing and/or storing biohazardous substances must have the appropriate biohazardous caution sign posted prominently. If infectious agents are used, a biohazard sign must be located outside the laboratory door to indicate the nature of the hazard, the biohazard level, special provisions for entry and contact information for the Principal Investigator and/or other responsible person(s). 3. Principal Investigators/Supervisors are responsible for ensuring that all hazard signs are current and accurate. Notify the Biosafety Officer if changes are necessary in laboratory door signage and/or equipment labeling. 4.8 Access/Security Controls Doors must be locked when laboratories are unoccupied and only authorized persons are permitted to enter laboratory working areas. Children under the age of 14 years must not be permitted to enter laboratory working areas. 4.9 Cell Culture All new cell lines introduced into TRU must be registered with the Biosafety Officer. Storage and retrieval of frozen cell cultures from liquid nitrogen require appropriate personal protective equipment. There are three major risks associated with liquid nitrogen (-196 °C): frostbite, asphyxiation and exposure. Gloves shall be worn that are thick enough to provide insulation, but flexible enough to allow manipulation of ampoules. When ampoules are submerged in liquid nitrogen, a high-pressure differential results between the outside and the inside of the ampoule. If the ampoule is not perfectly sealed, the pressure differential may result in inspiration of liquid nitrogen, which may cause the ampoule to explode violently when thawed. Wear eye protection, a face shield and earplugs are recommended Biological safety cabinets shall be kept clean and free of unnecessary equipment and material to ensure proper functioning of the cabinet. Liquid waste shall be decontaminated by chemical disinfectant (e.g., 10% hypochlorite). Vacuum collection flasks for liquid waste shall be kept outside the cabinet in a secure place and should contain an appropriate disinfectant .The collection flask shall also have a back-up trap to protect the central vacuum line. Decanting shall be done to minimize splashing. All flasks should be properly labelled. Decontamination of the biological safety cabinet shall be done with a liberal spray with supplied disinfectant followed by a wipe with 70% ethanol at the end of the procedure. Solid waste shall be placed in biological waste bags and the bags then sealed for autoclaving. Biohazard disposal containers with lids shall be used for primary disposal. Glass pipets shalld be placed in a pipet container with an appropriate disinfectant. Plastic disposable pipets must be disposed of in an appropriate container. Contaminated sharps shall be placed in the yellow biohazardous sharps containers 4.10 Personal Protective Equipment (PPE) The type and extent of clothing and equipment to be selected for any particular procedure depend on the research operations and levels of risk associated with them. At a minimum, a lab coat, closed-toe shoes, and gloves must be worn in any microbiology laboratory. Lab coats, closed-toe shoes, and gloves prevent biohazardous materials from contact with the skin, including areas where there might be cuts, abrasions, or dermatitis. The legs are a vulnerable area if uncovered, so it is inappropriate to wear skirts or shorts. Closed- toe shoes protect the feet from spills as well as injuries from dropped sharps. Soles must be non slip to avoid slips and falls, 4.10.1 Lab Coats The lab coat protects street clothing from contamination and prevents possible cross-contamination from any normal flora present on the skin. Lab coats must be worn by all personnel, including visitors, trainees and others entering orworking in the laboratory. Coats must be properly fastened. If contaminated, lab coats shall be decontaminated by autoclaving before being placed in the laundry. If decontamination is not possible, any contaminated coat shall be placed in the biohazard waste container. Lab coats are stored in area separate from street clothes. 4.10.2 Gloves Appropriate gloves must be worn for all procedures that might involve direct or accidental skin contact with biohazardous materials. Latex or vinyl gloves offer a high level of dexterity and a higher level of sensitivity; however, they don’t offer a great deal of protection from needle sticks, animal bites or sharps. All gloves will eventually permeate and shall therefore be changed periodically. If gloves become contaminated or torn, remove immediately and wash hands with soap. Some procedures may require double gloving. Gloves should overwrap the cuff and lower sleeve of the lab coat if double gloving is practiced Gloves must be removed prior to leaving the laboratory and decontaminated with other laboratory wastes before disposal. The following chart lists preferential gloves for different products. TYPE ADVANTAGES Natural rubber latex Low cost, good physical properties, dexterity Natural rubber blends Low cost, dexterity, generally better chemical resistance than natural rubber. Low cost, very good physical properties, average chemical resistance. Polyvinyl chloride (PVC) DISADVANTAGES FOR USE WITH: Poor against oils, greases, organic solvents, ethidium bromide. May cause allergic reactions. Physical properties often inferior to natural rubber. May cause allergic reaction. Bases, acids, alcohols, dilute aqueous solutions. Fair vs. aldehydes, ketones. Plasticizers can be stripped. Strong acids and bases, salts, aqueous solutions, alcohols, o greases and petroleum produc Bases, acids, alcohols, dilute aqueous solutions. Fair vs. aldehydes, ketones. Neoprene Average cost, average chemical resistance, average physical properties, high tensile strength, high heat resistance. Low cost, excellent physical properties, dexterity Poor vs. chlorinated hydrocarbons Oxidizing acids, alcohols, anilin phenol, glycol ethers, solvents oils, mild corrosives Poor vs. chlorinated organic solvents Good resistance to polar organics, high resistance to gas and water vapour Resists broad range of organics, good physical properties. Expensive, poor vs. hydrocarbons, chlorinated solvents Very expensive. Water sensitive, poor vs. light alcohols, acids and bases. Oils, greases, xylene, aliphatic hydrocarbons, perchloroethyle trichloroethane, ethidium bromide. Fair vs. toluene. Glycol ethers, ketones, esters, aldehydes, polar organic solve Fluroelastomer (Vitron®) Good resistance to organic and aromatic solvents. Flexible. Norfoil, Silver Shield™, 4H™ Excellent chemical resistance. Extremely expensive. Poor physical properties. Poor vs. some ketones, esters, amines Poor fit, stiff, easily punctures, poor grip. Nitrile Butyl Polyvinyl alcohol (PVA) Aliphatic and aromatic hydrocarbons, chlorinated solvents, ketones (except acetone), esters, ethers Aromatics and aliphatic hydrocarbons, chlorinated solvents, oils, lubricants, mine acids, alcohols. Use for Hazmat work. Good fo range of solvents, acids and ba 5. BIOLOGICAL SAFETY CABINETS (BSCs) Protection of the respiratory system is of major concern in any biological safety program because infectious organisms can readily enter the human body through the respiratory tract. Engineering controls (biological safety cabinets) are the primary barrier for inhalation of biohazard and should be use whenever possible. Respirators should only be used as secondary means of control. Any individual needing respiratory protection will be required to participate in the Respiratory Protection Program. The BSO will contact the Dean of Science who will ensure respirators will be made to individuals who require such. A biological safety cabinet is a ventilated cabinet that uses a combination of HEPA (high efficiency particulate air) filtration, laminar air flow and containment to provide personnel, product and environmental protection from particulates or aerosols involving biohazardous materials. It is distinguished from a chemical fume hood by the presence of HEPA filtration and the laminar nature of the air flow. The following is a basic description of the two classes of biological safety cabinets, their capabilities and the limitations of each class. 5.1 Class I Biological Safety Cabinet This is a ventilated cabinet which provides partial protection to the worker and environment but no protection to the work. These cabinets have unrecirculated airflow away from the operator that is discharged to the atmosphere after filtration through a HEPA filter. Chemical carcinogens and low level of radioactive materials and volatile solvents can be used in a Class I safety cabinet. 5.2 Class II Biological Safety Cabinet This is a ventilated cabinet that provides personnel, product and environmental protection. These cabinets have an inward airflow and HEPA-filtered supply and exhaust air. A class II biological safety cabinet shall be used whenever there is a risk of creating potentially infectious aerosols or droplets. Examples of processes that may create aerosols are: centrifuging, grinding, blending, vigorous shaking or mixing, opening containers of infectious materials that are at other than ambient pressure. Use of a centrifuge in the open laboratory may be carried out safely only if the samples have sealed heads or centrifuge safety caps are used. However, it is strongly recommended that centrifuging gets done in a contaminant hood at all times. Class II BSCs are the most commonly used BSC on campus. These cabinets provide personnel, environmental and product protection. (See Figure 3). Only BSCs that have hard ducts to the outside and provide a face velocity of 80 to 125 feet per minute should be used when working with volatile chemicals. Note: these cabinets are not designed to prevent ignition of volatile flammable chemicals 5.3 Working in a Biological Safety Cabinet • Turn the cabinet on for at least 10-15 minutes prior to use, if the cabinet is not always operating. • Disinfect work surface with suitable disinfectant followed by 70% alcohol. • Consider the materials necessary for the work to be conducted in the cabinet. • Place all required materials on an absorbent pad to avoid aerosol generation • Place items into the cabinet so they can be used efficiently without unnecessary disruption of the air flow, working with materials from the clean to the dirty side. • Wear appropriate personal protective equipment. At a minimum, this will include a buttoned laboratory coat and gloves. • Delay manipulation of materials for approximately one minute after placing the hands/arms inside the cabinet. • Minimize the frequency of hand movements in and out of the cabinet. • If it is required to move out of the BSC pull hands slowly and horizonatally from the cabinet. Allow time for the air flow disruption to diminish before putting hands back into the BSC • Do not disturb the airflow by covering any grillwork with materials. • Work at a moderate pace to prevent the air flow disruption that occurs with rapid movements. • Wipe the bottom and side of the hood surfaces with disinfectant when work is completed. • Leave the hood running for several minutes following the procedures before turning off the blower NOTE: BE VERY CAREFUL WHEN USING SMALL PIECES OF MATERIAL SUCH AS KIMWIPES IN THE HOOD. THESE CAN BE BLOWN INTO THE HOOD AND DISRUPT THE MOTOR OPERATIONS. ** THE BSC IN S365C IS T USE FOR STERILITY ONLY. FOR USE WITH POTENTIAL PATHOGENS USE THE BSC IN S367. NO CHEMICAL USE IS ALLOWED IN EITHER OF THESE BSCs. THE BSC IN S363 MAY BE USED WHEN VOLATILE CHEMICALS ARE REQUIRED TO BE USED IN THE BSC 5.4 Certification of BSC All biological safety cabinets must be certified on installation, when the filters are changed, before and after a move or transfer and annually. Cabinets must not be moved without first undergoing a decontamination process. The Biosafety Officer must approve any modifications to any biological safety cabinets. Cabinets must undergo certification following modification. The certification process is arranged for annually by the BSO. If problems are encountered in operating a biological safety cabinet, do not continue to use it; contact the BSO immediately. The certification of the biological safety cabinets is essential to their safe and effective use. 6. LABORATORY EQUIPMENT SAFE OPERATING PROCEDURES 6.1 Sonicators When used with infectious agents sonicators can release significant amounts of hazardous aerosols, and shall be operated inside a biological safety cabinet whenever possible. Sonicators are devices commonly used for disrupting cells and mixing samples. Vortexers are also used for mixing samples. The following safety measures should be used with a sonicator to reduce the chance of aerosol formation. Safe Operating Procedures include: • Loosely cap all samples. • Make sure there is enough water in the sonicator. • Avoid prolonged sonication. • Inspect all glassware to be used in the sonicator. Do not use chipped or cracked glassware. • Routinely replace the sonicator liquid. • Avoid sonicating volatile compounds. • When possible, use secondary containment (container within container within the sonicator). • Perform sonicating in isolated rooms and areas. • Make sure you have adequate ear protection. • Allow aerosols to settle for at least one minute before opening containers. 6.2 Centrifuges Safe use of centrifuges requires proper maintenance and operation. Failed mechanical parts or improper operation can result in release of projectiles, hazardous chemicals and biohazardous aerosols. Maintenance and repairs must be performed only by trained, qualified personnel. Centrifuges are a source of potential biological contamination due to the rapid speeds and relatively high pressure exerted by such devices. The following safety measures shall be used when using any centrifuge: Safe Operating Procedure includes: • Prior to starting, make sure the centrifuge is clean. Do not operate with any material spills in either the body or the rotor. • Ensure that the interlocking device prevents the lid from being opened when the rotor is in motion and the centrifuge from starting when the lid is open. • Make sure the centrifuge is level. If a portable model, make sure it is secure on the bench top before starting. • Inspect all equipment to be placed in centrifuge for cracks or weak areas. Ensure that the tube material provides the necessary chemical resistance and speed rating. • Use the lowest speed and time setting that will accomplish the job. • Avoid over-filling tubes. • Balance all loads. • Do not open the lid until it comes to a complete stop. • Wait for at least one minute before opening the lid to remove your sample. • Should a spill occur, disinfect immediately and dry completely before the next run. • Periodically inspect centrifuge. Check seal around top, baskets, rotors and wiring. • Avoid use of volatile materials when possible. • Plastic centrifuge tubes with seal-forming screw tops should be used whenever possible. • Centrifuges should not be placed into a biological safety cabinet if the motor produces strong air current because the air turbulence generated may disrupt the laminar airflow. 6.3 Vacuum If there is a vacuum system serving multiple areas, care should be taken that there are filters in the system, and that there is an overflow trap containing an appropriate disinfectant to prevent entry of contaminated material into the piping system and pumps. It is often best to use either a stand-alone pump-type vacuum system, or to use a water siphon vacuum system that is attached to a faucet (provided that measures are taken to prevent backsiphonage). 6.4 Gas Chromatograph Gas chromatography (GC) procedures involve the use of compressed gas cylinders and may involve the use of flammable solvents and toxic chemicals. Be familiar with the use and handling of compressed gas cylinders, and with hazardous properties, precautionary measures, and handling instructions for any hazardous materials being used. Refer to MSDSs (found on-line – use the MSDS icon on the desktop) or other reliable reference material. The following guidelines will assist in the safe operation of GCs: • Wear proper eye protection. GC columns are fragile and breakage could result in small projectiles during handling. As well, samples are prepared in various hazardous solvents that could damage the eyes upon contact. • When cutting a GC column, be sure that the cut is made away from the body. • Ensure that GC column cutters are capped or otherwise stored to prevent injury when not in use. • Discard small pieces of GC columns as sharps waste. • Ensure that the oven is allowed to cool before installing or removing a column or injector or prior to performing maintenance. • Ensure that gases are turned off prior to removing or installing a column. • Test for leaks after the installation of the column and whenever a leak is suspected. Use a technique that will not damage or sacrifice the integrity of the instrument. • Electron capture detectors (ECD) have a radioactive source and therefore need to be registered as part of the University’s Radiation Safety program. Contact Ron Smith ext.5544 for more information about Canadian Nuclear Safety Commission (CNSC) requirements. • Ensure that the instrument and gases are turned off and the power cord disconnected prior to performing maintenance. 6.5 Ultraviolet lamps Exposure to ultraviolet light (UV) may result in serious and painful injury to the eyes or skin depending on the wavelength and intensity of the light and the duration of exposure. • Label all UV light sources conspicuously with the following warning (or equivalent): “Warning – this device produces potentially harmful UV light. Protect eyes and skin from exposure.” • Ensure that the UV light source is shielded. • Ensure that appropriate PPE is worn and is sufficient to protect the eyes and skin. PPE shall include at least UV resistant face shield, gloves, and lab coat. • Shielding the equipment or the work area may be warranted. 6.6 Bunsen burners Bunsen burners are used for heating (fixing cells onto slides) and sterilization of loops and picks. Allow material to dry before sterilizing a loop or pick to avoid splattering of material and creating possible aerosol. Start flaming at top of loop or pick and move down toward the end. Do not use Bunsen burners in BSCs. 6.7 Pipetting aids Pipetting aids minimze the risk of creating aerosols. Dispensing liquid and subsequent mixing with a glass pipet can create aerosols. Use filtered pipets and pipets aidsand filtered pipet tips for micropipettors. Discharge liquids as close to the wall of the receiving vessel as possible or to the surface of media to avoid the creation of aerosols. Avoid forecfully aspirating oe expelling liquid from a pipette. 6.8 Standard Operation for Autoclaves In S365 I. ASSOCIATED RISKS Autoclaves are sterilizers using high pressure and high temperature steam. The potential safety risks for the operators are: • • Heat burns -from hot materials and autoclave chamber walls and door Steam burns -from residual steam coming out from autoclave and materials on completion of cycle • • • Hot fluid scalds- from boiling liquids and spillage in autoclave. Hand and arm injuries when closing the door. Body injury if there is an explosion II. Equipment to protect against scalds and burns: • • Heat-insulating gloves that provide complete coverage of hands and forearms. Closed-toed footwear. III. OPERATOR INSTRUCTIONS Training All operators must have successfully completed an authorized training session on the safe operating procedures of this autoclave. This requirement applies to both new and experienced personnel. A list of authorized users will be kept with the cycle records. Before Autoclaving can be done Ensure you do the Following Before turning on Autoclave • • • • • Before turning on the autoclave, drain the blue generator by : o opening the tap(labeled #1) at the bottom of the generator o opening the tap (labeled #2) on the side of the autoclave facing the wall to the lab o allow the water to drain for 5 minutes Close the taps that were opened to drain the generator Turn on the power supply switch on the wall beside the autoclave (labeled power switch) Turn on the water supply lever labeled water supply, (it is on when it is parallel with the water supply pipe and off when it is perpendicular to the pipe) Allow the pressure in the jacket to come up to 15 pounds before starting the autoclave (about 30 minutes) Material Preparation • • • Ensure that the material is able to be autoclaved samples containing solvents or substances that may emit toxic fumes should not be autoclaved. Glassware must be inspected for cracks prior to autoclaving. Prepare and package material suitably: Loose dry materials must be wrapped or bagged in steam-penetrable paper or loosely covered with aluminum foil. Wrapping too tightly will impede steam penetration, decreasing efficiency of the process. All containers must be covered by a loosened lid or steam-penetrable bung. Containers of liquid must be a maximum of 2/3 full, with lids loosened. Glassware must be heat-resistant borosilicate. Plastics must be heat-resistant eg: polycarbonate (PC), PTFE ("Teflon") and most polypropylene (PP) items. Items or containers must be tagged with autoclave tape to verify sterilization. Loosen all lids to prevent pressure buildup. Add water to containers as appropriate. • • Place items in containers to secure and contain spills: Items should be placed in a stainless steel or autoclavable plastic container for their stability and ease of handling. Place containers of liquid, bags of agar plates, or other materials that may boil over or leak, into a secondary pan in the autoclave. The pan must be large enough to contain a total spill of the contents. Bags must not be tightly sealed as steam cannot penetrate. Remove all labels from glassware prior to autoclaving Biohazard materials must be labelled as such and secured in containment vessels or autoclavable bags and processed as soon as possible according to requirements for the handling of infectious or biohazard materials. Loading Autoclave • • • • • Wear heat-insulating gloves, and closed toed shoes. Place material in autoclave. Do not mix incompatible materials. Do not overload; leave sufficient room for steam circulation. If necessary, place the container on its side to maximize steam penetration and avoid entrapment of air. Close and latch door firmly by raising the lid gently till it clicks into place. Do Not Let it Slam at the top as it will break the switch located there Operating Autoclave • • • • • Choose appropriate cycle ( e.g. liquid, dry unwrapped or wrapped etc) for the material. Set appropriate temperature for the cycle (if necessary, usually all loads are processed at 121° C Press the start button Do not attempt to open the door while autoclave is operating. The manuals for operation of the autoclave are located in the cupboard adjacent to small autoclave and under behind door panel of large autoclave Unloading Autoclave • • • • • • Wear heat-insulating gloves and closed toed shoes. Ensure the load is complete Wear gloves and stand back from the door as a precaution, carefully crack door open no more than 1 inch (2.5 cm) to release residual steam and allow pressure within liquids and containers to normalize. Allow sterilized material to stand for 10 minutes in the chamber. This will allow steam to clear and trapped air to escape from hot liquids, reducing risk to operator. Do not agitate containers of super-heated liquids or remove caps before unloading. After removal from the autoclave, place liquid agar in the water bath in the media area that should be turned on before starting the load. This will allow the media t cool to a temp ideal for pouring Maintenance and repair • No person shall operate the autoclave unless the autoclave is in good repair • Users are not to make repairs • Report possible malfunctions to Supervisor or Micro Lab Technician IV. CONTINGENCY PLAN Equipment malfunction • • • If the autoclave does not operate exactly as expected, do not attempt to fix the problem. A notice shall be placed on the autoclave indicating that it is not to be used until the problem is diagnosed and corrected. Record the problem in the autoclave log book. Contact _Carolynne or Shannon to report the problem. Repair of autoclaves shall be performed by qualified persons only.