Laboratory apparatus must be used only for its designed purpose unless appropriate safety modifications are made. Operating manuals should be consulted for detailed operating instructions for individual pieces of equipment.
| § ELECTRICAL |
All electrical equipment used in the laboratory must be grounded. Ground fault circuitt interrupters must be used whenever equipment is in a wet environment such as a cold room.
Electrical apparatus must be plugged into sockets which can be reached safely, without exposure to hazards.
Electrical apparatus used in a biosafety cabinets must be plugged inside the hood.
Electrical cords must be as short as practical and must be placed in such a way that the risk of tripping or spills is minimized.
Extension cords must be avoided. If unavoidable, ascertain that the extension cord is appropriate. Consult Facilities Managment or the Office of Office of Health and Safety for information.
Equipment, including electrical plugs and cords, must be kept in good repair. Electrical equipment must be unplugged before routine parts replacement or before making internal adjustments.
A qualified electrician must make electrical repairs.
Non-sparking electrical switches and motors are desirable in laboratory equipment to prevent combustion of any volatile flammable gases.
| § MICROWAVES |
Do not operate the oven if it is damaged. It is very important that the oven door close properly and that there is no damage to the door seals and sealing surfaces, hinges and latches.
Since there may be residual contamination, never use the laboratory microwave oven to heat food or drinks. Institute policy and federal regulations do not allow these items consumed or stored in the laboratory. Microwave in laboratory must be signed "Laboratory Use Only"
Do not use the microwave oven to heat up hazardous chemicals or radioactive materials.
Do not use ALUMINUM FOIL at anytime during the heating cycle. Metal utensils and utensils with metallic trim should not be used in the microwave oven.
Avoid heating materials in cylindrical-shaped containers. Liquids heated in certain shaped containers (especially cylindrical-shaped containers) may become overheated. When overheated, liquids may splatter during or after the heating cycle resulting in possible employee injury or damage to the microwave oven.
When heating liquids in screw-cap bottles, completely loosen the screw-caps to prevent pressure build-up within the container. This pressure build-up with a cap that is not sufficiently loose can cause the bottle to explode.
If steam accumulates inside or outside of the oven door, wipe with a soft cloth. This may occur when the microwave oven is operated under high humidity conditions and in no way indicates malfunction of the unit.
Be careful when removing containers from the microwave. Some containers absorb heat and may be very hot. Always use long sleeve laboratory coat, protective gloves and appropriate eye/face protection to minimize any possible injuries.
If materials inside the oven should ignite, KEEP OVEN DOOR CLOSED, turn oven off, and disconnect the power cord.
Do not attempt to tamper with or make any adjustments or repairs to door control panel, safety interlock switches or any other part of the oven. Repairs should be done by qualified service personnel only.
| § BIOLOGICAL SAFETY CABINETS (BSC) |
Primary barriers are important because most laboratory techniques are known to produce aerosol that can be readily inhaled by the laboratory worker. The majority of reported laboratory acquired infections, for which no specific cause has been identified, have been attributed to aerosol exposure (Pike 1967).
Selecting a Biological Safety Cabinet
- Factors influencing BSC selection
There are several types of safety cabinet with their own advantages and limitation; the principal investigator must carefully assess the program and match specific requirements to the appropriate contamination control cabinet. Pertinent factors are:
- Proposed Activity. Procedures that may cause aerosols are of particular
concerns.
- Risk of Biological Commodities. All known characteristics of the
commodity should be evaluated, e.g., infectivity, hazards of pathogenicity, concentration of viable agent, history of known laboratory acquired infections, classification of risk group.
- Control Objectives. The control protection desired should be
determined from the proposed activity and the specific agent.
- Product Protection only.
- Personnel Protection only.
- Personnel and Product Protection, and/or
- Environmental Protection
- Proposed Activity. Procedures that may cause aerosols are of particular
concerns.
- Three basic types of BSC
- Class I: Open fronted exhausts protective cabinets. There are
uncirculated laboratory fume hoods approved by the BSP for activities
involving the use of biological commodities.
- Class II: Vertical laminar flow cabinets. There are recirculating
laminar flow safety cabinets approved by the BSP for activities involving biological commodities.
- Class III: Totally enclosed exhaust protective cabinets. These are
completely enclosed system with negative pressure of gas tight construction, including glove boxes.
- Class I: Open fronted exhausts protective cabinets. There are
uncirculated laboratory fume hoods approved by the BSP for activities
involving the use of biological commodities.
- High Efficient Particulate Air (HEPA) Filter
- Definition
One of the most critical components of any biological safety cabinet is the high efficiency particulate air filter, which in effect stands between you and your experimental biological commodity. HEPA filters consist of continuous sheets of glass fiber paper pleated over rigid corrugated separators and mounted in a wooden frame. They are extremely delicate and the filter media should never be touched.
By definition, the HEPA filter has an efficiency of 99.97% for particles 0.3 microns in diameter. This size particle is used as the basis for filter definition because theoretical studies have shown that filtration efficiency should be at minimum for particles of this diameter with efficiency increasing for particles either larger or smaller. Experimental challenge of HEPA filter with various microbial agents including viruses has proven their effectiveness.
- Usage
HEPA filters are not effective, however, against chemicals in gaseous state that readily pass through these particulate barriers. Since most BSC units are partially recirculating, gaseous build up to a point of equilibrium may occur.
Chemical that readily vaporized, therefore should not be used in these cabinets if they meet one of the following conditions:
- Chemicals that alone or in combination attack filter
components or stainless steel. This must be ascertained for each chemical used in the cabinet.
- Chemicals that may become toxic to the operator, or any combination of two or more chemicals that may react and the resultant product becomes toxic to the
operator. If the cabinet is being correctly used, i.e., operator's arm only inside the unit with the view screen in a safe position and the blowers functioning;
toxicity or irritation should only occur through skin penetration, either directly or through wounds. A proper evaluation of the chemical toxicity should include not only information on single exposure, effects of concentration, but the effect of many small
exposures over time.
- Chemicals that alone or in combination are explosive or flammable. With recirculating build-up an explosion may be caused by ignition of the gas by a motor spark or burner in the work zone. Such explosions have occurred in the past. It is thus an extremely hazardous situation that should be carefully avoided.
- If any one of your experimental chemicals fits any one of the above categories, you should not be working in one of these BSC, but rather a total exhaust cabinet capable of handling the chemical burden.
- Chemicals that alone or in combination attack filter
components or stainless steel. This must be ascertained for each chemical used in the cabinet.
- Filter Life
The lifetime of a HEPA filter is governed by how and where you use your cabinet. Generally, under normal laboratory conditions, a filter will last from two to five years, but misuse of the cabinet and/or a heavy dust load within the laboratory will act to shorten any filter's lifespan.
Use of Bunsen burner other than the "Touch-O-Matic" type may damage filter media, especially if the blowers are not operating. Any destruction due to misuse of chemicals within the cabinet will also cut filter life.
The particulate load of the atmosphere within the laboratory is roughly inversely proportional to filter life. The higher the load, the shorter the lifetime. If your laboratory building is next to a construction site, or near nay area that creates large amounts of dust, the filter life will be shortened significantly. Even if the hood is located near the storage area where frequent opening of boxes and ruffling of packaging materials occurs, the resultant air burden will effect the filter life. (Keep laboratory work area in good housekeeping condition).
To ensure filter integrity and cabinet performance periodic exhaust and supply airflow measurements and filter leak checks should be taken at twelve-month intervals. This will make sure your filter continues offering your safety from you experimental materials.
- Definition
- Certification of BSC
To maintain the protective effectiveness of BSC, specific performance criteria must be met and maintained.
- All biosafety cabinets must be initially certified by an outside contractor, this will assure proper shipping, transporting, handling and installation. Subsequent certification will be done by BSP. Cabinets should be certified in accordance with NSF/ANSI Standard 49.
- The capability of BSC to protect personnel and the environment form
exposure to potentially hazardous aerosols is dependent on both the abilities of the laboratory worker to use the BSC properly and the adequate functioning of the BSC. A BSC should never be used to contain hazardous materials unless it has been demonstrated to meet certain minimum safety specification.
- Certification of the BSC for minimum safety specification are required
whenever:
- New cabinets has been purchased and installed, but before it is used. (An outside contractor does all initial certification).
- After cabinet has been moved or re-located,
- After HEPA filter replacement or maintenance repairs, and
- At least annually.
Limited certification is provided by BSP. Primarily, HEPA filter leak test, inflow (supply) and down flow (exhaust) velocity check. Full testing services can be arranged.
- The important features of a BSC are the air barrier at the front opening,
the supply filter, the exhaust filter and the air flow plenum.
- After locating the cabinet in the laboratory, a visual examination should
be made for gross damage. The cabinet should be leveled and when possible, connected to the building exhaust system.
- The certification process begins with halogen leak test or bubble test, to
assure that any positive pressure airflow plenums and cabinet joints are gas tight. This test should be performed whenever the cabinet is moved or after maintenance in which the cabinet was opened, i.e. after filters have been changed. (Note: This test is not normally done by BSP).
- The air velocity of the air entering the front opening should be determined; this air creates an air barrier that controls the escape of biological commodities from the work area.
- The certification should include a measurement of the airflow within
the BSC to assure that the velocity is uniform and unidirectional. The air prevents cross contamination in the work area.
- Certification should include a filter leak test to assure the filter is
properly installed and leak-free.
- All biosafety cabinets must be initially certified by an outside contractor, this will assure proper shipping, transporting, handling and installation. Subsequent certification will be done by BSP. Cabinets should be certified in accordance with NSF/ANSI Standard 49.
- Biological Safety Cabinet Program
- This program assists campus users in complying with NSF Standard 49, CDC and HDOA ventilation requirements for biosafety cabinet operations. EHSO also assists users in proper use of biosafety cabinets (BSC) and laminar flow clean benches (LFCB).
- When purchasing new biosafety containment equipment please call us for review of proper selection prior to completing of purchase orders. BSP has a reference library of popular models available.
- Please note that biosafety equipment will not be certified unless it is
used for biological containment. The Instutional Biosafety Committee (IBC) has discontinued the
certification/testing of laminar flow clean benches (10/2002), since most use of
this type of equipment is not for biological purposes, but for product
protection. If the equipment is used for manipulating biological commodities,
this is a inappropriate use and a biological safety cabinet should be used for
this type of manipulation. If there are external agencies that requires
certification of laminar flow clean benches for your work, i.e., Food and Drug
Administration and EPA-Pesticide, it will be the responsibility of the the PI
to meet these requirements.
- All biosafety cabinet must be initially certified by an outside vendor,
this will assure proper shipping, transporting, handling and installation. Subsequent certification will be done by BSP.
- This program assists campus users in complying with NSF Standard 49, CDC and HDOA ventilation requirements for biosafety cabinet operations. EHSO also assists users in proper use of biosafety cabinets (BSC) and laminar flow clean benches (LFCB).
- Flammable Gases in Biological Safety Cabinets
Overview
Certain biological safety cabinets (BSCs) recirculate air within the cabinet. Most BSCs at the UH are recirculating. The use of natural gas or other flammable gases within these BSCs may allow flammable gases to concentrate, potentially leading to an explosive atmosphere. The use of flammable gases within a BSC may alter the airflow pattern used to protect product and personnel. This outlines which BSCs recirculate air and the procedures to increase safety and prevent flammable gas explosions within BSCs.
ApplicabilityThis applies to the use of natural gas or other flammable gases inside biological safety cabinets that recirculate air. Natural gas within a BSC is typically "house" gas connected directly to the BSC.
ProcedureCertain types of BSCs are designed to contain, not exhaust, most of the air within a cabinet. This makes them prone to the buildup of materials within the cabinet. The following 4 types of cabinets are located at the UH:
BSC Type Former Name(s) % Recirculated Air Class II Type A1 Class II Type A 70 Class II Type A2 Class II Type A/B3 70 Class II Type B1 N/A 30 Class II Type B2 N/A 0 To determine the type of cabinet, locate the unique serial number on the cabinet. This area should also contain the BSC type. If unable to locate this information, contact EHS for further assistance.
If a gas leak occurs (e.g. valve left on or tube leak) inside a recirculating biological safety cabinet, over time the gas would become more concentrated and could reach explosive levels. Since it is within a BSC, the user may not detect the leak and, upon ignition, it could explode. Therefore, natural gas or other flammable gases should not be used within recirculating biological safety cabinets. Additionally, open flames can affect the airflow pattern of a BSC. According to the National Institutes of Health and the Centers for Disease Control and Prevention, "Open flames are not required in the near microbe-free environment of a biological safety cabinet. On an open bench, flaming the neck of a culture vessel will create an upward air current which prevents microorganisms from falling into the tube or flask. An open flame in a BSC, however, creates turbulence which disrupts the pattern of HEPA-filtered air supplied to the work surface."
Open flames should not be necessary in the near microbe-free environment of a biological safety cabinet. Alternatives to the use of flammable gases to disinfect include:
- The use of an electric bunsen burner.
- The use of a bact-cinerator.
Alternatives that avoid the need to disinfect instruments within a BSC include:
- the use of pre-sterilized inoculating loops and needles.
- pre-autoclaving forceps, scalpels, etc. in covered autoclavable plastic containers or the special sleeves supplied for this use by various companies. These can be taken into the BSC and used individually and placed in an autoclavable discard tray located in the BSC for used/contaminated utensils.
- the use of a Bunsen burner outside the BSC (> 2 feet away from the BSC) for some applications.
Flaming the necks of bottles is not necessary due to the protective airflow in the BSC. Using a flame for this purpose would disturb the airflow and may result in contamination of the tissue culture flasks.
ResponsibilitiesFacilities: New projects and maintenance requests will no longer add house natural gas to biological safety cabinets unless the BSC is verified to be a "total exhaust" cabinet by EHS. Natural gas lines to recirculating BSCs will be turned off as they are discovered. When applicable, natural gas lines to BSCs will be physically disconnected and capped during laboratory renovation projects.
BSC Users: Refrain from using natural gas and other flammable gases within recirculating BSCs.
References
Primary Containment for Biohazards: Selection, Installation and Use of Biological Safety Cabinets, 2nd edition. U.S. Department of Health and Human Services Public Health Service, Centers for Disease Control and Prevention and National Institutes of Health, September 2000.
|
| § LAMINAR FLOW CLEAN BENCHES (LFCB) |
- Definition
The term "laminar flow" as it applies to hoods or clean air equipment can mean many things. "Laminar Flow" typically means air flowing in one direction (unidirectional) with very low turbulence. In a horizontal "Clean Bench" air lows straight out of the hood towards the operator. In a true vertical flow clean bench the air flows directly down onto the work surface, then out into the room. Some people will call Class II BSC a "vertical flow" cabinet although that is technically incorrect.
- Use
This type of ventilation equipment is not suitable for work with biological commodities. Personnel are exposed to contaminated air, as the cabinet's positive pressure allows air to flow out of the cabinet. Such units are suitable only for use with known "clean" materials, where product protection is the only objective.
- Type
Horizontal (cross flow) and vertical (down flow) laminar flow clean bench, that forces air out of the front opening an into the room, should not be used in a biomedical laboratory. They are intended for product protection, not for the safety of the worker. Use of laminar flow clean bench in a biomedical laboratory may subject the worker to a potentially hazardous or allergenic substance. The use of potentially hazardous condition must be addressed to the workers.
- Regulation
Probably the most commonly reference standard for clean benches is Federal Standard 209-B covering Clean Room and Work Station Requirements, Controlled Environment. This standard was published back in 1973 and contained many useful definitions particularly of Air Cleanliness Classes. Federal Standard 209-B was superseded by 209-c and D. The current standard is Federal Standard 209-E. Federal Standard 209-E is still used today since is was the last 209 to include recommendations for air velocity testing and HEPA filter leak testing an appendix titled, "Non-mandatory Supplemental Guidance Information." On page 18, Section 40.3.4, 209-B recommends 90 feet per minute average, with uniformity within +20% across the entire area of the air exit. This has been interpreted over the years to mean the air velocity average should be between 72-108 feet per minute. Section 50.1 (a) on page 18 discusses in-place filter testing for HEPA filters and recommends that no penetrations exceeds 0.01% of upstream smoke concentration. A more current standard addressing clean air workstations is the Institute of Environmental Science (IES) Recommended Practice, January 1986, "Laminar Flow Clean Air Devices."
- Testing and Certification
Like Biological Safety Cabinets, most manufacturers recommend annual testing and certification. For hospital applications, JCAHO goes along with that for the most part. Pharmaceutical companies under FDA jurisdiction are required to perform semi-annual certification. Some State Boards of Pharmacy also require semi-annual certification. For R & D or electronics manufacturing use annual tests are recommended. IES, recommends testing at "regular periodic intervals, at a frequency consistent with location, function, and established guidelines," and "following potentially disruptive events, such as relocation of the device or replacement of the HEPA/ULPA filters."
IES recommends the following tests for certification in the field: Airflow velocity, HEPA/ULPA filter installation leak test, induction leak test/back streaming testing (when appropriate), lighting level (when appropriate), noise level (when appropriate), vibration (when appropriate). The "when appropriate" clauses recognize the necessary testing differences between a horizontal flow clean bench and a ceiling-hung laminar flow module that is 8 feet off the floor.
The Instutional Biosafety Committee (IBC) has discontinued the certification/testing of laminar flow clean benches (10/2002), since most use of this type of equipment is not for biological purposes, but for product protection. If the equipment is used for manipulating biological commodities, this is a inappropriate use and a biological safety cabinet should be used for this type of manipulation. If there are external agencies that requires certification of laminar flow clean benches for your work, i.e., Food and Drug Administration and EPA-Pesticide, it will be the responsibility of the the PI to meet these requirements.
Laminar flow units can be affected by the same problem as BSC, i.e., incorrect location in the work room, in proximity to high traffic areas or doors, room ventilation problems, or building electrical limitations.
- Use
| § VACUUM SYSTEMS |
A simple means to achieve protection against pathogens is illustrated in this diagram.
Dual aspirator flasks (A and B) are placed in series and a disposable in-line HEPA filter (C) is placed between the flasks and the source valve (D). Flask A typically is used to collect the contaminated fluids into a disinfectant such as bleach. Flask B serves as a fluid overflow collection vessel. Extending the tube to the bottom of the flask helps to minimize splatter. The filter should be replaced annually at a minimum and whenever there is evidence of failure or blockage.
When this arrangement is used for work that is done inside a biological safety cabinet (BSC), the flasks also should be located inside the BSC. If placement outside of the BSC is necessary, the flasks should be protected against breakage by being positioned in a leakproof secondary container.
| § CENTRIFUGES |
Hazards associated with centrifuging include mechanical failure and the creation of aerosols. To minimize the risk of mechanical failure, centrifuges must be maintained and used according to the manufacturer's instructions. Users should be properly trained and operating instructions including safety precautions should be prominently posted on the unit.
Aerosols are created by practices such as filling centrifuge tubes, removing supernatant, and re-suspending sediment pellets. The greatest aerosol hazard is created if a tube breaks during centrifugation. To minimize the generation of aerosols when centrifuging biohazardous material, the following procedures should be followed:
Use sealed tubes and safety buckets that seal with O-rings. Before use, inspect tubes, O-rings and buckets for cracks, chips, erosions, bits of broken glass, etc. Do not use aluminum foil to cap centrifuge tubes because it may detach or rupture during centrifugation.
Fill and open centrifuge tubes, rotors and accessories in a BSC. Avoid overfilling of centrifuge tubes so that closures do not become wet. After tubes are filled and sealed, wipe them down with disinfectant.
Add disinfectant to the space between the tube and the bucket to disinfect material in the event of breakage during centrifugation.
Always balance buckets, tubes and rotors properly before centrifugation.
Do not decant or pour off supernatant. Use a vacuum system with appropriate in-line reservoirs and filters. (For more information, call the ORCBS)
Work in a BSC when re-suspending sediment material. Use a swirling rotary motion rather than shaking. If shaking is necessary, wait a few minutes to permit the aerosol to settle before opening the tube.
Small low - speed centrifuges may be placed in a BSC during use to reduce the aerosol escape. High-speed centrifuges pose additional hazards. Precautions should be taken to filter the exhaust air from vacuum lines, to avoid metal fatiguing resulting in disintegration of rotors and to use proper cleaning techniques and centrifuge components. Manufacturer's recommendations must be meticulously followed to avoid metal fatigue, distortion and corrosion.
Avoid the use of celluloid (cellulose nitrate) tubes with biohazardous materials. Celluloid centrifuge tubes are highly flammable and prone to shrinkage with age. They distort on boiling and can be highly explosive in an autoclave. If celluloid tubes must be used, appropriate chemical disinfectants are necessary for decontamination.
| § BLENDERS, ULTRASONIC DISRUPTERS (SONICATORS), GRINDERS AND LYOPHILIZERS |
The use of any of these devices results in considerable aerosol production. Blending, cell disrupting and grinding equipment should be used in a BSC when working with biohazardous materials.
Safety Blenders
Safety blenders, although expensive, are designed to prevent leakage from the bottom of the blender jar, provide a cooling jacket to avoid biological inactivation, and to withstand sterilization by autoclaving. If blender rotors are not leak-proof, they should be tested with sterile saline or dye solution prior to use with biohazardous material. The use of glass blender jars is not recommended because of the breakage potential. If they must be used, glass jars should be covered with a polypropylene jar to prevent spraying of glass and contents in the event the blender jar breaks. A towel moistened with disinfectant should be placed over the top of the blender during use. Before opening the blender jar, allow the unit to rest for at least one minute to allow the aerosol to settle. The device should be decontaminated promptly after use.
Lyophilizers and Ampoules
Depending on lyophilizer design, aerosol production may occur when material is loaded or removed from the lyophilizer unit. If possible, sample material should be loaded in a BSC. The vacuum pump exhaust should be filtered to remove any hazardous agents or, alternatively, the pump can be vented into a BSC. After lyophilization is completed, all surfaces of the unit that have been exposed to the agent should be disinfected. If the lyophilizer is equipped with a removable chamber, it should be closed off and moved to a BSC for unloading and decontamination. Handling of cultures should be minimized and vapor traps should be used wherever possible.
Opening ampoules containing liquid or lyophilized infectious culture material should be performed in a BSC to control the aerosol produced. Gloves must be worn. To open, nick the neck of the ampoule with a file, wrap it in disinfectant soaked towel, hold the ampoule upright and snap it open at the nick. Reconstitute the contents of the ampoule by slowly adding liquid to avoid aerosolization of the dried material. Mix the container. Discard the towel and ampoule top and bottom as biohazardous waste.
Ampoules used to store biohazardous material in liquid nitrogen have exploded causing eye injuries and exposure to the infectious agent. The use of polypropylene tubes eliminates this hazard. These tubes are available dust free or pre-sterilized and are fitted with polyethylene caps with silicone washers. Heat sealable polypropylene tubes are also available.
| § EYEWASHES |
Biosafety requires an eyewash station where active manipulation of biological commodities are handled. Eyewash station must be within 10 seconds or 50 feet from the hazard. Nozzles spray should be not less that 33 inches nor more than 45 inches from standing level. Must stay on. Eyewashes should be routinely flushed in order to make sure they are free of dust and debris and assure that they are in proper working order. A flush log should be kept for each eyewash indicating the dates the flushes where preformed. A sign indicating location should be well lighted and higly visible. All eyewashes need to be easily accessable
| § PIPETTES AND PIPETTING AIDS |
Mouth pipetting is strictly prohibited. Mechanical pipetting aids must be used. Confine pipetting of biohazardous or toxic fluids to a biosafety cabinet if possible. If pipetting is done on the open bench, use absorbent pads or paper on the bench. Use the following precautions:
- Always use cotton-plugged pipettes when pipetting biohazardous or toxic fluids.
- Never prepare any kind of biohazardous mixtures by suction and expulsion through a pipette.
- Biohazardous materials should not be forcibly discharged from pipettes. Use "to deliver" pipettes rather than those requiring "blowout."
- Do not discharge biohazardous material from a pipette at a height. Whenever possible allow the discharge to run down the container wall.
- Place contaminated, reusable pipettes horizontally in a pan containing enough liquid disinfectant to completely cover them.
- Autoclave the pan and pipettes as a unit before processing them for reuse.
- Discard contaminated Pasteur pipettes in an appropriate size sharps container.
- When work is performed inside a biosafety cabinet, all pans or sharps containers for contaminated glassware should be placed inside the cabinet as well while in use.
- Always use cotton-plugged pipettes when pipetting biohazardous or toxic fluids.
- Never prepare any kind of biohazardous mixtures by suction and expulsion through a pipette.
- Biohazardous materials should not be forcibly discharged from pipettes. Use "to deliver" pipettes rather than those requiring "blowout."
- Do not discharge biohazardous material from a pipette at a height. Whenever possible allow the discharge to run down the container wall.
- Place contaminated, reusable pipettes horizontally in a pan containing enough liquid disinfectant to completely cover them.
- Autoclave the pan and pipettes as a unit before processing them for reuse.
- Discard contaminated Pasteur pipettes in an appropriate size sharps container.
- When work is performed inside a biosafety cabinet, all pans or sharps containers for contaminated glassware should be placed inside the cabinet as well while in use.
| § SYRINGES AND NEEDLES |
Syringes and hypodermic needles are dangerous objects that need to be handled with extreme caution to avoid accidental injection and aerosol generation. Generally, the use of syringes and needles should be restricted to procedures for which there is no alternative. Do not use a syringe and needle as a substitute for a pipette.
Use needle locking syringes or disposable syringe-needle units in which the needle is an integral part of the syringe.
When using syringes and needles with biohazardous or potentially infectious agents:
- Work in a biosafety cabinet whenever possible.
- Wear gloves.
- Fill the syringe carefully to minimize air bubbles.
- Expel air, liquid and bubbles from the syringe vertically into a cotton pad moistened with a disinfectant.
Needles should not be bent, sheared, replaced in the sheath or guard (capped), or removed from the syringe following use. If it is essential that a contaminated needle be recapped or removed from a syringe, the use of a mechanical device or the one-handed scoop method must be used. Always dispose of needle and syringe unit promptly into an approved sharps container. (See your laboratory specific Wastes Management Plan and Exposure Control Plan) Do not overfill sharps containers (2/3 filled = full).
Metal Sharps Containers for Use at UH
Hawaii Medical Vitrification requires that sharp containers be uniform in shape and size. Thus upon their suggestion ALL metal sharp containers used by the University of Hawaii community should be from the Becton-Dickinson and Company: These BD products can be obtained from both VWR and Fisher Scientfic. | |
 BD Multi-Use One Piece: Funnel Sharp Containers |
Convenient, vertical "point first" drop, these robust sharps collectors are pre-assembled and offer a one-way funnel valve to minimize needlestick and overfilling. Sizes include 3.3 quarts (qt), 6.9, 8.2, 5 gal.
|
 Bio-Chemical Mixed Wastes or Chemotherapy sharps Containers |
Collectors feature a wide entry port design for disposal of bulky chemotherapy waste. In addition, these collectors are designed to offer increased leak resistance. Non-autoclavable. 5 gallons only. |
| § LOOP STERILIZERS AND BUNSEN BURNERS |
Sterilization of inoculating loops or needles in an open flame generates small particle aerosols that may contain viable microorganisms. The use of a shielded electric incinerator or hot bead sterilizers minimizes aerosol production during loop sterilization. Alternatively, disposable plastic loops and needles may be used for culture work where electric incinerators or gas flames are not available or recommended. Wear eye protection.
Continuous flame gas burners should not be used in BSCs. These burners can produce turbulence which disturbs the protective airflow patterns of the cabinet. Additionally, the heat produced by the continuous flame may damage the HEPA filter.
The use of alcohol lamps are strictly discouraged and maybe prohibited by fire code.
 Bacti-cinerator |
 BackElectric burner |
Bacti-cinerator. Sterilizes needles, loops, and culture tube mouths in five to seven seconds at optimum sterilizing temperature of 815.6°C (1500°F). Eliminates microorganism spattering associated with flame sterilization. Ceramic funnel tube enclosed in stainless-steel perforated guard and cast-aluminum support stand.
Electric burner. Electric Bunsen burner. Temperature ranges from 800 - 1000ºC (1472 - 1832ºF). Replaces heating elements. Corrosion resistant metal housing.
Combine the advantages of the gas burner with the clean operation and control ease of electric heating. Conical shaped heating elements are easy to replace. Since the radiation emitted from the heater is directed upwards only, it can be used for such purposes as heating test tubes, crucibles, small flasks and beakers independent of their shape. Air circulation from the vented housing keeps the base cool enough to hold during operation. The top cowl deflects heat away from your hand.
Glass Bead Sterilizers
 Glass Bead sterilizer |
Glass Beads Sterilizers are not FDA approved sterilizers. However, they can be used in non health care facilities. Glass Beads sterilizers are a quick, easy and accurate alternative to traditional methods of sterilization. Once switched 'ON', the units are ready to use within 30 Minutes and ensure total sterilization. The constant temperature of 230° C ensures the total distruction of any pathogen and non-pathogen microorganisms in a few seconds.
Various models of sterilization instruments are glass bead sterilizer and hot air sterilizers which have been introduced and are being used in many laboratory and biotechnology / tissue culture for the past 30 years. Over the years, apart from the domestic market, an overseas market has also been identified and the units have been exported to several countries including Australia, Sri Lanka, Hungary, Spain and U.S.A.
Most Glass Beads Sterilizers are incorporated with an imported thermostat, which maintains the set temperature with an accuracy of ± 1% of the preset range. These units, having a stainless steel body, are compact enough to be placed on any Laminar Air Flow Work benches or any other workside tables in a clean room atmosphere.
| § REFRIGERATORS, FREEZERS AND MICROWAVES |
Refrigerators
Appropriate use of refrigerators in the laboratory is essential for the health and safety of laboratory personnel. The following stringent procedures must be implemented and maintained for safety use of refrigerators.
Refrigerators must never be used for the storage of food or beverages (non-laboratory use).
Personnel must avoid direct inhalation of refrigerator atmosphere. Because there is almost never an adequate ventilating device for the interior of refrigerators, the atmosphere in the refrigerator may contain a mixture of air combined with the vapor from flammable or toxic substances.
There must be no source of electrical sparks within the refrigerator compartment.
Modification of refrigerator units include removing any interior light that is activated by switch mounted o the door frame; moving the contacts of the thermostat controlling the temperature to a position outside the refrigerated compartment; and removing the contacts for any thermostat present that controls fans within the refrigerated compartment to the outside of the refrigerated compartment. An alternative to modification is to place a prominent sign warning against storage of flammable substances within the unit.
Where appropriate, purchase a unit that has been designated for "flammable storage" by the manufacturer.
"Frost free" refrigerator are not appropriate for laboratories because the modification problems. Drain tubes or holes direct water (and any flammable material present) to areas adjacent to the compressor and presents a spark hazard.
Uncapped containers are not permitted in refrigerators.
Containers capped with aluminum foil, corks, corks wrapped in aluminum foil, or glass stoppers are not permitted.
Potentially explosive or highly toxic substances in laboratory refrigerators are discouraged. Prominent warning signs are displayed on the outside of the unit. Laboratories using such materials should purchase and use an explosion-proof refrigerator.
Scheduled inventory and disposal of outdated or unnecessary materials must be maintained. Dispose of any materials that are not essential immediately.
Storage of some reagents is best accomplished by refrigeration. Refrigeration of certain laboratory reagents refers to refrigeration of materials at temperature between 4oC and 10oC. The best way to measure a refrigerator temperature is by keeping the thermometer in a clear glass bottles filled with water. The thermometer will be stable when read through the glass. Calibration and temperature should be documented at regular intervals using recording charts attached to the unit itself for easy access. Refrigerators and freezers are essential instrumentation I the laboratory. Stringent guidelines for use and maintenance of these instruments must be maintained.
Microwave ovens have become prominent laboratory instrumentation and as standard equipment used in today's laboratory, they must be included in any comprehensive quality control program.
A. Potential health hazards
The issue of health hazards arising from the use of "household" microwave units in laboratories is not unwarranted. Several points must be addressed when operating a "household-type" microwave in the laboratory. These potential hazards include the following:
Airborne concentrations of formalin vapors may be increased when using the microwave oven for fixation purposes (vapor concentration may well exceed the allowable PEL).
Invented oven chambers may release concentrated vapors when door is opened. (Chamber ventilation systems prevent build up of toxic or flammable vapors and any vented air form the oven should be passed through the laboratory ventilation system or charcoal filter, or the oven should be enclosed in a hood).
Procedures using alcohols and xylene are flammable and potentially volatile. Potential ignition of volatile solution vapors may occur by a spar (switch) that runs off the magnetron when the door is opened (unless switch is explosion-proof).
Solvent vapors may cause deterioration of door gaskets (if present) resulting in radiation leakage.
B. Controls
Because of the aforementioned potential hazards, microwave units have been developed for laboratory use. These units are costly and for laboratories that use non laboratory (household) units, some solutions are proposed to address these problems.
The use of zip-type bags, particularly those made for biological purposes, or at least, freezer-weight kitchen type, can be used to contain the fumes created when solutions are heated. (Thinner gauge bags will melt in the oven). By enclosing the container in completely sealed bags, fumes are contained.
After heating, the container I the bag is taken to a fume hood and safely opened, reducing exposure to the lab tech.
Enclosing containers in bags also contain any spill that might occur, protecting the lab person from solution contact and preserving the surface of the oven interior.
Minimization of the amount of solution used for many procedures, such as the use of micro-containers in place of coplin jars, further reduces the amount of vapor concentrations that may be produced.
Control of volatile solutions is accomplished by quality control calibration of the oven, knowledge of boiling points of solvents, and controlled heating of such solutions when microwaved.
The non laboratory or household microwave ovens manufactured currently do not have rubber-type gaskets at the door edge. If the oven does have gasket, a routine inspection by a radiation safety officer should be able to detect if the gasket is no longer sealing the door properly.
| § AUTOCLAVES, PRESSURE VESSELS AND RETORTS |
Steam sterilization of infectious waste utilizes saturated steam within a pressure vessel at temperatures sufficient to kill infectious agents potentially present in the wastes.
Treatment by steam sterilization, decontamination of the waste occurs primarily from steam penetration. Heat conduction provides a secondary source of heat transfer. Therefore, for effective and efficient treatment, the degree of steam penetration is the critical factor. For steam to penetrate throughout the waste load, the air must be completely displaced from the treatment chamber.
The presence of residual air within the sterilizer chamber can prevent effective sterilization.
The principal factors that should be considered when treating infectious wastes are:
- Type of wastes,
- Packaging and Containers,
- Volume of wastes and
- It's arrangement in the treatment chamber
Many infectious wastes that have multiple hazards should not be steam sterilized because of the potential for exposure of equipment operators to toxic, radioactive, or other hazardous chemicals. Infectious wastes that should not be steam sterilized include those that contain antineoplastic drugs, toxic chemicals, or chemicals that could be volatilized by steam.
Persons involved in steam sterilizing of infectious wastes should be educated in proper techniques to minimize personal exposure to the hazards posed by these wastes. These techniques include use of personnel protective equipment, minimization of aerosol formation, and prevention of spillage of waste during autoclave loading.
A record thermometer should be used to ensure that a sufficiently high temperature is maintained for an adequate time during the cycle. Failure to attain or maintain operating temperature may indicate mechanical failure.
All steam sterilizer should be routinely inspected (Note: Certification done by State Department of Labor and Industrial Relations, Division of Occupational Health and Safety, Boiler and Elevator Inspection Bureau for all autoclave with a separate boiler) and serviced. Monitoring the steam sterilization process is required to ensure effective treatment. The process should be monitored periodically to check that proper procedures are being followed and that the equipment is functioning properly.
Each autoclave should be checked on a quarterly basis or sooner, by using biological indicators (spores of Bacillus steraotherophilus) to determine that the autoclaved biowastes have been sterilized at the proper temperature, pressure and time. Temperature indicator chemical strips that change color indicate only the outside of the container was exposed to a certain temperature, they do not indicate that the waste has been sterilized. They do indicate, however, processing time rather than treatment effectiveness.
Biological materials requiring registration from the BSP must be autoclaved in the primary laboratory unless otherwise authorized by the BSO. (As a condition of import from the Hawaii Department of Agriculture).
All material to be autoclaved must be accompanied by a time, thermal, biological and color indicator any be must be placed in the autoclave by a member of the responsible research group who will see that temperature and pressure are up to desired levels and that unequivocal instructions for sterilization are posted prior to leaving the area.
Dead animals, animal wastes or tissue, must be placed in sealed polyethylene bags (biological commodities requiring registration must be doubled bagged) and put into a lidded, leak proof, metal container and labeled prior to autoclaving.
To assure that sterilization is being accomplished sterilization procedures and equipment must be certified before routine use and periodically thereafter (preferably during each cycle). Certification is usually accomplished utilizing instrumentation, chemical indicators, and biological indicators to verify that appropriate physical parameters are satisfactory to kill resistant commodities.
All records of time or thermal (chemical or biological) are to be kept on file for three years.
| § LABORATORY FURNITURE |
The lab furniture must be easily cleanable and able to withstand decontamination procedures should they become contaminated. Carpets and rugs in laboratories are not appropriate.
Bench tops should be impervious to water and resistant to acids, alkalis, organic solvents (disinfectants and sterilants) and moderate heat (Bunsen burners, alcohol lamps and microbe incinerators).
Laboratory furniture should be of sturdy construction with spaces between benches, cabinets and equipment accessible for cleaning. Laboratory furniture should be capable of supporting anticipated loading and uses.
Chairs and stools should be covered with a non-fabric and non-absorbent material. Chairs should be covered in a material like plastic, vinyl or leather finish that can be easily cleaned in the event of contamination or spill.
| § FLOW CYTOMETRY |
Flow cytometry is a method of quantifying structural or biochemical features of cells or other small particles by using a laser as an excitation light source and photodetectors for measurement. This is generally accomplished by either light scatter and/or fluorescence. Flow Cytometry may further be defined as a technology to measure properties of particles as they move, or flow, in liquid suspension.
In some flow (or sorting) cytometers, the liquid containing the particles is broken into droplets by the reciprocating motion of a nozzle. Individual particles are captured in small droplets, and then those droplets are electrostatically charged and deflected through a high potential. If a plug or other obstruction (bubbles, clumps of cells, etc.) occurs, then this stream of droplets can go awry, generating aerosols. There is a potential for aerosol exposure to individuals near a flow cytometer because modern sorting units operate with 14 to 40 pounds per square inch of pressure (some units can operate at 100 psi). Even non-sorting flow cytometers, which do not suspend cells in droplets, operate under pressures near 5 psi and can develop leaks and generate aerosols.
The following requirements must be followed when using a flow cytometer due to the potential for aerosol exposure to individuals:
- Used only in a negative pressure laboratory.
- Performed by trained individuals.
- Proper personal protective equipment (PPE) including gloves, lab coats, and safety goggles.
- Cleaned and properly disinfected after each use.
- The catch basin (traps) should have an adequate disinfectant (i.e. bleach) added when the unit is in use. Must have proper contact time prior to disposal down the drain.
- When possible, biological samples should be fixed (usually with 1% formaldehyde) before being run through the flow cytometer.
- For infectious, pathogenic, and/or toxic materials, flow cytometry must be conducted in a certified, totally ducted biological safety cabinet.
| § CRYOSTAT |
Frozen sectioning done on unfixed tissue pose a high risk because of the knife blade and techniques involved. Freezing of tissue does not inactivate infectious agents. Freezing propellants under pressure should not be used for frozen sections as they may cause the splattering of droplets of infectious material. Gloves and safety goggles should be worn during frozen sections.
The contents of the cryostat should be considered to be contaminated and should be decontaminated frequently with an appropriate disinfectant. The wastes trimmings and sections of tissue that accumulate in the cryostat should be considered to be contaminated, should be removed during decontamination and preferably autoclaved. The cryostat should be defrosted and decontaminated once a week with a tuberculocidal hospital disinfectant with a label claim for tuberculocidal disinfection.
Stainless steel mesh gloves should be worn when changing knife blades. Solutions used for staining frozen sections should be considered to be contaminated. They must be filtered and particulates autoclaved. The wastes stain should be disposed of as required by Hazardous Materials Management Program.
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Cryogenics is the study and use of materials at extremely low temperatures. According to the National Institute of Standards and Technology (NIST), the term "cryogenics" applies to all temperatures lower than -150°C (-238°F). Liquid nitrogen, liquid oxygen and liquid helium are commonly used substances in cryogenic work. Cryogenics are applied in various industries and fields of study, including tool and metal tempering, nuclear research, electromagnetism and many laboratory techniques. Surgeons have begun to use cryogenics to treat Parkinson's disease, to destroy brain tumors and to arrest the advance of cervical cancer. Rocket engines are fueled by liquid oxygen, as are cutting and welding torches. Anything as cold as these common cryogenic liquids is inherently dangerous and should be handled with extreme care. Special precautions are a must, so consider the following before starting any project that involves cryogenic materials.
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| § EQUIPMENT / INSTRUMENTATION DISPOSAL OR REPAIR |
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Biomedical Equipment Repair Services in Hawaii
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Equipment / Instrumentation potentially contaminated with biological commodities:
If no biological commodity contamination, remove all items, defrost and clean. If there is no evidence of spills or leaks, contact Facilities Planning and Management (FPMO).
If biological agents have spilled or leaked, clean with a 10% bleach solution (1 part bleach to 9 parts water). FPMO personnel can then be contacted.
Combination of chemicals/drugs and biological agents:
Remove all items and defrost. If there is no evidence of spills or leaks, contact FPMO.
If any chemicals, drugs and/or biological agents have spilled or leaked, follow the aforementioned protocols. Be careful not to combine incompatible substances such as bleach and ammonia. FPMO personnel can then be contacted.
Radioactive material and any combination of radioactive material with chemicals, drugs, or biological agents: Follow the reporting, surveying and tagging procedures as established by Radiation Safety.
Procedure For The Removal Or Repair Of Laboratory Refrigerators Or Freezers
The users of refrigerators and freezers are responsible for emptying, defrosting and decontamination (if needed) prior to removal or repair by FPMO.
Instructions for having your refrigerator or freezer repaired or removed:
- Read the decontamination protocols printed on page one of this sheet.
- Circle all items (below) that describe what type of storage the refrigerator/freezer was used for: food • drugs • chemicals • biological agents • radioactive material
- Apply the appropriate protocols for your situation.
- Sign on the line below indicating that you have read and completed the appropriate protocols.
- Place this signed sheet on the refrigerator or freezer door.
- Contact the appropriate shop:
- Removal of this sheet must be done by Physical Facilities personnel only.
Example of an information sheet that should be attached to refrigerator or freezer door.
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OUT OF SERVICE __________________________________ _______________ (signature) (date)
FOR DISPOSAL/REPAIR/SURPLUS |