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Pathogen Safety Manual also available:  Word  PDF
Introduction
Work involving pathogenic microorganisms requires training, practice and careful attention by the researcher or clinician. This synopsis covers the basic skill set and equipment needed for handling of potential pathogens at the BSL1 and BSL2 levels.
Italicized items indicate additional requirements for work at BSL2.
- Immediately notify the laboratory supervisor or Principal Investigator (PI) in case of an accident, injury, illness, or overt exposure associated with laboratory activities. When deemed necessary by the laboratory supervisor proceed to Curry Health Center for appropriate medical surveillance and/or treatment.
- For those intending to work with live viruses: The IBC application from the PI must be read and understood. Standard Operating Protocols should be reviewed and signed.
- Be aware that access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments or work with cultures or specimens is in progress. Laboratories must have doors to control access.
- Only personnel advised of the special hazards and meeting any specific entry requirements, i.e., appropriate immunizations, serum sampling, are permitted in the laboratory. Read and follow all biosafety procedures provided by the PI.
- Be aware that any possession or use of select biological agents or toxins as defined by the federal government requires registration and inspection; restricted lab access; written and strictly followed safety and security plans; personnel background checks and training; accurate records and/or reporting of agent use, transfer, loss, or destruction. Any plans for obtaining such materials must be discussed with and approved by the IBC.
- Ensure that when infectious agents are in use in the laboratory, a biohazard sign is posted on the lab access door. This sign identifies the agent(s) in use, their biosafety level, any required immunizations, the PI’s name and telephone number, and any Personal Protective Equipment (PPE) that must be worn in the laboratory.
- Understand that the PI must ensure that all laboratory personnel receive appropriate training on hazards associated with the agents involved; the necessary precautions to prevent exposures; and exposure evaluation procedures.
- Wash hands frequently and always after handling viable material or animals, after removing gloves, and before leaving the laboratory. A sink for hand washing is present in each laboratory.
- Consider foot, knee, or automatically operated hand washing sinks.
- Know the location of a readily accessible eyewash station.
- Do not eat, drink, smoke, chew gum, handle contact lenses, or apply cosmetics in the laboratory.
- Do not bring any food, medications, or cosmetics, into the laboratory for storage or later use. Food is stored outside the work area in cabinets or refrigerators designated specifically for that purpose. Do not bring animals unrelated to experiments into the laboratory.
- Do not pipette by mouth; only mechanical pipetting devices are permitted.
- Perform all procedures carefully to minimize the creation of splashes or aerosols.
- Establish and follow policies for safe handling of sharps.
- Use a high degree of caution when handling any contaminated sharp item, such as needles and syringes, slides, pipettes, capillary tubes, and scalpels.
- Restrict needles and syringes or other sharp instruments in the laboratory for use only when there is no alternative, such as for parenteral injection, phlebotomy, or aspiration of fluids from laboratory animals and diaphragm bottles. Substitute plasticware for glass whenever possible. Handle broken glassware with brush and dustpan, tongs, or forceps - not directly with hands.
Use only needle-locking syringes or disposable syringe-needle units (i.e., needle is integral to the syringe) for injection or aspiration of infectious material.
- Use, whenever possible, syringes which re-sheathe the needle, needleless systems, and other safety devices.
- Do not bend, shear, break, recap, or remove used needles from disposable syringes or otherwise manipulate such units by hand before disposal. Dispose of needles and syringes in the puncture resistant container provided in the laboratory for this purpose.
- Use of lab coats, gowns, or other designated laboratory uniform is recommended to prevent contamination or soiling of street clothing.
- Wear lab coats, gowns, smocks, or other provided protective garments while in the lab. When leaving the lab, remove and leave coats and other protective clothing in the lab for either disposal or laundering. Wear gloves. Protective eyewear must be worn for procedures that involve anticipated splashes of microorganisms or other hazardous materials to the face.
- Wear gloves when manipulating infectious materials or agents or when hands must otherwise contact contaminated surfaces. Double gloving is recommended. Remove and change gloves when overtly contaminated or when torn or punctured. Do not wear contaminated gloves outside the lab. Do not wash or reuse disposable gloves. Consider alternatives to latex gloves to prevent allergic response.
- Wear appropriate face protection (goggles, mask, face shield or other splatter guard) for anticipated splashes or sprays of infectious materials to the face when agents must be handled outside the BSC.
- Decontaminate equipment and work surfaces at completion of work, at the end of the day, and following spills of viable materials. If a spill occurs, cover the spill with paper towels and soak the towels with a 1 to 10 dilution of chlorine bleach or other suitable disinfectant. Allow the material to soak for approximately 20 minutes before discarding materials in biohazard bag. Bench tops are impervious to water and resistant to solvents, acids, alkalis, and chemicals used for surface decontamination. Laboratory surfaces and spaces between fixtures are designed to be easily cleaned; no carpets or rugs.
- Work on open bench tops is permitted; use of special containment equipment such as a biological safety cabinet is not generally required for agents assigned to BL1.
- Work in the open laboratory is permitted, except that a properly maintained biological safety cabinet is required whenever:
- Procedures with a potential for creating infectious aerosols or splashes are conducted. These may include centrifuging, grinding, blending, vigorous shaking or mixing, sonic disruption, opening containers of infectious materials whose internal pressures may be different from ambient pressures, inoculating animals intranasally, and harvesting infected tissues from animals or embryonate eggs. High concentrations or large volumes of infectious agents are used. Such materials may be centrifuged in open laboratory if sealed rotor heads or centrifuge safety cups are used, and if these rotors or safety cups are opened only in a biological safety cabinet.
- Be aware that air sampling studies have shown that most of the common manipulations of bacterial and viral cultures in research laboratories release aerosols of viable organisms. This must be considered when evaluating need for use of the biological safety cabinet or other physical containment device.
- Properly dispose of all regulated medical wastes (potentially biohazardous) and associated wastes.
- Cover containers of all cultures, tissues, specimens of body fluids, or other potentially infectious waste to prevent leakage during collection, handling, processing, storage, and transport.
- Have an insect and rodent control program in place. Ensure screens are fitted on exterior windows that open into the lab.
- Ensure that illumination is adequate for all activities, avoiding reflections and glare that could impede vision.
Types of BSCs
BSCs are classified as Class I, Class II or Class III cabinets. When properly maintained and operated, they effectively contain and capture microbial contaminants and infectious agents using HEPA (High Efficiency Particulate Air) filters. (See Figure 1.) Biosafety cabinets should not be confused with clean benches which only protect the material being worked with and are not suitable for work with infectious or toxic material. (Although clean benches, like BSCs, have HEPA-filtered air, with clean benches the air flows over the experimental material toward the user rather than being drawn away.) BSCs should also not be confused with conventional fume hoods that do not filter microorganisms.
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| Figure 1. Diagram of HEPA filter. These filters are typically constructed of continuous sheets of paper-thin filter medium, pleated to increase surface area, divided by aluminum separators and affixed to a frame. |
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| Figure 2. Class I Biological Safety Cabinet |
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| Figure 3. Class II Biological Safety Cabinet |
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Working in a BSC
- Turn the cabinet on for at least 10-15 minutes prior to use, if the cabinet is not left running.
- Disinfect work surface with 70% alcohol or other suitable disinfectant.
- Consider the materials necessary for the planned work in the cabinet.
- Place items into the cabinet so that they can be worked with 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.
- Adjust the working height of the stool so that the worker's face is above the front opening.
- Delay manipulation of materials for approximately one minute after placing the hands/arms inside the cabinet.
- Minimize the frequency of moving hands in and out of the cabinet.
- Do not disturb the airflow by covering any of the 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.
NOTE: Be very careful when using small pieces of materials such as kimwipes in the hood. These can be blown into the hood and disrupt the motor operations.
Certification of the BSC
Certification is a series of performance tests on the BSC to confirm that it will provide the user and experimental material the protection for which it is designed. The air flows, filters, and cabinet integrity are checked to ensure that the cabinet meets minimum performance standards. Certification is arranged and paid for by the PI and is contracted to an outside vendor such as Marischal Inc., (800) 491-1776.
BSCs intended for user protection must be certified:
- After they are received and installed (before use with infectious materials)
- After filter changes
- Annually
BSCs intended only for protection of the experimental material are certified at the discretion of the Principal Investigator.
BSC decontamination (using the paraformaldhyde gas production process) is also provided by an outside vendor and needs to be done:
- Before maintenance work requiring disassembly of the air plenum, including filter replacement
- Prior to cabinet recertification
- Before moving the cabinet to a new laboratory
Definitions
- Decontamination is a process or treatment that renders an instrument or environmental surface safe to handle. A decontamination procedure can be as simple as clean-up with detergent and water or as thorough as sterilization. Sterilization, disinfection, and antisepsis are all forms of decontamination.
- Sterilization is the use of physical or chemical processes to destroy all microbial life, including highly resistant forms, such as bacterial spores.
- Disinfection is the elimination of essentially all pathogenic non-spore forming microorganisms but not necessarily all microbial forms from work surfaces and equipment. Effectiveness is influenced by a number of factors, including: types and numbers organisms; amount of organic matter; the object being disinfected; the disinfectant being used; exposure time, temperature and concentration.
- Antisepsis is the application of a liquid antimicrobial to skin or other living tissue to inhibit or destroy microorganisms. Examples include hand washing with germicidal solutions or swabbing skin before an injection.
When to Decontaminate
All material and equipment contaminated with or containing potentially infectious agents should be decontaminated:
- Upon completion of procedures involving the use of biologically-active materials
- In the event of spills of such materials
- At least daily when in use
- Before being washed, stored, or discarded
In most UM laboratories, decontamination is accomplished by steam heat sterilization in an autoclave, or by surface application of or placement in a chemical disinfectant solution, such as 1:10 bleach solution or its equivalent.
Autoclave Use
Autoclaving (saturated steam under pressure of approximately 15 psi to achieve a chamber temperature of at least 250°F for a designated time) is the preferred and most convenient method to rapidly destroy all forms of microbial life. However, to do this, the autoclave process must reach proper temperature and time and also prevent the entrapment of air in the bag or container of treated material.
- Material to be sterilized must come into contact with live steam.
- Bags or containers should be left open during autoclaving or water (~200ml) should be added to sealed bags to generate steam.
- Heat indicator tape should be used with each autoclave load to indicate that sterilization has been completed.
- Autoclave sterility monitoring should be conducted on a regular basis using biological indicators (such as B. stearothermophilus spore strips) placed among treated materials and at locations throughout the autoclave. The spores, which are more resistant to heat than most microbials, provide validation of general microbial destruction when they are effectively inactivated (250°F for 15 minutes) by autoclave operation.
Chemical Disinfectant Use
- The most practical use of chemical disinfectants is for surface decontamination and, when used in sufficient concentration, as a decontaminant for liquid wastes prior to final disposal down the drain.
GENERAL RECOMMENDATIONS
- Liquid Decontamination
- Add liquid chlorine bleach to provide a final 1:10 dilution
- Let stand at least 20 minutes
- Discard down the drain
- Surface Decontamination
- Wipe with 1:10 dilution of chlorine bleach, or
- Wipe with iodophor disinfectant (per label concentration), or
- Wipe with 70% alcohol
In the event of an exposure to an infectious agent or material, the following guidelines should be used:
Intact skin
- Remove contaminated clothing
- Wash contaminated skin for 1 minute with soap and water
Broken, cut or damaged skin or puncture wound
- Remove contaminated clothing
- Wash contaminated skin for 5 minutes with soap and water
- Seek medical attention at Curry Health Center
Eye
- Immediately flush eyes for at least 15 minutes with water, preferably using an eyewash; if no eyewash is available, pour water on the eye(s) for 15 minutes, rinsing from the nose outward to avoid contamination of the unaffected eye.
- Hold eyelids away from your eyeball and rotate your eyes so that all surfaces may be washed thoroughly.
- Seek medical attention at Curry Health Center
Ingestion or Inhalation
- Seek medical attention at Curry Health Center
- Do not induce vomiting unless advised to do so by a health care provider
Spills and Preparing for Them
In the event of a spill of biological material, the individual(s) who caused the spill is responsible for the clean-up. If additional resources are needed, call EHRM at 243-4503 or refer to the emergency numbers posted in your lab.
- Minimize the consequences of any spill of biological material by performing all work on plastic-backed liner to absorb spills
- Have a simple spill kit on hand including:
- Chlorine bleach or some other concentrated disinfectant
- A package or roll of paper towels
- Autoclavable bags
- Rubber gloves
- Forceps or broom and dust pan for pick-up of broken glass
Spills Inside a Biological Safety Cabinet
- LEAVE THE CABINET TURNED ON.
While wearing gloves, spray or wipe cabinet walls, work surfaces, and equipment with disinfectant equivalent to 1:10 bleach solution. If necessary, flood the work surface, as well as drain pans and catch basins below the work surface, with disinfectant for a contact time of at least 20 minutes
- Soak up disinfectant and spill with paper towels. Drain catch basin into a container. Lift front exhaust grill and tray and wipe all surfaces. Ensure that no paper towels or solid debris are blown into the area beneath the grill.
- Clean up materials that have been completely disinfected may be disposed of in the conventional trash. If there is any doubt, place the clean up materials in the biowaste container. Wash hands and any exposed surfaces thoroughly after the clean-up procedure.
Small Spill of Material Outside of a Biological Safety Cabinet (spill that can be covered by a few paper towels)
- Wearing gloves and a lab coat, cover the spill with paper towels and gently apply disinfectant, proceeding from the outer edge of the spill to its center. Leave in place for 20 minutes.
- Pick up the towels and discard into a biohazard container. Pick up any pieces of broken glass with forceps and place in sharps container.
- Re-wipe the spill area with disinfectant and thoroughly wash hands after glove removal.
Large Spill of BL2 Material (>500ml) Outside of a Biological Safety Cabinet
- Hold your breath and leave the room immediately.
- Warn others to stay out of the spill area to prevent spread of contamination; post a sign stating: "DO NOT ENTER, BIOHAZARD SPILL," contact (name and phone number) for information."
- Remove any contaminated clothing and put into a biohazard bag for later autoclaving.
- Wash hands and exposed skin and inform your PI or supervisor about the spill
- Put on protective clothing (lab coat, gloves and, if indicated, surgical mask, eye protection, shoe covers) and assemble clean-up materials.
- Wait 30 minutes before re-entering the contaminated area to allow dissipation of aerosols.
- Cover the spill with paper towels and gently apply disinfectant, proceeding from the outer edge of the spill to its center. Leave in place for 20 minutes
- Collect all treated material and discard in a biohazard container. Pick up any broken glass with forceps or a broom and dust pan and place them into a sharps container.
- Re-wipe the spill area with disinfectant and wash hands thoroughly at completion of clean-up.
Biohazardous Waste (Regulated Medical Waste)
Some wastes associated with biological materials must be disposed of in special ways because they may have been contaminated with infectious organisms or agents.
- All sharps, e.g. glass implements, needles, syringes, blades, etc. coming from facilities using infectious materials
- Biologically-cultured stocks and plates, human blood or tissues
For disposal of these wastes, the lab personnel:
- Sterilize or disinfect waste materials associated with viral, bacterial or other agents infectious to humans (by autoclave or chemical treatment equivalent to 1:10 bleach solution).
- Place all biohazardous wastes, except for sharps, directly into a red bag. Do not use red bags for non-biohazardous waste.
- Place sharps into labeled sharps containers.
- When a Medical Waste box or bag is filled, seal the container and notify EHRM for pick-up.
Other wastes generated in these facilities that are not contaminated with biological agents or materials are not treated as biohazardous and may be discarded in the regular trash container, with recyclables, or into other specially designated waste containers. These include such items as recyclable and non-recyclable waste glass, gloves, unused plates or tubes, fly media or embryo plates, etc. (Note: broken glass must be placed in a separate glass container, not the regular trash. These containers are to be taken to the dumpster by lab personnel.)
In order to clarify how these various wastes are to be handled in laboratories using biological materials, please see the biological waste stream information below.
Animal Bedding Waste
Laboratory Animal Resources (LAR) staff will normally change all bedding for the researchers. Unless known to contain an infectious or toxic material, the bedding will be disposed of in the normal trash by the LAR staff. In the event the bedding represents an unusual risk, the LAR Director and EHRM Director will determine appropriate safeguards and collection and disposal practices in conjunction with the researcher.
Animal Carcasses
Freezers are provided in each LAR animal facility for storage of carcasses that have been bagged and sealed. The larger frozen carcasses are taken to the land fill by LAR staff and the bagged, small frozen carcasses are placed into the normal trash on trash collection day. The carcasses of animals containing infectious or toxic materials are double-bagged into biohazard-labeled bags and placed in a specially marked freezer in the Health Science Building. EHRM picks up this biohazardous material when requested by the LAR staff.
Animal Waste from BSL2 Animal Room
Animals housed in a BSL2 animal space are considered to be potentially infectious because as part of the research protocol they are infected with Biosafety Level 2 (BSL2) animal and/or human pathogens. Animal bedding, carcasses, and tissue are placed in biohazard bags by the research or LAR staff. All animal bedding is autoclaved before being placed in the normal trash. Bagged animal carcasses and tissue are placed in the provided storage freezer and removed by LAR staff to medical waste boxes for pick-up by EHRM as part of the medical waste stream.
Patient Care Waste Disposal
- All disposable wastes generated at Curry Health Center from patient rooms and as part of direct patient care are considered potentially infectious and are disposed of in the medical waste stream. Syringes, needles, and other sharps are placed in the provided sharps container which, when filled and sealed are placed in the provided medical waste boxes.
- Patient care waste generated at other sites on campus by medical response personnel (i.e. Public Safety) are placed in biohazard bags and brought to Curry Health Center for medical waste disposal or handled by responding EMS personnel.
- A program is in place to ensure that needles and syringes generated as part of personal diabetes care will not be an exposure hazard to others. Collection containers are available from Curry Health Center which, when filled, are returned to Curry Health Center for proper disposal in the medical waste stream.
All biohazardous waste must be disposed of by one of the following means. There are no exceptions. Call Environmental Health and Risk Management at 243-4503 for free pick up of your biohazardous waste.
Sharps
- All sharps must be collected in durable containers for offsite disposal with our biohazardous waste contractor. Special heavy wall red plastic biohazardous sharps containers, coffee cans or other puncture proof, stiff walled containers may be used for sharps collection. Plastic milk bottles, cardboard boxes or other containers that are likely to allow a needle to penetrate the container walls during handling may NOT be used. Under no circumstances may sharps be disposed of in the normal trash.
- Sharp containers are picked up by EHRM upon request.
Liquid Blood or other body fluids
- Liquid blood or other body fluids may be disposed of down the drain. If you know these materials contain agents that are infectious via airborne transmission, you must disinfect them with a bleach solution prior to drain disposal.
Solid Biohazardous Waste
- Solid biohazardous waste such as used bandages, gloves, counter pads, disposable labware pipette tips etc. may be treated in several different ways.
- Small quantities may be chemically disinfected and disposed of in the normal trash as long as no appreciable free liquid is present.
- Dry waste may be autoclaved and disposed of in the normal trash if the biohazard symbol is defaced on the bag prior to disposal. If biohazard bags are used that auto indicate that acceptable temperatures have been attained, then the bags and contents may be disposed of without manually defacing the biohazard symbol.
- Dry waste, like sharps may be disposed of through our offsite biohazardous waste contractor. NEVER put sharps in a biohazardous waste bag. You or someone else may experience a needle stick through the bag if all sharps are not contained as outlined above.
See UM Pathogen Shipping Manual
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