3. BIOSAFETY PRACTICES AND PROCEDURES

• Restricting access to laboratories using biological hazards per the BMBL and the UTK laboratory security policy. The PI is ultimately responsible for restricting access to approved personnel or visitors.
• Hand washing after handling biologicals and potentially hazardous materials, after taking off gloves and before leaving the lab.
• Avoiding hand-to-face (or mouth) contact. No eating, drinking, smoking, or applying cosmetics in the lab.
• Disinfecting work surfaces daily and decontaminating after spills.
• Prudent handling, management, and disposal of sharps.
• Using procedures that minimize the formation of aerosols and splashes; engineering controls for aerosol release may be required.
• Wearing appropriate personal protective equipment (PPE), e.g., lab coats, gloves, safety glasses, safety goggles, etc.
• Using primary and secondary containment when transporting materials.
• Disposal of all non-sharps solid biological waste in a proper container lined with an autoclavable bag for disposal. Liquid biological waste must be disinfected or autoclaved before sink disposal.

Other SMP to be used in laboratories handling biological materials include:

• Keeping personal items such as cell phones or tablets outside of the work area
• Disallowing outside pets, plants, or other personal effects/decorations from the work area
• Minimizing exposed skin, and thoroughly covering broken or abraded skin or skin with rashes
• Avoiding open toed shoes, sandals, flip flops or any footwear with exposed skin or holes while working in the laboratory
• Knowing the risks of lab activities and don the appropriate PPE
• Restraining long hair when operating equipment with exposed moving components or when working with chemicals or biologicals
• Providing information regarding changes to health status that could impact their health and safety in the research environment.

Biosafety Level 2 practices (BSL-2) consist of SMP plus:

• Hazard communication through door signs/placards and biohazard labelling
• More stringent PPE requirements, typically gloves, long-sleeved lab coat/smock, and protective eyewear.
• Aerosol control and containment
• Enhanced engineering controls:
  • Biological safety cabinets
  • Emergency eyewashes

• Integrated pest management program
• Medical surveillance programs as indicated by risk assessment

BSL-2 (& BSL-3) labs can be easily differentiated from BSL-1 labs in that the outer doors of areas where biological hazards are manipulated feature cautionary placards. The Biosafety Program-approved placard is exampled here.

Placards are available by contacting EHS Lab Safety Services at 865-974-5084.

At UTK, the universal biohazard symbol and the word “BIOHAZARD” are used to indicate higher risk areas where there is a risk to human health.  Therefore, BSL-2 door placards feature the universal biohazard symbol which alerts lab workers, visitors, maintenance & housekeeping personnel, vendors, and others who may need to enter the laboratory.

Placards also communicate entry considerations, e.g.:

• Entry requirements such as PI authorization;
• Health: immunocompromised individuals are advised of the increased risk and an individual risk- assessment can be conducted for these individuals; vaccine requirements may also be posted;
• Practices: PPE requirements & SMP highlights
• Doors should be kept closed during experiments when BSL-2 agents are in use.

Equipment used to process or store biohazards that may impact human health must be labeled with the universal biohazard symbol.  For example, hoods, freezers, incubators, and centrifuges, etc. are labeled if they are used with risk group 2 (RG2) agents or higher.  Also, transport containers and biohazard waste containers must display a biohazard label on the outermost part.

Hands must be washed:

• Anytime they may have become contaminated with infection-risk material
• After glove removal
• Before leaving the work area

Procedure:

1. Use cold-to-tepid water and soap (does not need to be antibacterial)
2. Lather for 20-30 seconds, paying attention to palms, backs of hands, finger webbing, cuticle beds, and wrists
3. Rinse and dry. Use paper towel to turn off water faucet drying your hands.

Hand Hygiene – A word about hand sanitizers:

In case of limited access to soap and water (e.g. field procedures), waterless hand sanitizers may be used as a temporary means of reducing contamination until a source of running water and soap can be reached. However, because of the variety of products available and organisms used in UT labs, EHS Lab Safety Services cannot adequately validate waterless hand sanitizers for efficacy in all cases.  Therefore, these products are NOT a replacement for hand washing with soap and water and should not be used as such.

When using a waterless sanitizer:

• Ensure that the active ingredients include > 60% alcohol (ethanol or isopropanol)
• Apply quantity recommended by manufacturer
• Thoroughly distribute as if you were washing your hands (paying attention to palms, backs of hands, finger webbing, cuticle beds, and wrists)
• Rub until completely dry
• Wash your hands with soap and water (as described on previous slide) once available.

Proper attire is a prudent practice in any research laboratory, including those where biological materials are handled.  Prior to working in the laboratory, ensure that you are wearing clothing that covers exposed skin and close toed shoes made of substantial, non-porous materials. Proper attire minimizes potential routes of entry, and is particularly important at BSL-2.

What is personal protective equipment?

Personal protective equipment, commonly referred to as “PPE”, is equipment worn to minimize exposure to hazards that cause serious workplace injuries and illnesses. These injuries and illnesses may result from contact with biological, chemical, radiological, physical, electrical, mechanical, or other workplace hazards. Personal protective equipment may include items such as gloves, safety glasses, chemical splash goggles, lab coats, and respiratory protection.

What can be done to ensure proper use of personal protective equipment?

All personal protective equipment should be safely designed and constructed, and should be maintained in a clean and reliable fashion. It should fit comfortably, encouraging worker use. If the personal protective equipment does not fit properly, it can make the difference between being safely covered or dangerously exposed. When engineering, work practice, and administrative controls are not feasible or do not provide sufficient protection, employers (e.g. PI or lab supervisor) must provide personal protective equipment to their workers and ensure its proper use.

Employers are also required to train each worker required to use personal protective equipment to know:

• When it is necessary
• What kind is necessary
• How to properly put it on, adjust, wear and take it off
• The limitations of the equipment
• Proper care, maintenance, useful life, and disposal of the equipment

Gloves

Gloves are required for all manipulations of RG2 organisms unless alternative protection is established in an SOP.

Gloves must be:

• Fluid resistant (a non-latex material, e.g. nitrile, must be available)
• Powder-free
• Disposable – do not reuse gloves unless they are utility gloves that are intended to be reused; dispose when compromised
• Free of defects, tears, or breaks
• The right size for your hands
• Removed before leaving the lab, touching phones, doorknobs, or other common contact surfaces or objects (fomites, see below)

Donning & Doffing Gloves

Lab coats

Protective lab coats are recommended to prevent contamination of personal clothing at BSL-1.  At BSL-2, they must be worn when working with hazardous materials.

Lab coats must be:

• Long sleeved – preferably with a gathered cuff
• Removed before leaving for non-laboratory areas
• Laundered appropriately – lab coats must be laundered routinely and when contaminated. Lab coats should never be cleaned in personal or public washers and dryers.  See the Biosafety web page for more information regarding proper laundering of lab coats

Eye Protection

Wear protective eye wear when conducting procedures that have the potential to create splashes of microorganisms or other hazardous materials.

Eye protection must be:

• Worn according to the laboratory hazard assessment
• Cleaned, stored, and replaced – according to manufacturer’s recommendations and prudent laboratory practices
• Appropriate for the task:
  • Safety glasses with side shields must be worn when there is a risk of particulate projectiles (broken glass shards)
  • Indirectly vented chemical splash goggles must be worn when there is a risk of aerosols, splashes, sprays, or splatters of infectious or hazardous liquid material coming into contact with the eyes or the tissues surrounding the eyes (bleach, liquid cultures, etc.)

Face Protection

Face protection must be used if splashes or sprays of infectious or other hazardous materials are anticipated.

Face protection must be:

• Worn according to the laboratory hazard assessment
• Cleaned, stored, and replaced – according to the manufacturer’s recommendations and prudent laboratory practices
• Appropriate for the task:
  • UV exposure requires a specially rated type of material for the face shield
  • Liquid exposure requires protection against potential splashes or sprays of infectious or hazardous liquid

Respiratory Protection

Additional PPE such as surgical masks may be worn for procedures with high probability for splashes, spray, splatter or droplets.  Work in rooms with infected animals or high risk inhalation hazards (infectious to humans) may require respiratory protection.  If respiratory protection if used voluntarily or as required PPE, the EHS department on the main campus of UTIA safety office on the Agricultural Campus must be contacted prior to voluntary or required use.  The respective office will assist with selection, training, fit testing, and other requirements.

Aerosols are solid or liquid particles suspended in the air (1-100 µm).  The fate of these particles is determined by relative size:

• Larger particles settle more rapidly becoming a risk for surface contact.
• Smaller particles can remain airborne for a long period of time, dehydrating and becoming “droplet nuclei” and spreading wide distances.
• Smaller particulates (1 to 10 um) are also more easily inhaled.

Common laboratory procedures that may produce aerosols include:

• Blending, macerating, or sonicating infectious material
• Stirring or vortexing liquids
• Opening lyophilized cultures, culture plates, ampoules, tubes and bottles
• Removing stoppers
• Pouring liquids
• Pipetting & blowing out pipettes
• Dropping culture containers
• Flaming inoculating needles, slides or loops
• Streaking or spreading inoculum
• Freeze-drying specimens
• Inserting a hot loop into a culture
• Breakage of culture containers
• Cage cleaning and changing animal bedding
• Intranasal inoculation of animals
• Animal or human necropsy
• Harvesting infected materials
• Carelessly removing protective gloves or PPE

Devices that have the potential to create aerosols:

Aerosol droplets are formed with virtually any activity that disrupts the surface tension of a liquid or applies mechanical force to a solid.  As liquid particles bearing infectious agents may remain in the air for extended periods, it is important to consider methods of aerosol control.

Using a combination of the appropriate safety equipment and safe procedures is the primary method to minimize the creation of and exposure to aerosols.

Lab safety equipment to protect personnel from aerosols

• The certified biological safety cabinet (class I or II) is the primary barrier to protect workers from aerosols. Other safety devices include safety centrifuges with automatic locking mechanisms or solid lids, bioseal rotors, safety centrifuge cups, safety blenders, safety sonicators.
• If aerosol production cannot be prevented or contained, respiratory protection may be required. Contact EHS Lab Safety Services at ehs_labsafety@utk.edu to determine if the use of a respirator is appropriate.
• Vacuum line trap and filter systems are used to protect the vacuum system from aerosols.
  

Safe work practices for centrifugation of biohazards

• Confining work with viable biologics to a designated work space
• Wear appropriate personal protective equipment according to the lab’s assigned biosafety level or documented risk assessment. If respiratory protection is indicated, special training and a medical evaluation may be required.  Contact ehs_labsafety@utk.edu for more information.
• Routinely inspect centrifuge to ensure that leakage is not occurring.
• Do not overfill centrifuge tubes. Wipe the outside of the tubes with disinfectant after they are filled and sealed.
• Centrifugation may be performed in a centrifuge that is contained within a specially designed biological safety cabinet or other physical containment device.
• If a centrifuge containment device is not available, internal aerosol containment devices (e.g., sealed canisters, safety cups or buckets with covers, heat sealed tubes or sealed rotors, etc.) may be indicated.
• Aerosol containment devices should be removed from the centrifuge and opened in a biological safety cabinet. If a biological safety cabinet is unavailable, a settling time based should be allowed. If there is no known settling time specific to the materials being centrifuged, an industry standard of 10 minutes is recommended.
  

Safe work practices for blending, sonicating, grinding, and lyophilizing of biohazards

• Operate blender, sonicator, and grinder in a biological safety cabinet if required for the biosafety level of the lab, or place a towel moistened with disinfectant over the top of blender, grinder, or sonicator.
• Segregate the sonication procedure to a specific area of the lab, keeping the sonication vessel covered to the extent possible
• Thoroughly disinfect the area when the process has been completed.
• Use safety blenders designed to prevent leakage.
• If leak-proof blender is not available, regularly inspect the bottom of the blender for leakage.
• Avoid glass blenders.
• Open equipment in a biosafety cabinet if required for the biosafety level of the lab. If not, allow aerosols to settle for at least 10 minutes before opening equipment.
• Filter lyophilizer vacuum pump exhaust through HEPA filters or vent into a biological safety cabinet.
• Autoclave or disinfect all equipment promptly after use.

Safe work practices for pipetting of biohazards

• Pipette all biohazardous materials in a biological safety cabinet if possible.
• Drain a pipette with tip against the inner wall of the receiving vessel. Never forcibly expel any hazardous material from a pipette.
• Place reusable pipettes horizontally in a pan filled with enough liquid disinfectant to completely cover them.
• Mouth pipetting is prohibited; mechanical pipetting devices must be used.

Other safety precautions

• Minimize air bubbles when filling a syringe. Place a pad moistened with disinfectant over the tip of the needle when expelling air. Perform work in a biological safety cabinet whenever possible.
• Use a shielded electric incinerator or hot bead sterilizer to sterilize inoculating loops. Disposable plastic loops and culture needles are good alternatives to open flames.
• If a spill occurs that may generate aerosols, follow the lab specific spill response plan. Ensure that all lab personnel are aware of the lab’s spill response plan.
• Wear gloves when handling infectious materials, or infected animals.

BSCs offer protection by drawing the air in at the face opening (A) and immediately drawing the air through the front grille and under the work surface.  The air is then blown through the rear air plenum (B) to the top of the cabinet where it is divided into two chambers.  Thirty percent of the air is exhausted out of the cabinet (C) through a high efficiency particulate air (HEPA) filter into the laboratory room.  The remaining (70%) of the air is directed through another HEPA filter down onto the work surface (D) in a laminar flow directional air pattern.  Air then splits to either the front grille or back grille (E).  Most BSCs at UTK operate in this manner.

EHS Lab Safety Services requires that BSCs used for BSL-2 containment be certified:

• Upon initial installation
• If physically moved or relocated
• At least annually

The certification verifies HEPA filter efficiency and calibration of the airflow that provided that protective inward airflow at the face of the cabinet.  Certification is performed by a contracted field certifier in accordance with prescribed parameters. Once certified, the BSC will be tagged by the contractor as illustrated here.

While biosafety cabinets are not required for worker protection in BSL-1 containment, if they are used, annual certification will help ensure that the intention of use is met, regardless of the operating biosafety level.

If your BSC is due for certification, or if you need a list of approved field certifiers, contact EHS Lab Safety Services at 865-974-5084.

1. Turn on blowers and allow cabinet to run for 5-10 minutes to cycle air.
2. Confirm that the BSC is within its annual certification.  Check the pressure/magnehelic to ensure the cabinet is operating within parameters of its most recent certification.
3. Check to ensure UV light source is turned off. Many BSCs use UV (germicidal) bulbs as a means of decontamination.  UV bulbs may cause severe burns to exposed areas such as the skin and eyes.  While newer models have safety interlocks in place to prevent operation of UV sources while the cabinet is in use, this is not always the case.  Speak to senior lab personnel before you use the BSC to ensure you understand the equipment.
4. Disinfect all internal surfaces with an effective disinfectant.  Allow the disinfectant to remain wet for the time recommended by the manufacturer (contact time).
5. Ensure that your BSC is not overloaded; avoid clutter or having materials in the cabinet that are not required for your procedure. Overloading the BSC disrupts the laminar flow that is integral to providing a sterile work environment inside the cabinet.  BSCs are not engineered to provide storage.  Placing items on the grate at the front of the cabinet allows non-HEPA filtered air to enter the work surface inside the cabinet.  This could result in contaminated cultures or the escape of concentrated pathogens.

• Set up the cabinet in clean, transfer, and disposal zones. Work from clean to dirty, and keep all materials inside the BSC until completion of procedures.  If necessary, set up a small biohazard bag or pipet disinfectant tray inside the cabinet to discard contaminated wastes.

• The airspeed velocity at the front of the cabinet is relatively low (100 fpm). In order to minimize infectious aerosol leaks:
  • Use deliberate motions when placing or removing hands and objects in the BSC. Move hands straight in and straight out.  Avoid rapid side-to-side radial arm motions.
  • Keep front and rear air intake grilles clear of obstructions
  • Avoid excessively disrupting the air curtain by keeping items and waste inside the cabinet to the extent that is reasonably allowable
• Avoid open flames in the BSC. According to the World Health Organization (WHO), open flames should be avoided in the near microbe-free environment created inside the BSC. They disrupt the airflow patterns and can be dangerous when volatile, flammable substances are also used. To sterilize bacteriological loops, microburners or electric “furnaces” are available and are preferable to open flames.
• If a spill occurs, leave the cabinet running and follow spill procedures

• UNDER NO CIRCUMSTANCES SHOULD YOUR HEAD BE INSIDE THE BSC WHILE IN USE!!

• Allow the cabinet to continue to run.
• Close all primary containers. Wipe down exterior surfaces of containers with appropriate disinfectant (soaked paper towel, gauze, or wipes).
• Close waste containers, wipe with disinfectant, and discard as for other biohazardous waste (see below).
• Thoroughly wet all interior surfaces with appropriate disinfectant. Allow to remain wet for contact time and clean. Note that bleach solutions are corrosive and will “pit” and rust stainless steel over time.  If bleach is used, a follow-up rinse with alcohol or mild detergent is recommended.
• Allow the cabinet to run for 5-10 minutes after disinfection.
• Turn off cabinet and activate germicidal lamp (optional). Germicidal lamps should be inactivated after 15-20 minutes.

For additional resources on the care and use of BSCs, see:

• CDC training video
• NIH training video
• ESCO Global training video

The Laminar Flow Bench:

• Also referred to as a “clean bench”
• Typically, these benches are used for mixing sterile solutions, prepping agarose plates, etc.
• Pulls room air using a blower mounted below a HEPA filter
• Filtered air is passed across bench space to produce a “clean zone”
• These cabinets provide product protection, but no personnel protection
• DO NOT USE IN CONJUNCTION WITH INFECTIOUS AGENTS, HUMAN/NHP MATERIALS OR HAZARDOUS CHEMICALS

The Chemical Fume Hood:

• Chemical fume hoods use directional air flow to remove harmful vapors and gases from the cabinet and are hard ducted to the building exhaust system.
• Cabinets are typically not equipped with HEPA filters and are not compatible with viable infectious agents
• Incoming air is not filtered, so product sterility is not feasible
• Chemical fume hoods are useful for applications such as formalin fixing of cells, as the chemical hazard greatly outweighs the biological. Use discretion.

3.9.1.1 Before Centrifugation

• Train each operator on proper operating procedures, review the user manual.
• Use only rotors compatible with the centrifuge. Check the expiration date for ultracentrifuge rotors.
• Check tubes, bottles, and rotors for cracks and deformities before each use.
• Make sure that the rotor, tubes, and spindle are dry and clean.
• Examine O-rings and replace if worn, cracked, or missing.
• Never overfill centrifuge tubes (don’t exceed ¾ full).
• Always cap tubes before centrifugation.
• Always balance buckets, tubes, and rotors properly.
• Check that the rotor is seated on the drive correctly, close the lid on the centrifuge, and secure it.
• When using swinging bucket rotors, make sure that all buckets are hooked correctly and move freely.

3.9.1.2 During centrifugation

• Keep the lid closed at all times during operation. Never open a centrifuge until the rotor has stopped.
• Do not exceed safe rotor speed.
• The operator should not leave the centrifuge untilfulloperating speed is attained and the machine appears to be running safely without vibration.
• Stop the centrifuge immediately if an unusual condition (noise or vibration) begins and check load balances.

3.9.1.3 After centrifugation

• Allow the centrifuge to come to a complete stop before opening.
• Wear gloves to remove rotor and samples.
• Check inside of centrifuge for possible spills and leaks, clean centrifuge and rotor thoroughly if necessary.
• Wash hands after removing gloves.

3.9.1.4 Centrifuging Infectious Materials or Human Samples

Follow the safety procedures above, plus:

• Place a biohazard label on the centrifuge.
• Always wear gloves when handling tubes or rotors.
• Avoid the use of celluloid tubes with biohazards. If celluloid tubes must be used, an appropriate disinfectant must be used to decontaminate them.
• Always use sealed safety cups, safety buckets, or sealed rotors with O-ring as secondary containment if available.
• Fill centrifuge tubes, load into rotors, remove from rotors, and open tubes within a biological safety cabinet if biological safety cabinet is available.
• Wipe exterior of tubes or bottles withdisinfectant prior to loading into rotor or bucket. Seal rotor or bucket, remove outer gloves, and transport to the centrifuge.
• If possible wait approximately 10 minutes after the run to allow aerosols to settle before opening the centrifuge. Check for possible spills or leaks. For spills of infectious materials, see centrifuge emergency procedures below.
• Decontaminate centrifuge interior, safety cups or buckets, and rotors if tube breakage occurs.
• Includecentrifugationprocedure and decontamination plan in lab SOPs.

3.9.2.1 Emergency Situations

The following events are considered an emergency:

• If there is a spill in the centrifuge
• If centrifuge malfunctions
• If there is a rotor failure
• If there is a tube breakage

3.9.2.2 Emergency Procedures

For emergencies with RG2 organisms or hazardous materials

• Turn centrifuge off immediately and keep centrifuge lid closed
• Notify others
• Evacuate the lab if necessary
• Close the lab door
• Post a biohazard spill sign at the lab door
• Leave for 30-minutes to reduce the risk of aerosols.
• For spill cleanup, the operator should wear proper PPE, remove debris, clean and disinfect centrifuge interior, rotors, safety cups or buckets following the manufacturer’s instructions
• Place any contaminated protective clothing, gloves, and all cleanup materials in a biohazard bag
• Wash hands and any exposed skin surfaces with soap and water
• Seek medical attention as necessary
• Report incidents to P.I. or lab supervisor and EHS if the incident involves chemical hazards. Call EHS Lab Safety Services at 865-974-5084 for assistance

• Outer surfaces of storage and sample containers
• Vortexes
• Centrifuges (housing and rotor)
• Outer & interior surfaces of incubators
• Interior surfaces of the BSC before & after procedures
• Common contact surfaces (doorknobs, keyboards, etc.)
• Any surface you may have touched with gloved hands

• When retrieving sample containers
• After removing from water baths
• Upon completion of procedures involving infectious agents
• Any time a spill occurs

1. Is the product appropriate for the organisms that are being targeted?
2. Is it safe for the given surface?
3. Are there personal hazards to consider that would warrant the use of PPE?  Remember that disinfectants typically work by targeting basic cellular structures/macromolecules (dissolve lipids, denature proteins, induce nucleic acid breaks, etc.).  Our cells share this biology; thus, disinfectants can act as hazardous chemical.  Depending on the type/class, these may cause skin/eye burns, sensitization of the skin (rashes) and/or respiratory tract (wheezing/asthmatic symptoms), or cancer (the aldehydes and ethylene oxide are recognized carcinogens).
4. Are there special instructions for the preparation of the mixture?
5. Does it need to be diluted?
6. What is the minimum time it must be in contact with contaminated surfaces to reduce the infectious potential to acceptable levels (also called the contact time).

Currently, there are a wide variety of disinfectant options available. The products generally fall into the following categories (listed from low-level to high-level disinfectants):

1. Quaternary Ammonium (Cavicide; Lysol)
2. Ethyl and isopropyl alcohol
3. Phenols (Clorox bleach-free, Vesphene)
4. Chlorine compounds and hypochlorite/bleach (Clorox)
5. Iodophors (Povidone-iodine solution)
6. Aldehydes: Glutaraldehyde (Cidex); formaldehyde; paraformaldehyde
7. Ethylene oxide

• Keep bench clutter to a minimum! From a disinfection/decontamination standpoint, poor housekeeping is problematic because:
  • Cross contamination from surface contact with infectious materials is a higher risk
  • Spill cleanup becomes more complicated, more time consuming, less effective, and poses a higher risk of exposure to infectious agents.
  • Surface disinfection is hindered by clutter

Needles are not to be recapped at BSL-2 unless specifically exempted by EHS.  Needle recapping is discouraged at BSL-1, but if necessary, the one-handed scoop technique may be used.

If research activities create the need to recap needles, your lab should submit the needle re-capping exclusion form to EHS.  In rare instances where re-capping is permissible, utilize the one-handed scoop method.  First, place the cap in a flat surface.  Second, using one hand, scoop the cap up with the needle.  Third, using the same hand, “click” the cap in place by pressing with your thumb at the base of the needle cap. 

INFECTIOUS WASTE COLLECTION, DISINFECTION, AND DISPOSAL

3.12.2.1 Liquids

Liquid wastes may be treated and disposed of by either of the following methods:

1. Chemical treatment of liquids with disinfectant; disposal via lab sink:
   • Add household bleach to the collection vessel so that the bleach makes 10% of the final volume.
   • Allow a contact time of at least 30 minutes.
   • Carefully pour the mixture down the lab sink, then thoroughly rinse down the sink with water.
   • Wear the appropriate PPE for handling bleach and bleach-treated liquids. Lab coat, gloves, and chemical splash goggles are recommended. A lab-specific hazard assessment will determine the need for protective eyewear.

OR

2. Autoclave treatment of liquids; disposal via lab sink:
   • Place the closed collection vessel in a secondary container and transport by cart to the autoclave facilities.
   • Treat by autoclave using the liquids cycle, generally 20-30 min.
   • Pour cooled, treated liquid down the lab sink followed by a thorough water rinse.
   • Never autoclave liquids that have had bleach added to them.

3.12.2.2 Sharps

Biohazardous sharps waste must be disposed in an FDA-approved container that is manufactured for the disposal of biohazardous sharps waste:  1) puncture resistant; 2) restricted opening disallowing retrieval of sharps; 3) a lid that can be securely closed once full; and 4)  labeled with the universal biohazard symbol.

Additionally:

1. All sharps containers must be permanently closed and disposed of when 2/3 to ¾ full or whenever items do not freely fall into the container. Never pack, tamp, or shake a sharps container to fit additional items.
2. Wipe down the exterior surface of the container with disinfectant prior to submission for disposal.
3. Disposal of biohazardous sharps will be accomplished through a medical waste disposal contractor coordinated through EHS on the main campus or through the UTIA Safety Office on the Ag campus and at the College of Veterinary medicine. Do not dispose of biohazardous sharps containers in the trash, regardless of treatment status.
4. Clean sharps may also be placed in the red sharps containers as necessary.

3.12.2.3 Solids

Non-sharps solid biohazardous waste must be collected for final treatment and disposal in a leak-proof container lined with an autoclavable bag of moderate thickness to prevent punctures.  The collection container must have a lid or other means of closure and the container must be labeled with the biohazard symbol regardless of the lab’s operating biosafety level.  For BSL-2 labs, bags must be red, orange, or embossed with the biohazard symbol.

Treatment of solid, non-sharps biohazardous waste may occur in two ways:

1. Autoclave the biohazard bags according to posted parameters for bagged waste, allow to cool to room temp, and place in the designated “Autoclave-treated waste” bins, located in or near the autoclave rooms.
2. Some departments have a contract for pickup and disposal of waste through a medical waste disposal contractor. In this case, the same procedures apply as above, except bags do not have to be autoclave prior to packaging for contractor pickup.  Follow the contractor’s procedures for final packaging and storage prior to removal.

NOTE:  If other methods of decontaminating and disposing of biohazardous waste are desired, validation data specific to the requested alternative method may be submitted to EHS for review.

3.12.2.4 Serological Pipettes

Pipette wastes may require creative approaches for accumulation prior to disposal.  Serological pipettes and micropipette tips are good examples, as they may not fit some biohazardous waste bins or may present a sharps hazard if they comingle with heavier wastes (e.g., agar plates).

Suggested practices:

• Line a cardboard box, 5 gallon bucket, or similar container with a biohazard bag and collect the pipettes with the tips             oriented in the same direction.
• Ensure the outside of the container has a biohazard label.
• When the bag is full, treat the pipettes by autoclave or disposal through the medial waste contractor as indicated
• Pipette tips may be placed in the sharps container or other puncture resistant collection device.

3.12.2.5 Pathological Waste

Pipette wastes may require creative approaches for accumulation prior to disposal.  Serological pipettes and micropipette tips are good examples, as they may not fit some biohazardous waste bins or may present a sharps hazard if they comingle with heavier wastes (e.g., agar plates).

Animal tissues, tissue trimmings, organs, or carcasses must be:

• Collected in leak proof, sealed bags. Bags must be red, orange, or embossed with the universal biohazard symbol if the pathological material contains an infectious agent, recombinant/synthetic nucleic acid, or biological toxin.
• Freeze and store tissues for disposal through the respective lab animal facility or satellite facility.
• Unless otherwise indicated by EHS, pathological waste is not to be autoclaved. Rather it will be shipped offsite for incineration or submitted to the College of Veterinary Medicine’s necropsy unit for alkaline hydrolysis.

NOTE:  Never discard pathological wastes into the trash! 

Any lab worker handling untreated biohazard waste must:

1. Complete training offered by EHS.
2. Complete and sign the lab-specific training form after being trained in lab biosafety practices by the PI or lab supervisor
3. Complete the Bloodborne Pathogens (BBP) training annually, if applicable
4. Complete Regulated Medical Waste training if packaging waste or signing waste manifests for UT’s regulated medical waste contractor.

Autoclaves pose physical (e.g., heat, steam and pressure) and biological hazards.  Each autoclave has unique characteristics and resulting hazards.  Review and understand the owner’s manual before using any autoclave for the first time and as needed thereafter.  Employ the following autoclave safety practices:

1. Before using, check inside the autoclave for any items left by the previous user that could pose a hazard (e.g., sharps).
2. Load properly (per manufacturer’s directions)
3. Loosen container caps
4. Place containers with liquid in a tray with solid bottom and walls to catch spills
5. Add water to the autoclave bags to facilitate steam generation. Add ¼ to ½ inch water to tray to ensure even heating
6. Check plastic materials to ensure they are compatible with the autoclave
7. Do not autoclave hazardous chemicals or radiological materials!!
8. Never place glassware directly on the autoclave bottom or floor
9. Make sure the door of the autoclave is fully closed and latched and the correct cycle has been chosen before starting the cycle
10. When removing items from the autoclave, wear the following PPE (at a minimum): heat resistant gloves, safety glasses with side shields, and a lab coat
11. For non-liquid loads, let liquids stand for a full hour before touching with ungloved hands. Be sure others in the area know a heat hazard is present.

3.12.4.1 Autoclave Use and Safety:  Validations

In accordance with local and state regulations, all biohazardous waste must be biologically-inactivated before it is disposed of as trash.  This can only be achieved if the waste is exposed to the right temperature for the right amount of time.  Optimally, the waste should be exposed to:  121°C, at a pressure of 15 PSIG for at least 20 minutes.

If you are an autoclave validation volunteer, employ the following autoclave validation practices:

1. 1. Validate quarterly using 3M Comply Thermalog TM Steam chemical integrator strips.  Strips may be obtained from EHS Lab Safety Services upon request.
   2. Place a full, medium-sized biohazard bag (e.g. 25” x 35”) into an autoclavable secondary container
   3. Add 1 cup (~250 mL) warm water to bag.
   4. Place a 3M Comply Thermalog TM Steam chemical integrator strip inside the bag near the center of the bagged contents.
   5. Place bag into the autoclave, leaving the top of the bag open for facilitate adequate steam penetration into the bag.
   6. Autoclave the biohazard bag for a minimum of 30 minutes at 250F/121C.  30 minutes is the recommended minimum, but sterilizations of >1 hour are not abnormal depending on the autoclave and load volume/contents.
   7. After cycle completion, note the status of the chemical integrator.  A successful test is achieved only if the blue indicator line reaches any portion of the “safe” window.
   8. Document the validation test.  Documentation should include the following:

• Date and time of test
• Load contents
• Parameter setting for autoclave
• Type of test and results of test
• Name of person performing test
• Failed attempts and remedial actions as necessary

1. If the validation was successful, the biohazard bag may be tied up and discarded into the designated white bins for final disposal.

Note: biological indicators (Geobacillus stearothermophilus spore) testing may be required in some circumstances.  Contact EHS for instructions. 

3.12.4.2 Autoclave Use and Safety:  Bagged Waste

Employ the following autoclave treatment protocol for biohazardous waste:

1. Use secondary containment (i.e., pan and/or cart with containment walls) for transporting waste bags to the autoclave for treatment to reduce the possibility of a spill during transport.
2. Add one cup of water to each bag to facilitate air displacement and enhance steam generation.
3. Leave bags open or loosely closed at the top to facilitate steam penetration.
4. Place bags in autoclavable secondary containment pan for autoclave treatment to reduce the possibility of a spill during treatment.
5. Follow waste cycle parameters established for the autoclave to assure effective decontamination of waste.
6. Unload waste after cycle is complete and chamber pressure has returned to 0 PSIG.  Do NOT override safety features to open the autoclave.
7. Use autoclave gloves, appropriate eye protection, and lab coat to avoid injury from contact with hot surfaces or liquids when removing waste from the autoclave.
8. Allow to cool, then tie or band the treated bags closed to reduce the possibility of a spill.
9. Discard in “Autoclave-treated wastes” bins located in or near the autoclave rooms.

CAUTION:  Do not autoclave wastes that are contaminated with hazardous chemicals or radiological materials!!

• Use shatter resistant containers with tight fitting lids or stoppers
• If handling multiple tubes, employ a tube rack or other device to prevent drops
• Use a leak-proof secondary container such as a cooler or tray to contain leaks or spills (critical for movement outside the lab)
• Always use a cart when transporting large volumes of materials or when moving from floor to floor

Cleaning up a biohazardous spill in a biosafety cabinet

1. Let the BSC run. Do not turn off.
2. Remove broken glass with forceps, tweezers or other tools and place glass in a sharps container.  Do not wipe up broken glass.
3. Cover spill with paper towels.
4. Pour (don’t spray) disinfectant to contaminated surface by pouring it around the periphery of the covered spill moving inward.  Allow the appropriate contact time for the disinfectant and agent.
5. After the contact time, wipe up the spilled material.
6. Reapply disinfectant to the affected area and after the appropriate contact time, wipe up the area. Repeat if necessary.
7. Perform disinfection before removing items.
8. Segregate contaminated cleanup materials into the appropriate biohazardous waste containers.

Cleaning up a biohazardous spill outside of a biosafety cabinet

1. Close off the area and allow aerosols to settle.
2. Notify others including supervisor.
3. Assemble all spill cleanup materials and review procedure.
4. Don appropriate PPE: laboratory coat, safety glasses or chemical splash goggles (depending on risk of splashes), Nitrile gloves.
5. Cover spill with paper towels.
6. Pour (don’t spray) disinfectant to contaminated surface that is covered with paper towels by pouring it around the periphery of the spill area moving inward.  Allow the appropriate contact time for the disinfectant and agent.
7. Wipe up spill. Dispose of the waste in the biohazardous waste containers.
8. Re-apply disinfectant to contaminated surface and allow it to stand for proper contact time.
9. Wipe up disinfectant, repeat if necessary.
10. Segregate contaminated cleanup materials into the appropriate biohazardous waste containers.

Cleaning up a biohazardous spill inside a centrifuge

1. Close off the area and allow aerosols to settle.
2. Notify others including supervisor.
3. Wait 30 minutes to allow the aerosols to settle.
4. Don appropriate PPE: laboratory coat, safety glasses and Nitrile gloves.
5. Remove rotors and bucket and place in Biosafety Cabinet.
6. Thoroughly disinfect the inside and outside of the centrifuge rotor, cups and accessories and allow proper contact time. After disinfection, move to sink for a thorough rinse, dry thoroughly.
7. For the centrifuge interior, follow the steps above for cleaning up a spill outside a Biosafety Cabinet.
8. Segregate contaminated cleanup materials into the appropriate biohazardous waste containers.

Note:  The response outlined above refers to spills that are <500 mL. For larger spills, please contact EHS.

In the event of a fire, follow the actions prescribed by UT fire policies.

In the event of a fire in the BSC, turn the blower off and close the sash to minimize the immediate spread of the fire and follow the actions prescribed by UT fire policies.

In the event of a building evacuation while you are working with materials at BSL-2, cover the materials or leave them in the BSC.  Remove your gloves and lab coat on your way out of the lab.

Medical emergencies can only be responded to successfully if they’re reported.  In many BSL-2 environments, the work may be done by one individual in a culture room or an isolated area of the lab where a person may not be readily visible.  Therefore, anyone who will be working at BSL-2 should assure that at least one other person in the lab group knows that they will be working, and the person notified should check on them frequently to assure that they are alright.

If a medical emergency arises for someone in your lab who is working with materials requiring BSL-2 containment, call EMS (by way of 911) immediately. While life safety is first and foremost, some basic actions should be taken to minimize the spread of contamination.

• Restrict foot traffic to the area.
• Provide emergency responders with information about the materials that the person was working with so that they know what contamination is present in the area.

For injuries not related to an exposure event (i.e. back strain, paper cut, etc.) follow the regular occupational injury reporting process.

In the event of biological exposure incident, follow the procedures outlined in Section 5 (Occupational Health) of the biosafety manual.  Remember to flush the exposed skin or mucous membranes for 15 minutes, report it to your supervisor and EHS at 865-974-5084 and to seek medical attention.

All personnel experiencing an exposure to potentially infectious materials must also complete the Biosafety Incident Report form and submit the completed form to EHS.   The form may be found at https://biosafety.utk.edu/emergency-response/, or by contacting EHS Lab Safety Services at 865-974-5084.  After initial investigation and filling out the form, contact EHS for communication and follow-up.

EHS encourages all lab personnel to report near miss events and unsafe conditions as well as exposures.  Follow the exposure guidelines above for near misses and unsafe conditions or call EHS at 865-974-5084.