Chapter 26

BIOSAFETY

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Appendix G. Biological Spills and Cleanup

imageG.1 Introduction and Scope

Hazards need to be assessed and a safe response must be implemented for each spill situation. This appendix provides general guidelines for decontamination and cleanup of various types of biological materials, including:

Note the following Biosafety Manual sections and guidelines for additional information related to biological spills and cleanup:

G.2  Pre-Cleanup Considerations

Text Box: Biological spill. Source: Berkeley Lab EHS.
Biological spill. Source: Berkeley Lab EHS.

Generally, you may clean a biological spill when the conditions listed below are present. If these conditions do not exist, request assistance from your supervisor or call the LBNL emergency and non-emergency telephone numbers listed in the Emergency Guide as appropriate.

Pre-cleanup conditions:

G.3  Biological Spill outside a Biosafety Cabinet   

  1. If you spilled a Risk Group 1 (RG1) material, or a small dilute amount of an RG2 material, remove any contaminated clothing, wash contaminated body areas with soap and water, and proceed to Step 6.
  2. If you spilled a significant amount (e.g., 100 ml or more) of an RG2 or higher material, hold your breath, leave the room immediately, and close the door.Text Box:
  3. Warn others not to enter the contaminated area. Get help as needed and call the LBNL emergency or non-emergency phone numbers in the Emergency Guide. If you leave the area, post a sign warning others to not enter the area.
  4. Remove and put contaminated clothing into a container for biohazardous waste disposal or autoclaving, and thoroughly wash hands and face.
  5. Wait 30 minutes before re-entering the area to allow dissipation of airborne biological materials (aerosols) created by the spill. Put on personal protective equipment (PPE) before re-entering the room.
  6. Put on the following PPE: lab coat or gown, safety glasses, and double gloves. If the risk of the material or contamination is high, wear additional appropriate PPE such as a respirator, jumpsuit with tight-fitting wrists, or shoe covers.
  7. Cover the spill with paper towels or other absorbent material to prevent liquid migration and aerosol production.
  8. Gently pour or squirt a freshly prepared solution of 10% household bleach or other appropriate disinfectant around the edges and then into the center of the spill area until the towels are soaked with the disinfectant.
  9. Let the disinfectant stay in contact with the spilled material for at least 10 minutes, and up to 20 minutes for larger volumes or RG2 materials.
  10. Use paper towels to wipe up the spill, working from the edges into the center of the spill. If sharps or sharp fragments such as glass might be in the spill, do not touch the spill materials with gloved hands. In this case, use a dustpan and squeegee or disposable cardboard to scoop up the spill materials and sharps.
  11. Clean the spill areas with paper towels soaked with disinfectant, and then with paper towels wetted with water.
  12. Dispose of or autoclave contaminated items. Dispose of contaminated items using biohazardous waste containers, biohazard bags, sharps containers, and other means specified in the Medical and Biohazardous Waste Generator’s Guide (PUB-3095). Reusable and autoclavable items may be decontaminated using an autoclave bag and pan in an on-site autoclave.
  13. Remove and dispose of PPE, or place coats in lab-coat laundry bin. Wash hands with soap and water.
  14. Report spill, exposure, and injury incidents to your work lead or supervisor and in accordance with Work Process D.10 of this program.

Text Box: Biological Spill Cleanup


Text Box: G.4  Biohazardous Spill inside a Biosafety Cabinet    

This procedure assumes the spill of biohazardous material of significant quantity or risk inside a biosafety cabinet (BSC).


  1. Ensure the BSC is operating and continues to operate during this procedure so as to prevent airborne contaminants from escaping the cabinet.
  2. Put on the following PPE: lab coat or gown, safety glasses, and chemical-resistant double gloves. Wear additional PPE (e.g., respirator or goggles) as needed based on the risk of the material, contamination, or splashing.
  3. Spray or wipe walls, work surfaces, and equipment with a disinfectant that is effective against the agents that may be present. A 1% solution of an iodophor decontaminant (Wescodyne or equivalent) is effective against most viruses, fungi, vegetative bacteria, and most nonencysted amoeba. A decontaminant detergent has the advantage of detergent activity, which is important because extraneous organic substances frequently interfere with the reaction between microorganisms and the active agent of the decontaminant.
  4. Flood the BSC’s top work-surface tray with disinfectant. In a Class II BSC, also flood with disinfectant the drain pans and catch basins below the work surface. Allow the disinfectant to stand for 10 to15 minutes.
  5. Remove excess disinfectant from the tray by wiping with a sponge or cloth soaked in disinfectant.In a Class II BSC, drain the BSC’s top work surface into the BSC catch basin, lift out the work surface and removable exhaust grilles, and wipe the top and bottom (underside) surfaces with a sponge or cloth soaked in disinfectant.Replace the work surface and grilles. Drain the disinfectant from the BSC base into an appropriate container. Place the container with disinfectant, gloves, cloth, or sponge in an autoclave pan, and then autoclave according to standard procedures.
  6. Report spill, exposure, and injury incidents to your work lead or supervisor and in accordance with Work Process D.10 of this manual.

Text Box:

G.5  Centrifuge Malfunction or Spill 

This procedure assumes that the following types of centrifuge events have occurred, especially if RG2 materials are involved: the spill of biological material in the centrifuge, significant mechanical failure (e.g., rotor failure), or centrifuge tube or container breakage. Evidence of such conditions might include noises during centrifuge operation or visual signs of failure or leakage when the centrifuge is opened. Note that breakage of tubes and leakage of fluid into the centrifuge wells or cups during centrifugation may release relatively few agents into the air. However, if a tube breaks and leaks in the centrifuge chamber, then aerosols and droplets may be created and dispersed.

In the event of a centrifuge malfunction or spill, follow the following steps:

  1. Turn centrifuge off immediately. Keep the centrifuge lid closed and latched.
  2. Notify others.
  3. Evacuate the laboratory if hazardous aerosols may have been generated. Close the door, post a biohazard spill sign at the lab door, and stay out of the laboratory for 30 minutes.
  4. For spill cleanup, the operator should wear PPE (i.e., gloves, lab coat, eye protection), remove debris, and clean and disinfect centrifuge interior, rotors, safety cups, or buckets in accordance with the manufacturer's instructions.
  5. Place any contaminated PPE and all cleanup materials in a biohazardous waste container. Wash hands and any exposed skin surfaces with soap and water.
  6. Report spill, exposure, and injury incidents to your work lead or supervisor in accordance with Work Process D.10 of this manual.

Text Box:

G.6  Radioactive and Biohazardous Spill 

This procedure assumes the spill of material outside a biosafety cabinet that has both radioactive and biohazardous concerns. In this case, the biological component of the spill should be inactivated prior to disposal of the spilled materials as radioactive waste. Call the Radiation Protection Group at extension 7277 or (510) 486-7277 for instruction and assistance.

 

Spill of RG1 Material or Small amount (e.g., Less Than 100 ml) of Dilute RG2 Material

 

  1. Warn others not to enter the contaminated area. Post a sign on the door as needed.
  2. Remove any contaminated PPE (e.g., lab coat, gloves) if there is a risk of exposure to biohazardous agents, and isolate PPE in a plastic bag or appropriate container.
  3. Contact the Radiation Protection Group (RPG) 24/7 at extension 7277 or (510) 486-7277 to report the incident. If RPG is responding to the spill location, wait until RPG arrives before proceeding with the steps below.
  4. Monitor yourself for radioactive contamination. If contaminated,wait for RPG assistance.
  5. Thoroughly wash your hands and face if there is a risk of exposure to biohazardous agents.
  6. Put on the following PPE: lab coat or gown, safety glasses, and double gloves. If the risk of the material or contamination is high, wear additional appropriate PPE such as respirator, jumpsuit with tight-fitting wrists, or shoe covers.
  7. Cover the spill with paper towels or other absorbent material to prevent liquid migration and aerosol production.
  8. Gently pour or squirt a freshly prepared solution of 10% household bleach or other appropriate disinfectant around the edges and then into the center of the spill area until the towels are soaked with the disinfectant.
  9. Let the disinfectant stay in contact with the spilled material for at least 10 minutes, and up to 20 minutes for larger volumes or RG2 materials.
  10. Use paper towels to wipe up the spill, working from the edges into the center of the spill. If sharps or sharp fragments such as glass might be in the spill, do not touch the spill materials with gloved hands. In this case, use a dustpan and squeegee or disposable cardboard to scoop up the spill materials and sharps.
  11. Clean the spill areas with paper towels soaked with disinfectant, and then with paper towels wetted with water.
  12. Place all contaminated materials into a plastic bag, and place the bag in the appropriate radiation waste container. Monitor for radiation contamination all potentially contaminated items that are not placed in the radiation waste container. Decontaminate and resurvey these items as necessary.
  13. Report spill, exposure, and injury incidents to your work lead or supervisor and in accordance with Work Process D.10 of this program.

 

Spill of Risk Group 2 Material Greater Than 100 ml

 

  1. If you spilled a significant amount (e.g., 100 ml or more) of an RG2 material, hold your breath, leave the room immediately, and close the door.
  2. Warn others not to enter the contaminated area.
  3. If possible, remain stationary and request assistance from others to contact the Radiation Protection Group (RPG). Contact RPG 24/7 at extension 7277 or (510) 486-7277 for assistance.
  4. Remove any contaminated PPE (e.g., lab coat, gloves) if there is a risk of exposure to biohazardous agents, and isolate PPE in a plastic bag or appropriate container.
  5. Thoroughly wash your hands and face if there is a risk of exposure to biohazardous agents.
  6. Proceed with the remaining steps after RPG’s arrival.
  7. Wait 30 minutes before re-entering the area to allow dissipation of airborne biological materials (aerosols) created by the spill. Put on PPE before re-entering the room.
  8. Follow Steps 6 through 13 noted in the previous section titled “Spill of RG1 Material or Small Amount (e.g., Less Than 100 ml) of Dilute RG2 Material.”

Text Box: G.7  Chemical and Biohazardous Spill

This procedure assumes that the spill of a material took place outside a biosafety cabinet, the material has both chemical and biological hazards, the chemical in the material is considered a hazardous waste, and the chemical has not already rendered the biological material nonviable or inactive.

  1. Prior to starting your research, determine which chemical disinfectant(s) and absorbent materials are compatible with the chemical(s) that may become biologically contaminated and whether the contaminated chemical(s) can be autoclaved.Autoclaves heat materials at high temperatures and pressures, and the autoclave operator may be exposed to chemical vapors when the autoclave is opened.
  2. If you spilled a significant amount (e.g., 100 ml or more) of an RG2 material, hold your breath, leave the room immediately, and close the door.
  3. Warn others not to enter the contaminated area. Get help as needed. If you leave the area, post a sign warning others to not enter the area.
  4. Remove and put contaminated clothing in container lined with a plastic bag for eventual decontamination, autoclaving, or disposal. Thoroughly wash hands and face. If clothing is chemically contaminated, autoclaving may not be advisable.
  5. If you evacuated the laboratory as stated in Step 2, call the LBNL emergency or non-emergency phone numbers in the Emergency Response Guide and wait 30 minutes before re-entering the area to allow dissipation of airborne biological materials (aerosols) created by the spill. Put on PPE before re-entering the room.
  6. Consult the LBNL Chemical Hygiene and Safety Plan for chemical spill response procedures. If the chemical(s) in the spill present a greater hazard than the biological agent(s), proceed with chemical decontamination first.
  7. Put on at least the following PPE: lab coat or gown, safety glasses, and chemical-resistant double gloves. If the risk of the material or contamination is high, wear additional appropriate PPE such as a respirator, a jumpsuit with tight-fitting wrists, or shoe covers.
  8. Cover the spill with an absorbent material or towel that will not react chemically with the spilled chemical. Towels will prevent liquid migration and aerosol production.
  9. Use a disinfectant that is compatible with the chemical(s) in the spill. Gently pour or squirt the disinfectant around the edges and then into the center of the spill area until the absorbent material or towel is soaked with the disinfectant.
  10. Let the disinfectant stay in contact with the spilled material for at least 10 minutes, and up to 20 minutes for larger volumes or RG2 materials.
  11. Use chemically compatible towels, dustpan, squeeges, or cardboard to scoop and wipe up the spill, working from the edges into the center of the spill. If there might be sharps or sharp fragments such as glass in the spill, do not touch the spill materials with gloved hands.
  12. Clean the spill areas with towels soaked with disinfectant, and then with towels wetted with water.
  13. If the chemical(s) are compatible with autoclaving, contaminated materials (paper towels, absorbent, glass, liquid, gloves, dustpan, squeegee, etc.) may also be placed into autoclave bags and an autoclave pan. Cover the pan with aluminum foil and autoclave according to standard directions. After autoclaving, the now-sterile materials may require being disposed of as hazardous chemical waste via the LBNL Waste Management Group.
  14. If the chemical(s) are not autoclavable (or if you do not know whether they are autoclavable), then transfer the disinfected materials into a screw-cap container, and place the container in the Satellite Accumulation Area.
  15. Report spill, exposure, and injury incidents to your work lead or supervisor and in accordance with Work Process D.10 of this section.

G.8  Cleanup of Small Dead Animals, Nests, or Droppings  Text Box:

The following general procedure should be used as a guideline for cleanup of small dead animals, nests, or droppings. This procedure may need to be adapted depending on the nature of the materials and situation. Contact the Facilities Division via the Facilities Work Request Center if assistance is needed due to a pest infestation or to the nature or size of the concern.

  1. Wear PPE such as reusable or disposable rubber gloves and safety glasses when handling decontaminant solutions, dead animals, or cleaning up contaminated materials. Use double disposable gloves if possible and appropriate. Determine if disposable or cleanable protective clothing is also needed.
  2. Clean up dead animals, nests, droppings or contaminated food by first spraying or soaking the item with an appropriate disinfectant such as 10% household bleach, Lysol®, or other appropriate janitorial disinfectant (see Appendix F, Section F.2.3 of this manual). Allow the disinfectant sufficient time to decontaminate the item (e.g., 10 minutes).
  3. If possible and appropriate, pick up the decontaminated item with an impervious barrier such as a plastic bag placed over the item. Place the decontaminated item into a plastic bag, tie the bag shut, place the bag into a second bag, and tie the second bag shut.
  4. Clean up localized gross surface contamination as needed by spraying or soaking with disinfectant and using disposable paper towels. Place waste materials in a plastic bag, remove outer contaminated disposable gloves, and double bag the waste materials.
  5. Dispose of the bags of waste in the general trash. Use an outside dumpster as needed to prevent odor problems.
  6. Clean contaminated surfaces or floors as needed. Use a solution of water, detergent, and disinfectant to mop floors or wipe surfaces. Steam clean or shampoo carpets and upholstered furniture. Do not vacuum or dry sweep surfaces before wet cleaning. Pour mop or cleaning wastewater into a drain that is connected to the building’s sanitary sewer system.
  7. Remove PPE, and then clean it or dispose of it.
  8. Remove any potentially contaminated clothing and launder separately with detergent and hot water.
  9. Wash hands with soap and water.

 

The state of California encourages the reporting of dead birds and squirrels to assist state agencies in tracking disease. This reporting is optional at LBNL and involves keeping the animal for 24 hours without decontamination or freezing. See the California West Nile Virus Web site for additional information and online reporting.

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