BL3 Standard Operating Procedures

I. Authorization to Use the Mullins' lab BL-3 Facility (B44C)

This document must be read and an agreement/authorization sheet signed and registered with Dr. Mullins.

Entry to the BL-3 Facility is restricted to individuals who have obtained prior approval. Authorization to use this facility is contingent on the user’s satisfactory completion of training, understanding of the safety procedures described below, and on his or her ability to follow these guidelines while using the facility. Individuals with little or no prior experience working in a BL-3 facility must have an experienced individual working with them until they become proficient in the safety techniques required.
Authorization to use the BL-3 facility requires review and understanding of the following information:
1. Practices outlined in the UW Biohazard Safety Manual, section IV on Biohazard Control Procedures, section VI on Occupational Health Program and section IX on Blood borne Pathogens.
1. The pamphlet from the CDC-NIH Biosafety in Microbiological and Biomedical Laboratories, pages 25-30, BL-3 Standard Microbiological Practices, Special Practices and Safety Equipment; and pages 116-121, Viral Agents: Retroviruses, Including HIV and SIV. The information in this booklet contains recent information on laboratory-acquired infection with HIV as well as guidelines for the use of biological safety cabinets, cleaning and decontaminating spills, and management of exposures.
2. The specific rules and procedures outlined in this document for work in the Rosen B44C BL-3 facility.
Authorization to use the BL-3 facility requires successful completion of the following:
1. Completion of theMullins B44C BL-3 Facility Usage Authorization Form. Make yourself a copy and turn in completed form to Mullins/Mittler BL3 Lab supervisor (who will make a copy for BL3 Entry Notebook and give copies to appropriate supervisors).
1. Completion of the EHS course Bloodborne Pathogen Exposure Control, required for laboratory employees working with human blood and blood byproducts. Covers risk assessment, personal protection, safe procedures, emergency response, and disposal of biohazardous waste. Annual refresher training required. Register online athttp://www.ehs.washington.edu/forms/pso/bpecrform.phpor call (206) 543-7201.
2. Completion of the EHS course, Biological Safety Practices (U Conjoint 420), scheduled upon request, autumn only. If a user already has extensive experience working in a BL-3 facility, this course does not have to be taken prior to beginning their work, but should be taken at the earliest possible convenience.
3. All users must have experience with tissue culture or working with other human pathogens prior to working with HIV and SIV. A laboratory orientation should be arranged with Dr. Mullins or the BL-3 lab supervisor. At this time, proficiency in biosafety techniques (use of the biosafety cabinets, microscopes, centrifuge safety cups, and proper handling of media and waste material, etc.) must be demonstrated.
4. Appointment with Occupational Health to cover the following:
5. 1. Hepatitis B immunization for work with blood or blood products.
  1. Counseling on working with HIV and SIV. This includes receiving a drug prescription for post exposure prophylaxis (PEP).
  2. Drawing of serum sample prior to initiating work in the BL-3 facility. This sample will be stored in the event that HIV seroconversion occurs in order to document the time of seroconversion. They will not be tested without the consent of the individual and then only after a documented exposure occurs. Every 6 months samples can be drawn for HIV testing (optional).

II. Entrance into the Facility

Food or drink is not permitted anywhere within the BL-3 laboratory or anteroom.
Sandals and shorts are not allowed in the BL-3 facility.
Whenever possible, laboratory personnel should avoid handling virus-containing material when he or she has cuts or skin abrasions including, for example, eczema on the hands, wrists or face. If work is necessary, and no replacement for that individual can be found to perform the work, cuts and/or abrasions on the hands and/or wrists should be thoroughly bandaged prior to donning gloves and entrance to the facility. A full-face shield, in addition to the facemask, should be worn to protect cuts and abrasions on the face. Immuno-compromised individuals should not use the facility.
The following dress requirements are to be followed by all individuals who use the facility regardless of the reason for entry: within the anteroom and prior to entry to the facility each user should put on one pair of nitrile gloves to cover at least two inches of sleeve (the integrity of the gloves can be checked for tears and punctures by flipping the gloves closed and observing that the air trapped inside is not leaking). The user should then don shoe covers, goggles (or glasses) and a face mask and enter the facility. The gown should be put on immediately upon entrance to the facility, followed by a second pair of latex long gloves. The inner pair of gloves should never be removed or replaced while in the facility unless a tear is suspected. Clean gowns will be provided in the anteroom. Contaminated gowns should be changed immediately. It should be emphasized that the reason for protective clothing is to protect the worker from inadvertent spills of infected media. The purpose of the shoe covers, for example, is to protect shoes from inadvertent spills, however, THE FLOORS (AND ALL SURFACES) SHOULD BE KEPT CLEAN OF SPILLS AND TRASH AT ALL TIMES. PROTECTIVE CLOTHING IS NOT MEANT TO NOR WILL IT EVER REPLACE APPROPRIATE AND PRUDENT WORK PRACTICES. Please note that the housekeeping staff are instructed not to enter this room, you are responsible for keeping it clean.
The air pressure in the BL-3 facility should be between -0.05 and -0.10. When the air pressure alarm sounds for a prolonged period of time after entry into the facility and closing the door, all work in the area should cease until the pressure has returned to normal.
Do not bring personal items into the lab.
Do not apply cosmetics while in the lab.
Workmen are only allowed to enter the facility if work is not ongoing and with the approval of Dr. Mullins or the lab supervisor. Workmen should be accompanied by someone authorized to enter the BL-3 facility who will show them the appropriate dress requirements. They should wear gowns and gloves but are not required to wear shoe covers, goggles or facemask, as they will not handle infectious materials.

III. Laboratory Procedures

General Considerations:
1. Failure to comply with the practices described below will result in suspension of access privileges.
1. Double gloves are required to provide protection against exposure to virus through gloves potentially weakened by alcohol, skin oils, other chemicals and overuse. EHS recommends changing your outer gloves every 30 minutes. At any time the outer gloves have been contaminated with culture supernatant, serum or infectious fluid, they should be removed and discarded into the autoclave bag inside the hood. A clean pair of outer gloves should be donned before continuing work. Gloved hands that have been working in the hood, should be sprayed with ethanol or changed prior to touching anything outside of the hood. It is recommended that you wash your hands free of oils and remove rings with sharp edges prior to putting on gloves. To take gloves off, take hold of the glove at the wrist and peel off inside out.
2. Chemical disinfectants for bloodborne pathogens:
  Care should be taken to minimize splashing when applying chemical disinfectants such as bleach, Wescodyne, or ethanol.
  Appendix Adescribes in detail our procedures for clean up of biohazard spills.
  - Chlorine Bleach(hypochlorite solution ~6-8% hypochlorite): Use 2% (1:50 dilution in water) for surfaces. Use 10% (1:10 dilution) for disposal of liquids containing potentially hazardous pathogens or for soaking heavily soiled tubes, vials etc. Use care - bleach will corrode stainless steel. We use bleach routinely to mop floors (1:50 dilution), for hood aspiration flasks (such that full trap is a final of at least 1:10 bleach), and to contain spills outside hood. Contact time should be at least 10 minutes.
  - Wescodyne(Iodophor compound - 0.175% Iodine): Use 1% (1:100 dilution in water) for surfaces. Use 2.5% (1:40 dilution) for disposal of liquids containing potentially hazardous pathogens or for soaking heavily soiled tubes, vials etc. All spills inside hood should be disinfected immediately with 2.5% Wescodyne followed by with 70% ethanol (Wescodyne is corrosive to stainless steel though much less so than bleach; it should not be used on aluminum or copper). We routinely use 2.5% Wescodyne solution in disinfectant buckets, pipet jars, and for spills and splashes inside or outside the hood. Contact time should be at least 10 minutes. Dilute solutions for soaking materials should be changed daily.
  - 70% Ethanol: Used to disinfected work surfaces, gloves, plastic surfaces, paper notes, instrument surfaces, and all surfaces where bleach or Wescodyne are inappropriate due to their corrosive and staining characteristics. DO NOT SPRAY ethanol directly into hood or into a spill; use a soaked paper towel.
3. Aerosols:
  1. Procedures with a high potential for creating aerosols, such as vigorous shaking and vortexing should be performed in the back third of the biological safety cabinet.
  2. Avoid pouring or decanting virus suspensions.
  3. Avoid vigorous pipetting and mixing. Do not forcibly expel the last drop of suspension from a pipette. Discharge pipetted material near the surface of fluid or down the wall of a tube.
  4. When opening culture tubes, bottles and flasks, manipulate them slowly.
  5. When resuspending liquid cultures, use a gentle swirling action to create a homogeneous suspension. Once cultures are resuspended, wait a few minutes before opening the container.
  6. Do not spray or pour disinfectants directly onto liquid spills. Place absorbent paper towels over spill, then pour 10% bleach or 2.5% Wescodyne around and onto towels.
4. GLASSWARE: Whenever possible, plasticware should be used. Avoid use of glassware.
5. SHARPS: Hypodermic needles, razor blades, glass Pasteur pipettes and other sharps which can easily penetrate the skin are forbidden in the BL-3 facility unless their use is absolutely necessary and specific permission for their use is obtained from Dr. Mullins.
6. MOUTH PIPETTING: Mouth pipetting is not permitted. Use of a mechanical pipetting device is required.
7. VACUUM: Vacuum lines must be protected with high efficiency particulate air (HEPA) filters and liquid disinfectant traps (containing 10% -100% bleach).
8. Avoid touching your face or any other exposed skin while in the BL-3 facility.
9. Before using the phone, change outer gloves to clean pair. Swab phone with 70% ethanol after use.
10. To avoid contamination of an entire notebook, write notes and data in pencil or ethanol resistant pen on single sheets of paper or paper towels. These can then be saturated with ethanol before removing from the BL-3 lab. Protocols brought into the BL-3 lab should be enclosed in a vinyl page protector so that they can be cleaned with ethanol.
Work in Biosafety Cabinets
1. The biosafety cabinet (class II, type A) is not designed for the use of volatile toxic chemicals and volatile radionuclides and therefore they cannot be used in this facility.
2. The height of the user’s chair should be adjusted to allow comfortable working and prevent a direct line of exposure to the user’s face during normal operation of the hood.
3. DO NOT TURN THE HOOD BLOWER OFF AT ANY TIME.
4. MANIPULATIONS OF POTENTIALLY INFECTIOUS MATERIAL shall be conducted in the laminar-flow biosafety cabinets as follows:
  - Each user must clean the hood surfaces with 70% ethanol before initiation of work and after completion of work.
  - Upon completion of work and your exit from BL3, the hood should be clean and free of all material except the followingPERMANENT HOOD RESIDENTS: tip boxes, pipettors, marker, aspiration flask, bleach bottle.BEFORE LEAVING THE BL3 CLEAN OUT YOUR WESCODYNE WASTE AND LEAVE EMPTY BUCKET BY SINK FOR NEXT USER.If you have extenuating circumstances for leaving other items in the hood LEAVE A NOTE WITH YOUR NAME, TIMEDATE, AND WHEN YOU EXPECT TO RETURN TO CLEAN UP!
  - Place bucket (no more than Â¾ full) of fresh 2.5% Wescodyne solution in hood.
  - Check aspiration flask. Aspirated liquid waste is collected and disinfected in a 2 L aspiration flask charged with 200 mL undiluted bleach. After aspiration of infectious materials, chase with bleach then 70% ethanol. Empty into sink when ~Â¾ full; recharge with 200 mL undiluted bleach.
  - Manipulations should be performed as far back from the front air grill as practical. Avoid placing anything on the front or rear grills of the cabinet since these will disrupt the airflow pattern and defeat the purpose of the cabinet.
  - Biological wastes must be exposed to 10% bleach or 2.5% Wescodyne for at least minutes before sink disposal. NOTE: Plastic tips and tubes/vials should be completely submerged and pipettes should be filled to just below the plug. Upon your final exit from the hood, decontaminated plastic pipettes should be transferred to pipette jar. The Wescodyne waste bucket should sprayed with ethanol, the contents strained over the sink, and plastics discarded in small biohazard bag.
  - Radioactive liquid waste will be disinfected with 10% bleach in a separate bleach trap and then transferred to the main lab and treated as general radioactive waste.
5. TRANSPORT OF POTENTIALLY BIOHAZARDOUS MATERIALS (tubes for centrifugation, culture dishes to be placed in incubators or observed under microscope, etc…) should not leave the hood without being properly sealed. Microscope slides containing infectious materials should be transported and observed within plastic petri dishes and examined while remaining within this container if possible.
6. AVOID AEROSOLIZATION: Care should be taken to minimize the splatter and/or aerosolization of infectious material inside the hood (e.g. pipette tips should be ejected only when the tip is present wholly within the confines of a plastic vessel containing appropriate disinfectant).
Centrifugation
1. Bench Top Centrifuge: Avoid tube collapse by matching the rated speed of the tube with the requirements of your experiment. Make sure to use the appropriate set of matched buckets for the type of tube being used.
  - All centrifugation conducted outside of the biosafety cabinets must be performed using sealed rotors or secondary buckets to contain potential aerosols, leaks, and tube breakage.
  - After centrifugation, the sealed rotor or buckets should be opened only inside a biosafety cabinet. These containers are disinfected in the hood by wiping out carefully with 70% ethanol. They cannot be autoclaved.
1. Microcentrifuge: When centrifuging infectious substances, the microcentrifuge should be transferred into the hood for use and surface decontaminated prior to removal.
Using the Microscope
1. Tighten caps on flasks of infectious culture before transporting to the microscope. Infectious cultures in plates or other containers without snugly fitting lids should be carried to the microscope in a tray.
1. When using the hemacytometer to count cells, enclose the hemacytometer in a clean/disinfected petri dish with lid for transport to the microscope and observation.
2. Change or spray gloves with ethanol before touching the microscope.
3. Disinfect the viewing platform of the microscope after each use.
4. If you must remove your safety glasses to look through the scope, don’t forget to replace them.
Waste Management
1. SHARPS:
  - Pipette tips and plastic pipettes are treated as quasi-sharps because they can puncture autoclave bags. Therefore, after decontamination and straining/draining, they must be double-bagged before autoclaving.
  - Metal sharps that will easily penetrate the skin are not allowed in the BL-3 facility without special permission. Upon completion of the work after at least a 30-minute exposure to bleach, the decontaminated sharps are transferred to a red sharps container to await autoclaving.
1. SOLID WASTE other than sharps generated inside the hood (vials, tubes, culture flasks, etc.) should be decontaminated, drained then placed directly into the large biohazard bag to await autoclaving. Solid wastes generated outside the hood (paper towels, outer gloves, shoe covers, etc.) are placed directly into the large biohazard bag to await autoclaving.
2. LIQUID WASTE collected in a bleach trap or a 2.5% Wescodyne solution within the biosafety cabinet must be exposed to disinfectant 10 minutes before sink disposal.
Waste Sterilization
1. All solid waste and glassware should be autoclaved for at least 60 minutes at 132Â°C. Liquid waste must be autoclaved for at least 60 minutes per gallon at 121Â°C. Laundry should be autoclaved for 30 minutes at 121Â°C.
1. All material to be autoclaved should have appropriate autoclave tape attached to confirm that the correct temperature was attained.
2. A chemical indicator (i.e. steristrip) is placed in the center of EACH LOAD to confirm proper decontamination conditions. Biological testing systems are required at monthly intervals. Records of the temperature, time, results of chemical indicators and biological testing systems must be maintained for each load of waste autoclaved for one year.
Identification and Storage of Materials
1. All equipment and materials in the BL-3 facility should be treated as if they are contaminated.
1. Storage vessels containing biohazardous materials including cultures in incubators and refrigerators and freezers should be labeled to identify their contents, owners, and date.
2. All equipment such as refrigerators, freezers, incubators, cell counters, centrifuges, etc. used for or containing biohazardous materials must be labeled clearly with the appropriate biohazards symbol.
Laboratory Transport
1. Live infectious materials, which are removed from the facility for storage in liquid nitrogen or -70Â°C freezers should be stored in nonbreakable, cryovials with rubber gasket seals. The vials are surface decontaminated with 70% ethanol after sealing and then transported to the freezers in nonbreakable, impermeable closed containers with biohazard symbols. These containers are assembled and then surface decontaminated prior to exit from the facility.

IV. Exiting the Facility

Prior to exiting the facility,each user should check to ensure that he or she has:
1. Appropriately cleaned and disinfected all work areas
1. Organized tools/supplies into correct storage location
2. Disposed of generated waste in the proper manner
3. 1. If the solid waste bag is Â¾ full, it must be secured with a single wrap with rubber band and placed in autoclave tray by door. If enough autoclave trays are filled, run autoclave.
  1. If the sharps container is full, it is to be surface decontaminated, transferred to the anteroom, tagged with autoclave tape and autoclaved.
  2. When pipette jar outside hood is Â¾ full, the pipettes are to be removed from the tank and allowed to drain in the sink. The pipettes are double-bagged before autoclaving.
4. Adjusted the water level in the water bath and confirmed that the temperature is set for 37Â°C
5. Turned off the aspirator and mechanical pipettor
6. Turned off the microscope and centrifuges
7. Wiped down all used switches, handles, and control panels with 70% ethanol
Before exiting the work area, users should remove their protective gear in the following order:
1. 1. Shoe covers (spray shoe soles with ethanol)
1. 2. Outer gloves (spray inner gloves with ethanol)
2. 3. Mask

EXIT THE WORK AREA

In the anteroom remove:
1. goggles
2. gown
3. inner gloves
Shoe covers, gloves and masks are disposable and should be placed in the solid waste bins. should be placed in the appropriate bin (yellow bags) in the anteroom to await autoclaving.
Hands should be washed in the anteroom prior to leaving the facility.

V. Emergency Procedures

Laboratory Accidents
1. Personal Injury or Exposure: All equipment in the BL-3 facility should be treated as if it were contaminated. Any cuts or abrasions obtained while in the facility should be immediately scrubbed for 10 minutes with 1% Wescodyne, soap and water. For mucous membrane exposure, thoroughly flush the surface for several minutes. There is an eye wash fountain located in the BL-3 facility. The user should then exit the facility using appropriate exiting procedures and seek immediate medical attention with the Occupational Health nurse practitioner, room NN256A; phone number (206) 548-4848. If the accident occurs after hours, go directly to the UW Medical Center emergency room. The occupational nurse/emergency room will evaluate the wound, draw a blood sample and give any necessary treatment; including HAART. The incident should be reported to Dr. Mullins or the lab supervisor and an Accident Report Form filed with EHS within 24 hours of the exposure.
1. Centrifuge Malfunctions: Centrifuge malfunctions should be handled in such a way as to avoid personal injury or exposure. If an imbalance occurs or a bucket is thrown from the rotor, the unit should immediately be turned off. Even in non-emergency situations, centrifugation is a procedure that is potentially hazardous when working with biohazardous material. Aerosols can be detected during the filling of centrifuge tubes, removal of caps or removal of contents. Breakage of tubes during centrifugation produces the greatest amount of aerosol. Safety sealed centrifuge cups will not allow the escape of contents or an aerosol in the event of an accident, provided that they are sealed properly and only opened in a biological safety cabinet.
2. Fire Alarm: If working with live materials, the cultures should be returned to the incubator and the facility should be exited using the appropriate exiting procedures.
3. Earthquake: Flying glass from the hood and sliding or falling heavy equipment are the primary dangers during an earthquake. Move immediately away from the front of the hood, freezer and stacked incubator. Stand flush against the wall. Watch for falling equipment and supplies. Some users may be able to duck under the knee space on the hood, which will lend some protection from overhead. After the earthquake, survey the area for biological or chemical spills; clean the spills only if the area is safe. If the area is not safe and the spill occurred in the hood, remove the lid of the dilute Wescodyne solution and overturn it in the hood. Leave as quickly as possible following the appropriate exit procedures. If there is time, leave a note on the door stating that a biological spill has occurred.

APPENDIX A - CLEAN-UP OF BIOHAZARDOUS SPILLS

Biohazardous Spills in the Biological Safety Cabinet
Chemical decontamination procedures should be initiated at once while the cabinet continues to operate to prevent escape of contaminants from the cabinet.
1. It is best to soak the spill into paper towels and pour 2.5% Wescodyne such that it seeps into the paper towels rather than pouring directly onto the spill and generating aerosols. Allow the detergent to act for 10 minutes then transfer soaked paper towels to an autoclave bag within the hood. Wipe walls, work surfaces, and equipment with detergent. The operator should be in protective gear during this procedure.
1. If the spill has spread to the catch basin underneath the front grill, wipe the grill with 2.5% Wescodyne, remove the grill and place it in the hood, soak up liquid in the catch basin with paper towels, and disinfect with Wescodyne as in “A” above. After paper towels have been removed, replace grill.
2. Seal and decontaminate the outside of the autoclave bag used to collect the soaked paper towels, remove from the hood and autoclave.
Biohazard Spill Outside of a Biological Safety Cabinet
1. Holding your breath leave the room immediately and close the door.
1. Warn others not to enter the contaminated area. Post a sign on the door if you leave the immediate vicinity.
2. In the anteroom, remove and properly dispose of contaminated garments and protective gear. Thoroughly wash hands and face.
3. Wait 30 minutes to allow dissipation of aerosols created by the spill.
4. Put on clean protective gear before reentering the room. If the spill was of high titer virus, the use of a respirator and tight fitting goggles should be considered.
5. Use paper towels to soak up the liquid. Then pour detergent around the spill such that it flows into the towels. To minimize aerosolization, avoid pouring directly into the spill.
6. Let stand 20 minutes to allow adequate disinfectant contact time.
7. Using a dustpan and sponge or small broom transfer all contaminated materials into an autoclave bag with as little hand contact as possible. If there is broken glass involved, transfer to a deep autoclave pan instead. DO NOT ATTEMPT TO PICK UP ANY BROKEN GLASS BY HAND!
8. Prepare a container of dilute detergent and sponge off the floor, removing as much liquid as possible.
9. Autoclave waste along with the dustpan and sponge. If the broom cannot be autoclaved, let it soak in 5% Wescodyne for 30 minutes or more and then rinse.
10. Report the spill to Dr. Mullins or the BL-3 lab supervisor.
Radioactive Biohazard Spill Outside a Biological Safety Cabinet
1. Follow A-D from above. Before clean-up procedures begin, contact a radiation safety officer (543-6328). The spill should be surveyed for external radiation hazard prior to determining the course of action.

Mullins' B44C BL-3 Facility

I, (print name) _______________________________________, have read, understand and will comply with the safety practices outlined in the University of Washington Biosafety Manual, the CDCâ€¢NIH guidelines for Biosafety Level 3 practices and the Mullins' lab rules and regulations regarding the Rosen B44C BL-3 facility.

Lab of Trainee (PI):__________________________________

Trainee Lab Trainer:__________________________________(assigned by PI)

Trainee Name:__________________________________

Signature:__________________________________

Mullins Lab Trainer:_________________________________________

Date Trained:____________________

Additional Comments:

The person named above has successfully completed training supervised by Dr. Mullins or the BL-3 lab supervisor and is now authorized to use the Rosen B44C BL-3 facility.

__________________________________ (James I. Mullins)

__________________________________ (Date)

For specifics on appropriate handling and waste procedures please see theonline chemical SOPsor our waste and spill notebook located in room 352.