Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 2. General Practices

1. Laboratory Entry

1. Gather all supplies from a BSL-2 laboratory for the experiment (e.g., cells, media, and consumables) prior to entry into the BSL-4 laboratory.
2. Complete the BSL-4 entry procedure (outlined in detail in reference⁷).

2. Preparation of a Class II Biosafety Cabinet in the BSL-4 Laboratory

1. Once inside the BSL-4 laboratory, ensure the daily internal checklist (Figure 1) has been completed. Complete the checklist if the checklist has not been previously filled out and indicate which viruses will be used. If the checklist has already been completed, add name and which virus will be used to the list.
2. Clean the Class II BSC by spraying down the entire inside of the BSC (including the sash) with 5% dual quaternary ammonium disinfectant solution (e.g., n-alkyl dimethyl benzyl ammonium chloride, n-alkyl dimethyl ethyl benzyl ammonium chloride or other disinfectant appropriate for the agent being used) and wipe down with paper towels^10,11. Spray 70% ethanol solution inside the cabinet and the sash to remove the tacky disinfectant solution.
3. If seated, set the chair in front of the Class II BSC at a comfortable height to ensure that the back of the cabinet can be reached and that the face of the laboratory specialist is situated above the front opening (Figure 2)¹¹.
4. Prepare a waste container for the biosafety cabinet. Ensure that the final concentration of dual quaternary ammonium disinfectant solution in the waste container is no less than 5%. Plan accordingly and make a 10% solution to which waste will be added that will dilute the disinfectant. In addition, place a spray bottle with 5% dual quaternary ammonium disinfectant solution inside the Class II BSC to spray any items prior to removal and gloved hands during and after completion of the assay.
5. Place the appropriate materials needed for the entire experiment in the Class II BSC as far back in the cabinet as possible to avoid repeated material introductions into the Class II BSC and disruption of the air flow¹¹.

3. Example: Plaque Assay

1. Retrieve virus-containing samples and control virus from the storage location by hand and thaw materials in an incubator at 37 °C.
2. Label the wells of the 6-well plates to be used according to the virus dilutions planned. Mark the lids and body of the plates to ensure successful matching if the lids and body are separated.
3. Bring materials from steps 3.1-3.2 into the Class II BSC. Place all items that have not or will not come in contact with virus on one side ("clean side") and waste on the other side ("dirty side"). If possible, keep "clean items" at least 30 cm apart from "dirty items" during aerosol-generating activities¹¹.
4. Work as close to the inside center of the Class II BSC as possible, as the inside center is designed to be the most effective position to protect oneself¹¹.
5. Remove 50 µl of virus sample and control virus and place into 450 µl of Dulbecco's modified Eagle's medium with 2% fetal bovine serum. Proceed with making serial dilutions as far out as needed (Figure 3A).
6. During the dilutions, mix the virus samples at least 5 times with a pipette tip slowly and carefully while trying to minimize air bubble generation in the samples. Change pipette tips after each addition to the next dilution well.
7. In the last dilution, after mixing the sample 5 times, discard 50 µl of virus sample into waste container to ensure equal volumes of dilutions.
8. When disposing of tips, rinse each tip with disinfectant solution from the waste bucket to decontaminate the inside and outside of the tip before expelling tip into the waste bucket.
9. Once the dilutions are made, set the dilution wells aside and begin aspirating media from the wells of cell culture plates, leaving 500 µl of media in each well of a 6-well plate.
10. When finished with the pipette tip, aspirate disinfectant solution from the waste bucket to the top of the pipette using a manual, adjustable volume, push-button pipettor, to ensure proper decontamination of the inside of the pipette. Leave the tip into the waste bucket until the end of the experiment.
11. Once the media have been removed from the plates, add 100 µl of the correct sample into the proper well in the pre-labeled plates in duplicate, changing tips each for each sample.
12. Upon completion of the plaque assay inoculations, spray off gloved hands with the disinfectant solution and use a paper towel soaked with disinfectant solution to wipe the outside of all plates before placing the plates back into the incubator by hand. Repeat this process until all plates are back into the incubator.
13. Rock the plates in a figure-8 motion to ensure proper dispersal of the sample over the cells every 15 min for 1 hr.
14. Upon completion of rocking plates, return plates into the Class II BSC along with a mixture of 2x Eagle's minimal essential medium and 1.6% tragacanth, a semi-solid overlay that is easier to manipulate than agarose, used for the overlay of the plaque assay.
15. Add 2 ml of the overlay mixture to each well in a 6-well plate and rock once again in a figure-8 motion for equal distribution of the overlay throughout the surface of the well. Repeat this process until every used well is overlaid with the overlay mixture.
16. Ensure that the tip of serological pipette does not touch any liquid in the wells to avoid cross contamination when performing the overlay procedure.

4. Waste Disposal and Cleaning of the Instruments and Biosafety Cabinet

1. Completely submerge all waste material in 5% disinfectant solution in the waste bucket for a contact time of at least 10 min. Disinfect pipette tips, serological pipettes, and other waste as described above. In addition, spray the waste bucket (inside and out) with 5% disinfectant solution let the solution remain in contact with the waste bucket for a contact time of 10 min.
2. While waiting for the contact time to elapse for the waste, soak a paper towel with 5% disinfectant solution, wipe down instruments such as micropipettes, and remove them from the BSC. Spray gloved hands with 5% disinfectant solution before bringing gloved hands out of the Class II BSC.
   1. After removing the micropipettes from the BSC, wipe them with a separate paper towel with 70% ethanol solution to avoid sticky buildup on the instruments. Spray any instruments that cannot be effectively wiped down with 5% disinfectant and leave in contact with the solution in the BSC for 10 min.
3. After sufficient contact time, remove all items from the BSC. Take items, including waste bucket containing waste materials, to the sink. Rinse items that can be reused to remove disinfectant residue. Return all items to their storage locations.
4. Clean the Class II BSC's work surface, cabinet sides and back, interior of glass, and sash¹ with 5% disinfectant solution followed by 70% ethanol solution.
5. Pull out and drain the serological pipettes from the waste bucket and place surface-disinfected serological pipettes into separate pipette trays for autoclaving (serological pipettes can present a sharps hazard and may tear through the trash bag. Place these pipettes in a hard-sided container before autoclaving).
6. Pour the contents of the waste bucket into a strainer placed in the bottom of the sink.
7. Bring a biohazard waste container that is lined with a red biohazard bag to the sink and dump the contents of the strainer into the red biohazard bag. Do not reach into the strainer to remove micropipette tips that may stick to the inside of the strainer. Hit the strainer along the inside of the waste container until all tips are removed, or use a pair of tweezers to remove tips from the strainer.
8. Rinse out the waste bucket and place to dry on the rack next to the sink.

5. Autoclaving Waste

1. Remove the biohazard bag from the biohazard waste container and place in an autoclave tray on a cart.
2. Leave biohazard bags open and place a piece of autoclave tape over the outside of the bag, connecting the sides of the autoclave tray to keep the bag secured in the tray.
3. Place two pieces of autoclave tape on the serological pipette tip tray and label it with one's initials and the date. Place the serological pipette tray on the cart with the autoclave tray.
4. Open the autoclave. Connect the provided autoclave loading/unloading cart to the autoclave and bring out the retractable autoclave platform to rest on the cart.
5. Remove the metal rod from the autoclave and open the top. Place a biological indicator vial containing spores of Geobacillus stearothermophilus into the metal rod to check that the autoclave sterilization cycle was completed successfully.
6. Place the metal rod into the center of the waste bag in the autoclave tray but ensure that the metal rod is still easily accessible.
7. Place autoclave tray filled with waste and serological pipette tray onto the retractable autoclave platform and push it back into the autoclave.
8. Detach the autoclave loading/unloading cart from the autoclave and close the autoclave door.
9. Start the autoclave.
10. Do not leave the autoclave until the cycle has started. The operating screen of the autoclave will indicate time remaining for that run.
11. After the autoclave run is completed, remove the biological indicator and evaluate for growth by heating in a specified incubator. If growth is detected on the biological indicator, re-run the trash in the autoclave, and assess anew indicator. If no growth is detected, remove the trash from the facility.

6. Example: Fixing and Staining of Plaque Assays

1. After the appropriate number of days (which is dependent on the virus used) has passed, return back into the laboratory and perform steps from sections 1 and 2 once again.
2. Carefully remove plaque assay plates from the incubator completed in step 3.1.2, and place into the Class II BSC by hand.
3. Pipette off all of the media and overlay material and dispense into disinfectant solution container and replace with a mixture of 10% neutral buffered formalin and 0.8% crystal violet^12,13. Let the mixture remain on the plates for 30 min to inactivate the virus on the plates.
4. After inactivation, carefully remove the neutral buffered formalin/crystal violet mixture and place in a separate waste container to be neutralized prior to disposal.
5. Spray gloved hands with disinfectant and wipe the outside of the plates before passing them out of the Class II BSC as described above.
6. Proceed to the sink, rinse the plates to remove excess stain, and then place the plates on a cart to dry.
7. Once the plates are completely dry, use a light box to count the plaques. Record all the counts and calculate virus titers from the standard equation (Figure 3B).

7. Removing Samples from the BSL-4 Laboratory

1. Inactivate any samples that will be manipulated under BSL-2 laboratory conditions. Follow one of two methods approved by the internal biosafety office at the IRF-Frederick using 10% neutral-buffered formalin (NBF) or Trizol LS (phenol, guanidine isothiocyanate, ammonium thiocyanate, sodium acetate, glycerol)^1,14. Transfer samples to a new clean tube or plate outside of the BSC prior to packaging for removal from the BSL-4 laboratory.
2. Heat-seal inactivated samples in tubes or plates in a heat-seal pouch containing sufficient 5% disinfectant or fixative solution to disinfect the inside of the bag and the outside of the sample tubes undergoing transfer out of the BSL-4 laboratory. Place this pouch into another pouch following same procedure.
3. Seal the second pouch and place the heat-sealed pouch into a dunk tank containing 5% disinfectant solution for at least 10 min to disinfect the outside of the heat-sealed pouch.
4. Fill out a dunk tank log book inside the lab by delineating the number and size of tubes, volume in tubes, agent used, inactivation method used, and room to where samples will be transferred.
5. Coordinate with a colleague on the outside of the BSL-4 laboratory to retrieve the pouch from the dunk tank and take samples to the BSL-2 laboratory.