The overall goal of this procedure is to demonstrate safe practices when working in a biosafety level 4 suit laboratory using the plaque assay as an example. This procedure demonstrates the modifications necessary to perform a standard plaque assay in a biosafety level 4 environment. Demonstrating the procedure with me with be Tracey Burdette a technician from the Integrated Research Facility.
Upon completing the daily internal systems check list, clean the class II biological safety cabinet by spraying down the inside, including the sash, with 5%dual quaternary ammonium disinfectant solution. Then, wipe down the cabinet with paper towels, being sure to leave the spray bottle containing the disinfectant solution inside of the cabinet to spray gloved hands during and after completion of the assay. Spray 70%ethanol solution inside the cabinet and the sash to remove the tacky disinfectant solution.
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%taking into account any additional liquid that is added. Place the waste container containing 5%dual quaternary ammonium disinfectant solution inside the class II biosafety cabinet.
Perform all work as close to the inside center of the class II biosafety cabinet as possible. The inside center is designed to be the most affective position to protect oneself. Remove 50 microliters of virus sample and control virus and place them into 450 microliters of Dulbecco's Modified Eagle's medium with 2%fetal bovine serum in dilution wells.
Proceed with making serial dilutions as far out as needed. Mix the virus samples at least five times with a pipette tip changing pipette tips after each dilution. Mix slowly to minimize air bubble generation in the samples.
Upon completion, set the dilution wells aside. 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 the tip into the waste bucket. Remove most of the media from the wells of six well cell culture plates.
Add 100 microliters of the correct sample into the proper well of the pre-labelled plates in duplicate. Upon completion, spray off gloved hands with the disinfectant solution. Use a paper towel soaked with disinfectant solution to wipe the outside of all plates before placing the plates into the incubator by hand.
Rock the plates in a figure-8 motion to ensure proper dispersal of the sample over the cells every 15 minutes for one hour. Add two milliliters of the overlay mixture to each well and rock once again in a figure-8 motion for equal distribution of the overlay throughout the surface of the well. Completely submerge all waste material in 5%disinfectant solution in the waste bucket for a contact time of at least 10 minuets.
Spray the inside and outside of the waste bucket the inside of the class II biosafety cabinet the exterior of equipment, and gloved hands with 5%disinfectant solution. Let the solution remain in the waste bucket and on other surfaces for a contact time of 10 minutes. Then remove the items from the biosafety cabinet and take them to the sink.
Place the used pipette into a pipette tray for autoclaving. Poor the contents of the waste bucket into a strainer in the sink. Then 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.
Place the biohazard bag on an autoclave tray on a cart. Loosely close the autoclave bags with tape permitting steam to enter the bag. And label one of the autoclave tapes with one's initials and date.
Remove the perforated metal rod from the autoclave and open the top. Place a biological indicator vial containing spores of Geobacillus stearothermophilus into the metal rod to serve as a check that the autoclave sterilization cycle completes successfully. Place the autoclave tray filled with waste into the autoclave.
Start the autoclave and check that the cycle has started. The operating screen of the autoclave will indicate time remaining for that run. After the required incubation period is completed carefully remove the plaque assay plates from the incubator and place them into the clean class II biosafety cabinet by hand.
Aspirate the medium and overlay material with a pipette and dispense into the disinfectant solution container. Replace the medium with a mixture of 10%neutral buffered formalin and 0.8%crystal violet to inactivate the virus for a contact time of 30 minutes. After inactivation, carefully remove the neutral buffered formalin/crystal violet mixture and place it in a separate waste container to be neutralized prior to disposal.
At the sink, rinse the plates to remove excess stain and then place the plates in a tray to dry completely. Use a light box to count the plaques. Record all the counts, and calculate virus titers from the standard equation.
For removal of inactivated samples from the BSL-4 lab place the tubes or plates in a heat seal bag. Spray the inside of the bag with 5%disinfectant or fixative solution, to disinfect the inside of the bag and the outside of the sample tubes. And then heat seal the bag.
Place this pouch into another pouch following the same procedure. Heat seal the second pouch and place the double-sealed pouches into a dunk tank containing 5%disinfectant solution for at least 10 minutes, to disinfect the outside of the pouches. Fill out a dunk tank log book inside the lab by delineating the number and size of the tubes the volume in the tubes, the agent used the inactivation method used, and the room to where samples will be transferred.
Following proper procedures within the biosafety level 4 laboratory is critical for ensuring the safe and effective completion of assays. By referring to the completed daily internal check-list laboratory staff ensure that equipment is fully operational. The virus sample is serially diluted to obtain plates that have 30 to 300 plaques per well and to determine virus titer.
A number of factors affect formation of plaques including virus tropism for host cell lines inoculation technique, conditions for virus growth appropriate dilution range, and overlay selection. Waste generated in the biosafety cabinet during the procedure is disinfected twice. The waste is properly disinfected once prior to removal from the biosafety cabinet.
Subsequently, waste is autoclaved prior to leaving the animal biosafety level 4 environment. By following these procedures no laboratory-acquired infections have been recorded during animal biosafety level 4 research at the Integrated Research Facility at Frederick. This technique can be done in three to four hours depending on the number of samples if the technique is performed properly.
While attempting this procedure remember to perform dilutions correctly count plaques accordingly, and comply with all safety precautions, to ensure a safe and successful experiment. After watching this video, you should have a good understanding of the physical limitations imposed by positive pressure suits, and the additional time required to complete a standard plaque assay.
