Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures.

The overall goal of this procedure is to demonstrate how to properly don and doff a positive pressure suite and to enter and exit a BioSafety level for four suit lab. For personnel who are new to high containment research this procedure outlines the steps involved in putting on a positive pressure suit, and entering a BSL-4suit laboratory. Through this technique the safety of the personnel at the NIH Integrated Research Facility at Fort Detrick is promoted by inspecting personal protective equipment and decontaminating the suite appropriately.

Demonstrating the procedure will be Ricky Adams, a technician from the NIH Integrated Research Facility at Fort Detrick. Enter the staff person's name, time of entrance, laboratory location, pathogens under study in the personnel entry/exit log book. Indicate the laboratory location and check that the laboratories have been cleared entry.

To ensure that laboratory room pressure is within negative pressure set-points that maintain proper directional airflow, consult the completed systems checklist. Obtain scrubs, socks, and towels from the storage room containing the facility's supplies located near the personnel entry/exit logbook. After passing through the security access control points to the outer change room, remove street clothing, undergarments, jewelry and watches.

Don scrubs and proceed to the suit room. Pick a positive pressure protective suit and visually inspect the suit for any holes, tears, puncture points, seam rips, or weak spots. Inspect the outer suit gloves thoroughly since gloves are the most likely item to be damaged.

Change gloves at least every seven calender days, or if damage to the gloves is identified. To replace the outer suit gloves on this model suit remove the duct tape from the existing suit glove, roll the o-ring onto the sleeve, and remove the old suit glove. Put a new glove onto the cuff of the suit and ensure that the glove covers the entire cuff and that the thumb is in the proper position.

Place the o-ring over the glove, seat the o-ring onto the groove on the cuff and use duct tape to secure the outer suit glove. Fold the clove cuff over the duct tape and o-ring, seal the glove with one final piece of duct tape and reinspect the newly attached glove. Apply a thin layer of zipper lubricant to the zipper at least once a week.

Next, cover each of the exhaust valves inside the suit with either duct tape or an aluminum pressure test cap and zip the suit closed. Connect the quick disconnect/connect attachment at the end of each breathing line to the hose connection on the suit. Inflate the suit until the arms and legs are firm and place the suit into an upright position.

Disconnect the air supply. Do not over-pressurize the suit, as excessive pressure can cause serious damage to the suit. Thoroughly inspect the suit for any indication of an air leak for approximately five minutes and inspect the suit for signs of deflation.

If the suit loses firmness, check the suit for leaks by re inflating the suit. Listen and feel for leaks in the suit that may occur around the suit seams, the zipper, and the junction of the visor with the suit. If needed, spray the suit with a soap solution to help visualize the location of the leak.

Monitor for the formation of bubbles that indicate a leak, paying particular attention to seams in the visor. Check that the exhaust valves are totally sealed. Unzip the suit and remove the covers from the exhaust valves.

Failure to remove the exhaust covers will prevent air form escaping and severely damage the suit. Enter the testing and repair information in the positive pressure suit integrity test log. Next, tape the socks to the legs of the scrubs.

Don inner Nitrile gloves and duct tape them to the cuffs of the scrubs. Don a second pair of Nitrile gloves over the inner gloves. If necessary, clean the inner face shield with glass cleaner for increased visibility.

Don the positive pressure suit, zip up the suit completely, and connect the breathing airline. Prior to BSL-4 Lab entry, ensure that the building automation system indicates normal zone pressure. To enter the BSL-4 suit laboratories disconnect from the breathing airline in the suit room, and push the request to access button for the chemical shower room.

To pass through the chemical shower room, close the air-pressure resistant, or APR door to the suit room and wait for the bladder to fully seal the door frame. Activate the second APR door, leading to the laboratory. Proceed into the BSL-4 laboratory and connect the suit to a breathing airline.

Close the APR door leading to the laboratory. Ensure that the chemical shower is automatically activated. After the shower cycle is completed the APR door can be opened again from the suit room.

Move freely around the laboratory by changing connections to airlines. Stay connected to an airline as much as possible, even when bending over. Observe the monitors various alarms, and current suite status.

To exit the BSL-4 suite laboratory, disinfect gloved hands and cuffs in detergent disinfectant cleaner in plastic dump tanks. If no dump tank is present, spray gloved hands thoroughly with disinfectant. Disconnect from the breathing airline and proceed to the chemical shower.

Enter the chemical shower by pushing the request to access button to open the APR door from the laboratory and close the door. Reconnect the suit to a breathing airline inside the shower. Once the APR door is closed, the chemical shower cycle should automatically start.

Check for glove and foot leaks by placing gloved hands and feet in the plastic tub containing detergent disinfectant cleaner solution kept inside the chemical shower. Check for tears, rips or weak portions on the outer glove and look for liquid trapped underneath the outer glove. If a leak is found wait until the shower cycle is complete, remove the outer suit glove and leave it in the plastic tub.

Scrub the suit and integrated suit boots with a long handled scrub brush or with hands to distribute the chemical disinfectant on the suit. Thoroughly rinse the suit of detergent disinfectant cleaner during the water cycle of the shower. Disconnect the breathing airline, disinfect and rinse the connection on the suit during the chemical and water cycle of the shower.

Upon completion open the APR door leading to the suit room and close it after exiting. Dry the exterior of the suit with a large towel. Unzip the positive pressure suit.

Remove one hand from the outer suit glove and examine the outside of the outer Nitrile glove carefully for moisture. Remove the other hand and repeat the check. Remove the suit, hang the suit up and continue to dry the exterior of the suit to prevent dry rot damage.

Spray the inside of the face shield with glass cleaner and wipe dry. After hanging the suit up to dry remove the duct tape and inner Nitrile gloves from the scrubs and dispose of these items in the bio-hazard waste trash bin. Proceed to the changing room, remove scrubs and socks, and enter into the personal shower.

Following the personal shower, enter the exit time and the personnel entry log book and indicate laboratory exit on the locator board. Staff have been carefully and thoroughly trained in these techniques to ensure safe and consistent practices inside the a bio-safety level four facility. By checking that the facility is functioning properly as indicated on the daily checklist, all of the necessary administrative and engineering controls are in place to maintain a safe environment.

The positive pressure suit provides an additional layer of protection for the staff member. Proper maintenance and use of these suits is integral for this protection. Strict adherence to these procedures and practices and proper use of engineering controls help prevent laboratory inquired infections and ensure containment of infections materials.

The NIH NIAID Integrated Research Facility at Fort Detrick has not documented a laboratory inquired infection to date. After watching this video you should have a good understanding of how to don and doff a positive pressure suit and enter and exit a bio-safety level four suit laboratory. These procedures illustrate generally in place at the IRF and may not be representative of the procedures at other BSL-4 labs.

When working in other biosafety level four labs comply with the protocol specific for that lab.