Name: safety precautions and operating procedures in an (a)bsl-4 laboratory: 2. general practices
Uploaded: 2016-10-03T13:00:00
Description: Watch this Scientific Journal Video about Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 2. General Practices at JoVE.com

The content of this publication does not necessarily reflect the views or policies of the US Department of Health and Human Services (DHHS) or of the institutions and companies affiliated with the authors. This work was funded in part through Battelle Memorial Institute's prime contract with the US National Institute of Allergy and Infectious Diseases (NIAID) under Contract No. HHSN272200700016I. M.R.H., D.P., L.B., K.J. and J.W. performed this work as employees of Battelle Memorial Institute. Subcontractors to Battelle Memorial Institute who performed this work are: S.M. as an employee of MRI Global; M.G.L. as an employee of Lovelace Respiratory Research Institute, Inc.; and J.H.K. as an employee of Tunnell Government Services, Inc.

1. Laboratory Entry
<ol>
	<li>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.</li>
	<li>Complete the BSL-4 entry procedure (outlined in detail in reference7).</li>
</ol>
2. Preparation of a Class II Biosafety Cabinet in the BSL-4 Laboratory
<ol>
	<li>Once inside the BSL-4 laboratory, ensure the daily internal checklist (Figure 1) has been completed. Complete the c...

<ol>
	<li>Shurtleff, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+regulations,+safety+considerations+and+physical+limitations+on+research+progress+at+maximum+biocontainment.">The impact of regulations, safety considerations and physical limitations on research progress at maximum biocontainment.</a> Viruses. 4, 3932-3951 (2012).</li><li>de Kok-Mercado, F., Kutlak, F. M., Jahrling, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NIAID+Integrated+Research+Facility+at+Fort+Detrick.">The NIAID Integrated Research Facility at Fort Detrick.</a> Appl Biosafety. 16, 58-66 (2011).</li><li>Keith, L., Moyer, B. R., Cheruvu, N. P. S., Hu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+imaging+in+BSL-3+and+BSL-4+envrionments:+imaging+pathophysiology+of+highly+pathogenic+infectious+diseases.">Preclinical imaging in BSL-3 and BSL-4 envrionments: imaging pathophysiology of highly pathogenic infectious diseases.</a> Pharmaco-imaging in drug and biologics development. , (2014).</li><li>Janosko, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+Precautions+and+Operating+Procedures+in+an+(A)BSL-4+Laboratory:+1.+Laboratory+Suite+Entry+and+Exit+Procedures.">Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Laboratory Suite Entry and Exit Procedures.</a> J Vis Exp. , (2015).</li><li>Baer, A., Kehn-Hall, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+concentration+determination+through+plaque+assays:+using+traditional+and+novel+overlay+systems.">Viral concentration determination through plaque assays: using traditional and novel overlay systems.</a> J Vis Exp. , e52065 (2014).</li><li>Gonzalez-Hernandez, M. B., Bragazzi Cunha, J., Wobus, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plaque+assay+for+murine+norovirus.">Plaque assay for murine norovirus.</a> J Vis Exp. , e4297 (2012).</li><li>Alfson, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+to+plaque-forming+unit+ratio+of+Ebola+virus+influences+disease+course+and+survival+in+cynomolgus+macaques.">Particle to plaque-forming unit ratio of Ebola virus influences disease course and survival in cynomolgus macaques.</a> J Virol. , (2015).</li><li>Shurtleff, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+of+the+filovirus+plaque+assay+for+use+in+preclinical+studies.">Standardization of the filovirus plaque assay for use in preclinical studies.</a> Viruses. 4, 3511-3530 (2012).</li><li>Blow, J. A., Dohm, D. J., Negley, D. L., Mores, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+inactivation+by+nucleic+acid+extraction+reagents.">Virus inactivation by nucleic acid extraction reagents.</a> J Virol Methods. 119, 195-198 (2004).</li></ol>

Work in a BSL-4 laboratory requires considerable time and additional attention to detail. Any type of work in this environment requires well trained, thorough, and conscientious individuals. The standard viral plaque assay provides an accurate model of a common procedure for working with high-consequence pathogens in the BSL-4 laboratory, as the assay involves several major concepts in which laboratory workers must be trained.
The first major concept is the proper use and application of safe p...

A correction was made to the authors section in: Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 2. General Practices
One of the authors names was corrected from:
Nicole Joselyn
to:
Nicole Josleyn

A correction was made to the authors section in: Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 2. General Practices

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

As safety of laboratory personnel handling high-consequence pathogens (no infection prophylaxes nor treatment options exist) is paramount, the US Department of Health and Human Services has established guidelines for facility construction and best practices for the safe conduct of work with pathogens in biomedical and clinical laboratories from a biosafety perspective1. Through legislation and regulation, many of the practices and procedures have become mandatory requirements that must be followed for work with these pathogens. In the US, pathogens that are easily transmitted from person to person, result in high case-fatality rates, and/or have the potential for major public health impact and bioterrorism, are categorized as National Institute of Health/National Institute of Allergy and Infectious Disease (NIH/NIAID) Priority A Pathogens and or Centers for Disease Control and Prevention (CDC) Bioterrorism Category A Agents2. In addition, high-consequence pathogens are classified as Tier 1 Select Agents if these pathogens are potential bioterrorism agents, have potential for mass casualties or devastating effects to the economy, critical infrastructure, or public confidence3.
BSL-4 operations, including access to institutes with BSL-4 laboratories, are more highly controlled than BSL-2/3 operations. For instance, it is substantially more difficult to gain access to a BSL-4 laboratory compared to a BSL-2 or BSL-3 laboratory due to substantial suit training requirements, extensive mentorship requirements, and additional medical biosafety prerequisites. In addition, there are typically more physical security barriers in a BSL-4 facility versus a BSL-2 or BSL-3 facility4-6. As outlined in our first article on BSL-4 entry and exit procedures, laboratory staff undergo extensive training and psychological screening to qualify for entrance into the BSL-4 laboratory7. Within the BSL-4 laboratory, risk of infection and mistakes are avoided or mitigated by following established procedures. Research must proceed carefully and deliberately, with minimal multitasking or distractions. Bending over in positive pressure suits is difficult, and the face shield may restrict procedures such as microscopy. Bulky gloves impede the performance of fine motor tasks, such as handling small items or labeling tubes. To minimize time spent in BSL-4 laboratories, laboratory specialists should review work procedures to identify steps that can be done ahead in a BSL-2 laboratory and then transport these materials into the BSL-4 laboratory for completion of the task(s). When removing materials for further processing in BSL-2 laboratory, materials are fixed and removed from the BSL-4 laboratory in a sealed secondary container. Examples of samples that may need to be removed include: fixed plates or tubes of infected material that will be analyzed by enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (IFA), or polymerase chain reaction (PCR).
In addition to greater physical limitations imposed by personal protective equipment required in BSL-4 laboratories compared to those in BSL-2 laboratories, procedures for inactivation of high-consequence pathogens in cell culture plates and waste disposal are stricter than those needed for less pathogenic viruses studied in a BSL-2 laboratory. At a minimum, these methods should meet CDC requirement. For example, contaminated cell culture plates and other materials can be inactivated with chemical reagents, such as neutral-buffered formalin. Treated cell culture plates or tubes are to be placed into heat seal pouches containing formalin and removed from the laboratory via a dunk tank filled with a liquid disinfectant. Waste buckets filled with disinfectant solutions and spray disinfectants are used for temporarily receiving waste generated during the experiment and for disinfecting gloves, cleaning biosafety cabinet surfaces and instruments, respectively. Quaternary ammonium disinfectant solution at the concentration listed is considered the gold standard for all US BSL-4 laboratories (Barr J, personal communication, 2015). Solid waste from a waste bucket is autoclaved to eliminate potential for contamination.
In an effort to visually demonstrate the workflow and limitations of general BSL-4 procedures, we used a standard viral plaque assay as an example of a commonly used viral procedure. While the viral assay procedure is described in general, we stress the biosafety procedures used to ensure safety of laboratory personnel in this protocol. Please refer to previous classical plaque assay visualizations for additional background on the plaque assay technique8,9.
The procedures presented here follow the BMBL specifications outlined by CDC1. However, the presented protocols are specific to the IRF-Frederick. Each BSL-4 facility has different standard operating procedures (SOPs) and methods of operation that impact the execution of experiments within the BSL-4 laboratory. Alternative procedures for waste stream management and execution of plaque assays may differ based on the management and operation of these laboratories. Nevertheless, a general understanding of the setup of a BSL-4 suit laboratory and procedures for performing work with Class II cabinets inside the BSL-4 environment will help scientists understand the constraints and safety implications when contemplating studies of high risk pathogens. Increased awareness of outside collaborators of the difficulties surrounding work in a BSL-4 laboratory can lead to adjusted expectations and greater ease in developing medical countermeasures in the research community.

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safety precautions and operating procedures in an (a)bsl-4 laboratory: 2. general practices

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory with non-high-consequence pathogens will take significantly longer in a BSL-4 setting. This increased time requirement is due to a multitude of factors that are aimed at protecting the researcher from laboratory-acquired infections, the work environment from potential contamination and the local community from possible release of high-consequence pathogens. Inside the laboratory, movement is restricted due to air hoses attached to the mandatory full-body safety suits. In addition, disinfection of every item that is removed from Class II biosafety cabinets (BSCs) is required. Laboratory specialists must be trained in the practices of the BSL-4 laboratory and must show high proficiency in the skills they are performing. The focus of this article is to outline proper procedures and techniques to ensure laboratory biosafety and experimental accuracy using a standard viral plaque assay as an example procedure. In particular, proper techniques to work safely in a BSL-4 environment when performing an experiment will be visually emphasized. These techniques include: setting up a Class II BSC for experiments, proper cleaning of the Class II BSC when finished working, waste management and safe disposal of waste generated inside a BSL-4 laboratory, and the removal of inactivated samples from inside a BSL-4 laboratory to the BSL-2 laboratory.

Following proper procedures within the BSL-4 laboratory are critical for ensuring safe and effective completion of assays. By referring to the completed daily internal checklist (Figure 1), laboratory staff ensure that equipment is fully operational. Proper body positioning in the center of the BSC ensures that the experiment is performed under optimum air flow conditions (Figure 2). The virus sample is serially diluted to obtain plates that have 30-300 p...

Watch this Scientific Journal Video about Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 2. General Practices at JoVE.com

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 2. General Practices

Performing viral assays in a BSL-4 laboratory is more involved compared to work in a BSL-2 laboratory due to required additional safety precautions. Here, we present an overview of practices and procedures used inside a BSL-4 laboratory illustrating proper Class II biosafety cabinet usage, waste management/disposal, and sample removal.

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

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory ...

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.

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Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

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

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 4. Medical Imaging Procedures

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 4. Medical Imaging Procedures

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 3. Aerobiology

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 3. Aerobiology

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol ...