<p class='jove_content'>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.</p>

<p class="jove_title">1. Laboratory Entry</p>
<ol>
	<li>Gather all supplies from a BSL-2 laboratory for the experiment (<em>e.g.</em>, cells, media, and consumables) prior to entry into the BSL-4 laboratory.</li>
	<li>Complete the BSL-4 entry procedure (outlined in detail in reference<sup>7</sup>).</li>
</ol>
<p class="jove_title">2. Preparation of a Class II Biosafety Cabinet in the BSL-4 Laboratory</p>
<ol>
	<li>Once inside the BSL-4 laboratory, ensure the daily internal checklist (<strong>Figure 1</strong>) 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.</li>
	<li>Clean the Class II BSC by spraying down the entire inside of the BSC (including the sash) with 5% dual quaternary ammonium disinfectant solution (<em>e.g.</em>, 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<sup>10,11</sup>. Spray 70% ethanol solution inside the cabinet and the sash to remove the tacky disinfectant solution.</li>
	<li>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 (<strong>Figure 2</strong>)<sup>11</sup>.</li>
	<li>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.</li>
	<li>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 flow<sup>11</sup>.</li>
</ol>
<p class="jove_title">3. Example: Plaque Assay</p>
<ol>
	<li>Retrieve virus-containing samples and control virus from the storage location by hand and thaw materials in an incubator at 37 &#176;C.</li>
	<li>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.</li>
	<li>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 (&#34;clean side&#34;) and waste on the other side (&#34;dirty side&#34;). If possible, keep &#34;clean items&#34; at least 30 cm apart from &#34;dirty items&#34; during aerosol-generating activities<sup>11</sup>.</li>
	<li>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 oneself<sup>11</sup>.</li>
	<li>Remove 50 &#181;l of virus sample and control virus and place into 450 &#181;l of Dulbecco&#39;s modified Eagle&#39;s medium with 2% fetal bovine serum. Proceed with making serial dilutions as far out as needed (<strong>Figure 3A</strong>).</li>
	<li>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.</li>
	<li>In the last dilution, after mixing the sample 5 times, discard 50 &#181;l of virus sample into waste container to ensure equal volumes of dilutions.</li>
	<li>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.</li>
	<li>Once the dilutions are made, set the dilution wells aside and begin aspirating media from the wells of cell culture plates, leaving 500 &#181;l of media in each well of a 6-well plate.</li>
	<li>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.</li>
	<li>Once the media have been removed from the plates, add 100 &#181;l of the correct sample into the proper well in the pre-labeled plates in duplicate, changing tips each for each sample.</li>
	<li>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.</li>
	<li>Rock the plates in a figure-8 motion to ensure proper dispersal of the sample over the cells every 15 min for 1 hr.</li>
	<li>Upon completion of rocking plates, return plates into the Class II BSC along with a mixture of 2x Eagle&#39;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.</li>
	<li>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.</li>
	<li>Ensure that the tip of serological pipette does not touch any liquid in the wells to avoid cross contamination when performing the overlay procedure.</li>
</ol>
<p class="jove_title">4. Waste Disposal and Cleaning of the Instruments and Biosafety Cabinet</p>
<ol>
	<li>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.</li>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Clean the Class II BSC&#39;s work surface, cabinet sides and back, interior of glass, and sash<sup>1</sup> with 5% disinfectant solution followed by 70% ethanol solution.</li>
	<li>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).</li>
	<li>Pour the contents of the waste bucket into a strainer placed in the bottom of the sink.</li>
	<li>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.</li>
	<li>Rinse out the waste bucket and place to dry on the rack next to the sink.</li>
</ol>
<p class="jove_title">5. Autoclaving Waste</p>
<ol>
	<li>Remove the biohazard bag from the biohazard waste container and place in an autoclave tray on a cart.</li>
	<li>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.</li>
	<li>Place two pieces of autoclave tape on the serological pipette tip tray and label it with one&#39;s initials and the date. Place the serological pipette tray on the cart with the autoclave tray.</li>
	<li>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.</li>
	<li>Remove the metal rod from the autoclave and open the top. Place a biological indicator vial containing spores of <em>Geobacillus stearothermophilus</em> into the metal rod to check that the autoclave sterilization cycle was completed successfully.</li>
	<li>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.</li>
	<li>Place autoclave tray filled with waste and serological pipette tray onto the retractable autoclave platform and push it back into the autoclave.</li>
	<li>Detach the autoclave loading/unloading cart from the autoclave and close the autoclave door.</li>
	<li>Start the autoclave.</li>
	<li>Do not leave the autoclave until the cycle has started. The operating screen of the autoclave will indicate time remaining for that run.</li>
	<li>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.</li>
</ol>
<p class="jove_title">6. Example: Fixing and Staining of Plaque Assays</p>
<ol>
	<li>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.</li>
	<li>Carefully remove plaque assay plates from the incubator completed in step 3.1.2, and place into the Class II BSC by hand.</li>
	<li>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<sup>12,13</sup>. Let the mixture remain on the plates for 30 min to inactivate the virus on the plates.</li>
	<li>After inactivation, carefully remove the neutral buffered formalin/crystal violet mixture and place in a separate waste container to be neutralized prior to disposal.</li>
	<li>Spray gloved hands with disinfectant and wipe the outside of the plates before passing them out of the Class II BSC as described above.</li>
	<li>Proceed to the sink, rinse the plates to remove excess stain, and then place the plates on a cart to dry.</li>
	<li>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 (<strong>Figure 3B</strong>).</li>
</ol>
<p class="jove_title">7. Removing Samples from the BSL-4 Laboratory</p>
<ol>
	<li>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)<sup>1,14</sup>. Transfer samples to a new clean tube or plate outside of the BSC prior to packaging for removal from the BSL-4 laboratory.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

<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> <em style='font-style: italic'>Viruses</em>. <b>4</b>, 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> <em style='font-style: italic'>Appl Biosafety</em>. <b>16</b>, 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> <em style='font-style: italic'>Pharmaco-imaging in drug and biologics development</em>. , (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> <em style='font-style: italic'>J Vis Exp</em>. , (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> <em style='font-style: italic'>J Vis Exp</em>. , 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> <em style='font-style: italic'>J Vis Exp</em>. , 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> <em style='font-style: italic'>J Virol</em>. , (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> <em style='font-style: italic'>Viruses</em>. <b>4</b>, 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> <em style='font-style: italic'>J Virol Methods</em>. <b>119</b>, 195-198 (2004).</li></ol>

<p class='jove_content'>The authors have nothing to disclose.</p>

<p class="jove_content">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.</p>
<p class="jove_content">The first major concept is the proper use and application of safe p...

<p class="jove_content">A correction was made to the authors section in: <em>Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 2. General Practices</em></p>
<p class="jove_content">One of the authors names was corrected from:</p>
<p>Nicole Joselyn</p>
<p class="jove_content">to:</p>
<p>Nicole Josleyn</p>

<p class="jove_content">A correction was made to the authors section in: <em>Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 2. General Practices</em></p>

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

<p class="jove_content">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 perspective<sup>1</sup>. 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 Agents<sup>2</sup>. 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 confidence<sup>3</sup>.</p>
<p class="jove_content">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 facility<sup>4-6</sup>. 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 laboratory<sup>7</sup>. 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).</p>
<p class="jove_content">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.</p>
<p class="jove_content">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 technique<sup>8,9</sup>.</p>
<p class="jove_content">The procedures presented here follow the BMBL specifications outlined by CDC<sup>1</sup>. 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.</p>

<table><tbody><tr><td>Micro-Chem Plus</td><td>National Chemical Laboratories</td><td>255</td><td></td></tr><tr><td>Ethanol</td><td>Fisher</td><td>BP2818500</td><td></td></tr><tr><td>2 ml 96-Deep Well Plates</td><td>Fisher</td><td>278743</td><td></td></tr><tr><td>10 ml Serological Pipette</td><td>Fisher</td><td>13-678-11E</td><td></td></tr><tr><td>25 ml Serological Pipette</td><td>Fisher</td><td>13-678-11</td><td></td></tr><tr><td>6-well plates</td><td>Fisher</td><td>140675</td><td></td></tr><tr><td>Crystal Violet</td><td>Sigma</td><td>HT90132-1L</td><td></td></tr><tr><td>10% Neutral Buffered Formalin</td><td>Fisher</td><td>22-050-105</td><td></td></tr><tr><td>Tragacanth</td><td>Fisher</td><td>50-702-2000&amp;nbsp;</td><td></td></tr><tr><td>20 &amp;mu;l Pipette Tips</td><td>Fisher</td><td>21-402-550</td><td></td></tr><tr><td>200 &amp;mu;l Pipette Tips</td><td>Fisher</td><td>21-402-561</td><td></td></tr><tr><td>1,000 &amp;mu;l Pipette Tips</td><td>Fisher</td><td>21-402-581</td><td></td></tr><tr><td>DMEM</td><td>Lonza</td><td>12-604Q</td><td></td></tr><tr><td>FBS</td><td>Sigma</td><td>F2442-500mL</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Lonza</td><td>17-602E</td><td></td></tr><tr><td>2x EMEM</td><td>Quality Biological</td><td>115-073-101</td><td></td></tr><tr><td>Pipettor</td><td>Drummond</td><td>4-000-101</td><td></td></tr><tr><td>1,000 &amp;mu;l Pipette&amp;nbsp;</td><td>Rainin</td><td>&amp;nbsp;L-1000XLS+</td><td></td></tr><tr><td>200 &amp;mu;l Pipette&amp;nbsp;</td><td>Rainin</td><td>&amp;nbsp;L-200XLS+</td><td></td></tr><tr><td>12-Well, Multichannel 200 &amp;mu;l Pipettor</td><td>Rainin</td><td>L12-200XLS+</td><td></td></tr><tr><td>8-Well, Multichannel 1,000 &amp;mu;l Pipettor</td><td>Rainin</td><td>LA8-1200XLS</td><td></td></tr><tr><td>Attest</td><td>Express Medical Supplies&amp;nbsp;</td><td>MMM12192</td><td></td></tr><tr><td>Autoclave</td><td>Getinge</td><td>GEB 2404 AMB-2</td><td></td></tr><tr><td>Autoclave Bag</td><td>Fisher</td><td>01-828E</td><td></td></tr><tr><td>2,000 ml Beaker</td><td>Fisher</td><td>02-591-10H</td><td></td></tr><tr><td>Autoclave Tray</td><td>Fisher</td><td>13-359-20B</td><td></td></tr><tr><td>Pipette Tray</td><td>Fisher</td><td>13-361-5</td><td></td></tr><tr><td>37 &amp;deg;C Incubator</td><td>Fisher</td><td>WU-39321-00</td><td></td></tr><tr><td>Biohazard Can</td><td>Rubbermaid Commercial</td><td>FG614500 RED</td><td></td></tr><tr><td>Autoclave Tape</td><td>Fisher</td><td>15-903</td><td></td></tr><tr><td>Autoclave Rod</td><td>Made by IRF Facility</td><td>N/A</td><td></td></tr><tr><td>Light Box</td><td>Fisher</td><td>S11552</td><td></td></tr><tr><td>Heat Sealer</td><td>Fisher</td><td>NC9793612&amp;nbsp;</td><td></td></tr><tr><td>Heat Seal Pouches</td><td>Fisher</td><td>01-812-25H</td><td></td></tr><tr><td>Biohazard Bag</td><td>Fisher</td><td>01-828E</td><td></td></tr></tbody></table>

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

<p class="jove_content">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.</p>

<p class="jove_content" fo:keep-together.within-page="1">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 (<strong>Figure 1</strong>), 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 (<strong>Figure 2</strong>). 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

<p class='jove_content'>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.</p>

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 ...

safety-precautions-operating-procedures-an-bsl-4-laboratory-2-general

Research

JoVE Journal

Immunology and Infection

17.9K Views.  National Institutes of Health (NIH). 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.

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

Combination of Adhesive-tape-based Sampling and Fluorescence <em>in situ</em> Hybridization for Rapid Detection of <em>Salmonella</em> on Fresh Produce

<p class="jove_content">This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence <em>in situ</em> hybridization (FISH) for rapid culture-independent detection of <em>Salmonella</em> spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a <em>Salmonella</em>-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow <em>in situ</em> growth of <em>Salmonella</em> microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of <em>Salmonella</em> spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of <em>Salmonella</em> on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.</p>

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

<p class="jove_content">This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of <em>Salmonella</em> using fluorescence <em>in situ</em> hybridization (FISH).</p>

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

<p class="jove_content">We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical large scale diagnostic testing. In the low-income countries inexpensive ART has transformed the prospects for the survival of HIV seropositive patients but there are doubts whether there is a need for the laboratory monitoring of ART and at what costs - in situations when the overall quality of pathology services can still be very low. The appropriate answer is to establish economically sound services with better coordination and stricter internal quality assessment than seen in western countries. This video, photographed at location in the National Health Laboratory Services (NHLS-SA) at the Witwatersrand University, Johannesburg, South Africa, provides such a coordinated scheme expanding the original 2-color CD4-CD45 PanLeucoGating strategy (PLG). Thus the six modules of the video presentation reveal the simplicity of a 4-color flow cytometric assay to combine haematological, immunological and virology-related tests in a single tube. These video modules are: (i) the set-up of instruments; (ii) sample preparations; (iii) testing absolute counts and monitoring quality for each sample by bead-count-rate; (iv) the heamatological CD45 test for white cell counts and differentials; (v) the CD4 counts, and (vi) the activation of CD8+ T cells measured by CD38 display, a viral load related parameter. The potential cost-savings are remarkable. This arrangement is a prime example for the feasibility of performing &gt; 800-1000 tests per day with a stricter quality control than that applied in western laboratories, and also with a transfer of technology to other laboratories within a NHLS-SA network. Expert advisors, laboratory managers and policy makers who carry the duty of making decisions about introducing modern medical technology are frequently not in a position to see the latest technical details as carried out in the large regional laboratories with huge burdens of workload. Hence this video shows details of these new developments.</p>

Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

<p class="jove_content">Anti-retroviral therapy to treat HIV/AIDS is monitored in South Africa on a large scale. Flow cytometry is combined for haematology (CD45), immunology (CD4) and viral-load linked CD38 assay. Recorded at NHLS-SA laboratories, Johannesburg, these modern methods are cost-efficient with heightened local internal quality control, serving as role-models for resource-limited diagnostics.</p>

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

<em>Trichuris muris</em> Infection: A Model of Type 2 Immunity and Inflammation in the Gut

<p class="jove_content"><em>Trichuris muris</em> is a natural pathogen of mice and is biologically and antigenically similar to species of <em>Trichuris </em>that 
infect humans and livestock<sup>1</sup>. Infective eggs are given by oral gavage, hatch in the distal small intestine, invade the intestinal epithelial cells (IECs) 
that line the crypts of the cecum and proximal colon and upon maturation the worms release eggs into the environment<sup>1</sup>. This model is a powerful tool to 
examine factors that control CD4<sup>+</sup> T helper (Th) cell activation as well as changes in the intestinal epithelium.  The immune response that occurs in 
resistant inbred strains, such as C57BL/6 and BALB/c, is characterized by Th2 polarized cytokines (IL-4, IL-5 and IL-13) and expulsion of worms while Th1-associated 
cytokines (IL-12, IL-18, IFN-&gamma;) promote chronic infections in genetically susceptible AKR/J mice<sup>2-6</sup>. Th2 cytokines promote physiological changes in 
the intestinal microenvironment including rapid turnover of IECs, goblet cell differentiation, recruitment and changes in epithelial permeability and smooth muscle 
contraction, all of which have been implicated in worm expulsion<sup>7-15</sup>.  Here we detail a protocol for propagating <em>Trichuris muris</em> eggs which can 
be used in subsequent experiments.  We also provide a sample experimental harvest with suggestions for post-infection analysis.  Overall, this protocol will provide 
researchers with the basic tools to perform a <em>Trichuris muris</em> mouse infection model which can be used to address questions pertaining to Th proclivity in 
the gastrointestinal tract as well as immune effector functions of IECs. </p>


Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

<p class="jove_content">Trichuris muris infection is an intestinal model of Th2 immunity where resistant mice generate a protective Th2 response and susceptible mice generate a pathological Th1 response.</p>

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

<p class="jove_content">Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative <em>ex vivo</em> imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.</p>
<p class="jove_content">Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their<em> in vivo</em> interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.</p>
<p class="jove_content">Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice  were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KI<sup>gfp/gfp</sup> mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for <em>in vivo </em>imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.</p>

<p class="jove_content">We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.</p>

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 <em>Burkholderia</em> Species

<p class="jove_content">The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases caused by these  organisms. Here we describe a procedure  for protein extraction from <em>Burkholderia</em> species based on mechanical lysis using glass beads in the presence of  ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate  buffered saline. This method can be used  for different <em>Burkholderia</em> species,  for different growth conditions, and it is likely suitable for the use in  proteomic studies of other bacteria.  Following protein extraction, a two-dimensional (2-D) gel electrophoresis  proteomic technique is described to study global changes in the proteomes of  these organisms. This method consists of  the separation of proteins according to their isoelectric point by isoelectric  focusing in the first dimension, followed by separation on the basis of  molecular weight by acrylamide gel electrophoresis in the second  dimension. Visualization of separated  proteins is carried out by silver staining.</p>

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

<p class="jove_content">Members of the <em>Burkholderia</em> genus are pathogens of clinical importance.  We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.</p>

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

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

<p class='jove_content'>Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor treatment options exist. The hallmarks of these laboratories are: custom-designed airtight doors, dedicated supply and exhaust airflow systems, a negative-pressure environment, and mandatory use of positive-pressure (&#8220;space&#8221;) suits. The risk for laboratory specialists working with highly pathogenic agents is minimized through rigorous training and adherence to stringent safety protocols and standard operating procedures. Researchers perform the majority of their work in BSL-2 laboratories and switch to BSL-4 suit laboratories when work with a high-consequence pathogen is required. Collaborators and scientists considering BSL-4 projects should be aware of the challenges associated with BSL-4 research both in terms of experimental technical limitations in BSL-4 laboratory space and the increased duration of such experiments. Tasks such as entering and exiting the BSL-4 suit laboratories are considerably more complex and time-consuming compared to BSL-2 and BSL-3 laboratories. The focus of this particular article is to address basic biosafety concerns and describe the entrance and exit procedures for the BSL-4 laboratory  at the NIH/NIAID Integrated Research Facility at Fort Detrick. Such procedures include checking external systems that support the BSL-4 laboratory, and inspecting and donning positive-pressure suits, entering the laboratory, moving through air pressure-resistant doors, and connecting to air-supply hoses. We will also discuss moving within and exiting the BSL-4 suit laboratories, including using the chemical shower and removing and storing positive-pressure suits.</p>

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

<p class='jove_content'>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.</p>

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

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

<p class='jove_content'>Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by high-consequence pathogens has not been possible. In 2014, the National Institutes of Health, National Institute of Allergy and Infectious Diseases, Integrated Research Facility at Fort Detrick opened an Animal Biosafety Level 4 (ABSL-4) facility to assess the clinical course and pathology of infectious diseases in experimentally infected animals. Multiple imaging modalities including computed tomography (CT), magnetic resonance imaging, positron emission tomography, and single photon emission computed tomography are available to researchers for these evaluations. The focus of this article is to describe the workflow for safely obtaining a CT image of a live guinea pig in an ABSL-4 facility. These procedures include animal handling, anesthesia, and preparing and monitoring the animal until recovery from sedation. We will also discuss preparing the imaging equipment, performing quality checks, communication methods from "hot side" (containing pathogens) to "cold side," and moving the animal from the holding room to the imaging suite.</p>

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

<p class='jove_content'>Here, we present an overview of the preparation and animal handling procedures required to safely perform medical imaging in an animal biosafety level 4 laboratory. Computed tomography of a mock-infected guinea pig illustrates these procedures that may be used to evaluate the disease caused by a high consequence pathogen.</p>

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

<p class='jove_content'>Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol releases or unexpected natural aerosol transmission. Specific laboratories designed to perform these experiments require tremendous engineering controls to provide a safe and secure working environment and constant systems maintenance to sustain functionality. Class III biosafety cabinets, also referred to as gloveboxes, are gas-tight enclosures with non-opening windows. These cabinets are maintained under negative pressure by double high-efficiency-particulate-air (HEPA)-filtered exhaust systems and are the ideal primary containment for housing aerosolization equipment. A well planned workflow between staff members within high containment from, for instance, an animal biosafety level-4 (ABSL-4) suit laboratory to the ABSL-4 cabinet laboratory is a crucial component for successful experimentation. For smooth study execution, establishing a communication network, moving equipment and subjects, and setting up and placing equipment, requires staff members to meticulously plan procedures prior to study initiation. Here, we provide an overview and a visual representation of how aerobiology research is conducted at the National Institutes of Health, National Institute of Allergy and Infectious Diseases Integrated Research Facility at Fort Detrick, Maryland, USA, within an ABSL-4 environment.</p>

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

<p class='jove_content'>As high-consequence pathogens can potentially infect subjects through airborne particles, aerobiology has been increasingly applied in pathogenesis research and medical countermeasure development. We present a detailed visual demonstration of aerobiology procedures during an aerosol challenge in nonhuman primates in an animal biosafety level 4 maximum containment environment.</p>

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

Remote Laboratory Management: Respiratory Virus Diagnostics

<p class="jove_content">An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a multitude of issues ranging from transportation, access, facilities, equipment, and communication to provider training. To address this need, we have developed an innovative, scalable, logistics-enhanced, mobile, laboratory facility for emergencies and epidemics in resource-constrained global settings. Utilizing a background in clinical operations as an academic medical center, we designed a rapidly-deployable, modular BSL-2 and BSL-3 facility with user-friendly software for tracking and management of drugs and supplies in remote regions during epidemics and outbreaks. Here, we present our intermodal, mobile, expandable shipping-container laboratory units. The design of the laboratory facilitates off-grid usage by minimizing power consumption and allowing alternate water sources. The unit's information communication technology (ICT) platform provides (i) user-friendly tablet-based documentation, (ii) enhanced tracking of patients and supplies, and (iii) integrated communication onsite with built-in telehealth capabilities. To ensure quality in remote environments, we have developed a checklist for a basic laboratory workflow and a protocol for respiratory viral diagnosis using reverse-transcription polymerase chain reaction (RT-PCR). As described, this innovative and comprehensive approach allows for the provision of laboratory capability in resource-limited global environments. </p>

<p class="jove_content">A rapidly-deployable, off-grid laboratory has been designed and built for remote, resource-constrained global settings. The features and critical aspects of the logistically-enhanced, expandable, multifunctional laboratory modules are explored. A checklist for a basic laboratory workflow and a protocol for a respiratory viral diagnostic test are developed and presented.</p>

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a ...

Medicine

Lab Techniques

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse=""><strong>Determining the Density of an Egg</strong><button class="expand">Expand</button>
	<p>Density is a measure of how compact a substance or object is, and it is calculated as mass divided by volume. When a dense object is placed in a liquid with a lower density, the object sinks; when the object is less dense than the liquid, it floats. When the densities are the same, the object will be suspended in the liquid. In this experiment, we&#39;ll determine the density of an egg by creating a solution with the same density. We&#39;ll measure the density of the salt solution using different volume measurement glassware and determine which is the most accurate.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-1">
		<div class="lab-step">To begin, wear the appropriate personal protective equipment, including close-toed shoes, long pants, long-sleeved shirt, lab coat, chemical splash goggles, and gloves.</div>
		</li>
		<li data-sub-note-item="1-2">
		<div class="lab-step">Measure the mass of the egg. Set a weigh boat on the balance and tare it. Carefully set the egg in the weigh boat while you record the mass in your notebook.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Prepare the salt solution that will be used to suspend your egg. Use the 600-mL beaker and fill it 3/4 of the way full with deionized water.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Gently place your egg in the beaker, and then add about one teaspoon of NaCl into the water. Stir the solution gently until the NaCl dissolves. Continue to add NaCl until the egg floats just below the surface.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Use different glassware to measure the volume of the salt solution. Weigh the 50-mL graduated cylinder on the top loading balance and record the mass. Then, measure 20 mL of your salt solution and record the volume in your notebook. <strong>Note: </strong>Place a blank sheet of paper behind the graduated cylinder to read the volume more clearly. Always view the measurement at eye level and measure the volume from the bottom of the meniscus. The lines on the graduated cylinder are 0.2 mL apart, meaning that you can read the volume to the hundredths place.
		<h4><b>Table 1: Measuring Mass</b></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Container</strong></td>
					<td style="text-align: center;"><strong>Mass of empty container (g)</strong></td>
					<td style="text-align: center;"><strong>Mass of full containter (g)</strong></td>
					<td style="text-align: center;"><strong>Mass of solution (g)</strong></td>
					<td style="text-align: center;"><strong>Percent error</strong></td>
				</tr>
				<tr>
					<td>50-mL graduated cylinder</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>10-mL volumetric pipette</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>50-mL beaker</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>20-mL volumetric flask</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/41/Table_1._Measuring_Mass.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Weigh the filled graduated cylinder and record the mass in your notebook. Then, pour the salt solution back into the 600-mL beaker.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Use the volumetric pipette to measure volume. First, weigh the empty 50-mL beaker and record the mass in your notebook. Then, carefully measure 10 mL of the salt solution using the volumetric pipette and dispense the salt solution into the beaker.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Measure another 10 mL and add it to the beaker. Weigh the filled beaker and record the mass in your notebook. Then, empty, rinse, and dry the beaker.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Use the volumetric flask to measure volume. Weigh the empty 20-mL volumetric flask and record the mass. Then, carefully fill the flask with the salt solution to just below the 20-mL fill mark. Do not fill it all the way. Use an eyedropper to finish filling the flask so that the bottom of the meniscus just touches the fill line on the neck of the flask. Then, weigh the full flask and record the weight.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Lastly, use the 50-mL beaker to measure volume. Weigh the empty beaker, then fill the beaker with salt solution to the 20-mL line and weigh the full beaker. Record the masses in your notebook.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Measure the displacement of the egg when it is submerged in the salt solution. First, remove the egg and look at the level of the salt solution in the 600-mL beaker. <strong>Note:</strong> If it is not at a marked line, add deionized water to the solution so that the meniscus is at a readable volume and record the volume.&#160;
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td><strong>Measurements</strong></td>
					<td><strong>Salt solution using 20-mL volumetric flask&#160;</strong></td>
					<td><strong>Egg</strong></td>
				</tr>
				<tr>
					<td>Mass (g)</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Displacement of water (cm)</td>
					<td style="text-align: center;">-</td>
					<td></td>
				</tr>
				<tr>
					<td>Inner diameter of 600-mL beaker (cm)</td>
					<td style="text-align: center;">-</td>
					<td></td>
				</tr>
				<tr>
					<td>Volume (cm&#179;)</td>
					<td style="text-align: center;">-</td>
					<td></td>
				</tr>
				<tr>
					<td>Volume (mL)</td>
					<td style="text-align: center;">20</td>
					<td></td>
				</tr>
				<tr>
					<td>Density (g/mL)</td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<strong><a href="https://www.jove.com/CDNSource/lm/labs/41/Table_2._Measuring_Density.xlsx" target="_blank">Click Here to download Table 2</a> </strong></div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Gently place the egg in the beaker.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Use a ruler to measure the new height of the solution level and record the height. Then, place the ruler across the top of the beaker to measure the inner diameter.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Calculate the new volume from these measurements. Subtract the initial volume to obtain the displacement of the egg (volume).</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">To clean up from the experiment, return the egg to your instructor and pour the salt solution down the sink. Rinse all used glassware with tap water and set them out to dry. Finally, return the sodium chloride to your instructor.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse=""><strong>Results</strong><button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">Calculate the mass of the solution in each of the different volumetric glassware by subtracting the empty glassware mass from the full glassware mass.</div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Keep track of uncertainty in your measurement by using significant figures. For example, when we look at the graduated cylinder measurement, the weight of the empty container is 90.92, and the weight of the full container is 111.59. The weight measurements have significant figures to the hundredths place, so it is important that significant figures are maintained throughout our calculations. Thus, the mass of the 20-mL salt solution is 20.67 g.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">The 50-mL beaker weighed 28.65 g when it was empty and 47.96 g when it contained 20 mL of salt solution. Therefore, the mass of 20 mL of the salt solution is 19.31 g.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Repeat the calculation to obtain the mass of 20 mL of salt solution that was measured using the 10-mL pipette and the 200-mL volumetric flask.<b> Note:</b> Generally speaking, volumetric measurements are more accurate when using a volumetric flask.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Look at the accuracy of the other methods compared to the volumetric flask in terms of percent error. Percent error is calculated by subtracting the theoretical value from the experimental value, then dividing by the theoretical value times 100.
		<ul>
			<li><img src="https://www.jove.com/CDNSource/lm/labs/41/41_Procedure_1b.jpg" /></li>
		</ul>
		</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Use the volumetric flask measurement as the theoretical value to determine the percent error for the other volume measurement methods. As expected, the volume measurement that uses the markings on the beaker is the least accurate.<strong> Note: </strong>We also know this because the known density of pure water is 1 g/mL. Thus, 20 mL of pure water weighs 20 g. The density of the salt solution must be higher than 20 g since salt was added to the water.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Calculate the density of the salt solution using the volumetric flask measurement. Divide the mass of the solution by the volume of the solution. Thus, the density of the salt solution is 1.053 g/mL.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Compare the calculated density of the salt solution to the density of the egg. The volume calculated using the displacement of water in a beaker was found to be 61.14 cm<sup>3</sup> or 61.14 mL. The weight of the egg was measured to be 60.15 g, so its density is 0.9838 g/mL. The disparity between the density of the egg and the density of the salt solution arises from error introduced in both calculations, primarily due to volume measurement readings.</div>
		</li>
	</ul>
	</li>
</ol>

Accuracy, Precision, Uncertainty, and Significant Figures - Procedure

Source: Smaa Koraym at Johns Hopkins University, MD, USA

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