The work was supported by a grant from the Creutzfeldt Jacob Disease Foundation. The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

All procedures involving animals were approved and in compliance with the Guide for the Care and Use of Laboratory Animals by the Creighton University Institutional Animal Care and Use Committee.
NOTE: A schematic overview of the prion safety laboratory swipe test is shown in Figure 1.
1. Selection of swabbing sites and preparation for surface swabbing
<ol>
	<li>Identify and label appropriate surveillance areas for swabbing. Refer to the template provided for documentation and reference (Figure 2 and Supplementary Figure 1).</li>
	<li>Prepare two 1.5 mL microcentrifuge tubes for each of the identified areas. Add 250 &#181;L of Dulbecco&#39;s phosphate-buffered saline to the first set of microcentrifuge tubes for use in step 3.2. Let the second set of microcentrifuge tubes remain empty for use in step 4.3.</li>
	<li>Retrieve foam-tipped swabs from storage in an area free of prion contamination.</li>
	<li>Prepare a squeeze bottle with Milli-Q water (MQ H2O) for use in step 2.1.</li>
</ol>
2. Positive and negative control swab preparation
<ol>
	<li>Prepare dilutions for positive and negative swab controls using relevant control types. Example: For positive control, prepare a 1% dilution of prion-infected brain homogenate in Dulbecco&#39;s phosphate-buffered saline (DPBS). For negative control, prepare a 0.1% dilution of uninfected (UN) brain homogenate (BH) from the same species as the positive control in DPBS.</li>
	<li>Using clean gloves, retrieve the appropriate number of swabs from the clean packaging and place the handle side down into a tube rack, taking care to space out foam swabs so that the tips do not contact other swabs or surfaces. For each control sample, prepare three swabs. Change gloves between each sample.</li>
	<li>Holding a clean foam swab by the handle, apply 50 &#181;L of respective positive and negative control samples to foam swab tips. Make sure to apply to both sides of the swab tip to ensure complete absorption. 
	NOTE: Always change gloves prior to removing a clean swab from the tube rack.</li>
	<li>Using scissors, cut off the excess handle of the swab (approximately &#189; of the length) and place the swab into the preloaded microcentrifuge tube with the foam tip portion pointed down. Ensure that the foam tip is submerged in the DPBS and that the handle is cut adequately to close the lid of the microcentrifuge tube completely.</li>
	<li>Continue applying control samples after changing gloves until all samples have been applied.</li>
</ol>
3. Surface swabbing
<ol>
	<li>Holding a clean foam tip swab by the handle, prewet the foam tip with MQ H2O and shake off the excess. Place the moistened foam tip of the swab onto the area chosen for surveillance and swab the area back and forth approximately ten times while simultaneously rotating the tip of the swab on the surface.</li>
	<li>Using scissors, cut off the excess handle of the swab (approximately &#189; of the length) and place the swab into the preloaded microcentrifuge tube with the foam tip portion pointed down. Ensure that the foam tip is submerged in the DPBS and that the handle is cut adequately to close the lid of the microcentrifuge tube completely.</li>
	<li>Discard gloves and place new gloves on before each respective swabbing site to minimize the probability of cross contamination.</li>
	<li>Repeat the procedure until all areas chosen for surveillance have been swabbed.</li>
</ol>
4. Swab extraction and vacuum concentration
NOTE: Turn on the vacuum concentrator 30 min prior to use to allow the instrument to warm up.
<ol>
	<li>Place microcentrifuge tubes into a circular tube rack. Place the microfuge tube rack into the cup horn sonicator water bath. Ensure the foam swabs in DPBS within the microcentrifuge tubes are below the surface of the water in the cup horn (the handle portion does not have to be submerged). Apply the following settings: 15 s total run time (5 s on, 5 s off) at ~75-85 watts.</li>
	<li>Following sonication, centrifuge the tubes for approximately 15 s to collect DPBS in the bottom of the tube prior to transfer.</li>
	<li>Using a P1000 pipette set to 250 &#181;L, carefully collect all liquid (swab extract) from the bottom of the microcentrifuge tube and transfer into the corresponding microcentrifuge tube from the second, empty prelabeled set. Use the pipette tip to squeeze any excess liquid from the foam tip. Discard empty tubes containing swabs.</li>
	<li>Set the following settings on the vacuum concentrator: Temperature: 45 &#176;C, Heat time: 15 min, Run time: 2 h, Vacuum: 5.1.</li>
	<li>Place the microcentrifuge tubes containing swab extracts into the vacuum concentrator, ensuring the tubes are balanced and all tube caps are open.</li>
	<li>Upon cycle completion, ensure that samples are completely concentrated (only the pellet remains). Store the pellets at -80 &#176;C until utilized for RT-QuIC. 
	NOTE: In some instances, additional vacuum concentration time may be required to ensure complete concentration. Remove all concentrated samples, leaving only the tubes that still contain liquid. Rebalance the remaining tubes within the concentrator and run at additional 1-h increments until samples are completely concentrated.</li>
</ol>
5. Preparation of swabbing controls for use in RT-QuIC
NOTE: The RT-QuIC controls should be performed prior to the assay of environmental swab extracts to ensure that contamination has not been introduced during the swabbing, extraction, or concentration procedures. For example layouts, see Figure 3 and Figure 4.
<ol>
	<li>Prepare an appropriate negative RT-QuIC plate control (e.g., uninfected brain homogenate [UN BH]) by diluting to the appropriate concentration in tissue dilution solution (N2-0.1%SDS/PBS). Prepare a positive plate control dilution by diluting prion-infected brain homogenate to a dilution of 10-3 in tissue dilution solution.</li>
	<li>Remove previously stored control swab extract pellets from -80 &#176;C and resuspend each with 50 &#181;L of MQ H2O by pipetting up and down approximately 10 times, followed by vortexing briefly. Allow samples to sit at room temperature (RT) while performing the remaining steps.</li>
	<li>Load 2 &#181;L of specified plate controls, including negative plate controls (tissue dilution solution alone and uninfected brain homogenate) and a positive plate control (infected brain homogenate).</li>
	<li>Load 2 &#181;L of each positive and negative swab extract technical replicate into a minimum of four replicate RT-QuIC plate wells. 
	NOTE: If negative swabbing controls exhibit RT-QuIC seeding above a laboratory&#39;s standard for a positive determination, this would indicate that contamination had been introduced during the swabbing, extraction, and concentration process and that the swabbing experiment should be performed again</li>
</ol>
6. Preparation of samples for use in RT-QuIC
<ol>
	<li>Prepare an appropriate negative plate control (e.g., uninfected brain homogenate, uninfected lymph node, etc.) by diluting to the appropriate concentration in tissue dilution solution (N2-0.1%SDS/PBS). Prepare positive plate controls by diluting infected brain homogenate to a dilution of 10-3 in tissue dilution solution.</li>
	<li>Remove previously stored swab extract pellets from -80 &#176;C and resuspend with 20-50 &#181;L of tissue dilution solution (depending on the suspected amount of contamination) by pipetting up and down approximately ten times, followed by vortexing briefly. Allow samples to sit at RT while performing the remaining steps.</li>
	<li>Load 2 &#181;L of specified plate controls, including negative plate controls (tissue dilution solution alone and uninfected brain homogenate) and a positive plate control (infected brain homogenate) into the RT-QuIC plate wells.</li>
	<li>Load 2 &#181;L of each swab extract into four replicate wells.</li>
</ol>
7. RT-QuIC analysis and results
<ol>
	<li>Perform RT-QuIC according to individual laboratory protocols (optional protocols38,40).</li>
	<li>Define the parameters required for determining the positivity of a sample (see representative results, Figure 5). 
	NOTE: These parameters are defined by each lab.</li>
	<li>Record results in the provided table and take appropriate action based on laboratory best practices (Table 1 and Supplementary Figure 2).</li>
</ol>

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G., 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=Inactivation+of+prions+and+amyloid+seeds+with+hypochlorous+acid.">Inactivation of prions and amyloid seeds with hypochlorous acid.</a> PLoS Pathog. 12 (9), e1005914 (2016).</li><li>Williams, K., Hughson, A. G., Chesebro, B., Race, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+chronic+wasting+disease+prions+using+sodium+hypochlorite.">Inactivation of chronic wasting disease prions using sodium hypochlorite.</a> PLoS One. 14 (10), e0223659 (2019).</li><li>Taylor, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoclaving+standards+for+Creutzfeldt-Jakob+disease+agent+[letter].">Autoclaving standards for Creutzfeldt-Jakob disease agent [letter].</a> Ann Neurol. 22 (4), 557-558 (1987).</li><li>Brown, P., Rohwer, R. G., Gajdusek, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newer+data+on+the+inactivation+of+scrapie+virus+or+Creutzfeldt-Jakob+disease+virus+in+brain+tissue.">Newer data on the inactivation of scrapie virus or Creutzfeldt-Jakob disease virus in brain tissue.</a> J Infect Dis. 153 (6), 1145-1148 (1986).</li></ol>

J.C.B. and Q.Y. are inventors on a patent application pertaining to prion surface swabbing technology.

The described prion safety swabbing method can be used to enhance existing prion safety measures. This method can monitor prion laboratory spaces and equipment, as well as shared laboratory spaces, for potential prion contamination. Importantly, this method can be adapted to test laboratory-specific disinfection techniques to verify decontamination of prion-contaminated surfaces. As various prion strains display different sensitivities to disinfection techniques, this method can confirm that these techniques are effective for current laboratory experiments, such as treatment with sodium hydroxide or sodium hypochlorite (bleach)43,44.
Key steps for this methodology include the identification of appropriate swabbing sites that will provide a sample of both high-traffic areas as well as areas to monitor that may be involved with downstream cross-contamination of experiments. Additionally, laboratories should utilize positive and negative controls that closely mirror those commonly used in a given lab or clinic. For example, if working with rodent prions, the positive and negative controls should match that species. Finally, surveillance data should be kept up to date and organized so that trends in positivity can easily be identified, thus allowing for mitigation and retraining of laboratory staff in the event of consistent positive results for a given area.
One limitation of this protocol is the potential generation of erroneous RT-QuIC results. Given the sensitive nature of RT-QuIC, the presence of residual detergents, salt, and other substances on laboratory surfaces can impact the outcome of the reaction39. Overall, given this observation, it is advantageous that surfaces remain free of substances that can interfere with RT-QuIC, such as residual salt, detergents, and dust. As improvements to the RT-QuIC methodology become available, we anticipate that many of the current limitations of RT-QuIC will be resolved. We, therefore, encourage the end user to keep abreast of the literature to take advantage of the latest improvements in RT-QuIC.
A key benefit of this method for laboratories researching prions is the ability to minimize contamination that may impact the results of sensitive amplification assays. Necropsy tools are commonly disinfected prior to being reused for future necropsies. The described swabbing method provides a method to assess necropsy tools for residual prion infectivity that may affect downstream results. This can provide an additional level of rigor to experiments to exclude the possibility that prion detection in tissues was not due to contamination from the necropsy tools.

Prion diseases are invariably fatal neurodegenerative diseases with no known treatment or cure. Prion diseases are caused by PrPSc, the misfolded and pathogenic form of the normal cellular form of the prion protein1,2,3,4,5, PrPC. Prion diseases are known to affect humans and several other animal species. One human prion disease, Creutzfeldt-Jakob Disease, CJD, has three known etiologies: sporadic, inherited, and acquired. Acquired CJD can occur as a result of accidental transmission (iatrogenic and occupational) and is thought to be the cause of Kuru in the Fore people of Papua New Guinea6.
Prion transmission has been associated with prion-contaminated medical devices and transplant materials7,8,9,10,11,12,13,14,15,16,17. Iatrogenic transmission of CJD can occur via blood, tissue, or from prion-contaminated surfaces18,19,20. For example, iatrogenic CJD can develop in patients following an electroencephalogram with electrodes previously used on an individual in the preclinical stage of CJD who then later succumbed to CJD21. More recent laboratory-based occupational transmission has also occurred where a laboratory worker contracted prion disease via a skin puncture with forceps used to handle brain slices from an animal infected with sheep-adapted BSE22,23. Such transmission scenarios could occur within clinical, laboratory, and diagnostic laboratory settings where prion samples are handled.
Prions resist common disinfection techniques and can persist and remain infectious on surfaces for extended periods of time24,25,26,27,28,29. Common disinfection techniques such as the use of ethanol, phenolic cleaners, hydrogen peroxide, various forms of radiation, and formaldehyde are inadequate for the inactivation of prions, allowing surfaces to retain infectivity30,31,32,33,34,35,36,37. These characteristics contribute to the transmission of prions during iatrogenic and occupational exposure.
Methods for the detection of environmental prions have only recently been developed. An environmental swabbing method coupled with real-time quaking-induced conversion (RT-QuIC) can assess residual prion infectivity from environmental surfaces as well as common laboratory surfaces following ineffective disinfection38,39,40,41,42. Here, we describe how this technique can be incorporated into a broader prion safety program. Overall, this method can allow for the monitoring of laboratory-dependent disinfection protocols, the investigation and proper documentation of contamination status, which can help ensure the validity of experiments by minimizing cross-contamination, the assessment of shared use spaces for prion contamination and allows for directional retraining of personnel based on commonly contaminated areas.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fisherbrand&#160;PurSwab Foam Swabs</td>
			<td>Fisher brand</td>
			<td>Catalog #14-960-3E</td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q IQ 7005 Ultrapure Water System</td>
			<td>MilliporeSigma</td>
			<td>Q7005T0C</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-centrifuge, 6000 rpm</td>
			<td>Southern Labware</td>
			<td>MLX-306</td>
			<td></td>
		</tr>
		<tr>
			<td>Omega plate reader</td>
			<td>BMG Labtech</td>
			<td>FLUOstar Omega plate reader</td>
			<td></td>
		</tr>
		<tr>
			<td>Q700 sonicator</td>
			<td>QSonica</td>
			<td>Q700-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant hamster prion protein</td>
			<td>MNPRO</td>
			<td>MNPROtein-Hamster</td>
			<td>syrian hamster, amino acids 90-231</td>
		</tr>
		<tr>
			<td>Savant speedvac&#160;</td>
			<td>Thermo Scientific</td>
			<td>SPD1030-230</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica nanospheres (50 nm)</td>
			<td>nano Composix&#160;</td>
			<td>SISN50-25M</td>
			<td></td>
		</tr>
	</tbody>
</table>

prion safety laboratory swipe test

Transmission of iatrogenic prion disease has occurred from contaminated neurosurgical tools, transplant materials, and occupational exposure to prion-contaminated laboratory tools. Prions cause disease by the templated misfolding of the normal cellular form of the prion protein, PrPC, into the misfolded and pathogenic form PrPSc and are invariably fatal. Reducing iatrogenic and occupational prion transmission is challenging. First, prions can bind to and persist on surfaces for long periods of time. Second, prions are highly resistant to inactivation. Given this, surfaces can retain infectivity for long periods of time following ineffective decontamination. Not only can this pose a potential occupational risk for prion laboratory workers, but it could potentially cross-contaminate laboratory experiments utilizing sensitive prion amplification techniques. The protocol described here for a prion safety laboratory swipe test includes steps for the identification and documentation of high-traffic laboratory areas, recommended swabbing controls to ensure the validity of results, steps to identify proper responses to positive surface swabbing sites, representative results from prion swipe testing, as well as potential artifactual results. Overall, the prion safety laboratory swipe test can be implemented as part of a broader prion safety program to assess decontamination of surfaces, monitor common spaces for prion contamination, and implement the documentation of prion decontamination status.

Written description of positive and negative results (including positive/negative plate and swab controls)
Negative control swabs are included in the surveillance swabbing to monitor for potential prion contamination that could be introduced during the swabbing, extraction, and concentration process. The first RT-QuIC plate performed for a given monthly surveillance should include the positive and negative swab controls. Successful negative controls fail to cross the positive fluorescence threshold (Figure 6A). This result would indicate that contamination had not been introduced during the experimental procedures. Successful positive control swab extracts would exhibit positive seeding in all replicate wells for a given sample (positive control swabs were contaminated with the hamster-adapted transmissible mink encephalopathy strain Hyper (HY TME) brain homogenate. The inclusion of a positive control dilution series allows for the determination of the sensitivity of prion detection for a given experiment (Figure 6A).
When examining surface swab extract samples, surfaces that fail to show seeding above the predetermined positive fluorescence threshold can be considered to be prion negative (Figure 6B). Conversely, prion-contaminated surface swab extracts will show seeding capabilities above the positive fluorescence threshold, although the maxpoint ratio (MPR) and time to fluorescence can vary compared to the included positive plate control (Figure 6B). The ability of the method to assess adequate disinfection is highlighted by the bleach-treated prion contaminated surfaces, which now fail to seed RT-QuIC (Figure 6B).
Importantly, while our laboratory defines a positive sample as a sample that passes the set positive fluorescence threshold in at least half of the replicate wells, it is necessary for each laboratory to set its own standards. The rate of amyloid formation (RAF) and time to fluorescence can also be used to help establish laboratory-specific thresholds for positivity.
We have observed surface artifact results that pass the positive fluorescence threshold but with altered kinetic curves and after an abnormally long time to fluorescence compared to positive control samples (Figure 6C). These results should be cautiously interpreted as they may be caused by the presence of dust or residual chemicals present on a surface. These findings highlight the necessity of general laboratory cleanliness, as well as criteria for differentiating between true positive and false positive seeding.
Disposal of solid and liquid biohazard waste should be done following a given institution&#39;s current biohazard disposal guidelines. Common prion disinfection techniques include treatment with sodium hydroxide, sodium hypochlorite (bleach), or autoclaving at 134 &#176;C for 18 min43,44,45,46.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67889/67889fig01.jpg" /> 
Figure 1: Schematic overview of prion safety laboratory swipe test. <a href="https://www.jove.com/files/ftp_upload/67889/67889fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67889/67889fig02.jpg" /> 
Figure 2: Sample swabbing site layout. <a href="https://www.jove.com/files/ftp_upload/67889/67889fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67889/67889fig03.jpg" /> 
Figure 3:&#160;Sample experiment design for swab controls included prion safety laboratory swipe test. (A) Sample layout for negative and positive swab extract controls. (B) Negative and positive swab extract controls should be resuspended with 50 &#181;L of H2O. A 10-fold dilution of the resuspended swab extract should be generated by diluting it into a tissue dilution solution. <a href="https://www.jove.com/files/ftp_upload/67889/67889fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67889/67889fig04.jpg" /> 
Figure 4:&#160;Sample experiment design for surface swab extracts from prion safety laboratory swipe test. (A) Sample layout for surface swab extracts. (B) Surface swab extract samples should be resuspended with 20 &#181;L of tissue dilution solution. <a href="https://www.jove.com/files/ftp_upload/67889/67889fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67889/67889fig05.jpg" /> 
Figure 5: Monthly prion safety laboratory swipe test. Flowchart used for interpreting results and determining appropriate response. <a href="https://www.jove.com/files/ftp_upload/67889/67889fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/67889/67889fig06.jpg" /> 
Figure 6: Representative surface swabbing experiment. (A) Surface swab control plate containing negative swab extract controls in triplicate (DPBS, uninfected hamster brain homogenate 10-4 and 10-10) and positive swab extract controls in triplicate (HY brain homogenate 10-3). (B) Representative swab extracts for benchtop, glass, and stainless steel (S.S.) surfaces that were swabbed prior to contamination, following contamination with HY 10-3, and following bleach treatment of contaminated surfaces. (C) Fluorescence tracing comparison of uninfected brain homogenate 10-4, HY brain homogenate 10-3, and the swab extract from the benchtop coated with a fine film of dust. Negative plate controls for panels A and B include a blank (tissue dilution solution) and uninfected hamster brain homogenate 10-4. Panel B includes the addition of uninfected brain homogenate 10-4 and the positive plate control of HY brain homogenate 10-3. A positive fluorescence threshold (illustrated by a red dashed line) was determined to be at 2. The maxpoint ratio (MPR) reported is the maximum fluorescence divided by the initial fluorescence reading obtained by the plate reader. Each point represents one technical well replicate for a given sample type. The mean and standard deviation are presented. <a href="https://www.jove.com/files/ftp_upload/67889/67889fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Sample swabbing site monthly documentation form. <a href="https://www.jove.com/files/ftp_upload/67889/Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary Figure 1: Swabbing site layout template. <a href="https://www.jove.com/files/ftp_upload/67889/Supplementary Figure 1.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Swabbing site monthly documentation form. <a href="https://www.jove.com/files/ftp_upload/67889/Supplementary Figure 2.pdf" target="_blank">Please click here to download this File.</a>

A method to assess commonly used areas in laboratory settings for prion contamination and effective decontamination is lacking. The protocol described here provides key fundamentals for implementing a laboratory prion safety swipe test that can easily be modified to meet the individual needs of specific laboratories.

Prion Safety Laboratory Swipe Test

Transmission of iatrogenic prion disease has occurred from contaminated neurosurgical tools, transplant materials, and occupational exposure to ...

Research

JoVE Journal

Immunology and Infection

348 Views.  Creighton University. A method to assess commonly used areas in laboratory settings for prion contamination and effective decontamination is lacking. The protocol described here provides key fundamentals for implementing a laboratory prion safety swipe test that can easily be modified to meet the individual needs of specific laboratories.

Video: Prion Safety Laboratory Swipe Test

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

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella 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 Salmonella-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 in situ growth of Salmonella 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 Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

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

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 Salmonella using fluorescence in situ hybridization (FISH).

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

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such as Chronic Wasting Disease (CWD) of cervids and scrapie in sheep. The Prion hypothesis asserts that this abnormal conformer constitutes most or all of the infectious prion. The role of the immune system in early events in peripheral prion pathogenesis has been convincingly demonstrated for CWD and scrapie 1-3. Transgenic and pharmacologic studies in mice revealed an important role of the Complement system in retaining and replicating prions early after infection 4-6. In vitro and in vivo studies have also observed prion retention by dendritic cells 7-10, although their role in trafficking remains unclear 11-16. Macrophages have similarly been implicated in early prion pathogenesis, but these studies have focused on events occurring weeks after infection 3,11,17. These prior studies also suffer from the problem of differentiating between endogenous PrPC and infectious prions. Here we describe a semiquantitative, unbiased approach for assessing prion uptake and trafficking from the inoculation site by immune cells recruited there. Aggregated prion rods were purified from infected brain homogenate by detergent solubilization of non-aggregated proteins and ultracentrifugation through a sucrose cushion. Polyacrylamide gel electrophoresis, coomassie blue staining and western blotting confirmed recovery of highly enriched prion rods in the pelleted fraction. Prion rods were fluorochrome-labeled then injected intraperitoneally into mice. Two hours later immune cells from peritoneal lavage fluid, spleen and mediastinal and mesenteric lymph nodes were assayed for prion rod retention and cell subsets identified by multicolor flow cytometry using markers for monocytes, neutrophils, dendritic cells, macrophages and B and T cells. This assay allows for the first time direct monitoring of immune cells acquiring and trafficking prions in vivo within hours after infection. This assay also clearly differentiates infectious, aggregated prions from PrPC normally expressed on host cells, which can be difficult and lead to data interpretation problems in other assay systems. This protocol can be adapted to other inoculation routes (oral, intravenous, intranervous and subcutaneous, e.g.) and antigens (conjugated beads, bacterial, viral and parasitic pathogens and proteins, egg) as well.

Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like allergic reactions 1. In clinical practice, diagnosis of drug hypersensitivity is mainly performed by patient history, as skin testing is not reliable and oral provocation testing bears life-threatening risks for the patient 2. Hence, evidence for an underlying IgE-mediated pathomechanism is hard to obtain. 
Here, we present an in vitro method based on the use of human basophils derived from drug-hypersensitive patients that mimics the allergic effector reaction in vivo. As basophils of drug-allergic patients carry IgE molecules specific for the culprit drug, they become activated upon IgE receptor crosslinking and release allergic effector molecules. The activation of basophils can be monitored by the determination of the upregulation of CD63 surface expression using flow cytometry 3. 
In the case of low molecular weight drugs, conjugates are designed to enable IgE receptor crosslinking on basophils. As depicted in Figure 1, two representatives of NSAIDs, PP and DF, are covalently bound to human serum albumin (HSA) via a carboxyl group reacting with the primary amino group of lysine residues. DF carries an intrinsic carboxyl group and, thus, can be used directly 4, whereas a carboxyl group-containing derivative of PP had to be organochemically synthesized prior to the study 1.
The coupling degree of the low molecular weight compounds on the protein carrier molecule and their spatial distribution is important to guarantee crosslinking of two IgE receptor molecules. The here described protocol applies high performance-size exclusion chromatography (HPSEC) equipped with a sequential refractive index (RI) and ultra violet (UV) detection system for determination of the coupling degree.
As the described methodology may be applied for other drugs, the basophil activation test (BAT) bears the potential to be used for the determination of IgE-mediated mechanisms in drug hypersensitivity. Here, we determine PP hypersensitivity as IgE-mediated and DF hypersensitivity as non-IgE-mediated by BAT.

Basophil activation test is a potent tool for the detection of IgE-dependent allergies in vitro. Here, an optimized protocol for basophil activation test is used to investigate drug hypersensitivity. A method for the efficient production of covalent drug-protein conjugates and their physicochemical characterization is described.

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like ...

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models of ALI are key in enhancing our understanding of disease pathogenesis. Lipopolysaccharide (LPS) derived from gram positive bacteria induces neutrophilic inflammation in the airways and lung parenchyma of mice. Efficient pulmonary delivery of compounds such as LPS is, however, difficult to achieve. In the approach described here, pulmonary delivery in mice is achieved by challenge to aerosolized Pseudomonas aeruginosa LPS. Dissolved LPS was aerosolized by a nebulizer connected to compressed air. Mice were exposed to a continuous flow of LPS aerosol in a Plexiglas box for 10 min, followed by 2 min conditioning after the aerosol was discontinued. Tracheal intubation and subsequent bronchoalveolar lavage, followed by formalin perfusion was next performed, which allows for characterization of the sterile pulmonary inflammation. Aerosolized LPS generates a pulmonary inflammation characterized by alveolar neutrophilia, detected in bronchoalveolar lavage and by histological assessment. This technique can be set up at a small cost with few appliances, and requires minimal training and expertise. The exposure system can thus be routinely performed at any laboratory, with the potential to enhance our understanding of lung pathology.

We describe a method for inducing neutrophilic pulmonary inflammation by challenge to aerosolized lipopolysaccharide by nebulization, to model acute lung injury. In addition, basic surgical techniques for lung isolation, tracheal intubation and bronchoalveolar lavage are also described.

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

Safety Precautions and Operating Procedures in an (A)BSL-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 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.

Safety Precautions and Operating Procedures in an ABSL-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.

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: 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.

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.

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 and Operating Procedures in an (A)BSL-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 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.

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

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.

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

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.

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

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.

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