The authors would like to acknowledge their gratitude to the U. S. Army Environmental Quality 6.1 Basic Research program, the US Army Institute of Surgical Research, and NSRDEC&#8217;s Continuous Product Improvement program and Expeditionary Base Camp TecD for funding this work. We are grateful to Adam Driks (Loyola University Medical Center) for the micrograph shown in Figure 6B.

1. The Portable Chemical Sterilizer (PCS)
<ol>
	<li>Equipment. The PCS is an innovative device for portable, energy-independent, point-of-use medical sterilization. For these purposes, a commercial PELICAN rigid plastic suitcase was embellished with special design features to accommodate sterilization (Figure 1).</li>
	<li>Equipment Design. a) A wide-mouthed reaction vessel receives the dry chemical reagents and water; b) two check valves installed in the case wall relieves pressure at 1 psi; c) a filtered inlet valve allows air to be pumped in for flushing the chamber; d) a circulating tube distributes air through the chamber post-sterilization; e) disposable dry scrubber (activated carbon) devices engrafted over the outlet check valves remove residuals and ensure the health and safety of the user and environment; and f) stilts in the base of the case accommodate a surgical instrument tray or other perforated tray and maximize gas-flow during flushing.</li>
	<li>Operation. Place the PCS on a level surface and open the lid. Place a surgical tray containing clean, non-sterile instruments and wrapped in blue autoclave paper on the stilts inside the case. Mix dry chemicals and water (e.g., 93 g sodium chlorite, 63 g sodium sulfite, 25 g sodium hydrogen ascorbate, and 300 ml water &#8211; other permutations are possible) in the wide-mouthed reaction vessel to initiate chemical reaction, then close and lock the lid. In under 2 min, the reaction produces copious chlorine dioxide sterilant, heat, and humidity. At 25 min, connect a battery-operated or hand-powered air pump to the inlet valve and flow air into the chamber for approx. 5 min (Figure 2).</li>
	<li>Cycle Completion and Re-use. Open the case and remove the surgical tray of sterile instruments. Dispose of the reaction vessel (water and benign chemical salts). The PCS is available for immediate re-use with another tray of surgical instruments and a fresh set of dry chemicals and water.</li>
	<li>Validating Sterilization with Bio-indicators. Place commercially available B/T Sure Biological Indicators containing spores impregnated on paper (~105 spores/unit) of either G. stearothermophilus (used for wet heat) or B. atrophaeus (used for ethylene oxide gas sterilization) inside the case for a sterilization cycle. At completion of the ClO2 sterilization cycle, remove and activate the indicators, then incubate indicators for 24-48 hr&#160;to validate sterilization.</li>
	<li>Validating Sterilization with Spore Suspensions. Place aqueous suspensions of G. stearothermophilus spores (~105 cfu/ml) inside the PCS for exposure to a ClO2 sterilization cycle. Recover G. stearothermophilus spores exposed to the chlorine dioxide treatments on Antibiotic Assay Medium with 1% soluble starch8 (no recovery indicates sterility). Examine refractility of treated spores with phase contrast microscopy (spores inactivated by ClO2 retain phase bright character14).</li>
	<li>Validating Sterilization of Hard Surfaces. Inoculate hard, non-porous surfaces made of glass or metal with aqueous suspensions (~105) of G. stearothermophilus spores. Place inoculated materials into the PCS for treatment with a ClO2 sterilization cycle. Sampling the treated surfaces with commercially available Difco HY-Check swabs and obtaining no-growth confirms sterility.</li>
</ol>
2. &#8220;D-FENS&#8221;
<ol>
	<li>Equipment. &#8220;D-FENS&#8221; is a collapsible handheld bottle fitted with a hand-operated spray-trigger device. The flexible plastic bottle has a gusseted bottom to stand-up when full, and the chemically-resistant plastic affords multiple re-uses per sprayer (Figure 3).</li>
	<li>Generate Aqueous ClO2 Solution. D-FENS uses 1-10 g total quantity of dry reagents to generate up to 800 ml of 50 &#8722; 500 ppm chlorine dioxide solution (e.g., 4.7 g sodium chlorite, 1.6 g sodium sulfite, and 1.3 g sodium ascorbate, with permutations possible). Use &#8220;kinetics control,&#8221; a novel 2-step mixing process comprising: i. Pre-concentration &#8722; dissolving all reagents in a small volume, and ii. Post-reaction dilution &#8211; diluting the solution to its final working volume, to generate aqueous chlorine dioxide solutions in the spray-bottle in 2-9 min. The disinfectant solution is sprayed as a fine mist or aerosol to disinfect or decontaminate intended surfaces. The chlorine dioxide solution in D-FENS remains stable for a minimum 8&#160;hr shift and any remaining solution can be emptied down sink or floor drains at the end of a shift to purge biofilms.</li>
	<li>Microbiological Validation &#8211; inoculating porous surfaces. Prepare Petri dishes of Baird-Parker Agar (BPA) containing egg yolk tellurite (EYT) and Yeast Extract (YE) and inoculate agar surface with 0.1 ml of a ~106 cfu/ml suspension of a 3-strain cocktail of Staphylococcus aureus (S. aureus A-100 that produces enterotoxin A, S. aureus ATCC 14458 that produces enterotoxin B, and S. aureus 993 that produces enterotoxin D)7. S. aureus was selected as the target organism because it produces distinctively obvious black colonies, if not inactivated.</li>
	<li>Microbiological Validation &#8211; Testing Porous Surfaces. Using the D-FENS spray bottle containing chlorine dioxide solution, spray the disinfectant solution onto the porous agar surface. Use consistent, steady force to dispense approximately equal volumes of solution per spray-trigger pulse. Rotate the plates 90&#186; between successive pulses to apply uniform coverage of the agar surface. Also use this technique with a glass hockey stick and applying light pressure to spread chlorine dioxide solution over the agar surface for mechanical abrasion (equivalent to wiping or scrubbing &#8211; see Figure 4).</li>
	<li>Microbiological Validation &#8211; Hard Surfaces. Inoculate sterilized custom stainless steel (type 304) coupons (4&#8221; x&#160;4&#8221;, with total surface area 10.16 cm2) with 0.2 ml volume of aqueous suspensions of bacterial cells (e.g., S. aureus, Escherichia coli, or Listeria monocytogenes) and spread inocula uniformly across coupon surface. Air-dry coupons for 30 min at room temperature in laminar flow hood. Pick up inoculated coupons with sterile forceps and immerse in 20 ml test solution containing chlorine dioxide in a 100 ml stomacher bag. After contact times of 0.5 to 5 min, quench the sanitizer solution by adding a small amount of sodium sulfite solid, then masticate solution for 2 min in a stomacher. Remove bag and in a laminar flow hood withdraw supernatant and serially dilute solution onto pre-made agar plates, then incubate for 24 hr to enumerate survivors.</li>
</ol>
3. The PCS for Fresh Fruits and Vegetables
The ability of reduced PCS treatments to kill harmful foodborne pathogens (E. coli and L. monocytogenes) on fresh produce was tested using a spot-inoculation method in which high levels of pathogens were spotted onto the exterior surfaces of tomato wedges.
<ol>
	<li>Inoculation. Inoculate the exterior surfaces of 25 gram samples of tomato wedges with either 105 CFU/g L. monocytogenes OSY-8578 or with 106 CFU/g E. coli ATCC 11229, then air-dry in a sterile bio-hood for 15 min.</li>
	<li>PCS treatment. After the inoculum dries, place the tomato wedges (wear sterile gloves) in the PCS and test under various conditions of chlorine dioxide concentration and exposure time. In some instances, place spore bio-indicators of G. stearothermophilus and B. atrophaeus inside the PCS to accompany the tomato wedges and validate the sterilization treatment (Figure 5).</li>
	<li>Microbial Recovery. After the PCS treatment, place tomato wedges in a stomacher bag with 50 ml of aqueous phosphate buffer (pH 7), then masticate for 2 min with a stomacher blender.</li>
	<li>Enumeration. Dispense the masticated solution as serially 10-fold dilutions onto agar plates Tryptic Soy Agar-Yeast Extract (TSAYE) and nutrient agar (NA) for L. monocyotgenes and E. coli, respectively, and spread with a glass hockey stick, cover and incubate plates at T = 35 &#176;C for 24-48 hr. Enumerate survivors using a colony counter to confirm microbial inactivation.</li>
	<li>Inactivating Polyphenoloxidase (&#8220;browning&#8221;) Enzymes. Place uninoculated apple slices in separate Petri dishes inside the PCS and expose to chlorine dioxide (Figure 5). After treatment, remove the Petri dishes and expose the apple slices in the ambient environment. Visual observation showed no browning for up to 1 week post-treatment.</li>
</ol>
4. &#8220;D-FEND ALL&#8221;
<ol>
	<li>Generate Aqueous ClO2 Solution. D-FEND ALL uses small quantities of dry chemicals (chlorite and SAMIA) in water to generate chlorine dioxide solution in 0.5-3.0 min. For example, mix 0.8-3.3 g of reagents in 15-1,200 ml of aqueous solution to produce a chlorine dioxide solution.</li>
	<li>Microbiological Validation &#8211; Textiles. Inoculate sterile strips of textile samples with aqueous suspensions of Bacillus anthracis Sterne or Bacillus amyloliquefaciens spores, and let textile strips air-dry in a laminar flow hood. Pick up strips with sterile forceps and immerse in 20 ml of chlorine dioxide solution in a 100 ml stomacher bag. At 10 min, quench process without affecting spores by adding a small amount of sodium sulfite solid, then masticate textile strip and solution for 2 min in a stomacher. Remove bag and in a laminar flow hood serially dilute solution onto pre-made agar plates, then incubate for 24 hr and enumerate to validate decontamination.</li>
</ol>

<ol>
	<li>Curtin, M. A., Taub, I. A., Kustin, K., Sao, N., Duvall, J. R., Davies, K., Doona, C. J., Ross, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ascorbate-induced+oxidation+of+formate+by+peroxodisulfate:+product+yields,+kinetics+and+mechanism.">Ascorbate-induced oxidation of formate by peroxodisulfate: product yields, kinetics and mechanism.</a> Research on Chemical Intermediates. 30 (6), 647-661 (2004).</li><li>Curtin, M. A., Dwyer, S., Bukvic, D., Doona, C. J., Kustin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+and+mechanism+of+the+reduction+of+sodium+chlorite+by+sodium+hydrogen+ascorbate+in+aqueous+solution+at+near-neutral+pH.">Kinetics and mechanism of the reduction of sodium chlorite by sodium hydrogen ascorbate in aqueous solution at near-neutral pH.</a> International Journal of Chemical Kinetics. 46 (4), 216-219 (2014).</li><li>Doona, C. J., Curtin, M. A., Feeherry, F. E., Kandlikar, S., Baer, D., Kustin, K., Taub, I., McManus, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Portable Chemical Sterilizer., U.S. Patent Number 7,625,533. , (2009).</li><li>Doona, C. J., Curtin, M. A., Taub, I. A., Kustin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemical Combination for the Generation of Disinfectant and Heat., U.S. Patent Number 7,883,640. , (2011).</li><li>Doona, C. J., Feeherry, F. E., Kustin, K., Curtin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Process for producing aqueous chlorine dioxide for surface disinfection and decontamination., U.S. Patent Application Number 8,337,717. , (2012).</li><li>Doona, C. J., Feeherry, F. E., Kustin, K., Feng, H., Grove, S., Krishnamurthy, K., Lee, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Combining sanitizers and nonthermal processing technologies to improve fresh-cut produce safety. In: Electron beam pasteurization and complementary food processing technologies. , (2014).</li><li>Feeherry, F. E., Doona, C. J., Taub, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+water+activity+on+the+growth+kinetics+of+Staphylococcus+aureus+in+ground+bread+crumb.">Effect of water activity on the growth kinetics of Staphylococcus aureus in ground bread crumb.</a> Journal of Food Science. 68 (3), 982 (2003).</li><li>Feeherry, F. E., Munsey, D. T., Rowley, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+inactivation+and+injury+of+Bacillus+stearothermophilus+spores.">Thermal inactivation and injury of Bacillus stearothermophilus spores.</a> Applied and Environmental Microbiology. 53 (2), 365 (1987).</li><li>G&#243;mez-L&#243;pez, V. M., Devlieghere, F., Ragaert, P., Debevere, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shelf-life+extension+of+minimally+processed+carrots+by+gaseous+chlorine+dioxide.">Shelf-life extension of minimally processed carrots by gaseous chlorine dioxide.</a> International Journal of Food Microbiology. 116, 221 (2007).</li><li>Mahmoud, B. S. M., Bhagat, A. R., Linton, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+kinetics+of+inoculated+Escherichia+coli+O157:H7,+Listeria+monocytogenes,+and+Salmonella+enterica+on+strawberries+by+chlorine+dioxide+gas.">Inactivation kinetics of inoculated Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica on strawberries by chlorine dioxide gas.</a> Food Microbiology. 24 (7-8), 736 (2007).</li><li>Mahmoud, B. S. M., Linton, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+kinetics+of+inoculated+Escherichia+coli+O175:H7+and+Salmonella+enterica+on+lettuce+by+chlorine+dioxide+gas.">Inactivation kinetics of inoculated Escherichia coli O175:H7 and Salmonella enterica on lettuce by chlorine dioxide gas.</a> Food Microbiology. 25 (2), 244 (2008).</li><li>Kim, Y. -. J., Lee, S. -. H., Park, J. i., Park, J. o., Chung, M., Kwon, K., Chung, K., Won, M., Song, K. 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+Escherichia+coli+O157:H7,+Salmonella+typhimurium,+and+Listeria+monocytogenes+on+stored+iceberg+lettuce+by+aqueous+chlorine+dioxide+treatment.">Inactivation of Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes on stored iceberg lettuce by aqueous chlorine dioxide treatment.</a> Journal of Food Science. 73 (9), (2008).</li><li>Park, E. -. J., Gray, P. M., Oh, S. -. W., Kronenberg, J., Kang, D. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+FIT+produce+wash+and+chlorine+dioxide+on+pathogen+control+in+fresh+potatoes.">Efficacy of FIT produce wash and chlorine dioxide on pathogen control in fresh potatoes.</a> Journal of Food Science. 73 (6), (2008).</li><li>Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Spores.">Bacterial Spores.</a> Industrial Pharmaceutical Microbiology. Supplement 10, (2011).</li><li>Setlow, P., Doona, C. J., Feeherry, F. E., Kustin, K., Sisson, D., Chandra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+Safety+and+Extended+Shelf+Life+of+Fresh+Produce+for+the+Military.">Enhanced Safety and Extended Shelf Life of Fresh Produce for the Military.</a> Microbial Safety of Fresh Produce. , 263-288 (2009).</li><li>Taub, I. A., Roberts, W., LaGambina, S., Kustin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+Dihydrogen+Formation+in+the+Magnesium&#8722;Water+Reaction.">Mechanism of Dihydrogen Formation in the Magnesium&#8722;Water Reaction.</a> Journal of Physical Chemistry. 106 (35), 8070 (2002).</li><li>Young, S. B., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+killing+of+Bacillus+subtilis+spores+by+hypochlorite+and+chlorine+dioxide.">Mechanisms of killing of Bacillus subtilis spores by hypochlorite and chlorine dioxide.</a> Journal of Applied Microbiology. 95 (1), 54 (2003).</li></ol>

We have no further disclosures.

This foundational R&#38;D has set new research and technical directions through collaborations with academia, other Government agencies, and industry that have led to the commercialization of novel, environmentally-friendly (&#8220;green&#8221;) technologies. Chlorine dioxide is the first method approved by the National Sanitation Foundation in 20 years for safer, faster, and more environmentally-friendly sterilization than conventional treatments. The PCS, D-FENS, and D-FEND ALL prototypes have been validated as bench-s...

The PCS technology proceeds from where no commercial device existed previously and generates the disinfectant chlorine dioxide (ClO2) that has a proven ability to kill vegetative pathogens on fresh produce3,6,9-13,15&#160;or to decontaminate bacterial spores.6,14,15,17&#160;The PCS has been laboratory validated specifically to effectuate sterilization against live cultures of&#160;Geobacillus stearothermophilus&#160;(GS) spores (see related reference8) and spore bio-indicators of&#160;G. stearothermophilus&#160;and&#160;Bacillus atrophaeus&#160;(BA)6,15,16. The PCS has also been adapted to operate with less stringent conditions to ensure Food Safety by inactivating the vegetative pathogens&#160;Listeria monocytogenes&#160;and&#160;Escherichia coli&#160;on fresh produce, such as whole tomatoes, and to extend the shelf-life of fresh-cut produce, for example, by inactivating the polyphenoloxidase browning enzyme in sliced apples6,15. To generate chlorine dioxide, the PCS uses novel effector chemistry that proceeds via oxidation-reduction reactions at near-neutral pH, thus eliminating the use of acids and the inherent difficulties of shipping, storing, handling, and disposing acidic wastes in far-forward military deployments1,2,4,17. In addition to the military, the PCS can also be used by Homeland Security/Defense; during natural disasters (Superstorm Sandy, tsunamis, hurricane Katrina) that incapacitate access to power, potable water, and waste removal; on-site by emergency first-responders; and in community hospitals or schools during power outages (blackouts and brown-outs).
The&#160;Disinfectant-sprayer for&#160;Foods and&#160;ENvironmentally-friendly&#160;Sanitation (D-FENS) also uses effector chemistry (3 chemical components) and a 2-step mixing process (i.&#160;pre-concentration followed by&#160;ii.&#160;post-reaction dilution) to generate aqueous chlorine dioxide, primarily in a collapsible spray bottle for decontaminating surfaces of Army materiel, food handling equipment and field feeding equipment in Army field kitchens and sanitation centers and Navy Galleys, medical units, showers, and latrines anywhere large numbers of deployed personnel co-exist in close proximity5,6. Validation testing showed that D-FENS eliminates the pathogen&#160;Staphylococcus aureus, a common foodborne pathogen, on porous surfaces14. &#8220;D-FEND ALL&#8221; (Disinfectant for&#160;ENvironmentally-friendly&#160;Decontamination,&#160;All-purpose) provides a simpler (2 chemical components), more convenient (1-step mixing) alternative with unmatched versatility for producing aqueous chlorine dioxide to decontaminate bacterial spores on textiles, for surface disinfection to promote sanitation and hygiene, and to improve water quality and safety, with particular advantages for applications requiring the rapid production of large volumes of dilute chlorine dioxide solutions using small quantities of starting materials for applications in novel graywater recycling technologies designed to generate clean, potable water for Expeditionary Base Camps2.
A variety of mechanisms exist in accordance with the Federal Technology Transfer Act to facilitate the transfer of federal technologies to nonfederal entities as a way to encourage the development and commercialization of technologies for the material benefit of the nation. Accordingly, with their burgeoning potential for many military and civilian uses, the PCS, D-FENS, and D-FEND ALL technologies have been patented and transferred to industry for commercialization via Patent Licensing Agreements and Commercial Evaluation Licenses. A slow, controlled release version of D-FENS (called &#8220;D-FENS Lite&#8221;) was Technology Transferred to commercial industry for incorporation into packaging materials to extend the shelf-life of fresh berries, and the PCS has also been Technology Transferred to academia and other government agencies for comparative testing with other technologies, for research on Food Safety with fresh produce commodities, and for enhancing undergraduate science education. The Technology Transfer of the PCS and its chemistry led to a commercial product approved for bio-hood sterilization with improvements in time, cost, and environmental protection compared to conventional formaldehyde treatments.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium chlorite</td>
			<td>Sigma-Aldrich</td>
			<td>244155</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium sulfite</td>
			<td>Sigma-Aldrich</td>
			<td>239312</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium ascorbate</td>
			<td>Sigma-Aldrich</td>
			<td>A7631</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Fisher Scientific</td>
			<td>D-16</td>
			<td></td>
		</tr>
		<tr>
			<td>BT Sure biological indicator (steam)</td>
			<td>Thermo Fisher Sci</td>
			<td>AY759X3</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ Test (EtO)</td>
			<td>SGM Biotech Inc</td>
			<td>EZG/6</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Hy-check</td>
			<td>Becton-Dickinson/ Difco</td>
			<td>290002</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic Soy Agar</td>
			<td>Difco</td>
			<td>236950</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutrient Agar</td>
			<td>Difco</td>
			<td>213000</td>
			<td></td>
		</tr>
		<tr>
			<td>Baird-Parker Agar</td>
			<td>Difco</td>
			<td>276840</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg Yolk-Tellurite</td>
			<td>Difco</td>
			<td>277910</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5% Yeast extract</td>
			<td>Difco</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto-Peptone</td>
			<td>Difco</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto-Tryptone</td>
			<td>Difco</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Difco</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Soluble starch</td>
			<td>Difco</td>
			<td>0178-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Lemco Beef Extract</td>
			<td>Oxoid</td>
			<td>L29</td>
			<td></td>
		</tr>
		<tr>
			<td>Masticator - Classic</td>
			<td>IUL Instruments</td>
			<td>Cat. No. 400</td>
			<td></td>
		</tr>
		<tr>
			<td>Stomacher bags</td>
			<td>Seward</td>
			<td>Stomacher &#8216;400&#8217; bags</td>
			<td></td>
		</tr>
	</tbody>
</table>

the portable chemical sterilizer (pcs), d-fens, and d-fend all: novel chlorine dioxide decontamination technologies for the military

There is a stated Army need for a field-portable, non-steam sterilizer technology that can be used by Forward Surgical Teams, Dental Companies, Veterinary Service Support Detachments, Combat Support Hospitals, and Area Medical Laboratories to sterilize surgical instruments and to sterilize pathological specimens prior to disposal in operating rooms, emergency treatment areas, and intensive care units. The following ensemble of novel, &#8216;clean and green&#8217; chlorine dioxide technologies are versatile and flexible to adapt to meet a number of critical military needs for decontamination6,15. Specifically, the Portable Chemical Sterilizer (PCS) was invented to meet urgent battlefield needs and close critical capability gaps for energy-independence, lightweight portability, rapid mobility, and rugged durability in high intensity forward deployments3. As a revolutionary technological breakthrough in surgical sterilization technology, the PCS is a Modern Field Autoclave that relies on on-site, point-of-use, at-will generation of chlorine dioxide instead of steam. Two (2) PCS units sterilize 4 surgical trays in 1 hr, which is the equivalent throughput of one large steam autoclave (nicknamed &#8220;Bertha&#8221; in deployments because of its cumbersome size, bulky dimensions, and weight). However, the PCS operates using 100% less electricity (0 vs. 9 kW) and 98% less water (10 vs. 640 oz.), significantly reduces weight by 95% (20 vs. 450 lbs, a 4-man lift) and cube by 96% (2.1 vs. 60.2 ft3), and virtually eliminates the difficult challenges in forward deployments of repairs and maintaining reliable operation, lifting and transporting, and electrical power required for steam autoclaves.

The easy-to-operate PCS was designed to achieve sterility by inactivating bacterial spore suspensions or bacterial spore bio-indicators in 30-minute treatments involving the controlled production of chlorine dioxide by unique effector chemistry. Specifically, microbiological validation studies verified that the PCS achieved sterility by inactivating bio-indicators containing spores (105 spores/ml) of either G. stearothermophilus or B. atrophaeus, that are intended to indicate sterilization by...

Watch this Scientific Journal Video about The Portable Chemical Sterilizer PCS, D-FENS, and D-FEND ALL: Novel Chlorine Dioxide Decontamination Technologies for the Military at JoVE.com

The Portable Chemical Sterilizer PCS, D-FENS, and D-FEND ALL: Novel Chlorine Dioxide Decontamination Technologies for the Military

The Portable Chemical Sterilizer (PCS) is a revolutionary, energy-independent, almost waterless sterilization technology for Army medical units. The PCS generates chlorine dioxide from dry reagents mixed with water on-site, at-will, and at point-of-use (PoU) in a plastic suitcase. The Disinfectant-sprayer for Foods and ENvironmentally-friendly Sanitation (D-FENS) and the Disinfectant for ENvironmentally-friendly Decontamination, All-purpose (D-FEND ALL) produce aqueous chlorine dioxide in a collapsible spray bottle and other potential embodiments. These versatile decontamination technologies kill microbes in myriad diverse Dual-use applications for military and civilian consumers.

The Portable Chemical Sterilizer (PCS), D-FENS, and D-FEND ALL: Novel Chlorine Dioxide Decontamination Technologies for the Military

There is a stated Army need for a field-portable, non-steam sterilizer technology that can be used by Forward Surgical Teams, Dental Companies, Veterinary ...

the-portable-chemical-sterilizer-pcs-d-fens-d-fend-all-novel-chlorine

Research

JoVE Journal

Bioengineering

14.4K Views.  United States Army-Natick Soldier RD&E Center, Warfighter Directorate. The Portable Chemical Sterilizer (PCS) is a revolutionary, energy-independent, almost waterless sterilization technology for Army medical units. The PCS generates chlorine dioxide from dry reagents mixed with water on-site, at-will, and at point-of-use (PoU) in a plastic suitcase. The Disinfectant-sprayer for Foods and ENvironmentally-friendly Sanitation (D-FENS) and the Disinfectant for ENvironmentally...

Video: The Portable Chemical Sterilizer PCS, D-FENS, and D-FEND ALL: Novel Chlorine Dioxide Decontamination Technologies for the Military

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different infectious diseases. In this article we demonstrate a method to produce a SAPN core containing a six-helix bundle (SHB) assembly that is capable of presenting antigens in a trimeric conformation. We describe the expression of the SHB-SAPN in an E. coli system, as well as the necessary protein purification steps. We included an isopropanol wash step to reduce the residual bacterial lipopolysaccharide. As an indication of the protein identity and purity, the protein reacted with known monoclonal antibodies in Western blot analyses. After refolding, the size of the particles fell in the expected range (20 to 100 nm), which was confirmed by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. The methodology described here is optimized for the SHB-SAPN, however, with only slight modifications it can be applied to other SAPN constructs. This method is also easily transferable to large scale production for GMP manufacturing for human vaccines.

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to develop practical technologies that couple microscopic cell screening with cell isolation at high purity for downstream genetic analysis. Here, the use of photodegradable poly(ethylene glycol) hydrogels for screening and isolation of bacteria with unique growth phenotypes from heterogeneous cell populations is described. The method relies on encapsulating or entrapping cells with the hydrogel, followed by culture, microscopic screening, then use of a high-resolution light patterning tool for spatiotemporal control of hydrogel degradation and release of selected cells into a solution for retrieval. Applying different light patterns allows for control over the morphology of the extracted cell, and patterns such as rings or crosses can be used to retrieve cells with minimal direct UV light exposure to mitigate DNA damage to the isolates. Moreover, the light patterning tool delivers an adjustable light dose to achieve various degradation and cell release rates. It allows for degradation at high resolution, enabling cell retrieval with micron-scale spatial precision. Here, the use of this material to screen and retrieve bacteria from both bulk hydrogels and microfabricated lab-on-a-chip devices is demonstrated. The method is inexpensive, simple, and can be used for common and emerging applications in microbiology, including isolation of bacterial strains with rare growth profiles from mutant libraries and isolation of bacterial consortia with emergent phenotypes for genomic characterizations.

The use of photodegradable hydrogels to isolate bacterial cells by utilizing a high-resolution light pattering tool is reported. Essential experimental procedures, results, and advantages of the process are reviewed. The method enables rapid and inexpensive isolation of targeted bacteria showing rare or unique functions from heterogeneous communities or populations.

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to ...

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.