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

Summary

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.

Abstract

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, ‘clean and green’ chlorine dioxide technologies are versatile and flexible to adapt to meet a number of critical military needs for decontamination^6,15. 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 “Bertha” 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.

Introduction

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 produce^3,6,9-13,15 or to decontaminate bacterial spores.^6,14,15,17 The PCS has been laboratory validated specifically to effectuate sterilization against live cultures of Geobacillus stearothermophilus (GS) spores (see related reference⁸) and spore bio-indicators of G. stearothermophilus and Bacillus atrophaeus (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 Listeria monocytogenes and Escherichia coli 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 apples^6,15. 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 deployments^1,2,4,17. 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 Disinfectant-sprayer for Foods and ENvironmentally-friendly Sanitation (D-FENS) also uses effector chemistry (3 chemical components) and a 2-step mixing process (i. pre-concentration followed by ii. 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 proximity^5,6. Validation testing showed that D-FENS eliminates the pathogen Staphylococcus aureus, a common foodborne pathogen, on porous surfaces¹⁴. “D-FEND ALL” (Disinfectant for ENvironmentally-friendly Decontamination, 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 “D-FENS Lite”) 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.

Protocol

1. The Portable Chemical Sterilizer (PCS)

1. 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).
2. 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.
3. 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 – 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).
4. 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.
5. 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 to validate sterilization.
6. 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).
7. 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.

2. “D-FENS”

1. Equipment. “D-FENS” 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).
2. Generate Aqueous ClO_{2 S}olution. D-FENS uses 1-10 g total quantity of dry reagents to generate up to 800 ml of 50 − 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 “kinetics control,” a novel 2-step mixing process comprising: i. Pre-concentration − dissolving all reagents in a small volume, and ii. Post-reaction dilution – 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 hr shift and any remaining solution can be emptied down sink or floor drains at the end of a shift to purge biofilms.
3. Microbiological Validation – 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.
4. Microbiological Validation – 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 90º 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 – see Figure 4).
5. Microbiological Validation – Hard Surfaces. Inoculate sterilized custom stainless steel (type 304) coupons (4” x 4”, 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.

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.

1. 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.
2. 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).
3. 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.
4. 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 °C for 24-48 hr. Enumerate survivors using a colony counter to confirm microbial inactivation.
5. Inactivating Polyphenoloxidase (“browning”) 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.

4. “D-FEND ALL”

1. Generate Aqueous ClO_{2 S}olution. 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.
2. Microbiological Validation – 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.

Results

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...

Discussion

This foundational R&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 (“green”) 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...

Materials

Name	Company	Catalog Number	Comments
Sodium chlorite	Sigma-Aldrich	244155
Sodium sulfite	Sigma-Aldrich	239312
Sodium ascorbate	Sigma-Aldrich	A7631
Potassium phosphate	Sigma-Aldrich	P0662
Dextrose	Fisher Scientific	D-16
BT Sure biological indicator (steam)	Thermo Fisher Sci	AY759X3
EZ Test (EtO)	SGM Biotech Inc	EZG/6
Difco Hy-check	Becton-Dickinson/ Difco	290002
Tryptic Soy Agar	Difco	236950
Nutrient Agar	Difco	213000
Baird-Parker Agar	Difco	276840
Egg Yolk-Tellurite	Difco	277910
0.5% Yeast extract	Difco	212750
Bacto-Peptone	Difco	211677
Bacto-Tryptone	Difco	211705
Agar	Difco	214010
Soluble starch	Difco	0178-17
Lab Lemco Beef Extract	Oxoid	L29
Masticator - Classic	IUL Instruments	Cat. No. 400
Stomacher bags	Seward	Stomacher ‘400’ bags