Patent Application
20170142962
The present invention relates generally to methods for killing or deactivating bacterial spores.
Spore formation is a sophisticated mechanism by which some Gram positive bacteria survive conditions of external stress and nutrient deprivation by producing a multi-layered protective capsule enclosing their dehydrated and condensed genomic DNA. When such bacterial spores encounter a favorable environment, germination can take place enabling the bacteria to reproduce, and, in the case of pathogenic species, release toxins to cause disease. Bacterial spores possess a coat and membrane structure that is highly impermeable to most molecules that are toxic to the spores. Therefore, spores are highly resistant to damage by heat, radiation, and many of the commonly employed anti-bacterial agents and processes, and generally can only be destroyed by some severe chemical procedures including bleach, oxidizing vapors such as hydrogen peroxide, chlorine dioxide and aqueous ozone as ozone vapor is not efficacious against spores.
People receiving medical care in hospitals and long term care facilities can acquire serious infections called healthcare-associated infections (HAIs). While most types of HAIs are declining, one—caused by the germ Clostridium difficile, (“C. diff”)—remains at historically high levels. C. diff is linked to 14,000 American deaths each year. Those most at risk are people, especially older adults, who take antibiotics and receive long term medical care.
C. diff is an anaerobic, Gram positive bacterium. Normally fastidious in its vegetative state, it is capable of sporulating when environmental conditions no longer support its continued growth. The capacity to form spores enables the organism to persist in the environment (e.g., in soil and on dry surfaces) for extended periods of time.
Current methods of killing or deactivating C. diff include applying bleach, liquid solutions containing hydrogen peroxide, and other biocidal compounds, and/or ultraviolet radiation (UV) to C. diff for a period of time longer than 3 minutes.
Anthrax spores, Bacillus anthracis (“anthrax”) is the pathogenic organism that causes anthrax. Anthrax is a disease that is frequently fatal due to the ability of this bacterium to produce deadly toxins. Anthrax also forms spores. Inhalation of anthrax spores is frequently fatal, particularly if treatment is not started prior to the development of symptoms.
Anthrax spores are also among the most difficult spores to kill or deactivate. Present methods of killing or deactivating anthrax spores involve using pressurized steam at elevated temperatures, or topical treatment with highly caustic concentrated sodium hypochlorite solutions or certain disinfecting foam products.
One of the reasons it is very difficult to kill or deactivate dry spores is due to their tendency to aggregate and form multilayered structures. In addition, the dry spores are extremely hydrophobic and adhere to surfaces and skin very strongly, making it very difficult to mechanically remove them.
U.S. Pat. No. 6,706,243 (“the '243 patent”) titled Apparatus and Method for Cleaning Particulate Matter And Chemical Contamination From a Hand and U.S. Pat. No. 7,008,592 (“the '592 patent”) titled Decontamination Apparatus And Method Using An Activated Cleaning Fluid Mist disclose examples of activating fluids that contain hydrogen peroxide by passing the fluids through a plasma generated by an AC arc as a means for killing bacteria on hands and objects. The '592 patent provided examples of activating hydrogen peroxide solutions containing 3.0 percent hydrogen peroxide, 1.5 percent hydrogen peroxide, 0.75 percent hydrogen peroxide, 0.3 percent hydrogen peroxide, and 0 percent hydrogen peroxide solutions (water) for their effect against bacteria, which is much easier to kill or deactivate than spores. After contacting the specimen with activated solution of 0.3 percent hydrogen peroxide, the culture showed slight growth of bacteria and the 0.0 percent hydrogen peroxide solution (water) showed significant growth of the bacteria culture, and thus, the '592 patent demonstrated no efficacy in killing bacteria with water absent hydrogen peroxide. In addition, spraying a mist of hydrogen peroxide, such as 3 percent or 1.5 percent, is undesirable. According to the Agency of Toxic Substances & Disease Registry, “Vapors, mists, or aerosols of hydrogen peroxide can cause upper airway irritation, inflammation of the nose, hoarseness, shortness of breath, and a sensation of burning or tightness in the chest.” In addition, “exposure to high concentrations can result in severe mucosal congestion of the trachea and bronchi and delayed accumulation of fluid in the lungs.” The '592 patent appears to suggest a user wear a mask or other filter to avoid inhaling the mist. See, col. 8, lines 44-48. The OSHA permissible exposure limit is 1 ppm (averaged over an 8-hour work shift. According to the AIHA ERPG-2 (emergency response planning guideline), the maximum airborne concentration below which it is believed that nearly all individuals could be exposed for up to an hour without experiencing or developing irreversible or other serious health effects or symptoms which could impair an individual's ability to take protective action is 50 ppm. Accordingly, activating fluids that contain hydrogen peroxide, such as, the 3 percent hydrogen peroxide disclosed in the '243 patent and '592 patent and dispersing them as a vapor or mist may not be advisable.
In addition, all of the examples in the '243 patent and the '592 patent utilize a non-thermal AC arc to generate plasma. Non-thermal AC arcs produce plasma using bare metal electrodes draw a high currents, typically in the range of about 1 to 100 amps. The temperature in the vicinity of the plasma may be greater than 200° C. Plasma temperatures in this range generate different species than plasma temperatures that are near room temperature. For example, it is believed that any ozone (O3) generated with higher temperature plasma reacts with generated NO immediately after generation to form NO2 which quenches any ozone formed. In addition, it is believed that various additives may be affected by the temperatures. For example, it is believed that volatile additives such, as, for example, alcohol will quickly evaporate with these temperatures. Further, such evaporation is likely to be inconsistent.
Exemplary methods and solutions for killing or deactivating spores are disclosed herein, An exemplary solution for killing or deactivating a spore includes water and a stabilizer. The solution is activated by a plasma gas to activate the solution. The plasma gas is generated in an ozone generation mode and the activated solution is activated to an activation level that is sufficient to kill or deactivate one or more spores. The activated solution remains at an activation level that is sufficient to kill or deactivate one or more spores for at least about 30 seconds.
An exemplary method of killing or deactivating a spore includes preparing an aqueous solution including at least one additive. The aqueous solution contains less than 0.3% H2O2 prior to being converted to an activated solution by exposing the aqueous solution to a plasma. The activated solution is applied to a surface containing one or more dry spores for a period of time.
Another exemplary solution for killing or deactivating a spore includes water; at least 0.75% by volume of a stabilizer; and less than 10% by volume of an additive. The one or more of the water, stabilizer and additive are activated by a plasma gas generated in an ozone generating mode and the one or more of the water, stabilizer and additive remain activated to a level sufficient to kill one or more spores for at least 30 seconds.
Yet another exemplary solution for killing or deactivating a spore includes water; at least 0.75% by weight of an alcohol; and less than 10% by weight of an additive and one or more of the water, stabilizer and additive are activated by a plasma gas that is operated in an ozone generating mode.
Another exemplary method of killing or deactivating a spore includes applying a fluid comprising an additive to a dry surface containing one or more dry spores; and applying plasma generated in an ozone generating mode to the surface for a period of time.
Another exemplary method of killing or deactivating a spore includes providing a fluid and additive that contains less than about 0.3 percent by volume of H2O2 and exposing a mist or vapor of the fluid and additive to plasma generated in an ozone generating mode to activate the mist or vapor. The activated mist or vapor is applied to a surface containing one or more dry spores for a period of time whereby the spores are killed or deactivated.
These and other features and advantages of the present invention will become better understood with regard to the following description and accompanying drawings in which:
Plasmas, or ionized gases, have one or more free electrons that are not bound to an atom or molecule. Plasmas may be generated using a variety of gases including, air, nitrogen, noble gases (He, Ar, Xe, Kr, etc), oxygen, carbon dioxide and mixtures thereof under an applied electric field. In addition, non-thermal cold plasmas provide high concentrations of energetic and chemically active species. They can operate far from thermodynamic equilibrium with high concentrations of active species and yet remain at a temperature that is substantially the same as room temperature. The energy from the free electrons may be transferred to additional plasma components creating additional ionization, excitation and/or dissociation. Fluid that is contacted with plasma becomes “activated” and is referred to herein as plasma activated fluid, and in some embodiments, the plasma-activated fluid is plasma-activated water.
In some embodiments, plasmas may contain superoxide anions [O2•−], which react with H+ in acidic media to form hydroperoxy radicals, HOO•; [O2•−]+[H+]→[HOO•]. Other radical species may include OH•, NO•, and NO2• in aqueous phase or the presence of air or gas. Treating water with plasma results in plasma activated water that may contain concentrations of one or more of ozone, H2O2, nitrates, nitrites, peroxynitrite, radicals and other active species.
Activating water with plasma to obtain plasma activated water is shown and described in U.S. Patent Application Publication 2014-0322096 A1, titled Sanitization Station Using Plasma Activated Fluid, and U.S. Patent Application Publication 2014-0100277 A1, titled Solutions and Methods of Making Solutions to Kill or Deactivate Spores Microorganisms, Bacteria and Fungus, both of which are incorporated by reference herein in their entirety. U.S. patent application Ser. No. 13/843,189, entitled Methods and Solutions for Killing or Deactivating Spores, filed on Mar. 15, 2013 and International Patent Application No. PCT/US2014/030361, entitled Methods and Solutions for Killing or Deactivating Spores, filed on Mar. 17, 2014, are also incorporated by reference herein in their entirety.
The direct plasma system 100 includes a high voltage wire 101 connected to an electrode 103, a dielectric barrier 108 and a housing 102. The direct plasma produced by the direct plasma system 100 is at or about room temperature. The applied voltage is in the range of 3 kV to 30 kV. The high voltage power source to supply high voltage to electrode 103 may be a high frequency AC power source, a pulsed DC power source, a pulsed AC power source or the like. The power supply can be pulsed with a duty cycle of 0-100% and pulse duration of 1 nanosecond up to 1 microsecond. Because of the dielectric barrier 108, the arc formation is avoided and peak amplitude of plasma current is significantly lower and typically less than 1 amp when the AC power source is used
The direct plasma system 100 is used to kill or deactivate spores 107 through the application of a fluid 105 and plasma 104 to the spores 107. In some embodiments, the fluid being activated contains a stabilizer to stabilize the reactive species that kill or deactivate the spores. The stabilizer stabilizes the reactive species and allows for the fluid 105 to continue to kill or deactivate spores after removal of the plasma 104.
The indirect plasma system 110 includes a high voltage wire 111 connected to an electrode 113, a dielectric barrier 120 and a housing 112. The indirect plasma system 110 also includes ground 119 attached to a screen, perforated material or mesh 114. The indirect plasma system 110 is used to kill or deactivate spores 118 through the application of a fluid 116 and plasma 115 to the spores 118. The indirect plasma produced by the direct plasma system 100 is at or about room temperature. The applied voltage is in the range of 3 kV to 30 kV. The high voltage power source to supply high voltage to electrode 113 may be a high frequency AC power source, a pulsed DC power source, a pulsed AC power source or the like. The power supply can be pulsed with a duty cycle of 0-100% and pulse duration of 1 nanosecond up to 1 microsecond. Because of the dielectric barrier 120, the arc formation is avoided and peak amplitude of plasma current is significantly lower and typically less than 1 amp when the AC power source is used. In some embodiments, the fluid being activated contains a stabilizer to stabilize the reactive species that kill or deactivate the spores. The stabilizer allows for the fluid 105 to continue to kill or deactivate spores after removal of the plasma 104.
In certain embodiments, the fluid includes water. In certain embodiments, the water includes tap water, distilled water, deionized water, potable water, or reverse osmosis water.
In certain embodiments, the additive comprises one or more compounds to reduce the pH of the fluid, increase the supply of reactive oxygen species (ROS), increase the supply of reactive nitrogen species (RNS), and increase the stability of reactive species, such as reactive oxygen and reactive nitrogen species (RONS). Exemplary additives to reduce the pH include acids. Exemplary additives to increase the supply of reactive oxygen species include enzymes and hydrogen peroxide (H2O2). If hydrogen peroxide is used, the concentration of hydrogen peroxide of the fluid being activated, is less than about 1% hydrogen peroxide. Exemplary additives to increase the supply of reactive nitrogen species include enzymes, nitrites, and transition metals.
Exemplary additives to stabilize reactive species include alcohols. In certain embodiments, the alcohol includes one or more of ethanol (EtOH), isopropyl alcohol, and n-propyl alcohol.
Other exemplary additives include bioactive oils. In certain embodiments, the nitrite includes sodium nitrite or nitrous acid. In certain embodiments, the bioactive oil includes one or more of cinnamaldehyde, carvacrol, coconut oil, grape seed oil, thyme oil and olive oil. In certain embodiments, the acid includes one or more of acetic acid, citric acid, nitrous acid, nitric acid, and hydrochloric acid (HCl). In certain embodiments, the transition metal includes one or more of zinc and cadmium. In certain embodiments, the enzyme includes one or more of superoxide dismutase and nitrate reductase. Although these additives may not stabilize the species, they act synergistically with the plasma activated fluid.
The additive can be present in the fluid to any extent necessary to provide improved killing or deactivation of spores. Where the additive includes an alcohol, the fluid preferably contains at least about 0.75%, including about 30%, including about 50%, including about 70% or more alcohol. Where the additive is an additive other than an alcohol, the fluid preferably contains no more than about 10% of the additive, including about 1%, including about 0.1%, including about 0.01%, including about 0.001%, and including about 0.0001% of the additive. Where the additive is an alcohol and is being used as a stabilizer, the fluid preferably contains at least about 0.75% of alcohol by volume.
The fluid can be applied to the spores in any form that allows for effective killing or deactivation of the spores. In certain embodiments, the fluid contains electrostatically charged droplets and is applied to the spores as individual droplets. In certain embodiments, the fluid forms a thin film of liquid on the spores. In certain embodiments, the thin film has a thickness of less than about 500 microns, including about 400 microns, about 300 microns, about 200 microns, about 100 microns, or less.
The surface may be any surface, such as, for example, table, a bed, etc. made of polymer, metal, rubber, glass, silicone, fabric material or the like. The surface may be a hard surface or a soft surface, such as, for example, linens, curtains and the like. In addition, the surface may be tissue or skin. After the fluid containing the additive is applied to the surface, the surface is treated with plasma at block 134 (
Treatment time may vary depending on the surface and the spore to be deactivated or killed. In certain embodiments, the surface is treated for about 5 minutes. In certain embodiments, the surface is treated for less than about 5 minutes. In certain embodiments, the surface is treated for less than about 3 minutes. In certain embodiments, the surface is treated for less than about 1 minute. In certain embodiments, the surface is treated for about 30 seconds or less. In certain embodiments, the surface is treated for about 5 seconds or less. In certain embodiments, the surface is treated for about 2 seconds. In certain embodiments, the surface is treated for more than about 5 minutes. After the surface has been treated with plasma, the methodology ends at block 136.
Treating the surface with plasma activates the fluid, such as water, which penetrates the shell of the spore and kills or deactivates the spores. In certain embodiments, the plasma contacts the spores directly between droplets or vapor and creates an opening for the activated fluid to penetrate the shell of the spore to kill or deactivate the spore.
In certain embodiments, the methodology 130 generates one or more reactive species in the fluid. In certain embodiments, the reactive species include one or more of reactive oxygen and reactive nitrogen species. In certain embodiments, the reactive nitrogen species includes peroxynitrite, which has a half-life of around 1 second. The misted fluid has a relatively large surface area compared with non-misted fluid in a container, and the large surface area allows the plasma to activate the misted fluid quickly and more effectively, as higher concentrations of reactive oxygen and nitrogen species such as ozone, hydroxyl radicals, superoxide, singlet oxygen, hydrogen peroxide, nitrites and nitrates are generated. It also allows the generation of peroxynitrite, which almost immediately contacts the spore surface, as opposed to having to migrate through a larger volume of water to make contact with the spores. Thus, peroxynitrite may contact the spore prior to its degeneration. It is desirable to stabilize the reactive species to improve the ability to kill or deactivate the spores and also prolong the activity of the reactive species. In certain embodiments, the fluid includes an additive that stabilizes one or more of the reactive species. In certain embodiments, the fluid includes an additive provides stable sporicidal species after activation by plasma. In certain embodiments, the stabilizing additive is an alcohol. In certain embodiments, the additive stabilizes a reactive oxygen species. In certain embodiments, the additive stabilizes a reactive nitrogen species. In certain embodiments, the additive stabilizes both reactive oxygen and reactive nitrogen species. In certain embodiments, the additive stabilizes peroxynitrite. In certain embodiments, the addition of alcohol to the fluid, such as water, provides stable sporicidal species, such as peroxy acid, after activation by plasma. In certain embodiments, the addition of alcohol to the fluid, such as water, provides stable sporicidal species which is more volatile than alcohol after activation by plasma. When alcohol is used as a stabilizer and plasma is generated in ambient air at atmospheric pressure, the plasma operates in an ozone mode in order to produce stable sporicidal species. The plasma operating in the ozone mode in ambient air conditions includes DBD with a power density lower than 0.25 (W/cm2) and corona discharges.
In the exemplary methodology 130, plasma is applied to the fluid on the surface and activates the fluid. Thus, the short live species immediately contact the spores. Stabilizers provide greater efficacy in such situations, when the plasma source is removed from the fluid as the reactive species last longer and can continue to kill or deactivate spores. In embodiments, where the fluid with an additive is first activated then applied to the surface, stabilizers become more important. It has been discovered that without the use of stabilizers, the life of the reactive species that are effective against spores is very short, such as a few seconds. Thus, it would be difficult to apply the fluid to effectively kill spores absent a stabilizer or absent applying the fluid immediately after activation or simultaneously with the activation.
Also shown in
The second 220 single-dielectric plasma system is also similarly configured. The combination of plasma working gas and a fluid containing an additive 221 are added to the second 220 single-dielectric plasma system. The working gas is the gas used to generate the plasma 228, and can be any of the gases used to generate plasma described above. The second 220 single-dielectric plasma system includes a high voltage electrode 222, dielectric materials 223, and a ground electrode 227. In the second 220 single-dielectric plasma system, the high-voltage electrode 222 includes a mesh or perforated material through which the plasma 228 is generated in the vicinity of the electrode 222 and the inner surface of the dielectric material 223. The second 220 single-dielectric plasma produced is at or about room temperature. The applied voltage is in the range of 3 kV to 30 kV. The high voltage power source to supply high voltage to electrode 222 may be a high frequency AC power source, a pulsed DC power source, a pulsed AC power source or the like. The power supply can be pulsed with a duty cycle of 0-100% and pulse duration of 1 nanosecond up to 1 microsecond. Because of the dielectric barrier 223, the arc formation is avoided and peak amplitude of plasma current is significantly lower and typically less than 1 amp when the AC power source is used. The second 220 single-dielectric plasma system also includes a nozzle 224 from which activated fluid 225 is released onto a contaminated surface 226. The contaminated surface 226 can be any of the various surfaces described above and can be contaminated with one or more C. diff, anthrax and other spores.
The methodology continues at block 234. At block 234, a plasma working gas mixed with the mist or vapor is passed through a plasma zone to activate the mist or vapor. The working gas can be any of the working gases described above and the plasma zone is made of non-thermal plasma, which can be generated using any of the plasma generators described above. As described above, in certain embodiments, activation of the mist or vapor with the plasma results in the fluid containing electrostatically charged droplets.
In certain embodiments, activation of the mist or vapor with the plasma results in the production of one or more reactive species including one or more reactive oxygen and reactive nitrogen species. In certain embodiments, the one or more reactive nitrogen species includes peroxynitrite. Because these reactive species help kill or deactivate spores, but otherwise may have a short half-life, in certain embodiments, it is desirable that the mist or vapor includes fluid with an additive that stabilizes one or more of these reactive species, such as an alcohol.
At block 236, the methodology continues with the application of the activated mist or vapor to a surface containing one or more dry spores for a period of time sufficient to kill or deactivate the spores on the surface. After the application of the activated mist or vapor to a surface, the methodology ends at block 238.
Application of the activated mist or vapor to the surface can result in the fluid forming individual droplets over one or more spores on the surface or can result in the fluid forming a film over one or more spores on the surface. The surface may be any surface, such as the various surfaces described above. Depending on the spore and the surface, the period of time sufficient to kill or deactivate the spore can vary, but generally application periods of time of less than 5 minutes, including about 3 minutes, about 1 minute, and about 30 seconds are sufficient.
Where killing or deactivation of spores relies, at least in part, on the generation of one or more reactive species, because of the short half-life of some species e.g., 1-second, the activated mist or vapor generally needs to be applied to the surface immediately after activation, or activated while on the surface to be treated. Where the mist or vapor includes a fluid with an additive that can stabilize the reactive species the activated mist or vapor may be applied to the surface some period of time after the mist or vapor is activated. Appropriate periods of time after activation include, but are not limited to, greater than about 15 seconds, including at least about 30 seconds, at least about 1 minute, at least about 2 minutes, at least about 3 minutes, and at least about 5 minutes after activation. The activated plasma mist or vapor with the stabilizer can be directly applied to a spore-containing surface after a period of time. In certain embodiments, the activated plasma mist or vapor is collected as a liquid. The liquid can then be applied to a spore-containing surface. In certain embodiments, liquids obtained from plasma activated mists or vapor have greater stability of reactive species than liquids directly activated by plasma and the mist or vapor from which the liquid is collected. In certain embodiments, a liquid containing a stabilizer obtained from plasma activated mist or vapor can be applied to a spore-containing surface greater than 1 minute, including greater than 3 minutes, including greater than 5 minutes after the mist or vapor is activated by plasma. Exemplary systems for generating plasma activated mist or vapor and collecting the plasma activated mist or vapor as a liquid are shown in
The direct plasma system 300 includes a high voltage wire 301 connected to an electrode 303, a dielectric barrier 308, a ground 306, and a housing 302. The direct plasma 304 produced is at or about room temperature. Because of the dielectric barrier 308, the arc formation is avoided and peak amplitude of plasma current is significantly lower and typically less than 1 amp when the AC power source is used. The direct plasma system 300 is used to kill or deactivate spores through the application of an aqueous solution with an additive 305, which has been activated by plasma 304, to one or more spores.
The indirect plasma system 310 includes a high voltage wire 311 connected to an electrode 313, a dielectric barrier 319 and a housing 312. The indirect plasma system 310 also includes grounds 314 and 318. The indirect plasma produced is at or about room temperature. The applied voltage is in the range of 3 kV to 30 kV. The high voltage power source to supply high voltage to electrode 313 may be a high frequency AC power source, a pulsed DC power source, a pulsed AC power source or the like. The power supply can be pulsed with a duty cycle of 0-100% and pulse duration of 1 nanosecond up to 1 microsecond. Because of the dielectric barrier 319, the arc formation is avoided and peak amplitude of plasma current is significantly lower and typically less than 1 amp when the AC power source is used. The indirect plasma system 310 uses plasma 315 to activate an aqueous solution with an additive 316 which may be present in a container 317. The activated aqueous solution with an additive 316 can then be used to kill or deactivate spores for a period of time after activation, provided that the additive 316 is a stabilizer.
In certain embodiments, activation of the aqueous solution with the plasma results in the production of one or more reactive species including one or more reactive oxygen and reactive nitrogen species. In certain embodiments, the one or more reactive nitrogen species includes peroxynitrite. Because these reactive species help kill or deactivate spores, but otherwise may have a short half-life, the aqueous solution includes a stabilizer to stabilize one or more of these reactive species, such as an alcohol.
At block 334, the methodology continues with the application of the activated aqueous solution to a surface containing one or more dry spores for a period of time. After the application of the activated aqueous solution to a surface, the methodology ends at block 336.
Depending on the spore and the surface, the period of time the aqueous solution is applied to the surface can vary, but generally application periods of time will be less than 5 minutes, including about 3 minutes, about 1 minute, and about 30 seconds.
Where killing or deactivation of spores relies, at least in part, on the generation of one or more reactive species, because of the short half-life, e.g. 1-second, of some species, the activated aqueous solution generally needs to be applied to the surface immediately after activation. Where the aqueous solution includes an additive or stabilizer which can stabilize the reactive species the activated aqueous solution may be applied to the surface some period of time after the aqueous solution is activated. Appropriate periods of time after activation include, but are not limited to, greater than about 15 seconds, including at least about 30 seconds, at least about 1 minute, at least about 2 minutes, at least about 3 minutes, and at least about 5 or 10 minutes after activation.
An exemplary methodology for killing or deactivating spores 450 is illustrated in
The following examples illustrate specific embodiments and/or features of the present disclosure. The examples are given solely for the purpose of illustration and are not to be construed as limiting on the present disclosure, as many variations thereof are possible without departing from the spirit and scope of the disclosure.
In the following examples, various treatments were applied to measure the ability of plasma-activated liquids containing various additives to kill or deactivate spores from C. diff bacteria. Briefly, a volume of 10 μl of C. diff spores in sterile water (containing approximately 108 colony forming units (CFUs)/ml) was added onto a sterile stainless steel disc and left to dry for 30 minutes. The contaminated surfaces were then exposed to a treatment described below. After treatment, the killing or deactivation capacity of the treatment was measured by estimating the number of surviving CFUs. Estimation of surviving CFUs was determined by placing the disc in test tubes filled with a neutralizer. The test tubes were sonicated for 1 minute and vortexed for 15 seconds to fully remove spores from the surfaces. The neutralizer solution containing spores was diluted and plated on Brain Heart Infusion Agar supplemented with 0.1% Sodium Taurocholate (BHIT). The agar plates were incubated under anaerobic conditions for 36-48 hours at 37° C. CFUs were estimated based on colony counts on the agar plates following incubation.
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Unless otherwise indicated herein, all sub-embodiments and optional embodiments are respective sub-embodiments and optional embodiments to all embodiments described herein. While the present invention has been illustrated by the description of embodiments thereof and while the embodiments have been described in considerable detail, it is not the intention of the applicants to restrict or in any way limit the scope of the appended claims to such detail. Additional advantages and modifications will readily appear to those skilled in the art. Moreover, elements described with one embodiment may be readily adapted for use with other embodiments. Therefore, the invention, in its broader aspects, is not limited to the specific details, the representative apparatus and/or illustrative examples shown and described. Accordingly, departures may be made from such details without departing from the spirit or scope of the applicants' general inventive concept.
The present invention claims priority to and the benefits of U.S. Provisional Patent Application Ser. No. 62/258,840 filed on Nov. 23, 2015 and titled METHODS AND SOLUTIONS INCLUDING ADDITIVES AND STABILIZERS FOR KILLING OR DEACTIVATING SPORES, which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62258840 | Nov 2015 | US |
