‘US 20071 cu») United States 9017 2) Patent Application Publication co) Pub. No.: US 2007/0190174 A1 Holladay et al. (54) ANTIVIRAL COLLOIDAL SILVER COMPOSITION Inventors: Robert J. Holladay; Logan, UT (US) Williams D. Moeller, Alpine, UT (US Comesponilence Address: VENABLE LLP P.O. BOX 34385 WASHINGTON, DC 20043-9998 (US) (73) Assignee: AMERICAN SILVER LLC, Alpine, UT ws) (21) Appl. Nox 10/838,262 (2) Filed Oct. 2006 Related US. Application Data (63) Coatimnation-n-par of application No, 10/641,938, filed on Aug. 15, 2003, now Pat. No, 7,135,195. (43) Pub. Date: Aug. 16, 2007 Continvation-in-part of application No. 09946834, filed on Sep. 4, 2001, now Pat. No. 6,743,348, uid application No, 09/946,834 is a continuation of ‘pplication No, 09/323,310, filed on Jun. 1, 1999, ‘ow Pat, No, 6.214.299 (0) Provisional application No. 60/475,657, filed on Jun. 3, 2008, Publication Classification (1) Inc. AGIK «@) U. on ABSTRACT We disclose a colorless compesition comprising silver par- ticles and water, wherein sa particles comprise an interior of elemental silver and an exterior of ionic silver oxide ‘wherein the silver particles are present in the water ata level ‘of about S40 ppm, and wherein he composition manifests significant antimicrobial properties inluding antiviral prop- fenies, Methods of use ofthe composition are described 34/38 (2006.01) a 4241618US 2007/0190174 AI ANTIVIRAL COLLOIDAL SILVER COMPOSITION [0001] ‘The present application is 2 continuation-in-part of ‘pplication Ser. No. 10/641,938, fled Aug. 15, 2003, now US. Pat. No. ‘whieh in tim ig @ continwation-in-pat ‘of application See, No. 09°946,834, fled Sep. 4, 2001, now US. Pat. No. 6.743348 and a non-provisional of and ‘laiming priority from provisional spplication 60/475,657, filed Jun. 3, 2003, which application is incorporated by reference herein; application Ser. No, 09/946,834, fled Sep. 4, 2001, now US. Pat. No. 6,743,348 js itself a continuation ‘of application Ser. No. 09323,310, fied Jun. 1,1999, now US. Pat. No, 6,214,299. AREA OF THE ART [0002] The present invention generally relates to colloidal silver, and more particularly «© a composition of colloidal silver and a method for using said composition as an agent ‘azsinst onzanisms harmful to the health of humans Particular avian influenza virus (“bind ih") DESCRIPTION OF THE PRIOR ART [0003] 1s well known that certain preparations of silver have germicidal properties. Silver was employed as a wer micide and an antibiotic before modem antibiotics were ‘developed. In previous centuries, users would shave silver particles into thee drinking wate, or submerge whole silver Pieces inthe drinking water, for the parpose of ingesting the silver by drinking the water. It seems likly thatthe practice ‘of cating with silver utensils (Le, silverware) resulted rom, a belief in the healthful properties of silver [0004] There may be many reasons why edministering silver suspended in solution would enhance an individual's health, It possible that such a solution operates t inhibit the growth of bacteria, virses, and other unwanted organ- jams, a8 Well ns eradicating such existing bacteria, viruses, and other omganisms. It is also possible that a solution of filver can have an anti-inflammatory ellect, suficient to reduce symptoms of asthma. [0005] The present invention describes the use of a silver ‘composition in water to teat certain human ailments. An ‘embodiment of the invention isa silver composition com= prising small particles of silver which comprise an interior ‘Of metalic silver and an exterior of dome silver which particles ae suspended in water, A preferred embodiment of the invention ss slver composition comprising particles of silver wherein more than 50% ofthe particles age less than 0.015 micrometers in size and the pariles are colloially suspended in water SUMMARY OF THE INVENTION [0006] The present invention is generally directed to the use of silver, aa level of 5 to 40 ppm in water, o kill orto disable microorganisms, such 3s avian influenza virus, ‘hich are hazardous to human beings. The present invention specifically is dirwted (© compositions comprising silver Paricles, said particles comprising an interior of elemental silver and an exterior of jonic silver oxide, and water, ‘herein the silver particles are placed in colloidal suspen- sion in the water at a level of 5-40 ppm total silver. An ‘embodiment of the present invention comprises silver par ticles in Water, at a concentration of $40 pp, wherein more Aug. 16, 2007 than $0%6 ofthe silver particles have a maximum dimension less than 0.015 micrometers. The composition of silver in Water of this invention isan elfective antimicrobial agent. ‘This invention i directed to silver compositions, of 5-40 ‘ppm silver in water, which are effective antimicrobial ‘agens, and to methods of using said silver compositions as Antimicrobial agents [0007] _A preferred embodiment ofthe present invention is rected to compositions of silver in water made using @ ‘modification of the device and methods deseribod in US. Pat, No. 6,214,209, which is a parent of the instant app cation and is incorporated herein by reference, [0008] ‘The device and process of US. Pat, No. 6.214.299 have boen modified and improved to provide the silver ‘composition of the present inveation, Essentially, the cigh silverione common electrode device as disclosed in the patent has heen modified and scaled to fit a 75-gallon water chamber. To start the process approximately 70 gallons of high purity water are placed in the chamber. To this is added approximately five gallons of silver composition produced ina prior production run. Tis is necessary bocause the high purity Water is insulicieally conductive for the process ‘ecur propery. The water chamber is equipped with an air input that allows a stream of air bubbles to be streamed trough the liquid during the processing. It has been di covered that this approach gives improved mixing as com- pared to the impeller mixer described in the pate. [0009] The electrode device is operated at approximately fen thousand volts altemating current (with each silver clectmde having an individal voltage supply) as described in the patent, It has been found that volages significantly lower thas this produce a composition with lager particles ‘not having the optimal properties described herein. Voltages sigaifically higher tend to produce a solution with signifi ‘ant ionic silver dissolved therein, The present composition comprises in excess of 97% metalic silver with essentially ‘no free ionic silver in solution, [0010] | Thesilver concentration i determined according 10 the methods explained below. Estentally, the device is ‘operated continously and samples are analyzed until the ‘desired silver conceatraton i stained, The 10 ppen com- position requires approximately one and one half days of ‘pemtion, The 22 pp solution requires approximately three days, and the 32 ppm composition requires approximately six days, The rate of the process appears to slow a the higher concentrations are tained. Higher concentrations take a probibitively long time with the ultimate highest heing about $0 ppm, a least within the current [0011] The compositions all have the size characteristics Aeseribed below and unlike conventional silver composi tions are completely coloress and stable to ight and tem- perature changes without use of any additives. The compo- Sitions are unreactive towards added hydrogen peroxide, has been found to have @ synergi inventive silver composition. Hydrogen peroxide is avai able in concentration of 30% waht (% weight per volume ‘or weight perceat [Wweht %) or higher. Although the higher jons are usable, the preferred concentrations are in the range of Ito 5% wahtUS 2007/0190174 AI [0013] A preferred embodiment ofthe present inventio directed to compositions comprising 5 to 40 ppm silver, said silver being primarily elemental silver, 1 t0 3. waht % hydrogen peroxide, and water. A preferred embodiment of the present invention is the use, and method of use, of ‘compositions comprising 10 to 40 ppm silver and I to 3 ‘waht % hydrogen peroxide in waters antimicrobial agents DETAILED DESCRIPTION OF THE INVENTION [0014] The following description is provided t ensbleany person skilled in the art to make and use the invention and sets Zorh the best modes contemplated by the inventor of irying out his invention. Various modifications, however, ‘will remain readily apparent to those skilled in the a, since the general principles of the present invention have been ‘defined herein specifically o provide an improved colloidal silver product with significant abilities to kill human pallo- tens Both in vivo and in vit, [0015] Generally the present invention represents a novel ‘approach to killing or disabling microonganisms which ure hazardous to human beings by the use of silver particles in Water, at @ concentration of $ to 40 ppm silver. Depending upon the application, the silver composition may be used internally or externally. Depending on the application, the silver composition may also contain hydrogen peroxide PREFERRED PMBODIMENTS [0016] _Non-limiting preferred embodiments are presented fn the fllowing [0017] _A composition comprising silver particles, coli dally suspended in wate, wherein the ota eontent of silver js between 5 and 40 ppm, which composition kills or disables mieroorganisms which are hazardous o the human body. [0018] A composition comprising silver particles, colloi- dally suspended in water, wherein the total eontent of silver js about 1022 ppm, which composition kills or disables ‘microorganisms which ate hazardous to the human body. [0019] A composition comprising silver particles, colli dally suspended in water, wherein the total eontent of silver js about 2222 ppm, which composition kills or disables ricroorganisms Which are hazardous to the human bod. [0020] A composition comprising silver particles, coli dally suspended in water, wherein the total eontent of silver js about 3223 ppm, which composition kills or disables ricroorganisis hich ure hazardous to the human body. 0021] 1 will be apprecintd thot spesiffing the wil amount of sier in a composition of particles docs not ‘completely specify the mater. As the prticles comprising the composition ate made stale, «given concentration of silver wil represent larger mer of patses. Inadaition, the toll surface area for a given silver concearaion wil increase. Therefor, parle size and range of partie size ‘sm important parameter for defining an elective inventive composition [0022] A funhor class of embodiments is any of the shovedssribed compeition, whercin more than $0% of the ser particles have # maxim dimension les than (0015 microneter Aug. 16, 2007 [0023] A further class of embodiments is any of the abovesdescribed compositions, wherein more than 75% of the silver panicles have a maximum dimension less than 0015 micrometer, [0024] A further class of embodiments is any: of the above-described compositions, wherein more than 9% of the silver particles have a maximum dimension less than (0.02 micrometers. [0025] A further class of embodiments is any of the boverdeseribed compositions, wherein more than 75% of the silver particles have «minimum dimension greater than (0.008 micrometers, [0026] A further class of embodiments is any of the above-described compositions, wherein more than 9% of the silver particles have # minimum dimension greater than (0.005 micrometers [0027] A further class of embodiments is any of the shove-deseribed compositions, wherein the silver particles comprise both silver in the 7oro-valent, that is, metallic, ‘oxidation state (Ag(0)} aad a coating of silver in an ionie oxidation solected from the group consisting of gt), Atl), and Ap(ID. {0028] A further class of embodiments is any of the hovesdescribal compositions, wherein the silver particles comprise both silver in the zero-valen, that is, metal oxidation slate [Ag(0)} and a couting of silver in an ionic oxidation selected ffom the group consisting of atl) gC), and Ag, [0029] Experimental evidence shows that AgO in the particles of the present invention is atleast partially in the orm of Ag,O,-—that is slver I oxide. Ina molecule ofthis material fo ofthe silver atoms are in the I” state (silver I) \while the other two silver molecules are in the 3° tate (silver ID). Under cerain conditions these molecules ean give rise {o silver atoms inthe 2* (silver 11) state [0030] _ further class of embodiments is the combination ‘of any of the above-deseribed embodiments with hydrogen peroxide, at a level of 1-3 wat % hydrogen peroxide in the final produet EXAMPLES [0031] 1. Formation of Composition [0032] Compositions of silver in water can be made according to procedures set forth in US. Pat. No. 6,214,209, incorporated by reference herewith, [0033] A prefered method for producing a composition comprising silver according to this invention utilizes a clecttchemical cell comprising electrodes and comprises the steps [0034] (2) placing a silver electrode in contact with a ‘quantity of high purity water; [0035] (6) conveying electrical current chrough the silver electmde to thereby separate particles of silver from said silver electrode in « manner suficieat to cause production of suspended silver particles within the water, and [0036] (c) agitating the water during ssid produetion of suspended silver particles to thereby disperse the silver particles into more uniform conoeateation within saidUS 2007/0190174 AI water su that i ticles can be produc [0037] Another preferred method for producing a compo- sition comprising silver utilizes an electrachemicel cell and ‘comprises the steps of: [0038] (a) establishing an electrical cieuit comprising a ‘current source, anda first conductor electrically connected to Said current source and a second conductor elececally ‘connected to said current source, whercin sai fist conduc tors disposed spaced apar from sad second conductor, and wherein at least one of the conductors is mode of elemental silver, [0039] (b) closing te circuit by placing the first conductor ‘and the second conductor in communication with a Mudie resistor: [0040] (c) operating the current source to supply alternate fing current simultaneously to the firt conductor and the second conductor such that voltage is increasing. and decreasing within the first and second conductors in alter- nating tandem to thereby cause silver particles to separate fom the first cleetrode and enter the Audie resistor and become disposed in suspension within sad Mudie resistor; ‘and [0041] (4) selectively adjusting the electrodes by moving them toward the Midi resistor to compensate for decrease in electrode length dve to gradual separation of silver Particles therefrom to thereby prevent arcing ftom occurring between the electrodes and said Audie esstor. [0042] The analysis of the silver content in the silver ‘compositions of this invention may be done by atomic absorption (AA), inductively coupled plasma/atomic emis- sion (ICPIAES), or other techniques known 10 one of ordinary skill in the art to be sensitive 10 silver in the appropriate concentration range. Ifthe particle ofthe silver ‘composition are small and uniformly’ sized (for example, 0.01 micrometers or less), a reasonably accurate assay may be obtained by running the colloid directly by AA of ICPIAES. This is because the sample preparation for AA Jonizes essentially all of the silver allowing its ready detec- [0043] IF the compositions comprise particles as large as (0.2 micrometers, ils preferred to use a digestion procedure ‘The digestion procedure is not necessarily ideal for silver ‘compositions that may have been manufactured or stored in ‘conta with halides or other anionie species that may react ‘with finely divided silver, or combined with protein or other pelatinous material. An embodiment ofthe digestion proce- ‘dure isa follows: [0044] 1 Take @ 10 ml aliquot of @ thoroughly mixed oF shaken silver composition to be analyzed, and place it in a ‘lean polycarbonate botle or other coniaier of suitable ‘material (generally, the bottle) with a tight fiting lid. size (0 30-100 ml is prefered [0045] 2 With a micropipette or dropper, add 0.1 mt of nitec acid, reagent grade tothe silver composition in the battle [0046] 3 With the fd ofthe bot tightly in place, heat the silver composition to 80° C. with mild agitation fora time sufficient to dissolve the silver—dissolution is essentially instantaneous her quantity of suspended silver par- per batch Aug. 16, 2007 [0047] 4 Allow the resulting mixture to cool to +001 femperature with the lid in place. Shake the bottle thor- ‘oughly [0048] 5 Uiilize AA, ICP/ABS, or equivalent meaas 40 ‘analyze the silver content ofthe silver mixture, Preferably, fone will utilize a freshly prepared standard or standards, preferably prepared according the equipment manufactur rs instnictions, with appropriate dilution as needed. [0049] 6 When reporting results, one must taken into account all dilutions during preparation, including the 1% tltion caused by addition of the nisi sci. [0050] 1. Analysis of Physical(Chemical Form of Silver [0081] A. Introduetion [0082] A sample of a composition, nominally containing 22 ppm silver in water, was analyzed by time-of ight secondary ion mass spectrometry (TOF-SIMS) in order to determine the form of silver in the composition. The eon- csi is thatthe bulk of the silver exists as silver (0 [that js, metallic silver] and that thre is surface coating which as on average a composition of silver (Il) oxide [AgO]- As ‘mentioned above silver (1) oxide is usually a stochiometic combination of silver (I) and silver (ID, [0083] _B. Experimental Procedure [0084] A few drops of the 22 ppm inventive silver com- position were evaporated 1 dryness ona silicon substrate at Ambient temperate, The residue was analyzed by TOF SIMS, and is denoted as the sample. A reference silver(I} joxide (AgO) material was analyzed by placing a few par ticles ofthe reference powder as received from the vendor fon a silicon substrate, and is denoted asthe reference {0085] The Time-ofFlight Secondary Ton Mas Spectrom try technique (IOF-SIMS) i based on the principle of bombarding a soli sample with a pulsed, finely focused ‘ean of primary ons, andthe analyzing the secondary ions produced from the sufice othe sample via a timeof fight fouss spectrograph. This analytical tecinigue is surfoce Sensitive, deeving its informstion from a layer that extends to approsimately 20 to 40 A (one Angsromel 104 micrometers) below the surface. The TOF-SIMS technique Js nomully used as asurvey tool ideal the composition of unknown samples. Its capable of quanifistion ifthe ‘appropriate mjeounalytclstodards are available fo ca bntion. This analysis was caro owt sing standard high sass-esolton conditions [0056] C. Results [0057] Negative ion mass were obtained for the Ag(I)O reference material and the product sample, respectively. The :mas spectral region for both spectra showed the presence of AgO— species. The data suggest that silver (II) is the Average oxidation state ofthe silver present on te surface of the sample particles. The silver oxide (AgO) signals exhibit significantly higher intensity in the reference sample com- pared 10 the product sample which is probably” because ‘metalic silver is dominant inthe sample. twill be appre- ciated that as the average particle size in the sample is ‘decreased the ratio of silver to silver oxide will also decrease ‘as more silver oxide will be presentUS 2007/0190174 AI [0088] 2. Size Analysis [0089] 1 is likely that the unusual effectiveness of the silver preparations described here is due tothe relationship hetwoen the surface propertievinner properties (i, oxide! metal) of the particles and the size distribution’ of the particles. The smaller the average particle size, the greater the surface area and the greater the contribution of the particular surface chemistry: However, if the particles are ‘excessively small there ca bea loss of stability andlor other ineractions that negatively affect the product. The silver ‘compositions of the instant invention are remarkable bocatise they are stable in essentially pare water without surfactants, ete. Also, the materials aro essentially colorless ‘while other colloidal silver preparations (particularly with Jarger particle sizes) usually show colors. These properties fare a reslt of the exact manufacturing conditions as dise ‘cussed above, [0060] Digital analysis of the composition showed that there is an average partile diameter of 0.0106 micrometers ‘with a range of 0.005 micrometer to 0.0851 micrometers However, size distbution analysis shows that more than 95% of the particles were between about 0.005 micrometers ‘and sbout 0.015 micrometers in diameter [0061] 3. Evidence of Eicacy of 22 PPM Silver Compo sition Against Bacillus Subiis [0062] A. Purpose of Example [0063] The purpose of this example isto demonstrate the ‘antimicrobial activity of the slver-based composition ofthe present invention on bacterial endospores from the test ‘organism Bacillus subtilis, This was accomplished by per- forming a standard Klltime assay using a suspension of B. subs endospores. Normally, bacterial endospores are resistant to killing. [0064] B. Material und Methods [0065] Test Organism. A. test suspension containing ‘encdospores from Bacillus subtis (ATT #19659) was pre= pared from a culture grown on nutrient agar, 19 which ditional sponilation enhancement ingredients were ade. Plates were harvested with sterile water and endospores were purified by repeated centrifagations and resuspensions in water. The final wash sas in 70% ethanol for 30 min, 0 ‘ensure the destruction of all vegetative bacteria. The spores were restspended in water containing 0.1% Tween 80 (brand of polysorbate surfactant) to prevent clumping. [0066] Neutalizer. The Neutralizer mixture consisted of 12.7% Tween 80 (brand of polysorbate), 6.0% TamolR SN (Grand of sodium salt of naphthalene-formaldehyJe conden- sate), 1.7% lecithin, 1% Peptone, and 0.1% Cystine. This Aug. 16, 2007 solution was intended to neutralize any’ chemicals so they subsequent growth of the bacteria, 1 Procedure: 4)A9.9 ml aliquot ofthe disinfectant (inventive 22 {0058 om silver composition in water was placed ina sterile 20 ime150 mm tbe, The tobe wae equa in a 20° C water bath [0069] b).A.9.9 ml aliquot ofthe disinfectant (inventive 22 ‘ppm silver composition, in water) was placed ina sterile 20 ‘mS0 mm tube. The tube was equilibrated in a 20° C. water bath, [0070] ©) At 30 min. 1 he, and 4 hr, one ml of organ isinfoctant suspension was removed to a tube containing fine ml of Neutalizer. The tbe was mixed thoroughly [0071] 4) After ‘wo min, the neuralized suspension was serially diluted 1:10, in physiological saline solution (PSS). [0072] ©) The number of viable organisms in selected ‘ition tubes was assayed by membrane fiztion, One aliquots were plated in duplicate. ‘The membranes were ‘washed with about 100 ml of sterile PSS and removed to [Nuteient Aga plates. The plates were incubated at 37°C. for 20 be [0073] 1) The number of colonies on each filter was ‘ounted and log reictons were computed, [0074] Controls: [0075] a) Titers of the test suspensions were computed by performing membrane filtmtion assays of selected 1:10 lutions ofthe test suspensions in PSS, [0076] §) A neutralizer contol was performed by ince Jating a mixture of 9 mf neutralizer and 1 ml of disinfectant ‘with 100 pl ofa dilution ofthe titer coatsining 100 cf. This produced about 10 cfu inthe ube, which was allowed to Sand for 20 minutes prioe to assay by membrane filtration using duplicate 1 ml samples [0077] C. Results [0078] Bacillus subrits Titer hie tan at [0079] late tanto dant act once nce at > mre Ine BsUS 2007/0190174 AI contin Dion of seis soins ues Fine tO" tot tot at ttt Newton Caso 1 10 [0080] D. Discussion [0081] Results of the titer showed a viable B. subs spore ‘concentration of 6.65x10" spores per ml in the original suspension. Inoculation of 9.9 mi of disinfectant with 100 ‘of this suspension produced an initial concentration of 6.65:10" spores per ml in the assay tube [0082] Results from these procedures allowed log reduc tions (LR) and Percent Kill (PK) values to be calculate. They are Tisted i the table below. Values were computed using the formulae: LR=-Log(S/So) and PK=(1~(SIS0))x 100, where Seconcentation of organisms ata specific time: and Sosthe intial concentration of organisms at time zero [0083] Neuteaization contol data showed thatthe disin- fectant was adequately neutnized. Actoal counts come spond to those resulting from dilution without appreciable killing, [0084] The disinfectant preparation tested here displayed 00 sporicidal activity against B. subs spores. subifs {sa common species used in sporicidal testing and belongs to the same genus as the organism that causes anthrax. Because of their peneti similarities, B.subilis spores have been used asa non-pathogenic surrogate for Baill anthra cis, the anthrax. bacterium, Therefore, these results are applicable to anthrax. It is expected that longer exposure ‘would result in additional killing [0088] 4. Evidence of Fiicacy of 10 PPM Silver and 1.0% H,0; Composition and 14 PPM Silver and 1.5% HO: Composition Against Bacilus Subsilis [0086] [0087] The purpose of this example isto demonstrate the antimicrobial ativity of two silver-based compositions of the present invention on bacterial endospores from the test ‘organism Bacilus subs. This was accomplished by per forming standard kill-time assays using a suspension of B. subiilisendospores, Viewed relative tothe previous example (employing 22 ppm silver, this example establishes the promoting effeet of hydrogen peroxide (H,0.) on the anti ‘microbial properties of silver compositions. Hydrogen per ‘oxide is sible in the presence of the silver compositions of the present invention, While hydrogen peroxide has sign A. Purpose of Example Aug. 16, 2007 eto cant antimicrobial properties itself, i is frequently broken ‘down by catalase of other microbial enzymes. However, the hydrogen peroxide is capable of weakening becteral cell ‘alls and enhancing entry ofthe silver particles before any enzymatic destruction of the hydrogen peroxide ean occ, [0088] B. Material and Methoxs [0089] 1 Test Organism. A test suspension containing endospores from Bacillus subiis (ATCC # 19659) was prepared from a culture grown on Nutrient Agar, 9 which ‘addtional sporulation enltancers were added, Plates were Jharvested with sterile water and endospores were purified by repeated centifugations and resuspensions in Water, The final wash was in 70% ethanol for 30 min, to ensure the eath of all vegetative bacterin. The spores were rexus- pended in water containing 0.1% Tween 80 (brand of polysorbate) to prevent clumping [0090] 2 Neutralizer. The Newalizer mixture consisted of 12.7% Tween 80, 6.0% Tamol® SN (brand of sodium sat of naphthalene-formaldehyde condensate), 1.7% lecithin, 1% Peptone, and 0.1% Cystine. This solution was intended 19 neutralize any chemicals so they would not affect subse- {quent growth ofthe bacteria, [0092] a) .9.9 ml aliquot of each of the disinfectants (Gaventve colloidal silver compositions: one containing 14 ppm silver and 1.5% 1,03; the other eontaining 10 ppm Silver and 1.0% HO.) was placed in a sterile 20 mim 80 ‘mm tube, The tubes were equilibrated in 20” C, waterbath [0093] b) Bach tube of disinfoctant was inoculated with 100 jl ofthe test onganism suspension at time zero, [0094] ) At 10 min, 30 min, 1 hr 2 he, 4 he, 6 hr, and 8 ‘nr, one mil of organisn/dsinfectant suspension was removed fo a tube containing nine ml of neutralizer, The tuhe was nixed thoroughly. [0095] 4) After two min, the neutralized suspension was serially diluted 1:10, in physiological saline solution (PSS). [0096] ©) The number of viable organisms in selected ‘ition thes was assayed by membrane filtration. One ml aliquots were plated in duplicate, The membranes were ‘washed with about 100 ml of sterile PSS and removed to Columbia Agar plate. The plates were incubated at 37° C tor 20 he, [0097] 1 The number of colonies on each filter was sounted and log rections computed, [0098] 4. Controls: [0099] a) Titers of the test suspensions were computed by performing membrane filtmtion assays of selected 1:10 lutions ofthe test suspensions in PSS.US 2007/0190174 AI [0100] b) A neutralizer fectant with 100 yl of the 1:10" produced about 2,000 cfu/ml fo stand for 20 minutes priar to elit samples. [0101] C. Resuts [0102] Titer of Bacillus subsitis Spores: Linu? mt ae colo ite y 2 [0103] = 5 nie wre es 30 min Tse INCOR = = owe me | [0104] Neueaization Control ne rey [o10s] ‘Soon coring 10 ppm iver an 1.0% 1,05 nto] was performed by inocu Jating a mixture of 9 ml of neuvalizer and 1 ml of ution of the titer. This the tube, which was allowed E10. Bath tubes were assayed by membrane filtration using duplicate 1 ml Aug. 16, 2007 ‘Sohton coming 10pm iver and 146 105 Dining of ails ou dicta sumone Tine 1x10! x i tae [0106] Neutratizaton Control: Nie mo NTC = oo name oom [0107]. Discussion [0108] ‘The data showed a viable subtilis spore concen- {ration of.59%10 spores per min the original suspension. Inoculation of 9.9 ml of disinfectant with 100 yl of this suspension produced an initist concentration of 2.59x10* spores per mi in the assay tube {0109} Resets from these proedres allowed fog. reduc- tions (LR) and Percent Kill (PK) alos to be calla They ae listed inte fllowing table. Values were comput sing the formulae: LRe-Log(S/S0) and PRK=(1-(S'S0))x 10; where. Ssconcentration of organisms aa speci tine, and Somthe inal concentration of orgasms a ime zero Since tere was no sigan il within 30min, the 10 min data was used forthe So vals, The 6 rand # hr exposure times did not produce counts high enough wo be retable ‘Therefore these data were not sein the near regressions Linear regressions were perfomed ot the log reduction values ting the fied Hie pos” command inthe Minitab tiie software package. The repression equstionspeo- dice an the times roguiredt elles six-log rection are shown along with te log ediction and percent kill values inthe folowing table Tine thet! eh ta aetna aettnon A reotthion "EneUS 2007/0190174 AI [0110] Reyression Analysis [0111] Equation for 14 ppm calculated line: ¥=-0.66708+ 1.32936x. Equation for 10 ppm calculated line -0.59690+1.03033x. These extations predict that the time for a G-log reduction is $02 hrs for the 4 ppm ‘composition and 6.35 hrs forthe 10 ppm composition, [0112] The neutralization control data showed that the ‘isinfectant was adequately neutralized. Expected counts ‘corresponded to those expected from the dition [0113] ‘The experimental disinfectant solutions tested ‘exhibited significant sporicidal activity against B. subsilis spores. The . subtifis sain used in these evaluations is the same one specified inthe AOAC sporicdal test Spores from Aug. 16, 2007 trent broth, The dilutions were calculated to cover the susceptible ranges for cach organism for each agent, standard amount ofthe test culture was added to each tube ‘and the tube returned to an incubator (3722° C.) for growth ‘The tubes were checked turbidometrically to determine bacterial prowih, Below the MIC concentration the tubes showed an increase in optical density with time indicating bacterial growth, The lowest concentration ofthe anibiot that showed no growih Was the MIC, The “ao growth” tubes were thea subcultired in fresh mevum, The “no growth tube with the lowest concentration of antibiotic that showed ro growth on subculturing was the MBC [018] B. Results: ait na rguien Terie Spin 13858 this organism represent a significant challenge for most " denotes thatthe concentration ‘edad to obiain the MIC or the MBC was higher than test parameters measured fr the test. For example, the highest concentration of tetracycline sed on S.pyogene was S ppm. ‘At that concentration there was still growth upon subcultur- ing ofthe “no growth” tubes, Therelor the MBC must be (greater than) 5 ppm. [0120] The MICIMBC of B: col stain © 157-17, which ‘has been associated with oubreaks of hemonagie diariea And colitis, was determined ina subsequent study. The MIC ‘was determined to be 2.5 ppm and the MBC was determined to be 5 ppm. [0121] C. Conctosion [0122] The 10 ppm silver composition of the present ‘invention was tested and found to be both bacteriostatic and bactericidal for all organisms tested. In ther studies, this composition was compared to other commercially available colloidal silver products and) found to have a superior activity toll other preparations tested (data not shown) The ‘mos interesting observation was the broad spectrum that the 10 ppm silver composition possesses. The antimicrobial activity that was observed was fairly constant independent fof the particular ongonism tested. With the exception of Sireptococeus faecalis and Streprococeus aureus (which had (MIC values of 10 ppm and 5 ppm, respectively), MIC values ranged between 1.25 ppm and 2.5 ppm for both praUS 2007/0190174 AI positive and gram negative organisms. The MBC values behaved similarly with values ranging from 1.25 ppm to $ pm with the exception of Streprococeus mutans, Sirepto- ‘coccus gordonii, ad Streptococcus faccals (which all had MBC values of 10 ppm) The data suggest that 10 ppm silver ‘embodiment of this invention exhibits an equal oF broader spectrum of activity than any one antibiotic tested, Anti olics generally have restricted antibacterial spectra limited to susceptible organisms, but as the data demonstrate, the silver ‘composition of the present invention is equally ellective inst both gram positive and gram negative organisms. ‘The data suggest that with the low toxicity associated with silver, in general, and the broad spectrum of antimicrobial activity ofthis silver composition, this preparation can be ‘effectively used as an altemative to antibiotics D. REFERENCE FOR PRECEDING EXAMPLE [0123] 1 US. EPA IRIS Repor for Silve-CASRN 7440- Da [0124] 2 Fox CL, Modak $ M. Mechanism of Silver Sulphadiazine Action on Burm Wound. Infections. Ant ‘microbial Agents Chemother. 5:582-S88.1974, [0125] 3, Furehner, 1, Richmond C R, and G A Drake, ‘Comparative Metabolism of RadionucTies in Mammals. IV. Retention of Silver-110 min the Mouse, Rat, Monkey, and Dog. Health Phys, 15:505-514.1968, [0126] 4. Gri, N. Silver and its Compounds in Disinfec tion, Sterilization, and Preservation, (Seymour S, Block, co) 2"" Edn, pp 395-407. 1977, [0127]. 5. Hindler, 1, and J H Jorgensen. Procedure in ‘Antimicrobial Testing in Diagnostic Microbiology. (CR. ‘Mahon and G Manuselis, eds) pp 63-91-1995. [0128] 6. Bvidence of FMicacy of 32 PPM Silver Compo- sition Against Pseudomonas Aeruginosa, Salmonella Chol- ‘eraesuis and Staphylococcus Aureus [0129] A. Methods [0130] Pseudomonas aeruginosa ATCC #15442, Salmo: nella choleraesuis ATCC i 10708 and Staphylococcus ‘aureus ATCC #6538 were tested using the AOAC (As80- ‘ciation of Official Analytical Chemists AOC Methods, vol 1, 15th elton, 1990, AOAC Arlington, Va.) oficial meth- ‘ds 955.14, 95815 and 964,02, Nuteient broth (NBAOAC), tubes were inoculated from the stock culture, and the tubes incubsted at 3742° C. Transfers to fresh tubes of nutrient broth were made for three successive days with the final transfer being incubated at 3722° C. for 48-54 hr, The Preudomonar clture was decanted into a fresh tube 10 remove the pellicle, The other cultures were vortexed for 3-4 seconds and allowed to stand for 10 min at room tempens- ture, Finally theeuftures were diluted 1:100 in peptone water (PEPW) to which equine serum was added to yield a 5% ‘otal organie challenge. Tet carriers (10-mm long polished 304 stainless stel cylinders with an 8 mm outside diameter ‘and 6 mm inside diameter) were soaked in challenge solu- tion for 1S min, removed, drained und dried at 3722° C. for 44022 min prior to use. [0131] Phenol Resistance. Five-one ml aliquots of each ‘luton ofthe test phenol were placed ino sterile test tubes tnd allowed to equilibrate in 8 2082° C. water bath. At 30 ‘Second intervals, 0.5 aal ofeach challenge culture was added Aug. 16, 2007 {0 the appropriate dilutions of pheoo!,aptated, and replaced into the water bath: After the appropriate exposure times of 5,10, and 15 minutes, 2 loopfl of suspeasion was removed om the assay tubes and transferred to tubes ofetheen bros (CETH), The tubes of LETH were incubated at 372° C for 2 days, [0132] Caner Titation. For tiation of carers, 10 ml blanks of peptone Tween® (brand of polysorbate) (PEPT) solution were prepared. Two cariers were placed into the individual tubes, representing the first 1:10 dilution, The tubes were agitated vigorously enough to get bacteria into solution and serial dilutions were made into 9 ml blanks of LETH medium. The dilution blanks were incubated at 372° C. The last tbe with growth indicated the log, titer of ‘organisms on the carrier. AOAC requires caries to have inimum populations of 1x10 efulcaries. [0133] Test of Silver Composition. Using sterile glass Pipettes, 10 ml aliquots of the prepared disinfectants were placed into sterile test tubes and allowed to equilibrate in a relfigerted water bath held at 202° C. Without touching the sides of the test tubes, one contaminated dried carrer was added at 30 second intervals to each tube of silver composition and placed back into the water bath, For eh ‘organism the disinfectant was tested against 60 dried eon- ‘aminated carriers at S and ID minute exposuee intervals Following exposure, the carriers wore removed from the sisinfoctant and transferred to a tube of LETH. The cultore tues were incubated at 372° C. for 2 days and scored as Positive (4) oF negative (0) for growth of the challenge organise, [0134] Controls. For each onanism, a dred contaminated carrer was added to a tube of LETH as a positive contrl ‘Uninaculated media tubes served as negative controls, After ‘incubation all negative tubes were spiked with 1-100 colony {orming units (ctu) of the corresponding organisms to dem- onstrate neutralization efficacy. To demonstrate growth pro- ‘motion of the medi, the negative control tubes were also inoculated withthe same 1-100 efi forall three organisms. The inoculating volumes were plated ia triplicate onto soybean casein digest agar (SCDA) to verify the inoculating tiers The tubes and plates Were incubated a 3782” C, unt ‘growth Was sen in all tubes, [0135] On the P. aeruginosa neutralization, the inital titer ‘of inoewlam was found to be >100 efi which was too high {or the protocol. Because all orignal tubes had been spikes 4 simlated test was performed with same Iot of mia used testing by placing cariers into disinfectant tubes from all three lots of silver compositions for 10 miaute. The carriers were sub-ransfered to LETH blanks, These tubes were thea spiked with 1-100 eft of organism, The tubes were inc- Dated as before and scored for grow oF no growth, New tubes of sterile media from the same lot were also inoculated a a growth promotion verification [0136] B. Results [0137] Init testing sing S. aureus demonstrated passing results for sample #1 and #2, but sample #3 failed. Upon investigation it was decided that sample #3 may have been ‘damaged prior to shipment. new botle was obtained from the same lot as sample #3, and the new bale was labeled as sample #4. The S. aureus challenge was repented using sample #4. AOAC guidelines state that for any one time point and organism only I carrer i allowed for growth for ‘each lot testedUS 2007/0190174 AI [0138] Positive controls demonstrated growth aad newae tive controls demonsirited no growth for all lots, time points, and organisms, [0139] Carrer sation was run in duplicate forall organ- Jsms. The reported titer is an average ofthe replicates. For all thee organisms. the average tite found on the cases ranged from 5.5x10* to 5.5410" efwlearriet. AOAC requires ‘carriers to have a mininnim of 10x10" efueareee [0140] For P. aeruginosa 3/180 carriers showed growth at the 5 min test point nd 2/180 eariers showed growth atthe 10 min test point. For S. aureus 16/180 curriers showed jarowth at the 5 min test point and 2/180 carriers showed frowth at the 10 mi test point, For S. choleraesuir 6/180 feartiers showed growth atthe 3 min test point and W180 ‘cartier showed growth atthe 10 mia test pot. [0141] The test Pseudomonas culture showed growth fol- Towing a 5, 10 oF 15 min treatment with 1:90 phenol and shoved growth fllowing a 5 or 10min treatment with 1:80 phenol but no growth following 1 min treatment with 1:80 Phenol. The Staphylococcus culture showed growth follm= {ng 5,10 oF 1S min teatment with 1:70 phenol and showed srcwth following Sor 10min treatment with 1:60 phenol but no growth following 2 15 min teaument with 1:60 phenol ‘The Salmonella culture showed growth following a 8, 10 oF 15min treatment with 1:100 phenol but no growth following 495, 10 o¢ 15 min treatment with 1:50 phenol, [0142] 7. Evidence of Fifectiveness of 32,22, and 10 PPM, Silver and 22 PPM Silver and { 396 1,0, and 10 PPM Silver tnd 10 ppm K,S,O, Against Salmonella and Escherichia Coli in Freshly inoculated Beef Samples [0143] A. Purpose of Example [0144] The purpose of this example isto demonstrate the ‘antimicrobial activity of the silver-based composition ‘embodiments of the present invention on samples of bec! flank steak inoculated on the exterior surface with a five inuin cocktail of Salmonella species. ot Escherichia cali OL '57:h7 ata high inoeufum sohtion level (110° fem) and ‘Separately a alow inoculm solution level (1x10* efwem") (foecolony forming unit. [0145] B. Material and Methods 0146] eof Samples. Beof tissue samples were obiined from slayahier houses within 8 hours of eviseraion. The rectus abdominin muscle was pooled of carcasses hangin in the chill cooler by making an incision between the 11 snd 12" sibs and then peeling the muscle out along the notre sam, Thessaly rercved samples were placed in plastic bags and on ce packs and were transported ba the same day to the laborsory, were the samples. wore prompily packed in a Malti-Vae (A-300) and placed in a 4° {C-cooler Samples sed fr testing had pT between $8 and 60 and were no more tan 36 hous post evsceration. From randomly selected rectus abdomints muscles, 1338 em Samples were cit and trated. Alter weatment. a 3.5 em” flame sterilized stainless steel coring device and surgical scalpel were utilized 0 aseptialy retrieve v0 meat cores per sampling interval from each sample. Tissue cores were Placed ina serie stomacher bag with 25 ml of 0.1% peptone nal ere mine fortwo ssnutes ina slomsocher (Lab Render 40), Serial ditions were prepared and spiral plated at 0 Aug. 16, 2007 es, 20 minutes, 1 hour, 4 hours, and 24 hours post tment on selective and recovery media [0147] Bacterial Cultures, Bacterial cultures were ‘blained from the Kansas State University (KSU) stock culture collection and were stored using the “Protected ead?” storage system, The following cultures were used for the Sulnonella specimen: S. lille (UGA), S. montevideo (UGA), 8. nphimurium (UGA), 8. agona (KSU 08 from CDC outbreak isolate), and S: mewport (RSU 06 CDC outbreak isolate). The following cultres were used for the Escherichia coli specimen: E. coli OStHTT (CDC 01,0 E.coli O1ST:H (USDASIS O11-82 RiP resistant pm), E.coli O1S7:H7 (ATCC 43895 HUS associated Type 18 I toxins Rif Res.) and E.coli ATCC#23740 (Genotype K-12 provotrphie Lambda). [0148] Stock cultures were cultivated by placing one impregnate bead iat a Sm solution of Difeo® Tryptie Soy [Broth (TSB) snd incubating for 24 hours at 38° C. Next, @ (0.05 mi Joop of the respective culture ws inoculated into & '5 mil solution of TSB and incubated for 24 hour at 3$° Ct ‘obiaina pare culture, Aer incubation, 1m] of the respective clture was inoculated into 49 ml TSP and incubated for 24 hours at 35° C. Following incubation, samples were centr- fuged (15.300xg at 4” C.), and the supernatant material decanted and the pellet was re-suspended with $0 ml of 0.1% peptone and centrifuged (15,300xg at 4° C.) a final time. The peptone was decanted and the remaining. peli was resuspended with 10 ml of 0.1% peptone, The five 10 im} bottles of respective culture were mixed together 10 create a $0 ml coektal containing 10” efi of Salmonella species. The cocktail was diluted to 10° efiml or 10" efwiml ‘using 0.1% peptone. Cultures were confined by cultivation ‘on selective and differential media, and biochemical analysis fof presummpive colonies using ADI 20E kis. [0149] | Method of Inoculation. Samples of beet ank steak (ectus abdominus muscle) were trimmed to 13x8 em (104 em) and were inoculated with a five stain cocktail of Salmonella species. or Escherichia coli O1ST:A? ata high ‘inoculum solution level (10° log efwlem) and separately at 4 low inoculum solution level (10" log efilen). This ‘inoculum was misted onto the tissue surface using plastic spray bottle with samples contained within a sealed incew- Jum chamber. The actual Salmonella species. coneeatration fon the, meat surface was approximately 5.0 and 3.4 log efwiem’ for the high and Jw level inoculum solution, respec- Lively. For £. cali O1ST:H7, the respective meat surface ‘noctlation levels were 4.2 and 3.9 log efalem= [0180] The beet samples were then hung vertically on Stainless steel hooks attached to a motorized track that pulled the beef samples through a model spray cabinet (Kansas State University, Pood Safety Laboratory) while speay treatments were applied. Treatments with either the silver compenitions of this invention of deionized water ‘were applied tothe beef at 20 psi from a distance of 13 e in the model pressure rinse cabinet for 20 seconds. The spray nozzle (BETE NFOS80 308) delivered approximately 20 ml ‘of solation to the surface ofthe bee! sample. The temper- ture of solutions and treatment application room, was Approximately 14°C. After treatment, duplicate 3.5 em? core samples were randomly drain fom te lateral surface ofthe beef sample at 0, 20, 60 and 240 minvtes. Samples were cultivated and enumerated om selective differential andUS 2007/0190174 AI 10 recovery medi Log reduetions were calculated by subtract- ing the log of efufem ofthe inoculatedreated samples at the specified sampling times (0, 20, 60, and 240 minutes) from the log, of elwem® of the inoculated /untreated samples at © minutes. Sample treatment included the use of 32 ppm silver, 22 ppm silver. and 10 ppm silver composi tions acconding to the present invention. Separately, combi nations of 22 ppm Ag with 1.5 waht % hytogen peroxide and 10 ppm Ag with 10 ppm peroxydisulfate (K,S,0,) were tested. fost) [0152] The se ofa composition of 32 ppm silver accond- Sing this invention produced a reduction in bacteria on bec? sak. Ia the following, this redition is expressed the Jog, ofthe ratio ofthe numberof bacteria inthe eotrl at ti O to the amount of bacteria in the teat spocinen at the sampling (se, trent) time [0183] For Salmonella, at the lower initial bacteria level (10%, the folowing log exluctions were recorded: 0.78 at 0 minutes, 1-11 at 20 minutes, 1.08 at 60 minutes, and 1.23 at 240 minutes. Thus, at 4 houts 240 minutes), the rato of the inital bacteria count in the control fo bacteria in the sample treated with 32 ppm silver is 10". For the higher initial bacteria level (10°), the following log reductions were recoeded: 0.86 at 0 minutes, 0.95 at 20 min, 0.98 at 60 min tnd 1.17 at 240 min, The resuls indicate that the 32 ppm silver embodiment of this invention shows an elfecive bactericidal effet for Salmonella on beef steak. It will be appreciated that disinfecting 2 meat surface is an extreme challenge for any disinfectant [0184] For col, forthe lower initial bacteria level (104), the following log redctions were recorded: 1.03 at 0 riinutes, 1.28 at 20 mites, 1-42 at 60 minutes, ad 1.58 at 240 minutes. For the higher initial bacteria level (10°), the following log reduetions were rocordd: 0.65 at 0 minutes, (0.60 at 20 minutes, 0.83 at 60) minutes and O87 at 240 rites. The results indicate thatthe 32 ppm silver embodi- ‘ent ofthis invention shows an effective bactericidal elect Jor pathogenie E: coli on beef steak [0185] D. Results with 22 ppm Silver Composition [0186] Results with Silver in Water For Salmonella atthe lower initial bacteria level (10%), the following log rednc- tions were recorded: OI at O minutes, 0.43 at 20 minutes, (048 at 0 minutes, and 0.68 at 240 minutes. For the higher ‘nial bacteria level (10°), the following log reductions were recorded: 0.24 at 0 minutes, 0.24 at 20 minutos, 0.42 at 60 ‘minutes and 0:61 at 240 minutes. The results indicate that the 22 ppm silver embodiment ofthis invention furnishes aa ‘effective bactericidal effect for Salmonella on beef steak. [0187] Results with Silver ia water and 1.5 weht % hydrogen peroxide. For Salmonella, for the lower initial bacteria level (10°), the following log reductions were rococdod: 0:34 at minute, 0.33 at 20 minutos, 0.36 at 0 mines, and 0.62 at 240 minutes, For the higher inital bacteria’ level (10°), the following log reductions were recorded: 0.28 at minutes, 0.14 at 20 minutes, 0:30 3 60 ‘minutes and 0.69 at 240 minates. The results indicate that the 22 ppm silver with 1.5 weht % hydrogen peroxide ‘embodiment ofthis invention provides an effective hacer ‘ida effect for Salmonella on beet steak C. Results with 32 ppm Silver Composition Aug. 16, 2007 [0188] F. Results with 10 ppm Silver Composit [0159] Results with Silver Composition in Water. For Salmonella, for the lower initial bacteria level (10"), the following log reductions were recorded: 0.38 at ) minutes, O41 at 20 minutes, 039 at 60 minutes, and 0.61 at 2 ‘minutes. For the higher initial bacteria level (10%), the {allowing log reductions were recorded: 0.24 (at O minutes, 021 at 20 minutes, 0.41 at 60 minutes and 0.54 at 240 ‘minutes, The results indicate hat the 10 ppm silver embodi- ‘ment of this invention provides an effective bactericidal clfot foe Salmonella on bet steak [0160] | Results with Silver Composition in Water with 10 ‘pm K,S,0,. For Salmonella for te lower intial bacteria level (10°, the following log reduetions were recorded: 0.26 a 0 minutes, 0.28 at 20 minutes, 0.35 at 60 minutes, and (0.58 at 240 minutes. For the higher intial bacteria level (10°), the following log reduetions were recorded: 0.08 at 0 es, 0.16 at 20 minutes, 0.21 st 60 minutes and 0.36 at 240 minutes. The results indicate thatthe 10 ppm silver with 10 ppm potacsinm peroxyclisullate(K,S,0,) embodiment of this invention provides an ellective bactericidal effect for Salmonella on bee! steak [0161] 8. Evidence of Etfectiveness of 10 PPM Silver for Treatment of Human Ailments [062] A. Purpose of Example [0163] ‘The purpose of this example is to demonstrate the Uuilty of siver-based composition embodiments of the present invention for treating a variety of human ailments ‘The stios inthis section were performed in Ghana, West Africa, atthe Air Force Station Hospital under the direction fe. Kwabiah, atthe Korie-Bu Teaching Hospital under the rection of Sr Sackey, and atthe Justab Clini/Materity Hospital under the direction of De. Abraham. In ‘ota, ‘ily-vight (58) patients were teated using a composition of the present invention comprising 10 ppm silver The com- position was used both internally and extemally as an Altemative to traditional antibiotics. The ailments treated Jnclided malaria, upper respimstory tract infections, urinary ‘root infection, sinusitis, vaginal yeast infections, eye, nose and ear infections, cuts, fungal skin infections, and Sexually transmitted diseases, such a gonorshea, [0164] B. Treatment Methods and Outcomes [0165] Abdominal pain and Dinerhea. The method com- prises the step of administering approximately 5-25 ml of silver composition, one to five times a day orally until there ‘was a response. One patient was treated with about 10 ml (about two teaspoons) of composition of the present invention three times in one day. The patient had a full recovery in one day, [0166] Bronchitis. The method comprises the step of ‘administering ea, 2-25 ml of silver composition orally, one to five times a day until there was a response. Two patients \were treated with about $ ml (about one feaspoon) each of ‘8 composition of the present invention for two times a day Torthree days. The patients had a full recovery in three days [0167] Vaginal Yeast (Candida). The method comprises the step of administering ca. 5-25 mi of silver composition, ‘ne to five times a day’ as vaginal douches until there was @ response, Five patients Were ireated with about 10 ml (aboutUS 2007/0190174 AI ‘0 teaspoons) each of @ composition of the present i tion for two times per diy. The patents showed recovery within six days [0168] Conjunctivitis. The method comprises the sep of ‘administering ca, several drops ofa silver composition, one to five times a day to the infocted eye until there was @ response. TWo patients were treated with several drops of a ‘composition of the present invention in each of the infected ‘eyes for wo times per day. The patients had a fll recovery aller one day. [0169] External eus and infection (ineluding Staphvlocor~ ‘cus skin infections, septic wleers and infected abscesses), ‘The method comprises the step of administering « silver ‘composition, one to five times a day to the infected ages uti there was a response. Six patients were treated with about $ ml (about one teaspoon) each ofa compesition of the present ‘invention on the infected areas for two times per day. The Patients showed a full recovery within three days [0170] External Osis, The method comprises the step of ‘administering a silver composition, one to five times a day to the infected ear until there was a response. Six patents ‘were trated with approximately two drops ofa composition ‘of the present invention into the infected ears for three times per da. The patents showed a fll recovery after about four days. [0171] Oxitis Media, The method comprises the step of ‘administering a silver composition, ane to five times a day to the infected ea until there was a response. One patient ‘a5 tented with approximately two drops of # composition ‘of the present invention comprising into the infected ear tne mes pr dy. Te patient shoved 9 ull soe a four days [0172] Fungal Skin Infection. The method comprises the Step of administering a silver composition, one o five times a day topically to the infected area until there was response, TWwo patiens were treated with about tea ml (460 teaspoons) each of a composition of the present invention three times per day. The patients showed a full recovery ‘within eight days [0173] Gonorshea, The method comprises the step of adiinistering silver composition tothe infected area until there was a response. Two patieals Were each treated With about ten mi ($0 teaspoons) ofa composition ofthe present Jnvention three times per day. The patients showed ua absence of symptoms within six day’, [0174] Malaria. The method comprises the stp of admin= ‘string a silver composition, one to five times a day orally to the patent untl there wae a response. Eleven patients were treated with about ten mil (KWo teaspoons) each of & ‘composition of the present invention three Himes per day. “The paients showed a resolution of symptoms within five days. [0175] Halitosis and Gingivitis. The method comprises the step of administering a silver composition, one to five times a day as. mouthwash until there was a response. Two paliets were each treated with the composition us a mouth- ‘wash. There was a fall resoTution of symptoms within three days (gingivitis) and within one day (haltoss) [0176] Pelvic Inflammatory Disease. The method com= prises the step of administering about 5-25 ml of silver Aug. 16, 2007 composition, one to five times a day 38 a vaginal douche ‘until there was a response, One patient was twated With about $ ml (approximately one teaspoon) of a composition fof the present invention two times per day. The patiat's symploms resolved within five days [0177] Pharyngitis. The method comprises the step of Administering a silver composition, one to five times a day ‘asa gangle until there vas a response, Four patients were cach treated with about ten ml (two teaspoons) of a com- position of the present invention tree times per day. The patients showed full recovery within six days. [0178] _Retrovirus Infection (HIV). The method comprises the step of administering a silver composition, comprising 5 ‘0 40 ppm silver oneto five times a day orally area uni there was a response, One patient exhibiting HIV (human ia rodeficency virus }was treated with about 5 ml (appeo rately one teaspoon) of a composition of the prese jnvention (wo times per day. The patients symptoms resolved within five days [0179] Sinusitis and Rhinitis. The method comprises the step of administering a silver composition, one to five times ‘aday tothe nose until there was a response, Six patients with ‘nasal infections (four with sinusitis and two with shinitis) ‘were each treated with approximately two drops of com- position of the present invention comprising in their nasal passages three times per day. The patients showed fll recovery within four days, [0180] Tonsillitis, The method comprises the step of ‘administering a silver compesition, one to five times a day ‘as gargle until there was a response, One patient was treated with a composition of the present invention three times per day. The patient showed fll recovery within seven days [0181] Upper Respirstory Tract Infection. The method ‘comprises the slep of administering a silver composition, ‘one to fivetimes a day orally until there was a response. Two patients were each teated with about $ ml (approximately ‘one teaspoon) of «composition of the present invention three times per day. The patients showed full recovery within six days, [0182] Urinary ‘Tract Infections. The method comprises the step of administering a silver composition, one 10 five times a day orally until there was a response. Three patients were each treated with ahout ten ml (0 teaspoons) oF @ composition of the present invention two to three tines per ‘ay. The patients showed full recovery within six days [0183] C. Discussion [0188] These results are consistent with the various in viteo tests reported herein. Essentially, the silver compos sion is extremely effective against a lane number of microbes in vito, However, the tests indicate that this effectiveness remains even in the presence ofa large amount ‘of organic material, The silver compositions are widely cfectve in vivo where the organic background is extremely high. Many other disinfecting agents are ineffective in the presence of large amount af organic material and/or are oo fustic oF toxic to be used it Vivo,US 2007/0190174 AI 12 [0188] 9, Evidence of Etfcaey of 10 PPM Silver Against ‘Tuberculosis Bacteria, [0186] A. Purpose [0187] The purpose of this example isto demonstrate the ‘eflicacy of a silver composition of the present invention ‘against the bacteria that cause tuberculosis, This example ‘describes the procedures fr evaluation of the presen inven- tion for tuberculocdal ellicaey. The methodology is based ‘onthe Tuberculocidal Activity Test Method as accepted by the EPA on Dec. 11, 1985, [Refer to United States Environ- ‘mental Protection Agency, 1986. Office of Pesticides and Toxie Substances, Data Call-in Notice for Tubercuolocidal Ecctiveness Data for All Antimicrobial Pesticides with Tuberculocidal Claims. (Received Jun. 13, 1986), [0188] B, Material and Methods [0189] Materials. The silver composition of the preseat ‘vention comprised 10 ppm silver in Water, The silver ‘composition was evaluated employing 2 liguid to liguid mateix against Mycobacterium bovis BCG (TMC 1028) This onanism causes tuberculesis in animals and can cause tuberculosis ia humans. Its used as a “stand-in” for M, tuberculosis, the major cause of human tuberculosis, as tests have showin it to have a similar susceptibility to AL tuber= ‘culsis. The test organism was exposed to the silver com= positon in duplicate at four exposure times and quantified sing membrane filtration, [0190] Procedure, vial of frozen stock culture was removed from storage and thawed. An equal volume of buffered gelatin (BUGE) was added to the cell suspension ‘and homogenized with 4 Tellon® (brand of polytetraluo- roethylene) sue grinder for I minute while kesping the ‘culture at 0 104° C. in an ie bath, The homogenized cell, fuspension Was diluted with saline Tween® 80 (brand of polysorbate) solution (STR0) t0 approximately 10” eum! [0191] Challenge Titration. Tenfold serial dilutions ofthe ‘culture were prepared in dilation blanks containing 9 eal of neutralizer broth (NEUB) through a 10~ dilution. Three 1 mi aliquots of the appropriate dilutions were membrane fkered by fist adding 10-20 mil physiological saline solu- tion (PHSS) to the filter housing and then adding 21 ml iquot ofthe appropriate dilution. The filter was then rinsed ‘with approximately 100 ml of PHS, The filters were aseptically removed from the filter housing and placed onto ‘THI agar plates. The plates were incubated in a humidified chamber at 3722° for 21 days. [0192] Positive Contol. A tube containing 9 ml of STS was prepared and equilibrated fo 20805° At time O, 1 al ‘of test organism culture was added to the tube (1:10 dil tion). The sample was held for 60 minutes, Tenfold serial dllutions were preparod in dilution blanks containing 9 ml of NEUB through 10-° dilution, Thee 1 ml aliquots of the appropriate dilutions were membrane filtered by fist adding 10-20 ml PHSS tothe filter housing and then adding a 1 ml liguot ofthe appropriate diliton, The filter ws rinsed with ‘approximately’ 100 2a] PHSS. The filters were aseptically removed from the fiter housing and placed onto HL agar plates. The plates were incubated in a humidified chamber at 3722" C. for 21 days. [0193] Tests. Two 28x150 mm tubes containing 9 ml of the test sample were equilibrated to 2020.5" C. ina water Aug. 16, 2007 bath. To each tube containing the test disinfectant (ie, silver composition), | ml of test organism culture was added. The tube was mixed by swirling and placed back into the water bth. At 15, 30, 45, and 60 minutes, 1.0 mi aliquots of the isinfoctmt-celt suspension were transfered to 9 ml of [NEUB and mixed thoroughly, Tenfold serial dilutions were prepared in dilution blanks containing 9 ml of NEUB {rough the 10° dilution, Three 1 ml aliquots ofthe appro- ns were mombranesftered. by frst adding ISS tothe filter housing and then adding 1 ml aliquot ofthe appropriate dilution. The filter was rinsed with approximately 100 ml PHSS. The filters were aseptically removed from the fier howsing and placed onto THI gar plates, The plates were incubated in « humidification eham- ber at 3722” C. for 21 days. [0194] Pheoot Conteol. To demonstrate minimum culture Viability and resistance, the culture was tested against a (0.8% phenol solution. 1 mi aliquot of test onganism culture as placed into 9 ml ofthe phenol solution equilibrated to 252015" C. and incubated for 20 minutes. After the expostre period, 1 ml from the phenoloranism solution was removed and added to 9 ml of NEUB. Teafold serial lutions were prepared in dilution blanks containing 9 ml of NEUB through 10-* dilution. Thee 1 ml aliquots of the ‘appropriate dilutions were membrane fered by fst adding 10-20 ml PHSS tothe fiker housing and then adding a 1 mi Aliquot ofthe appropriate dilution, The filer was ised with approximately 100 ml PHISS. The filters were aseptically removed from the fter housing and placed onto 7H agar plates, The plates were incubated in a humidified chamber at 3782" C. for 21 days. [0195] Newteatizaton veritication. A 1 mi aliquot of the ‘sinfectant was added to 8 ml of NEUB, The disinfectant neutralizer broth was allowed to equilibrate to the same temperature as the test samples. One ml of test organism culture was added to the mixture aad asixed thoroughly Incubation was continued for the approximate time it woud take (filter a sumple. Additionally, 1 ml aliquot of test ‘onganism Was added to 9 ml oF NEUB and mixed thoroughly (1:10 dilution), Tenfold serial dilutions of both tubes were prepared in dilution blanks containing 9 ml of NEUB ‘thought 10° dilution. Three I ml aliquots ofthe appropriate lutions were membrane filtered by first adding 10-20 ml PHSS tothe filter housing and then adding a 1 mi aliquot of the appropriate dilution, The filter was rinsed with appeoxi- ‘mately 100 ml PHSS. ‘he filters were aseptically removed {rom the filter housing and placed on THI agar plates, The plates were incubated in a humidified chamber st 372° for 21 days. [0196] C. Results {0197}, The starting titer for the chlenge colure was 417310’. The positive conto ter was 65x10” eft The mia se inthis ty elective demonstrated eiraliztion with 95.2% recovery ins dinfetan! neralizer solton when compared 9 ma Bank [0198] For the test plates, expected eounts were underes- ‘imated and therefore te reported counts exhibit “>” 10 ‘mark that the couat isan estimation aad tat aeeurate counts are beyond the limit of detection forthe dilutions plate. [0199] In caleulating the log and percent reductions ofthe isinfectant against ML. bovis, the estimated counts whiehUS 2007/0190174 AI have “greater than” counts resulted in “less than” log and percent reductions (“<"). The purpose of this is to demon- rate that the results are an. estimation and beyoud the ‘accurate limit of detection for the dilutions plated. All reductions were calculated using the postive control as the ‘nial starting liter ofthe organism, The results for log and pereent reductions are summarized below. As @ measire of the resistance of the challenge entre, the phenol resistance ‘of the ME bovis showed a =1.81 log reduction with 20, rinutes of exposure to 0.8% phenol [0200] Replicate One 30 minutes on aun [0201] Replicate Two: mines Sane mites Sat [0202] D. Conclusions [0203] The use of silver compositions of the present ‘invention i effective against therculosis bocteris, Amethod ‘comprising the step of administering silver compositions of the present invention is elective against tuberculosis organ- [0204] 10. Fvidence of Piicacy of 10 PPM Silver Against Candida Aibicans ATCC #10231, Trichomonas Vaginais ATCC #20238, and MRSA Staphvloceocus Aureus ATCC BAN [0205] A. Purpose of Example [0206] The purpose of this example is to illustrate the ‘ellicacy of silver compositions of the present invention against Candida albicans ATCC1O231, Trichomonas vagi- nalis ATCC 20235, and drug. resistant Staphylococcus ‘oureus ATCC BAA-. [0207] Candide albicans, yeast, and Trichomonas vaet- nalisis, 1 protozoa, can cause numerous health problems Including vaginal infections, diaper rash, and thrush. The results below show that silver compositions of the present Jnvention produced nearly a 100% kill of both organisms. The results show the utility of silver compositions of the present iavention in a feminine hygiene product and in & diaper rash prod, [0208] Staphylococcus aureus can cause serious blood poisoning when it enters a wound, Itonce was easly treated ‘with penicillin, but the organism bas now mutated to the point where itis totally resistant t0 penicillin. The next ‘defense on the antibiotic ladder has been methicili, but rmethicillia-resistant strains have become inereasinaly co Aug. 16, 2007 hospitals. These stsins are known as resistant Staplnlococeus aureus) and have been dubbed the “superbug” People who contact ‘MRSA can die ina matter of days. In dhe results reported in {his example, a silver composition of the resent invention was found to Kill 1.69% of the MRSA in just LO minutes, and 99.59% in an hour. The results show the utility of silver compositions of the present invention in killing MRSA, 2 Known infectious threat. [0209] B. Methods and Results [0210] Employing the USP Preservative Repid Challenge ‘Test witha composition ofthe present invention comprising 10 ppm silver in water, dhe following results were oblained. “These results show that silver compositions of the present invention can be effective against yeast infections, protozoa infotions, and drug resistant hacieria infections [0211] Candida albicans ACC #10231. The inital cone centration of Candide albicans yeast was 6 8x10" elvan (After contact for either 10 minutes, 30 minutes, { hour, or ‘one day with the silver composition, there were no colonies detected. [0212] Trichomonas vaginalis ATCC #30235, The initial jon of Trichomonas vaginalis protozoa was 6.0% ‘Aller contaet with the silver composition for either 10 minutes, 30 minutes, 1 hour, or one day, thore was 0% motility of 100 Organisms. That is, one hundred (100) Trichomonas vaginalis parasites were analyzed via micros- copy for motility of agella, None ofthe one-hundred (100) patasites demonstrated motility after only ten (10) minutes ‘of contact with the silver composition indicating inhibitory fr lethal properties ofthe silver composition on the pare sites. The outer membranes of twenty-five (25) percent of the parasites had ruptured after contact of one (1) day. [0213]. Staphylococcus aureus MRSA ATCC PBAA-4. ‘The intial concentration of methcilin-esistant Staphylo coccus aureus (MRSA) was 6.0x10" cfu/ml. After contaet ‘with the silver composition, there were 500,000 fim! Setocted after 10 minutes eontaet (91.6% killed), 70,000 cfwiml ater 30 minutes contact (98.8% killed), 30,000 fwiml ater I hour contact (99.5% killed), and fewer than 10 fw after one day contact (vitally otal kill) [0214] 11. Evidence of the Elieacy and Lack of Cytowox- icity of 10 PPM Silver, I4 PPM Silvers] 5% HO, and 22 PPM Silver in Inhibiting DNA Polymerase and Reverse ‘Transcriptase in the Context of Hepatitis B [0215] A. Purpose of Example [0216] The purpose of the example is 10 illustrate the elcacy of silver compositions of the present invention against hepatitis B. This example shows that silver compo sitions of the preseat invention have antiviral properties. Any agent used in antiviral therapy should exhibit Tite or no $0 cytotoxicity ofthe silver compositions was [0217] Hepatitis B is cawed by a DNA virus of the hepachaviridae family of virases. The Hepatitis B. Virus (HBV) isa3.2kb DNA vir, replicating almost exclusively in the liver eels (hepatocytes). Replication involves two main enzymes: DNA polymerase and reverse transcriptase ‘The resulis of this example show that silver compositions of the present invention interfere with replication involving.US 2007/0190174 AI 4 ‘ther DNA polymerase or reverse transcriptase, The results ‘of this example show that silver compositions ofthe present invention have antiviral properties. The results of this ‘example show that silver compositions of the present iaven- tion ean be effective against hepatitis B, [0218] As furter detail, when hepatitis B enters the body ‘ofa ew host, it infects the liver it gets past the he's Snumune system, In the infection, the vir taches tothe membre of a iver cell, ae the coe particle of the vis ‘enters the liver ell. The core particle then releases its eontents of DNA and DNA polymerase into the liver cell nucleo. Within the liver cell the viws replicates via reverse transcription and wanslaon processes, which involve reverse transeriplase and DNA polymers enzymes, The DNA polymerase causes the liver eel to make cops of hepatitis DNA. These copies ofthe vine ae release fom the liver cell membrane into the blood stream. From ther, they ca intet other liver ells and this replicate cectively. “The incubation period of the epatis B virus is about 6 (0 25 weeks Ge, me before physical and generally detectable histological or physica symptoms oocur. However, there sre several biochemical ad Histological eanges that octr in the early stages following infection withthe hepatitis B [0219] B. Matsals [0220] Sotutions comprising 10 ppm, 14 ppm, 22 ppm, ‘nd 32 ppm silver compositions according to the present Aislogure were used. The nucleotides 4ATP, dGTP. ACTP, fand [H}JTTP were oblained from standard commercial Sourves, as were the compounds lamivudine (a synthetic ‘otretroviral gent) and zidovudine (AZT). soated Hepa tits B vis was freshly obtained fom a person suffering from Hepatitis B infection and was taken up by Haline Institute, Mumbai INDIA (a WHO ented testing labors tor). Test eel cultures (Vero and Tep2) were grown as confluent monolayers by typical ell clare methods. [0221] C. Methods [0222] 1) Procedure for Test of DNA Polymerase Inhibi- [0223] Overall approach. Hepatitis B viral extracts from hhuman subjects ae incubated with radiolabelled! noclotides ‘nd anaetve inhibitor. Percent inhibition is eleulted based ‘onthe amount of de novo viral nucle eid synthesized with, respect 0 lamivudine 2s a positive contol and phosphate buifer saline (PBS) as @ negative conto [0224] Specific procedure. Isolated Hepatitis B virus was Iysed to extract free polymerase enzyme, which is fre fom ‘contaminating enzymes, A virus extract (25 ul) was added to 4 reaction mixture comprising ATP, dGTP, dCTP and CHLTTP nucleotides (5 ul. Active inhibitor @ pl) was ‘added to the mixture comprising vitus extract and nucle- ‘tides. The resultant mixture was incubated at 37°C. for 2 hours [0225] A separate negative control experi Jormed in which phosphate buffer saline (PBS used instead ofthe inhibitor @ 4) [0226] A separte postive control experiment was per- ormed in which a known DNA polymerase inhibitor Qi ‘of lamivudine at a concentration 3 mle) wae used instead ‘of the tested inhibitor (3 l). Aug. 16, 2007 [0227] The reaction was stopped by adding 25 yl EDTA, And 25 pl TCA (richloroacetie aid), The reetion mixture was then spotted on ionic paper (DEAE paper). The paper was washed three times with TCA and then with ethyl alcohol. The filter paper was air dried and put into scintillation vial with scintillation cocktail. Radioactivity ‘was measured by a liquid scintillation counter (Blue Sts). As a counting control, a blank silver composition was run ‘through the complete procedure without viral load, to chock ‘any potential interference in the scintillation counter method [0228] _A reference fortis method is P.S. Venkateswaran, 1. Millia, and B.S. Blumberg, “Elect of an extract from Phsllanthus nirari on hepatitis B and woodehuck hepatitis Viruses: in vito andi Vivo studies," Proe, Natl Acad. Sc, USA, 1987, 84, 274-278, which is incorporated herein by relerence. [0229] 2) Procedure for Test of Reverse Transcriptase Inhibition. {0280] _A commercial viral enzyme preparation of Molo- ney murine leukemia virus reverse tanscriptase (MoMULV) having Poly ANT (pvimer for RT) vas wsed. 50 ofthe MoMULY’ preparation was combined with a mixture of ATP, dGTP, UCTP and ['HRITTP nucleotides [0231] This mixture was combined with 3 ofthe iibi- tor tobe tested, and the resultant mixture was incubated at 37°C. for 2 hours [0232] A negative control experiment was performed in ‘hich phosphate buifer saline (PBS, 3) was used instead of the inhibitor. [0233] A positive control experiment was performed in whieh a known reverse transcriptase inbibitor (3 yl of AZT. ata concentration 0.625 microgranml) was used intend of the tested inbibitor [0234] The reaction was stopped by adding 25 ul EDTA, and 25 yl TCA. The reaetion mixture was then spotted on ionic paper (DEAE paper). The paper was washed three times with TCA and then with ethyl alooho. The filter paper ‘was ir dred and put in scintillation vial witha scintillation cocktail. Radioactivity was measured by aliquid scintlle tion counter (Bive Star) [0235] 3) Procedure for Testing Cytotoxicity [0236] Cells were prepared from healthy, confhient Vero And Hep2 cell cultures that were maintained by passage every 34 days, One day prior fo the test cells were released fom the cultures sing standard techniques and suspended ‘na growth mestum and dispensed into wells ofa microtiter plate and placed in a 5% CO, incubator at 3722° C. Aa aliquot (100 ju) ofeach test substance was introduced into well (in triplicate) with 100 of PBS as a contro, Every 24 hrs the wells were examined under high power of aa inverted microscope to check for any cytopathic elect (CPE).US 2007/0190174 AI [0297] D. Results [0238] Resulls for Test of Reverse Transcriptase Inkibi- Sample ion eave cnn (28) o postive comm (AZT) 3133 Sine 14 pp an 1.38 H202 1693 [0239] Results for Test of DNA Polymerase Inhibition Sine 10 pp ms [0240] Silver compositions of the present invention sre highly effective at inhibiting DNA polymerase [02st] Results for Test of Reverse ‘Transcriptase Inhibi- iver compositions of the present invention would be expected (0 be elective against human ailments propagated by viruses, such as hepatitis B. [0243] Results for Test of Cytotoxicity Sie 10 pp NOCHE Nore Sino ppm wih 1% 202 CPE poakive CPE postive Sie 2 pe NocPE. _NoCPE [0244] These results indicate thatthe silver compositos ‘essentially non-eytotoxie. As expected, hydrogen peroxide, ‘hich is known to be eytotoxie, shows @ cytotoxie effet, ‘Thus, the silver should be harmless to cells when used in Aug. 16, 2007 [0245] 12. Bvidence of Eticaey of Silver Composition as Water Disinfectant [0246] A. Purpose [0247] Tests were carried out to demonstrate the efficacy of he inventive composition in disinfecting drinking water [0248] B. Methods [0249] A sample of raw river water was spiked with two Joopfuls of Klebsiella oxtyoca, 100 al aliquots of this ofthis spiked water volution were brought to 0.05 ppm, 0.1 ppm, (02 ppm. 0.5 ppm. oF 1.0 ppan of inventive silver compos ‘ion. After an incubation of 5-60 minutes, the samples were sembrane flere, The filter wus ised with approximately 100 ml sterile water, The filters were aseptically removed {rom the fer housing and placed on coliform nutrient agar plates. The plates were incubated under growth conditions Tor 24 hours and counted ° ° ° ° ° ° ° ° too ta to TRC = wo nineror oom [0250] The silver composition proved to be surprisingly effective. Even at the shortest test time (20 min) allowed for incubation of the lowest concentration tested (0.05 ppm) there was @ complete kill of the bacteria. At 0.20 ppm and higher there was a complete Kil at S minutes, I seems clear that a complete kill takes Tess than $ minutes, [0281] 13. Evidence of EMieacy of 32 PPM Silver as Surface Disinfection [0252] The Environmental Protection Agency (EPA) has approved a 32 ppm silver composition ofthe present inven- tion as a broad spectrum surlace disinfectant for use ia hospitals, medical environments, residential homes, com- serial buildings, and businesses. It has been approved for ‘use against some of the most deadly pathogens including: Gram-positive bacteria, such as Staphylococcus aureus (presently considered to be the most deadly bacteria in U.S. hospitals), Gram-negative hactera, such 2s Salmonella chol- eraesuis (responsible for food poisoning) and nosocomial for hospitalacguired pathogens, such as Pseudomonas ‘aeruginosa (often found in burns and cus) [0253] Silver compositions ofthe present invention can be sprayed in and around occupied areas without endangering the lives of people. One ca disinfect surfaces selected from the group consisting of walls, tables, chairs, light fixtures, bathrooms, glass, porcelain, metal, glazed ceramic, enam- led and painted by means of spraying or by means ofUS 2007/0190174 AI tg with a silver compositio preferred method of disinfecting comprises the steps of cleaning the surface tobe disinfected, applying, by means of a spray, mop, sponge, or cloth, a composition ‘of the present invention, thoroughly wetting the area to be ‘disinfected allowing the surface to remain wet frat least 10 minutes ata temperature of atleast 20° C.(ime/temperature interrelation can be adjusted vin the Amenius equation oF ‘other means known to one of ordinary skill), an wiping the surface witha clean paper or cloth towel. Compositions for «isinfecting surfaces comprise those comprising 50 40 ppm silver. A prefered composition of the present invention for slisinfoeting surfces comprises (3223) ppm silver. Another prefered composition ofthe preset invention for isin Ing surfaces comprise (1022) ppm silver Another preferred ‘composition of the present invention for disinfecting sur- faces comprises (2222) ppm silver. [0284] 14. Evidence of Elicacy of Silver Composition as Super Disinfectant [0285] A. Purpose of Example [0286] The purpose of this example is to show the ant microbial activity of a silver compesition of the present invention (here 10 ppm silver, 14 ppm silver with 1.5 waht °% hydrogen peroxide, and 32 ppm silver) against the fest ‘organism Yersinia pestis, the etiologic agent of bubonic plague. By performing standard killtime assay using a ¥ pestis suspension, its demonstrated that silver eompositions fof the present invention are effective even against the bubonie plague bacteria, [0257] B. Material and Methods [0258] _¥. Pests, strain D27, was grown on a Columbia Agar plate for about 24 hours at 30° C. in a $% CO, incubator. Growth from the plate was scraped into suspen= son, using 3 mi of sterile HPLC water. The suspension was transferred to 2 50 ml conical centrifuge tube. The plate was then rinsed using an additional 2 ml of HPLC water, This rinse was added to the centrifuge tube. The tube was ‘centrifuged at 3,500 for § minutes. The supematant was discarded and the pellet was resuspended in I ml of HDLC water, to give a final concentration of approximately 10° cells per ml. [0259] The Method Involved the Following Steps [0260] 1.4.99 ml aliquot ofthe silver composition to be tested was placed ina sterile 20 mmc 0 mm tube. The tube ‘as equilibrated in a 20° C, water bath [0261] 2 The tube of silver composition was inoculated with 100 ofthe test organises suspension at time 2er0 10 form @ mixture. ‘The tube was immediately vortexed and returaed to the water bath [0262] 3 At2 min, 3 min, 4 min, and $ min for 10 ppm oF 32 ppm silver or 2 mia, 4 min, 6 mia and 8 mia for 14 ppm silver with 1.5% vv H,O,, 1 mi of onganismisilver mixtnre ‘vas removed to 99 mi of neutralizer ina 250 ml Erlenmeyer flask. The flask was mixed thoroughly [0263] 4 The ncutalized suspension was immediatly serially diluted 1:10 in physiological saline solution (PSS). [0264] 5 The number of viable organisms in selected dilution tubes and flasks was assayed by membrane filtra- tion, One ml aliquots were plated in duplicate. The me Aug. 16, 2007 branes were washed with about 150 ml (oe 250 ml i the sample was taken fom the neutralizer Mask) of sterile phosphate bulfeed saline and removed to Columbia Agar plates, The entire remaining contents (98 ml) of the 4 && 5 ‘in neuiniizer Masks were also plated. The plates were ‘incubated at 30° C. in 8 $% CO, incubator for 72 hours. [0265] 6 The mumber of colonies on each fier was ‘counted and log reductions were computes, [0266] C. Results [0267] The Resuits for 10 ppm Silver areas Follows Sin 5 ae ‘it 368 98 [0268] The calculated regression equation fr these data is "Y=2.396540.1696 x. This indicates that the time fora 6-log reduction is 21.2 minute [0269] The Results for $2 ppm Silver are as Follows: Tine __Uig Reston Pesce Kl [0270] ‘The Results for 14 ppm Silver with 1.5% wv HO, are as Follows Tine Lag Reicton Pesce Kil [0271] ‘The calculated regression equation for these data is YeI.37141,024 x. This indicates that the ime for @ 6-log reduction is 4.52 minute [0272] The silver composition of the present invention exhibited significam bactericidal activity against Y. pestis, the etiologic agent of bubonie plague. The 32 ppm compo: sition gave more than a 7 log reduction (essetially total Kill) inless than 2 min. The data show that the 10 ppm silver takes some 20 min to achieve a 6 Jog kil. The silver and hydrogen peroxide show significant synergism with a calculated 6 log Kull of under 5 min, This is mueb better than 10 ppm silver ‘lone, The level of 14 ppm silver was chosen because the ata of other experiments suggested that this level of silver ‘combined with hydrogen pemxide would achieve results ‘approaching those of the 32 ppm silver produetUS 2007/0190174 AI 7 [0273] 15, Data Summary repeated above. However, the results were obtained using the procedures explained shove so that one of ordinary skill in the art can readily replicated the results ‘composition on a wide varity of mictobes and human [0278] Human Diseases Cured By’ and Pathogens Killed [0274] The following table contains a summary of the ‘above resus in terns of the elfects ofthe inventive silver Aug. 16, 2007

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