Abstract:
A method that includes the steps: inoculating nutrient agar with bacterial stock to form a culture; incubating the culture to form a first incubated culture; incubating a portion of the first culture with nutrient agar to form a second culture; incubating a portion of the second culture to form a third culture; incubating the third culture to form an inoculated test plate; forming an inoculum by suspending bacteria from the inoculated test plate in a buffered test solution, adjusting the pH to ˜7 to 8, and adding organic soil at a concentration of approximately 10% to 30% by weight; inoculating a silver-containing surface region of a test carrier with a portion of the inoculum; incubating the inoculated test carrier; washing the test carrier in a neutralizing solution to form a residual test inoculum; and calculating the percent reduction in the number of surviving bacterial colonies in the residual test inoculum.

Description:
[0001]    This application claims the benefit of priority under 35 U.S.C. §119 of U.S. Provisional Application Ser. No. 61/908,401 filed on Nov. 25, 2013 the content of which is relied upon and incorporated herein by reference in its entirety. 
     
    
     TECHNICAL FIELD 
       [0002]    The present disclosure relates generally to methods of testing the antimicrobial efficacy of silver-containing surfaces including the composition and preparation of inoculums for such testing. 
       BACKGROUND 
       [0003]    Various health benefits associated with metallic silver have been developed over the last few hundred years. Various antiseptic, antibacterial, antiviral and other antimicrobial effects have been observed and documented associated with the use of silver in various articles. Silver vessels, for example, were used in the care of ill and wounded individuals in the Middle Ages. More recently, the incorporation of metallic silver particles and silver salts into a variety of materials including yarns, fabrics, and glasses has been described as a means of imparting antibacterial properties to articles containing these materials. Silver ions, for example, can be incorporated into the surface of prosthetic devices having glass, glass-ceramic or ceramic surfaces to impart these surfaces with antimicrobial properties. 
         [0004]    The U.S. Food and Drug Administration approved silver solutions as antibacterial agents in the 1920s. However, only recently scientists have begun to explore how, why and where silver works as an antimicrobial agent. Silver has been shown to have antibacterial, antifungal, antiviral, anti-inflammatory, antibiofilm properties. Many gram-negative bacteria such as  Acinetobacter, Escherichia, Pseudomonas, Salmonella, Vibrio  and gram-positive bacteria including  Bacillus, Clostridium, Enterococcus, Listeria, Staphylococcus , and  Streptococcus  are sensitive to silver. Silver is a fast-acting fungicide against a broad spectrum of common fungi including genera such as  Aspergillus, Candida  and  Saccharomyces . Silver has also been demonstrated to be effective against viruses such as Human Immuno Deficiency Virus (HIV-1). To date, silver ions are known to be effective against over 650 types of bacteria. 
         [0005]    With the wide use of antibiotics in today&#39;s world, antibiotic-resistant bacteria such as methicillin-resistant  Staphylococcus aureus  (MRSA), vancomycin-resistant  Enterrococcus faecium , etc. are commonly associated with nosocomial infections (i.e., infections resulting from receiving treatment in a healthcare service unit, but secondary to a patient&#39;s original condition). Such antibiotic resistant microbes are also very sensitive to the biocidal effect of silver. With the increasing cost of Hospital Acquired Infections (HAIs), antimicrobial intervention strategies to reduce contaminated surfaces and reduce incidence of cross contamination has become a major focus in the health care industry. 
         [0006]    In contemporary society, computerized and electronic “touch screens” are prevalent in various consumer products, e.g., automatic teller machines, gasoline pumps, mobile phones, etc. Generally, these touch screens possess glass, glass-ceramic and ceramic substrates. Recent studies suggest that touch screens harbor large quantities of microbes, bacteria and viruses harmful to humans. Recent developments indicate that silver ions incorporated into the substrates of these touch screens can impart antimicrobial properties to these products. 
         [0007]    In the United States, antimicrobial products are regulated as pesticides by the U.S. Environmental Protection Agency (“EPA”). To be registered with public health claims (stating protection for the user from bacteria or other microbial organisms that can lead to health impact), antimicrobial products must demonstrate antimicrobial efficacy using recommended test methods that are either a controlled in-use study or simulated in-use study. 
         [0008]    So far, the only test method using a simulated in use study and being approved by the EPA for public health claim, is the Test Method for Efficacy of Copper Alloy Surfaces as a Sanitizer (“Copper Test Protocol”). Although this method has become the reference method, there are still a number of significant scientific challenges in establishing valid test methods, and performance standards for product claims specific for the product end use (e.g., touch screen applications). Moreover, this standard method has been successfully used to assess the efficacy of copper alloys, but has failed to measure the efficacy of other antimicrobial technologies. 
         [0009]    No EPA-approved test methods exist today for surfaces incorporated with silver ions as an antimicrobial product. Particular methods are not available to characterize the efficacy of glasses, glass-ceramic, ceramic substrates and other surfaces incorporated with silver ions as antimicrobial products. Furthermore, data suggests that the Copper Test Protocol is not capable of measuring with robustness the antimicrobial efficacy of surfaces containing silver ions. There is therefore a need to develop a protocol suitable for testing the antimicrobial efficacy of surfaces containing silver ions. 
       SUMMARY 
       [0010]    According to a first embodiment, a method for testing the anti-microbial efficacy of a silver-containing surface region is provided. The method includes the steps: inoculating nutrient agar with a portion of a stock having a plurality of bacterial organisms to form a culture; incubating the culture to form a first incubated culture; incubating a portion of the first incubated culture with nutrient agar to form a second incubated culture; incubating a portion of the second incubated culture to form a third incubated culture; and incubating the third incubated culture for approximately 48 hours to form an inoculated test plate with a plurality of bacterial colonies. The method also includes the steps: forming an inoculum by suspending a portion of the plurality of bacterial colonies in a buffered test solution, adjusting the test solution to a pH of approximately 7 to 8, and adding an organic soil serum at a concentration of approximately 10% to 30% by weight to the test solution; inoculating a silver-containing surface region of a test carrier with a portion of the inoculum; and incubating the inoculated test carrier for at least approximately two hours. The method further includes the steps: washing the incubated and inoculated test carrier in a neutralizing solution to form a residual test inoculum; counting the number of surviving bacterial colonies per volume in the residual test inoculum; and calculating the percent reduction in the number of surviving bacterial colonies in the residual test inoculum relative to a residual control inoculum. 
         [0011]    According to a second embodiment, a method for testing the anti-microbial efficacy of a silver-containing surface region is provided. The method includes the steps: inoculating nutrient agar with a portion of a stock having a plurality of bacterial organisms to form a culture; incubating the culture to form a first incubated culture; incubating a portion of the first incubated culture with nutrient agar to form a second incubated culture; incubating a portion of the second incubated culture to form a third incubated culture; and incubating the third incubated culture for approximately 48 hours to form an inoculated test plate with a plurality of bacterial colonies. The method also includes the steps: forming an inoculum by suspending a portion of the plurality of bacterial colonies in a buffered test solution, adjusting the test solution to a pH of approximately 7 to 8, and adding an organic soil serum at a concentration of approximately 10% to 30% by weight to the test solution; inoculating a silver-containing surface region of a test carrier comprising an inorganic glass material with a portion of the inoculum; and incubating the inoculated test carrier for at least approximately two hours. The method further includes the steps: washing the incubated and inoculated test carrier in a neutralizing solution to form a residual test inoculum; counting the number of surviving bacterial colonies per volume in the portion of the residual test inoculum; and calculating the percent reduction in the number surviving bacterial colonies in the portion of the residual test inoculum relative to a residual control inoculum. In addition, the plurality of bacterial organisms is selected from one of the group consisting of  Staphylococcus aureus, Enterobacter aerogenes, Pseudomonas aeruginosa  and  Escherichia coli.    
         [0012]    According to a third embodiment, a method of preparing an inoculum for testing the anti-microbial efficacy of a silver-containing surface region is provided. The method includes the steps: inoculating nutrient agar with a portion of a stock having a plurality of bacterial organisms to form a culture; incubating the culture to form a first incubated culture; incubating a portion of the first incubated culture with nutrient agar to form a second incubated culture; incubating a portion of the second incubated culture to form a third incubated culture; and incubating the third incubated culture for approximately 48 hours to form an inoculated test plate with a plurality of bacterial colonies. The method also includes the step: forming an inoculum by suspending a portion of the plurality of bacterial colonies in a buffered test solution, adjusting the test solution to a pH of approximately 7 to 8 and adding an organic soil serum at a concentration of approximately 10% to 30% by weight to the test solution. Further, the plurality of bacterial organisms is selected from one of the group consisting of  Staphylococcus aureus, Enterobacter aerogenes, Pseudomonas aeruginosa  and  Escherichia coli.    
         [0013]    According to a fourth embodiment, an inoculum for testing the anti-microbial efficacy of a silver-containing surface region is provided. The inoculum includes an inoculum comprising (a) a plurality of bacterial colonies; (b) an organic soil serum at a concentration of approximately 10% to 15% by weight; and (c) a buffering solution. Further, the inoculum has a pH of approximately 7 to 8. 
         [0014]    According to a fifth embodiment, an antimicrobial glass is provided. The antimicrobial glass includes a glass substrate having a silver-containing surface region. The surface region is characterized by a log kill rate of 2 or greater as tested by the inoculum of the fourth embodiment. 
         [0015]    According to a sixth embodiment, an antimicrobial glass is provided. The antimicrobial glass includes a glass substrate having a silver-containing surface region. The surface region is characterized by a log kill rate of 2 or greater as tested by the method of the first embodiment. 
         [0016]    According to a seventh embodiment, an antimicrobial glass is provided. The antimicrobial glass includes a glass substrate having a silver-containing surface region. The surface region is characterized by a log kill rate of 2 or greater as tested by the method of the second embodiment. 
         [0017]    According to an eighth embodiment, an antimicrobial glass is provided. The antimicrobial glass includes a glass substrate having a silver-containing surface region. The surface region is characterized by a log kill rate of 2 or greater as tested by the method of the third embodiment. 
         [0018]    Additional features and advantages will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the embodiments as described herein, including the detailed description which follows, the claims, as well as the appended drawings. 
         [0019]    It is to be understood that both the foregoing general description and the following detailed description are merely exemplary, and are intended to provide an overview or framework to understanding the nature and character of the claims. The accompanying drawings are included to provide a further understanding, and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiments, and together with the description serve to explain principles and operation of the various embodiments. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0020]      FIG. 1A  is schematic depicting the steps involved in the Copper Test Protocol. 
           [0021]      FIG. 1B  is a schematic depicting the contents of the inoculum employed in the Copper Test Protocol. 
           [0022]      FIG. 2  is a schematic depicting the effects of modifications to the inoculum employed in the Copper Test Protocol when used to test the antimicrobial efficacy of a glass surface incorporating silver ions. 
           [0023]      FIG. 3  is a bar graph depicting the log kill rate versus inoculum pH level for inoculum formulations employed to test the antimicrobial efficacy of a glass surface incorporating silver ions according to one embodiment. 
           [0024]      FIG. 4  is schematic depicting the steps involved in a protocol for testing the antimicrobial efficacy of the surface region of an article having silver ions according to another embodiment. 
           [0025]      FIG. 5  is a bar graph depicting the log kill rate versus various inoculum preparations employed to test the antimicrobial efficacy of a glass surface incorporating silver ions according to an additional embodiment. 
           [0026]      FIG. 6  is a bar graph depicting the log kill rate versus the concentration of sodium bicarbonate in a Hank&#39;s Balanced Salt Solution (“HBSS”) employed to test the antimicrobial efficacy of a glass surface incorporating silver ions according to a further embodiment. 
           [0027]      FIG. 7  is a plot of the log kill rate versus the concentration of organic soil in an inoculum buffered with a Phosphate Buffered Solution (“PBS”) employed to test the antimicrobial efficacy of a glass surface incorporating silver ions according to an embodiment. 
       
    
    
     DETAILED DESCRIPTION 
       [0028]    Reference will now be made in detail to the present preferred embodiments, examples of which are illustrated in the accompanying drawings. Whenever possible, the same reference numerals will be used throughout the drawings to refer to the same or like parts. 
         [0029]    The Copper Test Protocol is incorporated by reference herein. As schematically depicted in  FIG. 1A , a copper test protocol  100  that follows the Copper Test Protocol can be used to test the antimicrobial efficacy of copper containing surfaces. In general, copper test protocol  100  includes steps  20  through  50  to prepare an inoculum  18 . Inoculum  18  is then employed to evaluate the antimicrobial efficacy of a copper-containing test carrier  6  relative to a control test carrier  6   a  in steps  60  through  90 . 
         [0030]    In step  20 , a tester obtains a stock  2  of bacterial organisms  10 . Bacterial organisms  10  can include any one of colonies of  Staphylococcus aureus, Enterobacter aerogenes, Pseudomona aeruginosa , and  Escherichia coli  for a given test sequence. Once the microorganism strain is selected for the bacterial organisms  10 , a tube  4  (or other suitable container) containing a broth  12  is inoculated with a portion of the bacterial organisms  10  in step  30   a . The broth  12  may consist of a Tryptic Soy Broth (“TSB”) formulation as understood by those with ordinary skill in the field. The inoculated broth  12  in step  30   a  is then incubated for 24±2 hours at 35-37° C. 
         [0031]    In step  30   b , a portion of bacterial organisms  10   a  obtained from step  30   a  (incubated for 24±2 hours) is introduced into a fresh broth  12 . The now-inoculated broth  12  with bacterial organisms  10   a  in step  30   b  is then incubated for 24±2 hours at 35-37° C. Finally, in step  30   c , a portion of the bacterial organisms  10   b  obtained from step  30   b  (incubated for 48±4 hours) is introduced into a fresh broth  12 . The inoculated broth  12  in step  30   c  with bacterial organisms  10   b  is then incubated for 24±2 hours at 35-37° C., thus forming an inoculated test broth  12   a  (see step  40 ). 
         [0032]    In step  40 , the inoculated test broth  12   a  is incubated for an additional 48±4 hours at 35-37° C. Organic soil  14  and surfactant  15  are then added to the inoculated test broth  12   a . For example, the organic soil  14  can be fetal bovine serum and a TRITON® X-100 formulation can be used as the surfactant  15  as readily understood by those with ordinary skill in the field. Preferable, the organic soil  14  and surfactant  15  are added at concentrations of 5% and 0.01% by weight, respectively. An inoculum  18  is thus formed from the inoculated test broth  12   a  as depicted in step  50  in  FIG. 1A . 
         [0033]    Inoculum  18  can then be employed to test the antimicrobial efficacy of copper-containing test carrier  6  and control test carrier  6   a  in steps  60  and  70 . In step  60 , a portion of the inoculum  18  is spread on copper-containing test carrier  6  and control test carrier  6   a  and dried. Under the copper test protocol  100 , test carrier  6  comprises a copper alloy and control test carrier  6   a  comprises a stainless steel. In step  70 , the dried portion of inoculum  18  is exposed on the test carriers  6 ,  6   a  for about 120 minutes. 
         [0034]    Next, in step  80 , the exposed test carriers  6  and  6   a  are transferred separately into a neutralizer solution  8  and sonicated to obtain a residual test inoculum  18   a . The number of surviving bacterial colonies from the residual test inoculum  18   a  associated with each of the test carriers  6  and  6   a  is then counted by standard techniques. For example, the residual test inoculum  18   a  can be spread on a Tryptic Soy Agar (“TSA”) plate or a 5% sheep Blood Agar Plate (“BAP”) for purposes of bacterial colony counting. The acceptance criterion for copper protocol  100  includes an assurance that the minimum bacterial recovery on the control test carrier  6   a  (without Cu) is equivalent to 2×10 4  CFU/carrier (see Equation (1) below). 
         [0035]    Finally, in step  90 , various calculations can be conducted using the raw data obtained from step  80  associated with copper-containing test carrier  6  and control test carrier  6   a . For instance, the percent reduction in the number of surviving bacterial colonies per volume in the residual test inoculum  18   a  associated with each of the copper-containing test carriers  6  can be calculated according to standard methods as commonly understood in the art. Equations (1) through (5) below can be used to calculate such percent reduction values associated with each bacterial microorganism  10  tested on copper-containing test carrier  6  and control test carrier  6   a.    
         [0036]    In Equation (1) below, the number of bacterial microorganism  10  colonies can be calculated in terms of colony forming units per carrier (CFU) as follows: 
         [0000]      CFU/carrier=( x   colonies /agar plate)×(dilution)×(vol neutralizer solution) /vol plated   (1)
 
         [0000]    where x colonies /agar plate is the average number of bacterial colonies counted on each agar plate, dilution is the dilution factor, vol neutralizer  solution is the volume of neutralizer solution used in the testing and vol plated  is the volume of material plated on the agar plates. As such, Equation (1) can be used to calculate the CFU/carrier values associated with the copper-containing test carrier  6  and control test carrier  6   a.    
         [0037]    In Equation (2), the geometric mean number of surviving bacterial microorganisms  10  can be calculated for the copper-containing test carrier  6  as follows: 
         [0000]    
       
         
           
             
               
                 
                   
                     mean 
                     
                       control 
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                        
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                        
                       
                           
                       
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                   = 
                   
                     antilog 
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                      
                     
                         
                     
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                           log 
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                             2 
                           
                         
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                           log 
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                             Y 
                             N 
                           
                         
                       
                       N 
                     
                   
                 
               
               
                 
                   ( 
                   3 
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         [0000]    where Y 1 , Y 2 , Y 3 , etc. are the CFU/carrier values for each successively tested copper-containing test carrier  6 , and N relates to the number of such tests. Typically, N is set to 5 tests for the copper-containing test carrier  6 . 
         [0038]    In Equation (3), the geometric mean number of surviving bacterial microorganisms  10  can be calculated for the control test carrier  6   a  as follows: 
         [0000]    
       
         
           
             
               
                 
                   
                     mean 
                     
                       test 
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                   = 
                   
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                             3 
                           
                         
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                             4 
                           
                         
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         [0000]    where X 1 , X 2 , etc. are the CFU/carrier values for each successively tested control test carrier  6   a , and N relates to the number of such tests. Typically, N is set to 3 tests for the control test carrier  6   a.    
         [0039]    Equation (4) below relies on the data generated in Equations (2) and (3) and provides the percent reduction in bacterial organisms tested on a given copper-containing test carrier  6 : 
         [0000]      % reduction=[(mean control test carrier −mean test carrier )/mean control test carrier ]×100  (4)
 
         [0000]    where the mean control test earner  is obtained from Equation (2) and the mean test carrier  is obtained from Equation (3). As such, the % reduction value reflects the relative degree of bacterial killing or anti-microbial efficacy of a copper-containing test carrier  6  relative to a control surface, e.g., test control carrier  6   a.    
         [0040]    It should also be understood that a “log kill” relates to the % reduction obtained in Equation (4) according to the following relation given by Equation (5) below: 
         [0000]      log kill=−(log(1−% reduction))  (5)
 
         [0000]    As such, a 99% reduction in bacterial organisms for a copper-containing test carrier  6  is equivalent to a log kill of 2.0. 
         [0041]    The Copper Test Protocol can be employed to test the anti-microbial efficacy of a copper-containing surface to support claims that the tested surface kills greater than 99.9% of the particular bacterial organism tested. However, recent work by the named inventors suggest that the Copper Test Protocol is not effective at assessing the anti-microbial efficacy of silver-containing surfaces, such as glass sheet and films having a surface region containing silver ions. For example, Corning Ag IX glass having a surface region with silver ions was tested with the Copper Test Protocol. In these tests, % reduction levels against  S. aureus  was only approximately 70 to 80% or a log kill of approximately 0.5 to 0.7. In essence, the Copper Test Protocol cannot be used to readily ascertain the anti-microbial efficacy of surfaces containing silver ions relative to control surfaces. 
         [0042]    Some understanding of how silver ions function with an antimicrobial effect is necessary to develop new protocols for assessing the antimicrobial efficacy of silver-containing surfaces. Different classes of bacteria have different membrane structures. These membranes may contain peptidoglycan layers in addition to phospholipids and lipopolysaccharides outer layers. It is believed that small silver ions can associate with and penetrate membranes causing a structural change in the membrane. This causes increased cell permeability, and transport of silver through the inner cytoplasmic membrane. Silver ions act as antimicrobial agents by strongly binding to critical biological molecules (proteins, DNA, RNA) and disrupting their function(s). Silver chelates with thiol groups in proteins (containing cysteine amino acids) disrupting the activity of vital enzymes critical to cellular signaling needed for bacterial growth. In addition, silver complexes with the adenine, and guanine bases in nucleic acid (DNA and RNA). This causes disruption of DNA replication and cell division (bacteriostatic effect) ultimately leading to metabolite efflux resulting in cell death (bactericidal effect). Ultimately the modes of action depend on the concentration of silver ions present and the sensitivity of the microbial species to the silver ions. Contact time, temperature, pH and the presence of free water likewise impact the rate and extent of antimicrobial activity afforded by the silver ions. 
         [0043]    Disclosed herein are embodiments of methods of testing the antimicrobial efficacy of silver-containing surfaces, including the composition and preparation of inoculums for such testing. These embodiments were developed in view of the foregoing silver-related antimicrobial mechanisms and to better mimic the end-use applications for silver-containing surfaces. These embodiments also hew to principles of the Copper Test Protocol where possible, recognizing that they are structured to be qualified as test protocols by the EPA. But the embodiments also significantly depart from the Copper Test Protocol in view of the particular testing environment presented by articles containing a silver-containing surface region and to better mimic in-use bacterial contamination (e.g., fingerprint fomites in touch screen applications). 
         [0044]    As depicted in  FIG. 1B , an inoculum  18  prepared in the copper test protocol  100  at step  50  contains various constituents and reflects significant changes from its initial formulation. For example, the glucose, casein peptone and soya peptone, originally present in the TSB employed in the broth  12 , are not present at significant levels in the inoculum  18 . This is because these sugars and proteins are consumed as food by the bacterial organisms  10 ,  10   a  and  10   b  added to the broth  12  during steps  30   a - 30   c . At the same time, various metabolites and bacterial debris are formed in the inoculum as the bacterial organisms  10 ,  10   a  and  10   b  are cultured within the broth  12  in steps  30   a  through  40 . The formation of these metabolites and bacterial debris, and consumption of the sugars and proteins, tends to reduce the pH of the inoculum to approximately 5.5 to 6. It is believed that the release of metabolites during bacterial growth inhibits the ability of the inoculum  18  derived from the copper test protocol  100  to perform as an agent for testing the anti-microbial efficacy of a silver-containing surface. 
         [0045]    As depicted in  FIG. 2 , various modifications were made to the inoculum  18  obtained at step  50  in the copper test protocol  100  using  Staphylococcus aureus  as the bacterial organism  10  (see (A) in  FIG. 2 ), thereby producing modified inoculums  19   a ,  19   b  and  19   c  (see (B)-(D) in  FIG. 2 ). Inoculum  19   a  was prepared by removing the metabolites and bacterial debris present in inoculum  18 . This was accomplished by centrifuging the live  Staphylococcus  bacterial organisms  10   b  in the inoculum  18  using commonly understood procedures in the art, followed by refreshing the broth with a further quantity of TSB. The resultant inoculum  19   a  is depicted at (B) and possesses a pH of approximately 7.3, significantly more neutral than the pH level of inoculum  18 . Inoculum  19   b  was prepared in a process comparable to that used for inoculum  19   a , except that its broth was refreshed with a salt solution having glucose and no protein constituents (e.g., casein and soya peptone). Inoculum  19   c  was also prepared in a process comparable to that used for inoculum  19   a , except that its broth was refreshed with a salt solution having no glucose or protein constituents. Inoculums  19   b  and  19   c  both possess a pH of approximately 7.3. 
         [0046]    These modifications depicted in  FIG. 2  were made for the purpose of assessing the impact of changes to the formulation and processing of inoculum  18  with regard to testing the anti-microbial efficacy of an article containing a silver-containing surface region. As shown in  FIG. 2 , the inoculum  18  produced a log kill of 0.51 when tested on a glass test carrier having a surface region with silver ions. In comparison, inoculums  19   a ,  19   b  and  19   c  each had substantially higher log kill values, 1.41, 1.48 and 1.83, respectively. Based on the results from this experiment, it is believed that increasing the pH level of the inoculum will improve testing efficacy as evidenced by the increase in log kill values. It is also believed that bacterial debris in the inoculum may form chelates with the silver ions in the test carriers (e.g., test carrier  6 ), reducing the ability of the silver to kill or otherwise degrade the bacterial organisms  10   b  within inoculums  18 ,  19   a ,  19   b , and  19   c . As such, inoculum  19   c  with no sugar, proteins and bacterial debris demonstrated the highest log kill values in the experiment. 
         [0047]    As depicted in  FIG. 3 , a further experiment was conducted to further assess the effect of inoculum pH level on log kill for a glass substrate having a surface region with silver ions. Various inoculums were prepared in the experiment comparable to the inoculum  19   c  depicted in  FIG. 2 , refreshed with buffered salt solutions to obtain five inoculums with a pH ranging from 5 to 8. Two solutions had a pH of 6, one with a citrate buffer and the other with a phosphate buffer.  FIG. 3  demonstrates that only the inoculums having a pH of 7 to 8 exhibited log kill values greater than 1. As such, it is important to maintain a pH level close to physiological, neutral conditions to obtain high testing efficacy when evaluating the antimicrobial efficacy of silver-ion containing systems. It should also be noted that the log kill values near and below 1.0 for the inoculums with a pH of less than 7 are essentially noise. Hence, the results from the phosphate and citrate buffered inoculums having a pH of 6 shown in  FIG. 3  are inconclusive. 
         [0048]    As schematically depicted in  FIG. 4 , the Ag Protocol  200  was developed in view of the experiments depicted in  FIGS. 2 and 3 , among other findings and studies. Ag Protocol  200  can be used to test the antimicrobial efficacy of silver-containing surfaces. In general, the Ag Protocol  200  includes steps  120  through  150  to prepare an inoculum  118 . Inoculum  118  is then employed to evaluate the antimicrobial efficacy of an Ag-containing test carrier  106  relative to a control test carrier  106   a  in steps  160  through  190 . The Ag-containing test carrier  106  can be any article having an exposed surface with a surface region containing Ag ions. Preferably, the article used for test carrier  106  is a 1 in.×1 in. square of high-strength substrate glass (e.g., a display device substrate glass strengthened through ion exchange processes), such as Corning Gorilla® glass, having a surface region containing Ag and alkali metal ions. Typically, the control test carrier  106   a  will be the same underlying substrate material of Ag-containing test carrier  106 , but lacking Ag. 
         [0049]    In step  120 , a tester obtains a stock  102  of bacterial organisms  110 . Bacterial organisms  110  can include any one of  Staphylococcus aureus, Enterobacter aerogenes, Pseudomona aeruginosa , and  Escherichia coli  for a given test sequence. Once the microorganism strain is selected for the bacterial organisms  110 , a tube  104  (or other suitable container, such as a plate) containing nutrient agar  112  is inoculated with a portion of the bacterial organisms  110  in step  130   a . The nutrient agar  112  is a formulation for culturing bacterial organisms as understood by those with ordinary skill in the field. The inoculated nutrient agar  112  within tube  104  in step  130   a  is then incubated for 24±2 hours at 35-37° C. 
         [0050]    In step  130   b , a portion of bacterial organisms  110   a  obtained from step  130   a  (incubated 24±2 hours) is introduced into fresh nutrient agar  112 . The inoculated nutrient agar  112  within tube  104  in step  130   b  is then incubated for 24±2 hours at 35-37° C. Finally, in step  130   c , a portion of bacterial organisms  110   b  obtained from step  130   b  (incubated for 48±4 hours) is introduced into fresh nutrient agar  112 . The inoculated nutrient agar  112  within tube  104  in step  130   c  (with bacterial organisms  110   b ) is then incubated for 24±2 hours at 35-37° C., thus forming a cultured nutrient agar  112   a.    
         [0051]    In step  140 , the cultured nutrient agar  112   a  is incubated for an additional 48±4 hours at 35-37° C., thus developing bacterial colonies  110   c . As described earlier, the cultured nutrient agar  112   a  may now exist in a slightly acidic condition (pH˜5.5 to 6). 
         [0052]    In step  150 , an inoculum  118  is formed by suspending a portion of the bacterial colonies  110   c  from the cultured nutrient agar  112   a  (obtained at step  140 ) in a buffered test solution  116  within container  104   a . By collecting only a portion of the bacterial colonies  110   c  in the nutrient agar  112   a , bacterial debris and metabolites are not placed into the buffered test solution  116 . In addition, bacterial colonies  110   c  can be re-suspended directly in buffered test solution  116  to obtain a more physiological condition. Other approaches as understood by those with ordinary skill can also be used to adjust buffered test solution  116  to a pH of approximately 7 to 8. 
         [0053]    Further, organic soil  114  is added to the buffered test solution  116  at a concentration of 10 to 30% by weight. Preferably, the organic soil is added at a concentration of 10 to 15% by weight. Further, the organic soil  114  may comprise fetal bovine serum. In the Ag Protocol  200  the organic soil  114  serves the same purpose as the soil  14  in the copper test protocol  100 . That is, the soil  114  is incorporated into the solution to better mimic real-world conditions in which the article having the silver-containing surface region contains various soiling (e.g., fingerprint oils, mucus, blood, and other organic detritus). On the other hand, no surfactant (compare surfactant  16  in step  40  of the copper test protocol  100 ) is added in step  150  to the buffered test solution  116 . Other data suggests that the use of a surfactant in the buffered test solution  116  and its presence in the inoculum  118  would tend to reduce the wettability of the inoculum  118  on test carriers  106 ,  106   a  when hydrophobic silver-containing surfaces are tested. Non-uniform spreading of the inoculum  118  on test carriers  106 ,  106   a  will result in non-robust measurements of the antimicrobial efficacy of the carrier  106 . 
         [0054]    Inoculum  118  can then be employed to test the antimicrobial efficacy of a silver-containing test carrier  106  and control test carrier  106   a  in steps  160  and  170 . In step  160 , a portion of the inoculum  118  is spread on a 1 in.×1 in. square of silver-containing test carrier  106  and a 1 in.×1 in. square of control test carrier  106   a  and dried. In the Ag Protocol  200 , test carrier  106  comprises a silver-containing surface region and a substrate material, and control test carrier  106   a  comprises the substrate material (lacking Ag). In step  170 , the dried portion of inoculum  118  is exposed on the test carriers  106  and  106   a  for at least about two hours. Preferably, the exposure of inoculum  118  on test carrier  106  and  106   a  is conducted for at least four hours. 
         [0055]    Next, in step  180 , the exposed test carriers  106  and  106   a  are transferred separately into a neutralizer solution  108  and are each sonicated to obtain a residual test inoculum  118   a  that corresponds to the carriers  106  and  106   a . The number of surviving bacterial colonies from the residual test inoculum  118   a  associated with each of the test carriers  106 ,  106   a  is then counted by standard techniques. For example, the residual test inoculum  118   a  can be spread on a TSA plate or a 5% sheep BAP for purposes of bacterial colony counting. The acceptance criterion for Ag Protocol  200  is similar to the one described in the copper test protocol  100 . That is, the minimum bacterial recovery on the control carriers (without Ag) should be equivalent to 2×10 4  CFU/carrier. 
         [0056]    Finally, in step  190 , various calculations can be conducted using the raw data obtained from step  180  associated with silver-containing test carrier  106  and control test carrier  106   a . For instance, the percent reduction in the number of surviving bacterial colonies per volume in the residual test inoculum  118   a  associated with each of the silver-containing test carriers  106  can be calculated according to standard methods as commonly understood in the art. Equations (1) through (5) above can be used to calculate such percent reduction values associated with each bacterial microorganism  110  tested on the silver-containing test carrier  106  and control test carrier  106   a.    
         [0057]    The test carrier  106  utilized in the Ag Protocol  200  is an article that possesses a silver-containing surface region (configured for anti-microbial effects). The test carrier  106  is preferably an inorganic glass, ceramic or glass-ceramic material having a silver-containing surface region. The test carrier  106  may also comprise a hydrophobic layer, such as a polymeric coating, over or under the silver-containing surface region. In test carrier  106  configurations with a hydrophobic layer beneath the silver-containing surface region, the substrate beneath the silver-containing surface region and the hydrophobic layer can comprise metals, composites, ceramics and/or polymeric materials. 
         [0058]    The buffered test solution  116  employed at step  150  of the Ag Protocol  200  (see  FIG. 4 ) can comprise various commercial formulations to achieve a neutral and stable pH. As outlined in Table 1 below, the buffered test solution  116  can comprise TSB (“Fresh TSB”), PBS, HBSS, a modified HBSS with no calcium and magnesium salts (“HBSS—no Ca/Mg”), and Eagle&#39;s Minimal Essential Medium (“EMEM”) solutions. For each of the buffered test solutions  116  listed in Table 1, the constituents reflect the condition (e.g., pH from approximately 7 to 8) of the solution at step  150 . 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Fresh TSB 
                 PBS 
                 HBSS 
                 HBSS - no Ca/Mg 
                 EMEM 
               
               
                   
               
             
             
               
                 pH 7.3 
                 pH 7.05 
                 pH 7.75 
                 pH 8.02 
                 pH 7.40-7.90 
               
             
          
           
               
                 5 
                 g/l NaCl 
                 8 
                 g/l NaCl 
                 8 
                 g/l NaCl 
                 8 
                 g/l NaCl 
                 6.8 
                 g/l NaCl 
               
               
                   
                   
                 0.2 
                 g/l KCl 
                 0.40 
                 g/l KCl 
                 0.40 
                 g/l KCl 
                 0.40 
                 g/l KCl 
               
               
                   
                   
                 0.61 
                 g/l Na 2 HPO 4   
                 0.048 
                 g/l 
                 0.048 
                 g/l Na 2 HPO 4   
                 0.14 
                 g/l Na 2 HPO 4   
               
             
          
           
               
                 2.5 
                 g/l KH 2 PO 4   
                 0.19 
                 g/l KH 2 PO 4   
                 Na 2 HPO 4   
                 0.060 
                 g/l KH 2 PO 4   
               
             
          
           
               
                   
                   
                   
                   
                 0.060 
                 g/l KH 2 PO 4   
                   
                   
                 0.2 
                 g/l CaCl 2   
               
               
                   
                   
                   
                   
                 0.140 
                 g/l CaCl 2   
               
               
                   
                   
                   
                   
                 0.100 
                 g/l MgCl 2   
                   
                   
                 0.097 
                 g/l MgSO 4   
               
               
                   
                   
                   
                   
                 0.100 
                 g/l MgSO 4   
                 0.352 
                 g/l NaHCO 3   
                 1.5 
                 g/l NaHCO 3   
               
               
                 17 
                 g/l casein peptone 
                   
                   
                 0.352 
                 g/l NaHCO 3   
               
             
          
           
               
                 3 
                 g/l soya peptone 
                 AA, Vitamins 
               
             
          
           
               
                 2.5 
                 g/l glucose 
                 1.0 
                 g/l glucose 
                 1.0 
                 g/l glucose 
                 1.0 
                 g/l glucose 
               
               
                   
               
             
          
         
       
     
         [0059]      FIG. 5  depicts the results from an anti-microbial efficacy experiment conducted using the buffered test solutions  116  depicted in Table 1 according to the Ag Protocol  200  with respect to a test carrier  106  comprising an inorganic glass substrate having a silver-containing surface region. For comparison purposes,  FIG. 5  also depicts anti-microbial testing results for conducting the Ag Protocol  200  in a way that mimics copper test protocol  100 . In particular, the procedures for re-suspending bacterial colonies in fresh, buffered test solution  116  are bypassed with the use of copper test protocol  100 . Hence, the inoculum  118  used for comparison purposes in  FIG. 5  (“Comp. TSB/EPA Cu”) comprises a TSB solution, comparable to the “(A)” composition shown in  FIG. 2 . 
         [0060]    As the results in  FIG. 5  demonstrate, the log kill values for all but the Comp. TSB/EPA Cu group exceed 1. All the test solutions enabling an antimicrobial measurement&gt;1 log kill have neutral pH and contain buffering systems. Only the Fresh TSB and the Comp. TSB/EPA Cu versions of the buffered test solution  116  depicted in  FIG. 5  contain proteins. Hence, the other buffered test solutions  116  tested in this experiment and depicted in  FIG. 5  do not contain proteins, reflecting the possible insight that proteins can detrimentally chelate with the silver ions in the surface region of the test carrier  106 . Further, the PBS, HBSS, HBSS—no Ca/Mg and EMEM versions of the buffered test solution  116  possess no glucose or lower glucose levels compared to the TSB variants. 
         [0061]      FIG. 5  demonstrates that the use of the HBSS and EMEM versions of buffered test solution  116  can achieve log kill values exceeding 2, significantly higher than the other versions of buffered test solution  116  tested. As such, buffered test solution  116  preferably comprises a solution comparable to HBSS, HBSS—no Ca/Mg and EMEM. These high log kill values associated with the HBSS and EMEM versions of buffered test solution  116  are indicative of high levels of anti-microbial testing efficacy for the Ag Protocol  200 . 
         [0062]    It should also be understood that only the HBSS and EMEM versions of buffered test solution  116  depicted in  FIG. 5  and Table 1 contain sodium bicarbonate. The EMEM version of buffered test solution  116  had the highest log kill value, approaching an average of 2.5, and possessed a significantly higher level of sodium bicarbonate compared to HBSS and HBSS—no Ca/Mg. In view of these findings, another experiment was conducted to ascertain the effect of sodium bicarbonate in the buffered test solution  116  on the anti-microbial testing efficacy of a test carrier  106  having a silver-containing surface region. In this experiment, versions of buffered test solution  116  were created and used in tests according to the Ag Protocol  200  containing varying levels of sodium bicarbonate constituents. An HBSS solution was formulated comparably to the HBSS version depicted above in Table 1, but modified with the specified concentration levels of sodium bicarbonate ranging from 50 mg/1 to 6000 mg/l. As  FIG. 6  demonstrates, log kill appears to correlate with concentration of sodium bicarbonate, with log kill values exceeding 2 for the HBSS buffered test solution  116  containing 350 mg/1 of sodium bicarbonate or more. As such, buffered test solution  116  should preferably contain sodium bicarbonate at a concentration of 350 mg/1 or more. 
         [0063]    As noted earlier, organic serum  114  is added during step  150  of the Ag Protocol  200  for purposes of simulating soiling present on articles (e.g., electronic touch screen surfaces) employing the silver-containing surface region of test carrier  106 . As depicted in  FIG. 7 , an increase in organic serum content within the inoculum  118  tends to increase measured log kill values. The experiment used to generate the results depicted in  FIG. 7  was conducted according to the Ag Protocol  200  in a fashion similar to the experiments employed to generate the results depicted in  FIGS. 5 and 6 . In this experiment, however, the levels of organic serum  114  were varied during step  150  in the preparation of inoculum  118 . Further, a PBS buffering solution  119  was employed in this experiment. 
         [0064]    As  FIG. 7  indicates, the increasing levels of organic serum tend to increase the log kill values. Organic serum levels as low as 10% tend to produce log kill values exceeding 1. Preferably, the organic serum  114  level employed in the Ag Protocol  200  is set at a concentration of 10 to 15% by weight. It is believed that higher organic serum  114  values that exceed 30%, while likely to produce even better log kill values, are not representative of the human “soil” encountered in the natural applications of the articles having silver-containing surface regions, such as those employed as test carrier  106 . It should also be understood that limiting the organic serum  114  levels ensures that the Ag Protocol  200  is fairly comparable to the Copper Test Protocol, which typically relies on an organic serum concentration of 5% by weight (e.g., organic soil  14 ). 
         [0065]    In another study, the Ag Protocol  200  and the copper test protocol  100  were employed to generate anti-microbial efficacy test results for test carriers  106  having a surface region containing silver ions and control test carriers  106   a  for four different bacterial organisms (e.g., bacterial organisms  110   c  developed at step  140 ). Consistent with the discussion above, a buffered test solution  116  was employed with a formulation comparable to EMEM for the Ag Protocol  200 . The results from this work are listed below in Table 2. The table clearly demonstrates the superiority of the Ag Protocol  200  for testing the anti-microbial efficacy of articles having a surface region containing silver ions, particularly for  Staphylococcus aureus  and  Escherichia coli . 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                 Microorganism 
                 Ag Protocol 200 
                 Copper Protocol 100 
               
               
                   
                   
               
             
             
               
                   
                 
                   S. aureus 
                 
                 99.8% 
                 70% 
               
               
                   
                 
                   E. aerogenes 
                 
                 99.999%  
                 99.98%   
               
               
                   
                 
                   P. aeruginosa 
                 
                 &gt;99.999%   
                 99.4%   
               
               
                   
                 
                   E. coli 
                 
                 99.7% 
                 60% 
               
               
                   
                   
               
             
          
         
       
     
         [0066]    It will be apparent to those skilled in the art that various modifications and variations can be made without departing from the spirit or scope of the claims.