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[Akiko Yamamoto](https://orcid.org/0000-0002-9182-4886), Yuki Ito

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[Effect of Humidity on Antibacterial Activity of Copper and Its Alloy Surfaces](https://mdr.nims.go.jp/datasets/67a1bc3e-028f-424d-a5f7-8437fa51e591)

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Effect of Humidity on Antibacterial Activity of Copper and Its Alloy SurfacesEffect of Humidity on Antibacterial Activity of Copper and Its Alloy SurfacesAkiko Yamamoto1,+ and Yuki Ito21Research Center for Macromolecules and Biomaterials, National Institute for Materials Science, Tsukuba 305-0044, Japan2Innovation Center, Monozukuri and R&D Strategy Div., Mitsubishi Materials Corporation, Kitamoto 364-0028, JapanCopper and its alloys are well known to have bactericidal activity and applied to touch surface to suppress the transmission of pathogensvia material surface. The touch surface is exposed to real life conditions, which are generally lower in humidity than that in laboratory testingconditions. The influence of humidity on the antibacterial activity of copper and other materials was reported as contradictory results due todifferent inoculation methods and contact periods. In this study, the antibacterial activity of copper (C1020) and its alloys (C7060, C7150,MONEL400, and antibacterial stainless steel) was systematically investigated by a method simulating droplet transmission under controlledhumidity (60 and 35%RH) with Escherichia coli or Staphylococcus aureus. Corrosion rate of C1020 was estimated by electrochemicalimpedance spectroscopy under the controlled humidity. As a parameter of antibacterial activity, the contact time to reduce the viable bacteria tobe 1% of the inoculated ones, T0.01, was decided based on the test results. The all materials increased their T0.01 with reduction in humidity,indicating the decrease in their antibacterial activity. In case of C1020, the smallest T0.01 for E. coli was observed at 90% RH as 6.96min, whichincreased to 67.4min at 35%RH. At both high and low humidity, the materials with high copper contents (C1020, C7060, and C7150) had thesmaller T0.01 than those with lower copper contents. S. aureus, which has higher tolerance to the environment with low water activity thanE. coli, resulted in greater increase in T0.01 with reduction in humidity (In case of C1020, T0.01 at 90 and 35%RH for S. aureus were 8.50 and230min, respectively). The corrosion rate of C1020 decreased with reduction in humidity, suggesting the importance of copper ion release at thesurface for their antibacterial activity. [doi:10.2320/matertrans.MT-M2025099](Received July 7, 2025; Accepted September 27, 2025; Published December 25, 2025)Keywords: antibacterial tests, copper and copper alloys, relative humidity, droplet transmission, electrochemical impedance spectroscopy1. IntroductionEmergence of antimicrobial resistance (AMR) in bacteriaand other pathogens become a global threat as the reviewpaper published in 2014 estimated about 0.7 million deathsevery year due to the drug-resistant infections [1]. In 2019,the global deaths associated with bacterial AMR is estimatedas 4.95 million, including those attributable to AMR bacteriaof 1.27 million [2]. Rapid increase in global antibiotic useincreases the AMR strains, which overtakes the pace of novelantibiotic discovery. Running out of an effective antibioticfor the infection treatment drawbacks the greatest medicaladvances in 20th century; successful treatment for majordeadly illness such as pneumonia and tuberculosis, as wellas infection control for routine surgery and childbirth. Toavoid this situation, unnecessary and excessive antibiotic useshould be terminated not only in the medical/health carefields but also in an agricultural field based on the conceptof “one health” [1, 3]. In addition, it is important to have themeans to inhibit the spread of drug-resistant pathogenswithout antibiotics.Copper surface is well known to have bactericidal activityso called “contact killing” [4–7]. Though its exact mechan-ism is not fully understood, it can rapidly eliminate a widerange of pathogenic microorganisms such as gram negative/positive bacteria, fungi, spores, and viruses [4, 5, 8].Therefore, the application of copper and its alloys to touchsurface is considered to be one of the countermoves tosuppress the spread of infective pathogens. Fields trials ofcopper application to touch surface in hospitals and elderlycare facilities reported reductions in bioburden on the surface[9–14], however, evidence for significant decrease inhealthcare associated infection (HAI) is still limited [9, 10].The meta-analysis of 3 clinical studies revealed theintroduction of copper and its alloys has limited reductionin HAI by around 25% [10]. This suggests the necessity ofthe improvement in the antimicrobial activity of copper andits alloys for touch surface application under real lifeconditions.One of the key factors for the successful touch-surfaceapplication is the environment. Most of the laboratoryantimicrobial tests use bacterial suspension to inoculate ontomaterial surface. For example, ISO22196 indicates a 0.4mLportion of the bacterial suspension (2.5–10 © 105 cells/mL)contacting to the specimen surface area of 40mm © 40mm,which is controlled with a cover of a non-toxic polymer film[15]. Then, the inoculated specimen is kept at 35°C underthe relative humidity (RH) over 90% [15], which is a fullywet condition. However, in the hospital environment, thetemperature and humidity are generally 20–30°C and 40–50%RH [16] varying with weather, and pathogens areapplied as droplets or by direct contact to the touch surface.For the successful reduction in HAI, the materials having asufficient antimicrobial activity under the real-life environ-ment of the touch surface should be employed.Focusing on the difference between the laboratory testingenvironment and that in real life ones, several researchersevaluated the materials antibacterial activity at the ambienttemperature and low humidity. H.T. Michels et al. reportedthat the antibacterial activity of copper and its alloyscontacting 24 h with methicillin resistant Staphylococcusaureus (MRSA) were not influenced by the temperatures(³35°C or ³20°C) and relative humidity (³90%RH,³20%RH, or ³24%RH), whereas that of Ag-containingantibacterial material decreased with decrease in temperatureand relative humidity [17]. J. Li et al. reported contradictoryresults; the antibacterial activity of the ordinary copper foilcontacting 1 h with Escherichia coli increased with reductionin humidity from 90 to 30%RH whereas that of nano-structured copper foil decreased [18]. A. Mayr et al. reported+Corresponding author, E-mail: YAMAMOTO.Akiko@nims.go.jpMaterials Transactions, Vol. 67, No. 1 (2026) pp. 83 to 91©2025 The Japan Institute of Metals and Materialshttps://doi.org/10.2320/matertrans.MT-M2025099that the antibacterial activity of copper alloy (C71500)contacting 4 h with S. aureus decreased with reduction inhumidity from 50 to 35%RH [19]. The available studies arelimited for different testing materials at different relativehumidity with different contact periods. Furthermore, differ-ent bacterial inoculation methods may contribute to thecontradictory results. C.E. Santo et al. tested the antibacterialactivity of copper with “dry” (106 CFU applied by swab)and “moist” (109 CFU in 40 µL phosphate buffered saline)inoculation, reporting the “faster” killing in the formercondition [20]. Due to the large difference in inoculatednumber of bacteria, it is unclear that this faster killing is dueto less bacteria contacting to copper surface or to the “dry”condition. J. Jann et al. attempted to evaluate the materialantibacterial activity with a bacterial transfer from a moist gelor a dried nylon filter [21], but the results are complicatedpartially due to the ambiguity in the actual transferrednumber of bacteria to the testing material surface.In the present study, our primary purpose is to investigatethe effect of humidity on the antibacterial activity of copperand its alloys mimicking the real-life condition applied totouch surface. Test specimens were prepared with intentionalstorage in ambient atmosphere for long time after polishing.The antibacterial activity of test specimens was systemati-cally investigated using E. coli (gram negative bacteria) andS. aureus (gram positive bacteria) at different contact periodsupto 30min under controlled humidity. To avoid theambiguity, a small portion of the bacterial suspension wasdirectly applied to each of the testing material surface,simulating the droplet transmission. Electrochemical impe-dance measurement was introduced to evaluate the corrosionrate of copper under relatively low humidity.2. Experimental Procedure2.1 Testing materialsMaterials used are oxygen-free copper (C1020), threekinds of copper-nickel alloys (C7060, C7150, andMONEL400), and antibacterial stainless steel (NSSAM3,abbreviated as ABSS). Copper alloys are provided by thecourtesy of Mitsubishi Materials Corporation. All samplesare commercially available. Chemical compositions of thesematerials are shown in Table 1.All materials were cut into 15–20mm squares and 0.05–2mm thick for the antibacterial assay. Alloy specimens wereground by SiC paper up to #1200 (³5 µm), followed byrinsing with ultrapure water. C1020 specimens were in formof thin plate (0.05mm), so they were used as received. Thecut and polished specimens were stored under ambientatmosphere of the laboratory (22–24°C and 40–60%RH)over 7 days, considering a real-world condition for indoortouch surface application. In prior to the antibacterial assays,the specimen surfaces were cleaned with a detergent (a mixedsolution of sodium α-dodecan-1-yl-ω-(sulfonatooxy)poly-(oxyethylene) and fatty acid alkanolamide), followed byrinsing with ultrapure water and air-dry. For the antibacterialassay by the film method, the bottom surface of eachspecimen was covered with a thin silicone film to avoid thecontact to the collecting solution of survived bacteria.2.2 Antibacterial assay under controlled humidityThe antibacterial assay procedure was schematicallyshown in supplementary Fig. S1. The bacterial cell lines,Escherichia coli (ATCC 8739, 0483-PEC, Microbiologics,Inc.) and Staphylococcus aureus (ATCC 6538, 0485-PEC,Microbiologics, Inc.), were employed and prepared followingthe protocol supplied with the kit. Each of the testingmaterials was placed on the bottom of the glass dishindividually. A bacteria suspension was prepared in aphosphate buffer supplied with the kit adding Tween 80[Polyoxyethylene (20) Sorbitan Monooleate, FUJIFILMWako Pure Chemical Corporation] to be 0.5%. Thissurfactant was added to improve the spreadability of thebacteria suspension on the material surface, which influencesthe contact area and drying time of the suspension.A 1 µL portion of the bacteria suspension (containing³1 © 105CFU) was spread about 10mm square on thespecimen surface using a tip of the digital pipette. As acontrol surface, the bacteria suspension was spread on thebottom of a glass dish in the same manner. Each specimenwas dried at the ambient temperature and humidity in thebiosafety cabinet for 5min and then, incubated at 25 « 1°Cunder the controlled humidity as 35–90%RH for additional 5,10, and 30min. For ABSS, the incubation period was set as5, 120, and 1440min based on our previous study [22]. Thehumidity in the incubator was monitored by a hygrometer(Weathecom II electronic thermos and hygrometer EX-502,EMPEX Instruments, Inc.) and was controlled with the opensurface area of the water except the case of 35% RH, inwhich a silica gel pack was used instead of water.After the incubation, the testing material surface waswiped with a polyester swab containing 20 µL of sterileNutrient Broth ‘Eiken’ (abbreviated as NB, Eiken ChemicalCo. Ltd.). The swab was applied 20 times horizontally and 20Table 1 Chemical compositions of the testing materials (mass%).A. Yamamoto and Y. Ito84times vertically, alternately up to 5 sets (100 times in total)covering the loaded area of the bacteria suspension. The headof the swab was cut into a 480 µL portion of NB and vortexedfor 30 sec to extract collected bacteria. Then, the number ofviable bacteria in the collected solution was estimated usinga water-soluble tetrazolium salt (WST) [23, 24]. The WSTpenetrates into cells and forms a soluble formazan dye bysuccinate-tetrazolium reductase in the mitochondrial respira-tory chain, which is only active in viable cells. Therefore, theconcentration of the formazan dye correlates to the numberof viable cells. A brief explanation of the procedure is asfollows; a 180 µL portion of the NB with collected bacteriawas poured into a well of a 96-well microplate, followed bythe addition of 20 µL of the mixture of 5mM WST-1 [2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tet-razolium, monosodium salt, Dojindo Laboratories] and0.2mM 1-methoxy PMS (1-Methoxy-5-methylphenaziniummethylsulfate, Dojindo Laboratories) solution. NB (withoutbacteria) was poured into the blank well. Then, themicroplate was sealed and incubated at 35°C up to 16 hwhile the absorbance at 450 nm was measured every 20minfor E. coli. For S. aureus, the interval for the absorbancemeasurement was set as 30min during 24 h of incubation.The difference in absorbance between the sample well andthe blank well was calculated and find the incubation timewhen this difference reaches to 0.5 (TAbs0.5). This incubationtime was utilized to estimate the number of viable bacteriain the collected solution, NVBcol (CFU/mL), based on thecalibration curve prepared in NB as shown in Fig. S1(c). TheNVBcol was used to calculate the survival rate of bacteria,SRB as following equation;SRB ¼ NVBcol � 0:5=INBmatwhere INBmat indicates the inoculated number of bacteria perspecimen surface (³1 © 105CFU). Then, the contact time toreduce the SRB to 0.01, T0.01 was estimated using the probitregression. All the experiments were performed in triplicate.2.3 Antibacterial assay by film methodIn order to compare the antibacterial activity under a fullywet condition, antibacterial assay by a film method [22] wascarried out for C7060 and MONEL using the same bacterialcell lines, E. coli and S. aureus. Briefly, a 50 µL portion ofbacterial suspension (³1 © 106CFU) in 0.9% NaCl wasplaced onto a specimen surface and covered by a poly-ethylene film of 10mm square. Then, the specimens wereincubated at 35 « 1°C and the humidity over 90%RH up to1440min. After incubation, the bacteria on the specimensurface were collected into a 1mL portion of 0.1mMethylenediamine-N,N,NA,NA-tetraacetic acid, disodium salt,dihydrate (EDTA-2 Na) in 0.9% NaCl solution by pipetting.The viable bacterial number was decided using 5-cyano-2,3-ditolyl-2H-tetrazolium chloride (CTC) staining kit(-Bacstain- CTC Rapid Staining Kit for Flow cytometry,BS01, Dojindo Laboratories, Kumamoto, Japan) followingits instruction. All the experiments were performed at least induplicate and in triplicate when necessary.2.4 Electrochemical impedance analysisIn order to investigate the corrosion rate of the C1020under the controlled humidity, electrochemical impedancemeasurement was performed using a pair of parallel copperplates as electrodes [25]. Figure 1 indicates the details of thespecimen and equivalent circuit model used for data analysis.A commercially available 100 µm thick C1020 foil was cutinto 10mm wide with one side covered by a 50-µm-thickpolyimide insulating tape. Then, the covered sides of twopieces were faced to each other and embedded into a low-viscos resin using a mold of the inner diameter of 25.4mm.Then, the specimen surface was polished by SiC paper upto #1200 and the periphery of the resin was covered by theinsulating tape with a rim of 0.5mm in height.A 500µL portion of an electrolyte, 0.7M Na2SO4 waspoured onto the specimen surface which gives the salt densityof 10mg/cm2. Then, the specimen was placed in theincubator and electrochemical impedance spectroscopy(EIS) was performed immediately with AC amplitude of10mV in the frequency range of 10¹2–105Hz using apotentiostat equipped with a frequency response analyzer(VersaSTAT4, Princeton Applied Research). This result inthe electrolyte was indicated as the 100% humidity. Then,the humidity in the incubator was controlled to be 30 « 5%RH and left overnight to dry the moisture. The EIS wasperformed in the next day, followed by the increase in thehumidity to be 50 « 5, 60 « 5, 75 « 5, and 90 « 5% RH.EIS measurement was carried out at least 0.5 h after thehumidity is stable in the designated range. The RH in theincubator was controlled and monitored in the same mannerto the antibacterial test under controlled humidity.The obtained data were analyzed by assuming theequivalent circuit shown in Fig. 1(b) [26]. At the highfrequency, capacitance components approach to zero asnegligible, while at the lower frequency, capacitancecomponents approach to infinity. Therefore, when we definethe impedance at high frequency range (100 kHz) and atlow frequency range (10mHz) as Zhigh and Zlow, respectively,these can be expressed as following equations;(a)(b)100μmresinInsulating tape(0.5mm in height)100μm25.4mm10mmcopper foilFig. 1 Schematic explanation of the specimen (a) and equivalent circuitmodel (b) for the electrochemical impedance spectroscopy. Rs: the sumof the electric resistance of electrolyte and the EIS system used for themeasurement. Rc: charge transfer resistance. Cdl: double-layer capaci-tance.Effect of Humidity on Antibacterial Activity of Copper and Its Alloy Surfaces 85Zhigh ¼ RsZlow ¼ 2Rc þ RsWhen we consider the difference between these impedancesas Zdiff, it can be written as follows;Zdiff ¼ Zlow � Zhigh ¼ 2RcSince the corrosion rate, Icorr is proportional to the reciprocalnumber of Rc asIcorr ¼ k=Rcwhere k is a constant, 1/Zdiff can be employed as a corrosionparameter to monitor the change in the corrosion rate. EISmeasurements were performed in triplicate.2.5 Statistical analysisThe data obtained by antibacterial assay under controlledhumidity for copper and copper alloys were statisticallyanalyzed by parallelism test of 2 regression lines using astatistical analysis software (Kyplot 6.0, KyensLab. Inc.).The logarithm of bacteria viability of a material at a certainhumidity was plotted against the logarithm of the contacttime to obtain regression line, which is tested for equality ofslope or vertical line separation against another regressionline of another dataset such with different materials orhumidity. Results of the parallelism tests were described inthe supplementary Tables S1–5.3. Results3.1 Effect of relative humidity on antibacterial activityof copper and its alloysThe antibacterial activity of C1020 against E. coli wasexamined under the various humidity and shown in Fig. 2.At 75% RH, the SRB was almost equal to that of 90% RH. Atlower humidity, however, the SRB tended to increase withreduction in humidity. For example, no bacteria survivedafter 15min contact (5min drying and 10min incubation) at90% and 75% RH whereas the SRB after 35min contact(5min drying and 30min incubation) drastically increasedwith reduction in humidity as 60%, 50%, and 35% RH. Inother words, the slope of the SRB–contact time curvedecreased with reduction in humidity, indicating the decreasein the antibacterial activity of C1020, which agrees with theprevious reports for C7150 [19] and a nanostructured copperfoil [18].Figure 3 indicates the antibacterial activity of copper andits alloys examined at 60% and 35% RH against E. coli. In alltested materials, the antibacterial activity reduced at the lowerhumidity than at the higher. At 35% RH, no material cansucceed to reduce the SRB less than 0.001 (99.9% reduction)within 35min of contact (5min drying and 30minincubation). The order of antibacterial activity does notchange from that at 60%RH; C7060 > C1020 ² C7150 >MONEL ³ ABSS.In similar to E. coli, the antibacterial activity of copper andits alloys reduced at 35%RH for S. aureus as shown in Fig. 4.At the lower humidity, the reduction rate of SRB retarded incomparison with those at the higher humidity. The order of1 10 100 1000Bacterial viabilityContact time, T/minE.coli90%RH75%RH60%RH50%RH35%RHDetective limit10010-110-210-310-410-510-6Fig. 2 The antibacterial test results of C1020 under controlled humidityusing Escherichia coli (mean « sd). The contact time includes the dryingtime (5min) of the bacterial suspension before the sample is placed ina humidity-controlled chamber. The obtained results were statisticallyanalyzed by the parallelism test of 2 regression lines (see supplementallyTable S1). (online color)1 10 100 1000 10000Bacteria viabilityContact time, T/minC1020C7060C7150MONELABSSE.coli, 60%RH1 10 100 1000 10000Bacterial viabilityContact time, T/minC1020C7060C7150MONELABSSE. coli, 35%RH10110010-110-210-310-410-510110010-110-210-310-410-5(a)(b)Fig. 3 The antibacterial test results of copper and its alloys undercontrolled humidity using Escherichia coli (mean « sd). The contacttime includes the drying time (5min) of the bacterial suspension beforethe sample is placed in a humidity-controlled chamber. The obtainedresults were statistically analyzed by the parallelism test of 2 regressionlines (see supplementally Tables S2 and S3). (online color)A. Yamamoto and Y. Ito86antibacterial activity was as follows; C7150 ³ C7060 >C1020 > ABSS ³ MONEL.As the parameter of the antibacterial activity level, the timeto reduce the SRB to 0.01 (T0.01, min) was estimated by probitregression and shown in Table 2 together with the T0.01values examined by the film method referring to JISZ2801:2012 (ISO 22196). The bacterial survival ratio–contact time curves for C7060, C7150, and MONELobtained by the film method was displayed in supplementaryFig. S2. Table 2 also contains the T0.01 values of MONELand ABSS against E. coli at 90% RH; their survival ratio-contact time curves were displayed in supplementary Fig. S3.The T0.01 increased with reduction in humidity for all thematerials tested and bacteria used. In the film method,bacterial suspension will not dry out because of thepolyethylene cover film, therefore it can be referred as theresult in a fully wet condition. Except ABSS, the T0.01 at60%RH are close to those by the film method, but the T0.01 at35%RH is larger than those at 60%RH or by the film method.These data suggest that the lower humidity such as 35%RHdrastically reduces the antibacterial activity of copper andits alloys.Figure 5 plots T0.01 against the Cu content in the alloycomposition. For E. coli, C7060 has the smallest T0.01 both at60% and 30% RH, and T0.01 increased with decrease in Cucontent. For S. aureus, C7150 has the smallest T0.01 both at60% and 35% RH, and T0.01 increased with decrease andincrease in Cu content. These data indicate the dependanceof T0.01 on Cu content in the alloy composition. Cu-Ni alloywith Ni content up to 30% has superior antibacterial activityin similar or even better level of pure copper (C1020). Cu-Nialloys with Ni content over 30% decrease its antibacterialactivity with decrease in Cu content. This trend suggests thedependance of the antibacterial activity of Cu-Ni alloy in theCu content, which agrees with the general tendency of Cualloys; the higher Cu content gives the higher antibacterialactivity [22].1 10 100 1000 10000Bacterial viabilityContact time(min)C1020C7060C7150MONELABSSS.aureus, 60%RH1 10 100 1000 10000Bacterial viabilityContact time, T/minC1020C7060C7150MONELABSSS.aureus, 35%RH10110010-110-210-310-410-510110010-110-210-310-410-5(a)(b)Fig. 4 The antibacterial test results of copper and its alloys undercontrolled humidity using Staphylococcus aureus (mean « sd). Thecontact time includes the drying time (5min) of the bacterial suspensionbefore the sample is placed in a humidity-controlled chamber. Theobtained results were statistically analyzed by the parallelism test of 2regression lines (see supplementally Tables S4 and S5). (online color)Table 2 Time to reduce the viable bacteria to 1/100 of the inoculated ones on the material surface (T0.01/min).*“Film” indicates the results obtained by the film method (under fully wet condition).**Values from [22].T0.01 of C1020 at 50%RH, 75%RH for E.coli and at 90%RH for S.aureus are 23.8, 7.05 and 8.50 min, respectively.Effect of Humidity on Antibacterial Activity of Copper and Its Alloy Surfaces 873.2 Effect of relative humidity on corrosion of copperThe examples of the electrochemical impedance spectraof C1020 under various humidity were shown in Fig. 6. Itclearly shows the increase in impedance with reduction inhumidity. In order to evaluate the change in corrosionrates, the reciprocal of the difference in impedances at low(10mHz) and high (100 kHz) frequencies, 1/Zdiff wascalculated and plotted against humidity in Fig. 6(b). Itindicates the decrease in the corrosion rates with reductionin humidity, which agrees with the results of other metalsevaluated by a similar manner [27].The plot of the T0.01 of C1020 against 1/Zdiff is shown inFig. 7. When the 1/Zdiff was larger than 10¹7 (/³·cm2),which corresponded to the humidity over 75%RH, the T0.01stayed in a similar level (³10min). However, when the1/Zdiff was smaller than 10¹7 (/³·cm2), which correspondedto the humidity under 75%RH, the T0.01 increased, indicatingthe decrease in its antibacterial activity. This trend suggeststhe importance of the corrosion rate of the copper on itsantibacterial activity in middle to low humidity environment.4. DiscussionThe effect of a water content in media on the bacterialgrowth had been studied and reviewed by microbiologists. Awater activity (aw) is a parameter to indicate the ratio of freewater in the total water content of food or cultural media. Thereduction in aw influences bacterial growth via increase inlag phase and decrease in growth rate. The maximum growthrate of bacteria is generally observed between aw 0.990³0.995 [28]. The minimum aw for growth depends on the type0 20 40 60 80 100T 0.01/minCu content(%)S.aureus0 20 40 60 80 100T 0.01/minCu content(%)E.coli35%RH60%RH35%RH60%RH104103102101100104103102101100(a)(b)Fig. 5 Correlation of the antibacterial activity and copper content in thealloy composition. (online color)0 20 40 60 80 100Z diff-1/Ω-1 cm-2Relative humidity(%)Impedance, Z /Ωcm2Frequency, f/Hz0.7M Na2SO4, C1020101010910810710610510410310210110010-110-2      10-1       100        101        102        103         104        10510-310-410-510-610-710-810-910-10Soln.30%RH50%RH60%RH75%RH90%RH(a)(b)Fig. 6 Typical results of electrochemical impedance spectroscopy ofC1020 under controlled humidity (a), and the average 1/Zdiff plottedagainst relative humidity (b), n = 3, mean « sd. (online color)1101001000T 0.01/minZdiff-1 /Ω-1cm-2E.coliS.aureus10-9 10-8 10-7 10-6 10-5 10-490%RH100%RH60%RH35%RHFig. 7 Correlation of antibacterial activity (T0.01) and corrosion parameter(1/Zdiff) of C1020 under controlled humidity. (online color)A. Yamamoto and Y. Ito88of bacteria; 0.950 for the gram-negative bacteria E. coliwhereas a relatively low value of 0.860 for the gram-positivebacteria S. aureus [28]. Based on these data, the reductionin humidity is expected to assist the antibacterial activity ofchemicals/materials, more effectively for E. coli thanS. aureus, since low humidity itself suppresses the bacterialgrowth.In the present study, the bacteria suspension was applied asa 1 µL portion spreading over about 1 cm2 of the specimensurface with 5min of drying time at an ambient temperatureand humidity (³24°C and 45–50%RH), simulating thedroplet transmission of pathogens. After the drying time,the specimens were incubated under controlled humidity for acertain period of time before the quantification of survivedbacteria. As summarized in Table 2, T0.01, the time to reducethe viable bacteria to 1/100 of the inoculated ones (which isequivalent to log10 reduction of 2) increased with reduction inhumidity. In detail, T0.01 of C1020 for E. coli decreased from9.33 to 6.96min with reduction in humidity from a fully wetcondition (film) to 90%RH, but it increased with furtherreduction in humidity to 35%RH. The same trend was alsoobserved for S. aureus; T0.01 of C1020 decreased from 9.86to 8.50min from “wet” to 90%RH, and then, increased to230min at 35%RH. C7060 and C7150 had the similar trendfor both bacteria; the minimum T0.01 values were observed at60%RH. This first phase of decrease in T0.01 with reduction inhumidity may be attributed to the inhibition of bacterialgrowth due to the decrease in available water, as describedin the previous paragraph. However, further reduction inhumidity resulted in the increase in T0.01, as a second phase.This change cannot be explained simply by the effect of awon bacterial growth; it may be attributed to the change inmaterial surface by the reduction in humidity.For the bactericidal activity of copper and its alloys knownas “contact killing”, their corrosion and ion release play animportant role [4, 29]. Accumulation of copper inside thebacteria contacted to copper was also confirmed [20]. If thehumidity influences the corrosion reaction of copper, i.e. ionrelease, it should result in change of copper bactericidalactivity. However, it is hard to measure the corrosion rate orion release of the copper contacting to a small amount ofbacterial suspension. As described above, the 1 µL portionof the bacteria suspension was spread over 1 cm2 of thespecimen surface, which is calculated to form 10 nm thinlayer on the specimen surface. In the present study, thecorrosion rate of copper under various humidity wasevaluated by the electrochemical impedance measurementwith a thin electrolyte layer. This method is often employedfor the evaluation of atmospheric corrosion in steel and othermetals [25, 26]. As shown in Fig. 6, a corrosion rateparameter, 1/Zdiff of C1020 decreased with reduction inhumidity from 100% (in 0.7M Na2SO4 before drying) to35%RH. Figure 7 summarized the correlation between theantibacterial activity (T0.01) and corrosion rate (1/Zdiff) ofC1020. When the 1/Zdiff was over 10¹7 (³¹1·cm¹2), thereare a little change in T0.01 as ³3min, which corresponds tothe first phase mentioned earlier. In the humidity range lessthan 75%RH, however, T0.01 increased more clearly withdecrease in 1/Zdiff, which corresponds to the second phase.The first phase can be described as the range where thecorrosion rate of C1020 is high enough to have efficientbactericidal action against bacteria, resulting in less differ-ence in T0.01, or even in slight decrease of it from the “wet”condition to 90%RH probably due to the aw effect on thebacterial growth. In the second phase, the corrosion rate ofC1020 is lower than the required level to have the efficientantibacterial activity, resulting in the increase in T0.01 withdecrease in the corrosion rate. This suggests the decrease inthe corrosion rate of C1020 has a greater effect on thebacterial growth than aw, the latter only inhibits bacterialgrowth but not bactericidal. Nevertheless, the aw still hasvisible influence in the second phase; the T0.01 of C1020showed more remarkable increase for S. aureus than forE. coli, where the former has the relatively low minimum awfor growth, indicating its relatively high tolerance for lowhumidity environment. The Fig. 8 schematically illustratesthe influence of the humidity on antibacterial activity ofcopper surface. At high humidity, the copper releases enoughions to kill the bacteria on its surface, but at low humidity,the amount of released ions decreased, resulting in reductionof its antibacterial activity. However, the thinner electrolytelayer formed on copper surfaces at low humidity limitsavailable free water for bacterial growth. Therefore, thesurvival ratio of the bacteria on copper surface is thecombination of the damage by released copper ions and lowaw. In other words, the bacteria having higher tolerance forlow humidity environment result in more survival on coppersurface at low humidity.As is discussed by other researchers, the inoculationmethods (and their environmental conditions) are alsoinfluential for evaluating the copper antibacterial activity onlow humidity environment. Several researchers performedaerosol inoculation to mimic the airborne transmission.McDonald et al. reported that the inoculation by nebulizationtakes longer time as 30min, giving some damages onbacterial cells during nebulization [30]. At the end ofnebulization period, the number of bacteria on copper surfacereduces to 1/10 of that on stainless steel surface [30]. EvenCopper(b) Low humidityCopper(a) High humidityCu2+/Cu+ ionsBacteria ROS Electrolyte layer Fig. 8 Schematic explanation of the bacterial damage on copper surface at(a) high and (b) low humidity. ROS: reactive oxygen species. At highhumidity, the corrosion rate of copper is high, releasing many ions givingthe damages on bacteria. At low humidity, the corrosion rate of the copperis low, but low availability of water suppresses the bacterial growth.(online color)Effect of Humidity on Antibacterial Activity of Copper and Its Alloy Surfaces 89so, the reduction rate in the survived bacteria on coppersurface was much less by nebulization than by a dropletapplication with a 1 µL portion of bacteria suspension underthe incubation condition of 20°C and 40%RH [30]. Thismay suggest that the nebulization allowed less water contactto the copper surface, resulting in less corrosion and ionrelease. Interestingly, the difference on antibacterial activitybetween the nebulization and a droplet application wasalmost negligible for stainless steel [30], that also suggeststhe importance of copper ion release under low humiditycondition.Even by the nebulization method, the lower humidityduring the incubation after nebulization reduced antibacterialactivity of copper and its alloys [16], which agrees with theresults of the present study, indicating the criticalness ofcorrosion on the antibacterial activity of the copper. Asdescribed in Table 2, the influence of the humidity onantibacterial activity (T0.01) depends on the types of alloys.In Fig. 5, T0.01 was plotted against the copper content in thetesting materials. For E. coli, the values of T0.01 had theminimum peak at copper content of 90% (C7060) whereasthose for S. aureus had the minimum peak at 70% (C7150).After the minimum peaks, the decrease in copper contentincreased T0.01 for both bacteria. This suggests the depend-ance of antibacterial activity on the copper content in thealloy especially in the lower range, agreeing with theprevious study [22]. This is another evidence showing thepredominant effect of released copper ions on the anti-bacterial activity of copper and its alloys. For the applicationof copper and its alloys to high touch surface in low humidityenvironment, it is important to select the one having thereasonably high corrosion rate for its antibacterial activity.It also suggests that the humidity control near the materialsurface may efficiently improve the antibacterial activity ofcopper and its alloys for touch surface application.In the present study, test specimens were prepared withlong exposure to ambient environment after polishing tomimic the real-life conditions as indoor touch surface. Thiscondition predicts the formation of relatively thick oxidelayer on copper surface, composed of Cu2O and CuO [31].Further exposure to the droplet of bacterial suspension andlow or high humidity environment may influence thecomposition of the oxide layer. For further elucidation ofthe mechanism for the influence of humidity on copperantibacterial activity, it is mandatory to perform surfaceanalysis of the copper surface to clarify the change in oxidelayer composition, which can be planned as our future study.5. ConclusionThe effect of humidity on the antibacterial activity ofcopper and its alloys was systematically investigated by theinoculation method simulating the droplet transmission. Thestudy found that the antibacterial activity of copper increasedby the slight reduction in humidity, and thereafter, itdecreased with further reduction in humidity. The retardationin copper corrosion in low humidity environment is apredominant cause of the decrease in its antibacterial activitythan the reduction in water activity, aw. S. aureus, which hasa lower minimum aw for growth than E. coli, has relativelyhigh survival ratio on the copper surface at low humidityenvironment than E. coli.All the tested materials, including copper containing ABSSdecreased their antibacterial activity with reduction inhumidity. Even in low humidity environment, the alloyswith higher copper contents had the higher antibacterialactivity than those with low copper contents. For successfulapplication to high-touch surface, copper alloys with highcopper content with reasonably high corrosion rate in lowhumidity environment should be selected.AcknowledgmentsThe authors appreciate Dr. Komei Kato and his colleaguesfrom Mitsubishi Materials Corporation, Dr. Yukio Yagi fromthe Hardhealth Corpolation, and Dr. Yasutaka Hakamazukafrom NIMS for their valuable advises and discussion on thisresearch. The authors also appreciate Ms. Masuko Tsuda andMs. Akemi Kikuta from NIMS for their technical assistanceon antibacterial tests. This work was supported by MitsubishiMaterials Corporation.REFERENCES[1] Review on Antimicrobial Resistance: Antimicrobial Resistance:Tackling a Crisis for the Health and Wealth of Nations, (Review onAntimicrobial Resistance, London, 2014) pp. 1–16.[2] C.J.L. 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