3398 IEEE TRANSACTIONS ON PLASMA SCIENCE, VOL. 38, NO. 12, DECEMBER 2010

Pseudomonas aeruginosa Biofilm Inactivation:Decreased Cell Culturability, Adhesiveness to

Surfaces, and Biofilm Thickness Upon High-PressureNonthermal Plasma Treatment

Anna J. Zelaya, Gregory Stough, Navid Rad, Kurt Vandervoort, and Graciela Brelles-Mariño

Abstract—Bacterial biofilms are more resilient to standardkilling methods than free-living bacteria. Pseudomonas aeruginosaPAO1 biofilms grown on borosilicate coupons were treated withgas-discharge plasma for various exposure times. Almost 100% ofthe cells were inactivated after a 5-min plasma exposure. Atomicforce microscopy was used to image the biofilms and study theirmicromechanical properties. Results show that the adhesivenessto borosilicate and the thickness of the Pseudomonas biofilms arereduced upon plasma treatment.

Index Terms—Atmospheric pressure plasma, biofilms, biofilmremoval, sterilization.

I. INTRODUCTION

B IOFILMS ARE microbial communities responsible forundesirable effects such as disease and biofouling.

Cooperative interactions among members of the biofilmmake conventional methods of controlling microbial growthoften ineffective. Therefore, there is a need to develop newsterilization techniques. The use of gas-discharge plasmas isa good alternative since plasmas contain a mixture of reactivespecies, free radicals, and UV photons well-known for theirdecontamination potential against free microorganisms. Wehave reported the use of plasma to inactivate Chromobacteriumviolaceum biofilms [1]–[5]. We are presently studying biofilm

Manuscript received August 6, 2010; revised September 19, 2010; acceptedSeptember 21, 2010. Date of publication October 28, 2010; date of currentversion December 10, 2010. This work was supported by the U.S. NationalInstitutes of Health under Grant SCORE SC3 1SC3GM088070-01 and in partby the National Science Foundation Nanotechnology Undergraduate EducationProgram under Award 0406533 for the atomic force microscopy. The work ofA. J. Zelaya was supported by a fellowship from the U.S. National Institutes ofHealth RISE Program 2R25GM061190-05A2.

A. J. Zelaya is with the Biological Sciences Department, CaliforniaState Polytechnic University, Pomona, CA 91768 USA (e-mail: ajzelaya@csupomona.edu).

G. Stough and K. Vandervoort are with the Physics Department, CaliforniaState Polytechnic University, Pomona, CA 91768 USA (e-mail: egstough@csupomona.edu; kvandervoort@csupomona.edu).

N. Rad is with the Physics Department, California State University, Fresno,CA 93740 USA (e-mail: Navidkrad@yahoo.com).

G. Brelles-Mariño is with the Biological Sciences Department, CaliforniaState Polytechnic University, Pomona, CA 91768 USA and also with theCenter for Research and Development of Industrial Fermentations, (CCT LAPLATA-CONICET), Facultad de Ciencias Exactas, Universidad Nacional deLa Plata, La Plata, Argentina (e-mail: gbrelles@csupomona.edu; gbrelles@biotec.org.ar).

Color versions of one or more of the figures in this paper are available onlineat http://ieeexplore.ieee.org.

Digital Object Identifier 10.1109/TPS.2010.2082570

inactivation of the opportunistic pathogen Pseudomonas aerug-inosa PAO1.

P. aeruginosa is a gram-negative organism that preys onvictims with compromised immune systems, patients on respi-rators, and causes infections of burned tissue and colonizationof catheters and medical devices. It is also, together with Burk-holderia cenocepacia, the main cause of mortality in patients[6] with cystic fibrosis. P. aeruginosa is an extremely versatilebacterium and lives almost anywhere (in water, soil, plants, andanimals) and can use almost anything for food.

Pseudomonas biofilms have been intensively studied. Manyof the genes involved in biofilm formation and its regulation[7] and the physiological events leading to biofilm formation[8] are known. Different strategies were also used to con-trol/inactivate Pseudomonas biofilms: use of biocides, antibi-otics, or the combination of both [9]–[13]; use of chelators [14];use of compounds such as furanone and signal molecules suchas N-acyl homoserine lactones [15], [16]; and modification ofsurfaces [17], [18] just to mention a few.

In this paper, we present data on plasma-initiated inactivationof P. aeruginosa biofilm grown on borosilicate and microme-chanical properties of the biofilms through force versus distancecurves.

II. MATERIALS AND METHODS

A. Biofilm Growth

P. aeruginosa one-, three-, and seven-day-old biofilms wereproduced in batch culture using the CDC biofilm reactor (Bio-Surface Tech., MT). The biofilms were grown on borosilicate(glass) coupons in tryptic soy broth (TSB) at 37 ◦C with agi-tation. After the selected growth time, the coupons were asep-tically removed from the reactor, and unbound bacteria wereremoved by rinsing the coupons twice with saline. Couponswere air-dried prior to being subjected to gas-discharge plasmafor various exposure times (5, 10, 15, 30, and 60 min) understerile conditions. A control without plasma treatment (0-minexposure time) was included. After treatment, the coupons wereplaced in a wet chamber and incubated with 50 μL of sterilesaline for 10 min. Biofilms were then scrapped off the couponsand suspended in 1 mL of sterile saline, serially diluted, andsuspensions were plated in duplicates on an agarized solidTSB medium. Plates were incubated at 37 ◦C and evaluatedfor colony-forming-units (CFU) formation by counting the

0093-3813/$26.00 © 2010 IEEE

ZELAYA et al.: PSEUDOMONAS AERUGINOSA BIOFILM INACTIVATION 3399

colonies. Data (CFUs/mL) were transformed to percentagesassigning the control a 100% of survival. Short-exposure-timeexperiments (0, 1, 2, 3, and 5 min) were carried out as describedabove for three-day old biofilms.

B. Plasma Generation and Conditions

The gas-discharge plasma was produced using a commer-cially available inductively coupled Atomflo 300 reactor (SurfxTechnologies, CA) that delivers an atmospheric plasma jet[19]. The reactor consists of two perforated rectangular platesseparated by a gap of 1.6 mm across. The upper aluminum elec-trode is connected to a 100-W RF power supply (13.56 MHz),and the lower electrode is grounded. The size of the plasmashowerhead is 0.63 cm wide by 2.54 cm across. For the ex-periments, an atmospheric-pressure plasma jet was generatedusing a He flow of 20.4 L/min, a secondary gas flow (N2)of 0.15 L/min, and an input power of 35 W. Both gases wereindustrial grade. The plasma applicator was mounted such thatthe showerhead was 4 mm away from the biofilm.

C. AFM

Three-day-old biofilms were grown on glass coupons, treatedwith plasma for 0, 5, 30, and 60 min, and processed as indicatedabove. The coupons were rinsed twice and air-dried, and atomicforce microscopy (AFM) images were obtained in air in contactmode using the Quesant Instrument’s universal scanning probemicroscope. Commercial silicon cantilevers from MikroMaschwere employed with spring constants from 0.1 to 0.5 N/m.For each coupon, at least four widely separated regions wereimaged to obtain a representative sample and ensure repro-ducibility. Images consisted of 500 lines of 500 points per linefor a total of 250 000 pixels of data.

To ascertain the micromechanical properties of the biofilm,force–displacement curves were obtained. The procedureconsisted of bringing the AFM tip in contact with the sampleand then moving the sample upward in a set distance whilemonitoring the deflection of the cantilever. At each samplinglocation where force–displacement curves were obtained,the tip was brought in and out of contact at a rate of 0.5 Hzto the maximum set sample deflection (1.8 μm), and thedisplacement curve was recorded upon the fifth trial. Thistechnique helped to reduce hysteresis that was often observedin the first few trials. The process was then repeated so thatat least five force–displacement curves were recorded at eachsampling location. For comparing samples with differentplasma treatments, all of the force–displacement data wererecorded on the same day using the same cantilever. Thismethod ensured control for humidity and cantilever-dependentfactors (such as spring constant) that can influence the shape ofthese curves [20].

III. RESULTS AND DISCUSSION

The percentage of remaining culturable cells versus plasmaexposure time for borosilicate-grown P. aeruginosa biofilms isshown in Fig. 1. At time zero, the percentage of culturablecells is 100% and corresponds to the control without plasma

Fig. 1. Percentage of culturable cells versus plasma exposure time. P. aerug-inosa biofilms were grown on borosilicate for (a) one day, (b) three days,and (c) seven days and were subjected to plasma for various exposure timesand processed, as indicated in materials and methods. Results are the averageof at least four independent experiments. Platings for colony counting wereperformed in duplicates. The bars represent the standard error of the mean.(b-2) Inset to part b.

treatment. The graph shows that regardless of the biofilm age,there is a clear decrease in the percentage of cells versus time.Seven-day-old biofilms do not seem to be more resilient thanyounger biofilms, and there are no significant differences inthe percentage of remaining culturable cells for the differentsampling dates. Similar results were reported for C. violaceumbiofilms grown for four or seven days on polystyrene microtiterplates [1]–[5]. In the case of P. aeruginosa biofilms, the de-crease in the percentage of cells is even more dramatic since

3400 IEEE TRANSACTIONS ON PLASMA SCIENCE, VOL. 38, NO. 12, DECEMBER 2010

Fig. 2. AFM images of P. aeruginosa bacterial biofilm treated with gas-discharge plasmas for 0 (column I), 5 (column II), and 60 min (column III). The topand bottom rows consist of 40 µm × 40 µm and 10 µm × 10 µm scan areas, respectively. Cross sections of each 10 µm × 10 µm scan are included, with thelocation of the cross section indicated by a horizontal line on the image.

there are almost no culturable cells after a 5-min treatment withplasma. The inset to Fig 1(b) shows that most of the inactivationoccurs before biofilm exposure to plasma of less than 1 min.

To rule out the effect of temperature on biofilm inactivation,we measured the temperature of the gas reaching the couponsurface by placing a thermometer on its surface. Equilibriumtemperatures of 31 ◦C were reached within a few minutesand remained constant over time. Therefore, temperature is notresponsible for biofilm inactivation.

In a previous work, we studied the chemistry of the generatedplasma by spectroscopy, and we reported the presence of NOγ-bands around 250 nm and an OH band around 309 nm [3].These reactive species have direct impact on microorganisms,particularly on their outermost membranes [21]–[23]. The pres-ence of these radicals can, therefore, compromise the functionand viability of the membrane and the cell wall. The plasma

conditions chosen for our experiments were those that maxi-mized OH and NO emissions and produced stable plasma [3].

Cell concentration (in log CFU/mL) for biofilms not sub-jected to plasma treatment is 5.96±0.24, 7.12±1.52, and5.82±0.42 for one-, three-, and seven-day-old biofilms, respec-tively. As cell concentration is slightly higher for three-day-oldbiofilms, experiments for AFM imaging were carried out withthose biofilms.

Fig. 2 displays typical AFM images for P. aeruginosabiofilms grown on glass coupons and treated with plasma fordifferent exposure times, as indicated in materials and methods.The upper and bottom rows display 40 μm × 40 μm and10 μm × 10 μm area scans, respectively, and cross sections ofthe 10 μm × 10 μm area scans are included. For each image,removal of any overall background tilt was performed. Thisprocedure involved subtracting a plane determined from the

ZELAYA et al.: PSEUDOMONAS AERUGINOSA BIOFILM INACTIVATION 3401

Fig. 3. P. aeruginosa biofilm force–displacement curve for the control sampleon the glass coupon. Data for (dashed line) tip-sample approach and (solid line)tip-sample retraction are shown. The negative displacement of the cantileverthat occurred due to tip adhesion to the biofilm upon retraction is designated asthe adhesive step.

average slope between the top and bottom edges and rightand left edges of the scan. There are no obvious qualitativedifferences that we interpret from these images. From the crosssections of the 10 μm × 10 μm images, the overall thicknessof the biofilm can be determined as the distance betweenthe lowest features (the flat surface of the glass coupon) andthe highest features (the “peak” of the biofilm). Examiningthese differences yields biofilm thicknesses of ∼750, 700, and450 nm for the 0-, 5-, and 60-min plasma treatment samples,respectively. A more precise method for quantifying the surfacetopography of the biofilms was also employed by examining the40 μm × 40 μm images, the largest scan area obtainable fromthe AFM. For each scan, the average height of the surface fea-tures was determined as relative to the lowest point in the scan(assigned the value of zero height). Using this method from the8, 4, and 4 regions, sampled for the control, 5- and 60-minplasma treated samples yielded mean values of the averageheights of 1123, 1190, and 940 nm, respectively. Therefore, thetrend of reduction of the average height after a long period ofplasma treatment (60 min) was consistent with the reductionof biofilm thickness seen in the images of Fig. 2 and thereduction in culturable cells in Fig. 1. However, there was awide variability in the average heights measured for each of thesamples, and the reported mean values differed by less than onestandard deviation.

Fig. 3 shows a typical force–displacement curve obtained onthe P. aeruginosa biofilm grown on the glass coupon for thecontrol sample. The curve displays the same general featuresthat were exhibited in all of our measured force–displacementcurves. Upon (dashed line) approach, the tip encounters thesample’s surface at the origin of the graph and deflects upwardwith a slope that increases. Upon (solid line) retraction, the tiproughly retraces the approaching curve with some hysteresisthat mainly occurs around the middle of the first quadrant ofthe graph. Upon (in the third quadrant of the graph) furtherretraction, the tip adheres to the surface until it breaks free,and the points retrace the approaching data along the nega-tive x-axis of the graph. For the purposes of analysis, twosections of the curve were considered. For approaching, the

Fig. 4. P. aeruginosa biofilm force–displacement curve slopes for (control,first eight bars) 0- and (bars 11–15) 30-min plasma-treated samples. Eight andfive different regions were examined on the 0- and 30-min treated samples,respectively. The height of each bar on the graph corresponds to the mean slopeof five curves obtained for each region. Error bars represent plus and minus onestandard deviation about this mean.

slope of the curve for positive sample displacements up to0.2 μm was determined. For significantly higher sample dis-placements, the data are less reliable since the optical detectionof the cantilever deflection becomes increasingly nonlinear.Others have performed similar analyses on bacteria, employingforce–displacement curves over comparable scales [24]–[26].For retracting, the height of the adhesive step, as indicated inFig. 3, was measured.

The slope of the force–displacement curves can be used todetermine an elastic constant or stiffness of the biofilm. Thederivation involves the analysis of the relative compression dueto the contact between two effective springs, the cantilever andthe biofilm. The biofilm stiffness, S, is related to the unitlessslope, m (cantilever displacement/sample displacement), andthe cantilever spring constant, k, through the following: [25],

S = km

1 − m. (1)

Therefore, when using the same cantilever for compar-isons between different plasma treatments, relative changesin bacterial stiffness are a function of the slope of theforce–displacement curves only.

Fig. 4 shows comparisons between the initial slope of theforce–displacement curve for the 0-min treatment (control) and30-min plasma-treated samples on glass coupons. Curves at anumber of regions for each sample were obtained. Althoughthere appears to be an overall reduction in the slope of thedisplacement curves after 30 min of plasma treatment versusthe control sample, the results differ by just slightly more thanone standard deviation. Specifically, the mean slope value of theeight regions measured for the control sample is 0.947 with astandard deviation of 0.115. For the 30-min treated sample, themean slope value for the five regions measured is 0.754 witha standard deviation of 0.062. Therefore, this reduction in theslope is consistent with a reduction in the stiffness constant, S,of the biofilm and indicates softening of the biofilm with plasmatreatment.

From the same force–displacement curves obtained for thesample regions shown in Fig. 4, adhesive step data were ex-tracted and displayed in Fig. 5.

It is apparent from this graph that there is a wide variabilityin adhesive step values over the various regions of each sample.Even considering this variability, there is an obvious overall

3402 IEEE TRANSACTIONS ON PLASMA SCIENCE, VOL. 38, NO. 12, DECEMBER 2010

Fig. 5. P. aeruginosa biofilm force–displacement curve adhesive step datafor (control) 0- and 30-min plasma-treated samples. Eight and five differentregions were examined on the 0- and 30-min treated samples, respectively. Theheight of each bar on the graph corresponds to the mean adhesive step heightof five curves obtained for each region. Error bars represent plus and minus onestandard deviation about this mean.

reduction in this adhesion with plasma treatment. The meanadhesive step height of the eight regions measured for the con-trol sample is 0.516 μm with a standard deviation of 0.249 μm.For the 30-min treated sample, the mean adhesive step heightfor the five regions measured is 0.061 μm with a standard de-viation of 0.039 μm. Therefore, there is an order of magnitudereduction in adhesion with 30 min of plasma treatment, and thetwo means differ by well beyond one standard deviation. Thisreduction of adhesion with plasma treatment indicates that thebiofilm would exhibit less adhesion to surfaces, prohibiting itsretention.

We previously reported that C. violaceum biofilm-formingcells undergo sequential morphological changes after plasmatreatment. Bacterial cells may undergo modifications rangingfrom minimal changes to putative loss of cell walls. In anothercontribution, we verified the relative “roughness” of cells byexamining an image’s cross sections and analyzing the standarddeviation of the surface height. These surface features areconsistent with cells undergoing damage [4], [27]. The presentstudy goes beyond those reports suggesting that the architectureand the stability of the biofilm as a whole may be impacted byplasma treatment.

IV. CONCLUSION

Our results clearly show that bacterial biofilms can be in-activated by using gas-discharge plasma. The architecture andstability, together with cell culturability, are impacted by theplasma treatment. These results are evidences of the poten-tial for plasma as an alternative sterilization method againstbiofilms. However, based on our previous results, [4] we areaware that viability experiments should always be carried outbefore drawing the conclusion that plasma is useful to kill cellsbased solely on measurement of culturable cells. It is widelyaccepted that the lack of culturability does not imply that thereare no viable cells in the sample. When cells are viable butnonculturable (VBNC), they are unable to produce coloniesin an agarized medium but they are still alive and may retainpathogenicity. The VBNC state is a survival mechanism ofbacteria facing environmental stress conditions that has beenreported for many gram-negative organisms [28]–[30]. Bacteria

enter into this dormant state in response to one or more environ-mental stresses, which might otherwise be ultimately lethal tothe cell. Research is being carried out in our laboratories to tryto better understand the mechanism leading to cell inactivation.

ACKNOWLEDGMENT

The authors would like to thank S. S. Lwin for the technicalsupport and Dr. Nina Abramzon for allowing the authors to useher plasma reactor.

REFERENCES

[1] G. Brelles-Mariño, J. C. Joaquin, J. D. Bray, and N. Abramzon, “Gasdischarge plasma as a novel tool for biofilm destruction,” in Proc. 2ndInt. Workshop Cold Atmos. Pressure Plasmas, 2005, pp. 69–72.

[2] K. Becker, A. Koutsospyros, S. M. Yin, C. Christodoulatos, N. Abramzon,J. C. Joaquin, and G. Brelles-Mariño, “Environmental and biologicalapplications of microplasmas,” Plasma Phys. Control. Fusion, vol. 47,no. 12B, pp. B513–B523, Dec. 2005.

[3] N. Abramzon, J. C. Joaquin, J. D. Bray, and G. Brelles-Mariño, “Biofilmdestruction by RF high-pressure cold plasma jet,” IEEE Trans. PlasmaSci., vol. 34, no. 4, pp. 1304–1309, Aug. 2006.

[4] J. C. Joaquin, J. D. Bray, C. Kwan, N. Abramzon, K. Vandervoort, andG. Brelles-Mariño, “Understanding plasma assisted biofilm inactivation,”Microbiology, vol. 175, pp. 724–732, 2009.

[5] G. Brelles-Mariño, “Bacterial biofilm inactivation by gas-discharge plas-mas,” in Biological and Environmental Applications of Gas DischargePlasmas, G. Brelles-Mariño, Ed. Commack, NY: Nova, 2010.

[6] B. Tümmler and C. Kiewitz, “Cystic fibrosis: An inherited susceptibil-ity to bacterial respiratory infections,” Mol. Med. Today, vol. 5, no. 8,pp. 351–358, Aug. 1999.

[7] M. Whiteley, M. G. Bangera, R. E. Bumgarner, M. R. Parsek,G. M. Teitzel, S. Lory, and E. P. Greenberg, “Gene expression inPseudomonas aeruginosa biofilms,” Nature, vol. 413, no. 6858, pp. 860–864, Oct. 2001.

[8] D. G. Davies, “Regulatory events in biofilm development,” in MolecularEcology of Biofilms, R. S. McLean and A. W. Decho, Eds. Norwich,U.K.: Horizon Scientific Press, 2002.

[9] R. J. Gillis and B. H. Iglewski, “Azithromycin retards Pseudomonasaeruginosa biofilm formation,” J. Clin. Microbiol., vol. 42, no. 12,pp. 5842–5845, Dec. 2004.

[10] C. T. Huang and P. S. Stewart, “Reduction of polysaccharide produc-tion in Pseudomonas aeruginosa biofilms by bismuth dimercaprol (Bis-BAL) treatment,” J. Antimicrobiol. Chem., vol. 44, no. 5, pp. 601–605,Nov. 1999.

[11] T. Mikuniya, Y. Kato, R. Kariyama, K. Monden, M. Hikida, andH. Kumon, “Synergistic effect of fosfomycin and fluoroquinolonesagainst Pseudomonas aeruginosa growing in a biofilm,” Acta Med.Okayama, vol. 59, no. 5, pp. 209–216, Oct. 2005.

[12] G. Owusu-Ababio, J. A. Rogers, D. W. Morck, and M. E. Olson, “Efficacyof sustained release ciprofloxacin microspheres against device-associatedPseudomonas aeruginosa biofilm infection in a rabbit peritoneal model,”J. Med. Microbiol., vol. 43, no. 5, pp. 368–376, Nov. 1995.

[13] G. Tanaka, M. Shigeta, H. Komatsuzawa, M. Sugai, H. Suginaka, andT. Usui, “Effect of Clarithromycin on Pseudomonas aeruginosa biofilms,”Chemotherapy, vol. 46, no. 1, pp. 36–42, Jan./Feb. 2000.

[14] E. Banin, M. L. Vasil, and E. P. Greenberg, “Iron and Pseudomonas aerug-inosa biofilm formation,” Proc. Nat. Acad. Sci., vol. 102, pp. 11 076–11 081, 2005.

[15] M. Hentzer, K. Riedel, T. B. Rasmussen, A. Heydorn, J. B. Andersen,M. R. Parsek, S. A. Rice, L. Eberl, S. Molin, N. Høiby, S. Kjelleberg, andM. Givskov, “Inhibition of quorum sensing in Pseudomonas aeruginosabiofilm bacteria by a halogenated furanone compound,” Microbiology,vol. 148, pp. 87–102, Jan. 2002.

[16] D. Davies, M. Parsek, J. Pearson, B. Iglewski, J. W. Costerton, andE. P. Greenberg, “The use of signal molecules to manipulate the behaviorof biofilm bacteria,” Clin. Microbial. Infect., vol. 5, pp. 5S7–5S8, 1999.

[17] D. J. Balazs, K. Triandafillu, P. Wood, Y. Chevolot, C. van Delden,H. C. Harms, C. Hollenstein, and H. J. Mathieu, “Inhibition of bacter-ial adhesion on PVC endotracheal tubes by RF-oxygen glow discharge,sodium hydroxide and silver nitrate treatments,” Biomaterials, vol. 25,no. 11, pp. 2139–2151, May 2004.

ZELAYA et al.: PSEUDOMONAS AERUGINOSA BIOFILM INACTIVATION 3403

[18] J. D. Bryers and B. D. Ratner, “Bioinspired implant materials befuddlebacteria,” ASM News, vol. 70, no. 5, pp. 232–237, 2004.

[19] J. Y. Schutze, S. E. Jeong, J. Park, G. S. Selwyn, and R. F. Hicks, “The at-mospheric plasma jet: A review and comparison to other plasma sources,”IEEE Trans. Plasma Sci., vol. 26, no. 6, pp. 1685–1694, Dec. 1998.

[20] R. Jones, H. M. Pollock, J. A. S. Cleaver, and C. S. Hodges, “Adhesionforces between glass and silicon surfaces in air studied by AFM: Effectsof relative humidity, particle size, roughness, and surface treatment,”Langmuir, vol. 18, no. 21, pp. 8045–8055, Oct. 2002.

[21] M. Laroussi, I. Alexeff, and W. Kang, “Biological decontamination bynon-thermal plasmas,” IEEE Trans. Plasma Sci., vol. 28, no. 1, pp. 184–188, Feb. 2000.

[22] M. Laroussi and F. Leipold, “Evaluation of the roles of reactive species,heat, and UV radiation in the inactivation of bacterial cells by air plasmasat atmospheric pressure,” Int. J. Mass Spectrom., vol. 233, no. 1–3, pp. 81–86, Apr. 2004.

[23] M. Laroussi, “Low temperature plasma-based sterilization/decontamination of biological matter,” in Proc. 2nd Int. WorkshopCold Atmos. Pressure Plasmas, 2005, pp. 18–27.

[24] Y. J. Oh, W. Jo, Y. Yang, and S. Park, “Influence of culture conditionson Escherichia coli O157:H7 biofilm formation by atomic force mi-croscopy,” Ultramicroscopy, vol. 107, no. 10/11, pp. 869–874, Oct. 2007.

[25] L. Zhao, D. Schaefer, and M. R. Marten, “Assessment of elasticity andtopography of Aspergillus nidulans spores via atomic force microscopy,”Appl. Environ. Microbiol., vol. 71, no. 2, pp. 955–960, Feb. 2005.

[26] Y. J. Oh, N. R. Lee, W. Jo, W. K. Jung, and J. S. Lim, “Effects ofsubstrates on biofilm observed by atomic force microscopy,” Ultrami-croscopy, vol. 109, no. 8, pp. 874–880, Jul. 2009.

[27] K. G. Vandervoort, N. Abramzon, and G. Brelles-Mariño, “Plasma in-teractions with bacterial biofilms as visualized through atomic forcemicroscopy,” IEEE Trans. Plasma Sci., vol. 36, no. 4, pp. 1296–1297,Aug. 2008.

[28] J. D. Oliver, “Formation of viable but nonculturable cells,” in Starvationin Bacteria, S. Kjelleberg, Ed. New York: Plenum, 1993, pp. 239–272.

[29] R. R. Colwell and A. Huq, “Vibrios in the environment: Viable but non-culturable Vibrio cholera,” in Vibrio Cholerae and Cholera: MolecularGlobal Perspectives, T. Kaye, Ed. Washington, DC: ASM, 1994.

[30] D. B. Rozak and R. R. Colwell, “Survival strategies of bacteria inthe natural environment,” Microbiol. Rev., vol. 51, no. 3, pp. 365–379,Sep. 1987.

Anna J. Zelaya received the B.S. degree in environ-mental biology from the California State PolytechnicUniversity, Pomona, in 2010, where she is currentlyworking toward the M.S. degree in the Departmentof Biological Sciences. She is currently a scholarin the MBRS-Research Initiative for Scientific En-hancement Intensive-Graduate Program funded bythe National Institutes of Health and does researchunder the supervision of Dr. G. Brelles-Mariño.

She is currently with the Biological Sciences De-partment, California State Polytechnic University.

Gregory Stough is currently working towardthe B.S. degree in the Physics Department,California State Polytechnic University, Pomona. Heis a CCRAA apprentice doing research under Dr. E.Salik and works with Dr. K. Vandervoort during theacademic year.

Navid Rad received the B.S. degree in physics fromCalifornia State Polytechnic University, Pomona,in 2010 and did research under the guidance ofDr. K. Vandervoort. He is currently working towardthe M.S. degree in particle physics at California StateUniversity, Fresno.

Kurt Vandervoort received the B.S. degree inphysics from Humboldt State University, Arcata,CA, in 1986 and the M.S. and Ph.D. degrees inphysics from the University of Illinois at Chicago,in 1988 and 1991, respectively.

He was a Postdoctoral Fellow with Argonne Na-tional Laboratory, Argonne, IL, from 1991 to 1993and was an Assistant and then Associate Profes-sor in physics with Western Carolina University,Cullowhee, NC, from 1993 to 2002. Since 2002, hehas been an Associate and currently a Full Professor

in physics with California State Polytechnic University, Pomona. His presentresearch interest includes atomic force microscopy as applied to biologicalsystems.

Graciela Brelles-Mariño received the B.S. degreein chemistry and the M.S. and Ph.D. degrees inbiochemical sciences from the Universidad Nacionalde La Plata, La Plata, Argentina, in 1984, 1985, and1993, respectively.

She was with the University of Puerto Rico,Arecibo, Puerto Rico, from 1996 to 2001. She wasa Postdoctoral Fellow with the Consejo Superiorde Investigaciones Cientificas, Granada, Spain, in1999–2000 and a Research Associate with the Lab-oratoire des Interactions Plantes-Microorganismes,

Toulouse, France, in 2001–2002. Since 2003, she has been an Assistant Profes-sor in microbiology and then an Associate Professor with the California StatePolytechnic University, Pomona. She is also currently an Associate Professorwith the Center for Research and Development of Industrial Fermentations(CINDEFI), Facultad de Ciencias Exactas, Universidad Nacional de La Plata.Her research interest focuses on biofilm inactivation by atmospheric-pressureplasmas.