Investigation of the impact of latex components on the survival of Pseudomonas aeruginosa.

Control of biological spoilage is critical to the success of any coatings formulation. However, problems with bacterial resistance and biocide toxicity require changes in waterborne resin preparation and the resulting formulated product. Despite the importance of controlling bacteria-induced spoilage, very little research exists in the field. Critical aspects of biocide/bacteria/coatings component interactions must be understood in order to improve the design of future biocides specific for coatings formulations.

This research examines the impact of several common latex components and model biocide interactions on the survival of Pseudomonas aeruginosa. Systems were characterized using both traditional microbiological techniques and a novel high-throughput fluorescence technique. P. aeruginosa is recognized as the most problematic bacterium throughout the coatings industry because of its tendency to quickly develop resistance to both antibiotics and biocides. Designed experiments comparing formulations of 10% surfactant (by weight) reveal that formulation combinations rich in ionic surfactants promote bacterial growth compared to formulations with higher concentrations of nonionic surfactants. Studies also reveal that the ionic nature of the surfactant components has a strong impact on both the rate of P. aeruginosa survival as well as biocide efficiency, with gentamicin sulfate activity being strongly inhibited in formulations containing high concentrations of sulfated surfactant.

Keywords: Biocides, fluorescence spectroscopy, high-throughput screening, latexes, colloids, emulsions, waterborne, biofouling/antifouling, latex, water-based statistical design

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Water-based polymer systems are attractive targets for microbial invasion because of the inherent material properties of the latex components. During the manufacturing process, polymer latex systems are exposed to bacteria and other microbes during material handling and cleaning processes. Microbes that are able to produce cellulase enzymes attack dispersion components in the wet state, cellulose derivatives in particular, resulting in a loss of system properties commonly recognized as "spoilage." (1) In latex coatings, these cellulase enzymes are destructive at concentrations as low as [10.sup.-5] enzyme units per gram latex. (2) Because of this potent action, it is recognized industry-wide that an intelligent understanding of latex component/biocide/bacteria interactions is needed. Studies of these complex systems by conventional methods require tedious, labor-intensive techniques.

Polymer latex components generally include surfactants, cellulose-derived rheology modifiers, and many other additives in addition to the polymer itself. There are a myriad of potential molecular structures and addition levels for each component. High-throughput screening (HTS) techniques have successfully been adopted in the study as well as optimization of water-based coatings systems by several researchers. Wicks and co-workers have explored the use of such methods in water-based chemistry in several studies designed to optimize additive packages. (3) Developing these concepts to address the interdisciplinary overlap of polymer coatings and microbiology has proven useful in our studies. The work presented here uses such methods to address the impact of nonpolymer components on bacteria growth and survival in the absence of polymer particles.

During the final formulation stage, biocide compounds are often added to inhibit or terminate microorganism propagation in polymer latexes. (4) The optimization of such additives is paramount to the success of the final application. A number of small molecule biocides are commercially available, and any combination of these may be found in modern coatings formulations. Protection against microbial growth is needed in both the final polymer film and also in the liquid latex. Roughly 27% of all biocide sales are specifically for the protection of the liquid latex. (2) Well studied in model bio-systems, these small molecule biocides have potent action against a broad spectrum of bacterium; however, the development of resistance to industrial biocides is a real concern. (5) Very little work has been reported regarding the influence of latex components on both the ability of bacteria contaminants to survive within the latex and the activity of biocides intended to eliminate this growth.

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Work by Vieira and co-workers have revealed the benefits of system-specific biocides and the importance of polymer particle/biocide interactions. (6) Vieira et al. determined the optimal cationic character of two biocides specifically interacting with polymethylmethacrylate (PMMA)/polystyrene (PS) particles in water. Both the PMMA and PS particles were modified to have negative surface charges. These surface charges resulted in preferential interactions with the biocide molecules, drastically changing the small molecule antimicrobial behavior. While this work is insightful, this model study contained no surfactant, thus only polymer particle/biocide interactions were addressed. For practical application to stabilized latex systems it is necessary to examine the impact of nonpolymer components on biocide activity as well.

Typical small molecule biocides are not system-specific, and interactions with latex components such as surfactants, stabilizers, and thickening agents affect the efficacy of the biocide. In these studies we demonstrate that component interactions are crucial to the efficacy of the model biocide. This study investigates complex mixtures of polymer dispersion components, microbes, and antimicrobial agents in order to understand the impact of dispersion components on the ability of an antimicrobial agent to thoroughly express activity. It is our expectation that understanding the impact of component/component, component/polymer, and component/biocide interactions will allow for the design of more system-specific, potent biocides. High-throughput methods were developed to efficiently address the large number of components and the complexity of the mixtures involved in this work.

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Based on a 10-year study completed by Rohm and Haas, Pseudomonas sp. is recognized as the most common bacterial cause of latex contamination. (7) Gentamicin sulfate is a well-established antibiotic that is used as a benchmark for making comparative assessments of antimicrobial activity. (8) In this work, model additives used to stabilize vinyl acrylic latex were examined to discover the impact of these components on the minimum inhibitory concentration (MIC) of gentamicin sulfate against Pseudomonas aeruginosa. These evaluations were completed on P. aeruginosa using gentamicin sulfate blended into several model dispersant systems (i.e., surfactant, hydroxyethyl cellulose, and pH buffers). Evaluation of these systems included both traditional microbiological agar plate streak methods and novel high-throughput fluorescence methods developed specifically for this project. (3)

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Fluorescence methods were developed as a rapid screening for the presence of P. aeruginosa within latex component systems. In summary, the native fluorescence of P. aeruginosa was exploited to develop an accurate pass/fail response. A liquid sample containing live P. aeruginosa bacterium was first excited at 360 nm, and subsequent fluorescence emission was collected between 380-500 nm. The data in Figure 1 depicts the change in fluorescence for a kettle charge system before microbial growth and after 48 hr of incubation at 37[degrees]C. The native fluorescence of P. aeruginosa originates from pyoverdin, a protein located on the external membrane. (9) Upon cell death, outer membrane is compromised, quenching the pyoverdin fluorescence. Fluorescence of pyoverdine is proportional to concentration, so it is possible that this method may be developed into an assay for analysis of bacteria population (i.e., density of bacteria present in a sample). (10) Traditional microbiological screening methods to assess bacteria viability (i.e., agar streak plating) require long incubation times and are also labor intensive. The fluorescence techniques described in this work provide a fast and convenient alternative to these traditional techniques for our specific system. The fluorescence technique developed for this work can be applied to other systems after baseline data is established. It is important to note that the fluorescent moiety of P. aeruginiosa has been shown to undergo a reversible photobleaching effect in our screening tests. To address this effect, samples must be placed in the dark for a predetermined amount of time before fluorescence emission collection can be repeated on a sample.

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Results of initial studies prompted the investigation of specific surfactant end-moieties, and the impact of these end-moieties on the growth and survival of P. aeruginosa as well as the antimicrobial activity of gentamicin sulfate. High-throughput preparation, designed experiments, and fluorescence techniques proved useful for these evaluations as well. All studies were carried out using a 96-well plate layout shown in Figure 2; the maximum volume of each sample well was 500 [micro]L.

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EXPERIMENTAL

Materials, Compounds, and Sources

Sodium dodecyl benzene sulfonate LDS 22 (linear, M.W. = 194, 23% active in water) and ethoxylated nonylphenol Igepal CO 897 (M.W. = 1982, 40% active in water) were provided by Rhodia, Inc. Ethoxylated nonylphenol NP-9 Tergitol (denoted hereafter as NP-9N) was purchased from Aldrich. Phosphated ethoxylated nonylphenol Ultraphos 9 (denoted NP-9P) was provided by MFG Chemicals and sulfonated POE nonylphenol sodium salt (denoted NP-9S) was purchased from Chem Service, Inc. Hydroxyethyl cellulose Cellosize QP 300 (M.W. = 200,000) was provided by Dow Corporation. DIFCO[TM] minimal Davis broth without dextrose (dipotassium phosphate, 7.0 g; monopotassium phosphate, 2 g; ammonium sulfate, 1.0 g; sodium citrate, 0.5 g; magnesium sulfate 0.1 g), dissolved in each final solution per liter of water (denoted herein as trace elements), was purchased from Becton, Dickinson and Company. Gentamicin sulfate was purchased from Alexis Corporation. Tryptic soy agar (TSA) and tryptic soy broth (TSB) were also purchased from Becton, Dickinson and Company.

Measurements

Fluorescence spectra were obtained using a Tecan Safire 96-well multifunctional plate reader (Tecan U.S., Durham, NC). TSA was autoclaved immediately after mixing, poured into 5-cm diameter culture dish plates and allowed to solidify. Agar streak plating was completed using 100 [micro]l of inoculated test sample evenly spread onto a TSA culture plate. Inoculated TSA culture plates were allowed to incubate at 37[degrees]C for an average of 18 hr.

"Kettle Charge" Preparation

Formulation of a model dispersant system includes two surfactant molecules. LDS 22 provided a model anionic surfactant and CO 897 was used as a nonionic cosurfactant. For viscosity modification, hydroxyethyl cellulose (HEC) was added. To adjust pH, ammonium acetate was included in the formulation. The major phase of the formulation was distilled water, and in some cases minimal Davis broth without dextrose was added to simulate the trace elements commonly found in industrial process water conditions. The components mentioned were combined in different ratios in order to create an environment conducive to emulsion polymerization, commonly referred to as the "kettle charge." A typical kettle charge formulation used in this study would be comprised of approximately 90% water, 9% total surfactant, 0.8% trace element solution, and 0.1% HEC.

Bacterial Culture

P. aeruginosa was secured from registered stock and cultured in tryptic soy broth (TSB) to promote growth. Stock culture was frozen at -80[degrees]C. For each test, the bacterial inoculum was prepared from frozen stock culture to ensure that resistant bacteria mutants did not arise. To prepare a test inoculation, bacteria suspensions were washed in phosphate-buffered saline and then resuspended in sterile water according to standard microbiological protocol. Optical density measurements were used to estimate the number of the bacteria in each P. aeruginosa solution used in testing. Where indicated, aliquots of test solutions were plated on TSA.

High-Throughput Analysis of Latex Component Effect on Bacterial Growth and Gentamicin Sulfate Efficacy

This designed experiment examines the impact of four factors on the efficacy of gentamicin sulfate against P. aeruginosa. Common latex components of nonionic CO 897 surfactant, anionic LDS 22 surfactant, and HEC compose three factors in the study. As a fourth factor, the impact of industrial process water was simulated by a trace mineral broth with a high concentration of phosphate compounds similar to antiscaling agent residuals that often contaminate industrial plants. This work provided a statistical relationship between the components that comprise this dispersant formulation and the time-dependant growth of P. aeruginosa in that formulation.

These initial studies employed a Box-Behnken 4-factor/3-level design executed using 96-well titer plates and the following four factors: type of surfactant component, concentration of HEC, gentamicin sulfate, and trace minerals. (11) The Box-Behnken design has fewer runs than a full 3-level factorial, and was set up and evaluated using commercially available statistical software packages, Design Expert 6.10.0 and JMP 5.0. Mixture formulations were governed by the Box-Behnken design and prepared under sterile conditions in the 96-well plate layout. This experimental design was completed using both manual and robotic pipetting techniques under sterile conditions. After 48 hr of incubation, each well was examined for microbial growth/survival.

High-throughput fluorescence techniques were developed in order to quickly screen these experimental mixtures for live bacteria content. Traditional agar plating techniques were used to verify fluorescence results. Baseline emission was determined by obtaining fluorescence spectrum for each test solution, and then each sample was inoculated with 20 [micro]L of P. aeruginosa suspended in water (roughly 7 X [10.sup.3] cfu). The fluorescence technique was repeated immediately after inoculation (t = 0). To examine the impact of microbial propagation on system fluorescence, emission was also recorded at additional time intervals such as at t = 24 hr and t = 48 hr.

SURFACTANT FUNCTIONAL GROUP INVESTIGATION

A mixture study was designed to examine effects of the ethoxylated nonylphenol-based surfactant end group on the growth of P. aeruginosa. For this study, the ethoxylated nonylphenols will be denoted NP-9N (nonionic), NP-9P (phosphated), and NP-9S (sulfonated), as shown in Figure 3.

Standard kettle charge solutions were prepared to study each nonylphenol molecule. Solution components include water (major phase) hydroxyethyl cellulose, ammonium acetate, and trace elements all at standard test concentrations, as well as the surfactant of interest to create a 2% surfactant solution for ionic surfactants and a 0.2% solution for the nonionic surfactant. NP-9N was used in lower concentrations due to solubility limitations, and ongoing tests address issues prompted by this difference in concentration. After mixing, each solution was autoclaved at 121[degrees]C for 20 min to ensure sterile testing conditions. Ionic surfactant solutions at 10% were also evaluated using the same methods.

Functional Group Effects on Gentamicin MIC

A standard MIC test was designed to establish the existence of surfactant functional group/bacteria/gentamicin interactions. This MIC test was performed on kettle charge solutions of 0.2% NP-9N, 2.0% and 10.0% NP-9S, and both 2.0% and 10.0% NP-9P solutions. Using pipetting techniques, each well was allotted 200 [micro]L of kettle charge solution and 20 [micro]L of P. aeruginosa suspension (roughly 7 X [10.sup.3] cfu). Aliquots (20 [micro]L) from a four-fold dilution of gentamicin sulfate solution (0.25 [micro]g/mL-0.091 [micro]g/mL) were successively added to six different test wells to create a gradient study of gentamicin performance.

After establishment of MIC results, the primary study was designed such that microbial growth in the surfactant environment could be statistically evaluated via both traditional methods (agar streak plate) and high-throughput fluorescence responses. The centroid mixture design was completed according to Figure 4.

Eight replicates of the designed study were prepared via the manual sterile pipetting of 260 [micro]L of surfactant mixture solutions into a 96-well glass plate. Characteristic fluorescence emission spectrum (380 nm-500 nm) of each test well was collected after excitation at 360 nm to determine emission background. The test wells were then inoculated with 20 [micro]L of P. aeruginosa suspended in D1 water (approximately 5 X [10.sup.5] cfu/mL). Fluorescence emission spectra were again taken to verify inoculation. The entire 96-well plate was then lightly sealed to prevent cross-contamination and placed into a humid container to prevent evaporation from the wells, as shown in the schematic in Figure 5. The test plates were subsequently incubated (37[degrees], no shaking) for several days. Fluorescence spectra were collected at 24-hr intervals throughout the incubation period in order to monitor the survival and propagation of P. aeruginosa. This design layout was also repeated for solutions containing constant levels of HEC, trace elements, and biocide.

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Impact of Interactions between Model Surfactants and Latex Components on P. aeruginosa Growth

A second Box-Behnken experiment was designed to evaluate the impact of interactions between the model surfactant end group and the other latex nonpolymer components on bacteria growth. No biocide was used in this series of experiments. Factors in this designed experiment include surfactant end group, concentration of HEC, and the concentration of free trace elements. Each numeric factor was varied over three levels. The minimum concentration of each component was zero, and the maximum concentration was at the upper limit of practical formulation use levels. Each Box-Behnken series contained 48 unique solutions. The design was replicated three times, resulting in 144 total test solutions contained on two 96-well plates. Each sample consisted of 225 [micro]L volume component solution and 25 [micro]L P. aeruginosa in water, for a total sample volume of 250 [micro]L. The 96-well plate was sealed using a polystyrene sheet coated with pressure-sensitive adhesive to ensure no cross-contamination between testing samples. The 96-well plate was allowed to age at 37[degrees]C for 225 hr. Beginning at zero hours, the plate was removed from the incubator every 24 hr and a fluorescence spectrum was obtained for each well. Total time required to obtain all 144 spectra was approximately 10 min each day; spectra were obtained under the same conditions previously discussed in this work. To confirm the fluorescence spectra results, random test wells were selected for evaluation via agar plating.

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STUDY RESULTS

High-Throughput Analysis of Latex Component Effect On Bacterial Growth and Gentamicin Sulfate Efficacy

The high-throughput fluorescence techniques developed to quickly screen dispersion component solutions for bacterial growth were used to examine the presence of bacteria in the system. To validate these fluorescence emission results, each test well sample was also plated onto standard tryptic soy agar (TSA) culture media. After 18 hr of incubation, the plates were visually inspected for growth. Each plate was given a score based on the number of visible colonies seen on the TSA surface. Scores of 0 (no growth), 1 (0-150 cfu), 2 (150-250 cfu), 3 (250-350 cfu) or 4 (>350 cfu) were assigned to each plate and introduced to the statistical model as such. Analysis of the data from this Box-Behnken experimental design revealed an excellent correlation between peak fluorescence emission and the number of viable cells present in samples taken after 48 hr of incubation. The results of both fluorescence and culture plating data are shown in Figure 6.

The profile plot in Figure 6 summarizes the effect of each factor on the two responses recorded, growth and fluorescence emission intensity. The results in Figure 6 also suggest that after 48 hr bacteria growth is promoted by the anionic sulfur-containing surfactant used to formulate this model dispersant package. Results also indicate the anticipated inhibitory effect on bacteria survival induced by the gentamicin sulfate. Almost no effect of HEC can be seen above the minimum level tested, and trace elements are shown to have a minimum optimal concentration. The influences of single components are summarized in Figure 7. It is important to emphasize that this study was only evaluated up to the 48-hr time point.

Surfactant Functional Group Investigation Results: Impact of Latex Components on MIC of Gentamicin Sulfate

Previously reported MIC results indicate that the functional group of the surfactant has a major effect on the efficacy of the model biocide within the system studied. (12) At all surfactant concentrations tested, sulfur-containing solutions had strong microbial growth at all gentamicin concentration levels. Conversely, no microbial growth was detected at any gentamicin level for solutions containing high phosphorus-surfactant concentration. Also noteworthy, for this NP-9P solution no growth was seen in the inoculated control. As revealed by agar streak plate results, P. aeruginosa was only able to survive in solutions low in NP-9P concentration, thus indicating phosphorus-containing surfactants either have biocide activity at high concentrations or that the NP-9P favorably interacts with the antimicrobial agent, promoting antimicrobial behavior.

Surfactant Functional Group Investigation Results

Fluorescence results for the surfactant functional group mixture design reinforce trends described in the MIC results. In this portion of the study, bacteria survival and growth were affected only by the surfactant functional group, as all other variables were held constant. Each sample was incubated for a total of 240 hr with spectroscopic evaluation every 24 hr. Gentamicin was not a factor in the following results.

Solution composition obviously impacts P. aeruginosa growth, as indicated by fluorescence emission and standard culture dish plating (Figure 6). However, upon closer examination of the data it is clear that ionic content cannot be considered a balanced factor. Results of this ternary mixture study indicate the effect of each ionic component on bacteria survival and resulting fluorescence emission after 72, 144, and 168 hr. Results were evaluated via ANOVA analysis for the mixture cubic model. [R.sup.2] for the model is 0.87. The ternary plot shown in Figure 8 illustrates the strong impact of the sulfur functional group on microbial survival, as well as synergistic impact of NP-9S in mixture combinations after 72 hr of aging. Higher levels of NP-9S respond to 360 nm excitation with significantly higher fluorescence emission than those solutions containing the same ratio of NP-9P at this time point. Solutions with 100% nonionic surfactant and 100% phosphate terminated surfactant do not support abundant bacteria populations in comparison to the solutions rich in sulfonated surfactant.

After another 72 hr of incubation, the relative impact of solution components on bacteria survival had not changed significantly, however the overall concentration of bacteria, as indicated by fluorescence response, had as much as quadrupled in all solutions, as seen in Figure 9.

Evaluation of the same mixture design after 168 hr of incubation revealed that the importance of the NP-9S component was no longer critical, as the design space affording the highest levels of bacteria growth spanned the entire spectrum of NP-9S concentrations, as shown in Figure 10. Overall fluorescence at 168 hr was significantly lower than the 144-hr time point, indicating the system was no longer able to support the large population of bacteria seen in 144-hr samples. This probably resulted from the consumption of available carbon sources.

IMPACT OF INTERACTIONS BETWEEN MODEL SURFACTANTS AND LATEX COMPONENTS ON P. AERUGINOSA

This Box-Behnken design was intended to investigate the surfactant end group interactions with latex components and the resulting impact on microbial growth. Each of the 46 mixtures dictated by the Box-Behnken design was evaluated via fluorescence and confirmed in random samples using culture plating techniques. Concentration of viable P. aeruginosa population in a sample was correlated to mixture composition via quadratic analysis. Coefficients calculated from the emission analysis after 168 hr of incubation are generally representative of all post 36-hr time points, and are shown in Figure 11. Coefficients were calculated using a quadratic analysis and inverse transformation, and the average resulting [R.sup.2] = 0.82 for all time points was evaluated. The large amount of data uncovered by this designed experiment revealed several trends that are of interest.

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Static Impact of Compositional Variations

Specifics of mixture composition have a strong impact on microbial growth. There are very few differences in fluorescence emission results for each mixture solution immediately after inoculation with a small amount of P. aeruginosa (t = 0). Differences in emission become apparent after the inoculated mixture has been incubated for at least 18 hr, at which point the different mixture solutions have emission spectra which indicate the ability of each mixture to support bacteria propagation and survival. The impact of the ionic surfactants (NP-9S and NP-9P) on fluorescence emission after 72 hr of incubation are shown is Figure 12. Solutions containing the high concentrations of the NP-9S surfactant support the highest concentration of viable bacteria.

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Figure 13 shows the statistical evaluation as previously mentioned, but the nonionic surfactant component is absent from the formulation. Without the nonionic surfactant, results again indicated that solutions with a high concentration of NP-9S surfactant support the highest concentration of viable bacteria. However, the concentration of living P. aerguinosa was much lower than the mixture shown in Figure 12, as indicated by the lowered maximum emission intensity, which decreased from 946 to 586 (relative intensity at NP-9S = +1, NP-9P = -1).

The lower concentrations of viable bacteria detected in mixtures without nonionic surfactant are reflected in the statistical analysis of all mixtures after 72 hr of incubation. Statistical evaluations of all mixtures void of trace elements reveal negligible ability to support viable P. aeruginosa. Figure 14 represents the evaluation of bacteria growth at the various levels of the two ionic surfactants, but these solutions are without nonionic surfactant and trace elements. The importance of the trace element additive, which mimics industrial production contaminants, is depicted in Figure 15.

The HEC component was expected to be a critical component after 72 hr of incubation. However, the impact of HEC was not well pronounced beyond the value of standard deviation.

Dynamic Impact of Mixtures Over Time

As previously mentioned, each solution mixture was incubated and the bacteria was allowed to propagate within the system. As solutions support bacteria propagation, growth, and survival, solution components were metabolized and were eventually exhausted. This dynamic environment causes changes in solution properties, as well as the ability of the solution to support continued microbial growth. Observations made over time via statistical quadratic analysis of the Box Behnken studies indicate that the concentration of bacteria that is able to be supported by a certain solution composition changes with time. This is demonstrated in the comparison of Figure 12 and Figure 16, both of which predict bacteria growth as indicated by fluorescence emission intensity at the mid-levels of HEC, trace elements, and nonionic surfactant. After both 216 and 72 hr of incubation, optimal growth conditions were found in solutions with high concentrations of sulfonated surfactant. However, if the solutions were allowed to incubate for another 144 hr, solutions high in sulfonated surfactant support nearly double the bacteria population seen at 72 hr, compared to solutions low in NP-9S, which maintain a static bacteria population between 72 hr to 216 hr.

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CONCLUSIONS

Using the native fluorescence of P. aeruginosa, we have determined solution characteristics critical to supporting and suppressing microbe survival and propagation. The rate at which this microbial degradation occurs is directly correlated with the composition of the contaminated solution. The presence of trace elements in the solutions proves to be a strong supplement to bacteria survival and propagation at all time intervals tested. Solutions prepared without trace elements typically supported only a fraction of the bacteria population seen in identical solutions that included trace elements, highlighting the impact one would expect from contamination of production facilities.

Ionic surfactants were shown to be a critical component, especially if the formulation was without trace elements and/or cellulose components. While the phosphate endgroup of the NP-9P surfactant served as a critical nutrient source for microbes, it was hypothesized that the lower pH resulting from solutions rich in this surfactant inhibited microbial growth. The need for further work on the role of different surfactants on microbial survival within these systems is apparent. An evaluation of the impact of ionic surfactants on bacteria survival over time in a pH-controlled environment will be completed to further investigate the dynamic formulation environment under more controlled conditions. An important extension of this work will complete the evaluation of these systems in the presence of the polymer particle-containing latex synthesized with the surfactants described in this investigation.

ACKNOWLEDGMENTS

The authors would like to thank Adam Hathorne and Ayesha Alam for their assistance. Funding was provided by the National Science Foundation Integrative Graduate Education and Research Traineeship (IGERT) (NSF DGE-0333136) and NSF Partners for Innovation (NSF Award 0227827).

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(6) Vieira, D., Lincopan, N., Mamizuka, E., Petri, D., and Carmona-Ribeiro, A., "Competitive Adsorption of Cationic Bilayers and Chitosan on Latex: Optimal Biocidal Action," Langmuir, 19, 924-932 (2003).

(7) Sadasivan, L. and Gandhi, U., "A Multifunctional Isothiazolone Biocide for Latex Coatings," Proc. 80th Annual Meeting of the Federation of Societies for Coatings Technology, New Orleans, LA, October 30-November 1, 2002.

(8) Lambert, R., Joynson, J., and Forbes, B., "The Relationships and Susceptibilities of Some Industrial, Laboratory, and Clinical Isolates of Pseudomonas aeruginosa to some Antibiotics and Biocides," J. Appl. Microbiology, 91, 972-984 (2001).

(9) Olmo, A., Caramelo, C., and Sanjose, C., "Fluorescent Complex of Pyoverdine with Aluminum," J. Inorg. Biochem., 97, 384-387 (2003).

(10) Elliot, R., "Some Properties of Pyoverdine, the Water-Soluble Fluorescent Pigment of the Pseudomonads," Appl. Microbiol., 212, 393-395 (1958).

(11) Schmidt, S.R. and Launsby, R.G., Understanding Industrial Designed Experiments, Air Academy Press, Colorado Springs, CO, 2000.

(12) Rhoades, A., Wicks, D., and Elasri, M., "Fluorescence-Based Study of Bacteria Survival in Water-Based Latex," Proc. 31st International Waterborne, High-Solids, and Powder Coatings Symposium, New Orleans, LA, 2004.

Alicyn M. Rhoades, ([dagger]) Douglas A. Wicks,** and Mohamed O. Elasri ([double dagger]) -- The University of Southern Mississippi*

Presented, in part, at the 31st Annual International Waterborne, High-Solids and Powder Coatings Symposium, New Orleans, LA, 2004.

* 118 College Dr., Hattiesburg, MS 39406.

([dagger]) School of Polymers and High Performance Materials.

** Author to whom correspondence should be addressed.

([double dagger]) Dept. of Biological Sciences.