Scholarly Work - Center for Biofilm Engineering
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/9335
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Item Coupon position does not affect Pseudomonas aeruginosa and Staphylococcus aureus biofilm densities in the CDC biofilm reactor(Elsevier BV, 2024-08) Buckner, Elizabeth; Buckingham-Meyer, Kelli; Miller, Lindsey A.; Parker, Albert E.; Jones, Christopher J.; Goeres, Darla M.The CDC Biofilm Reactor method is the standard biofilm growth protocol for the validation of US Environmental Protection Agency biofilm label claims. However, no studies have determined the effect of coupon orientation within the reactor on biofilm growth. If positional effects have a statistically significant impact on biofilm density, they should be accounted for in the experimental design. Here, we isolate and quantify biofilms from each possible coupon surface in the reactor to quantitatively determine the positional effects in the CDC Biofilm Reactor. The results showed no statistically significant differences in viable cell density across different orientations and vertical positions in the reactor. Pseudomonas aeruginosa log densities were statistically equivalent among all coupon heights and orientations. While the Staphylococcus aureus cell growth showed no statistically significant differences, the densities were not statistically equivalent among all coupon heights and orientations due to the variability in the data. Structural differences were observed between biofilms on the high-shear baffle side of the reactor compared to the lower shear glass side of the reactor. Further studies are required to determine whether biofilm susceptibility to antimicrobials differs based on structural differences attributed to orientation.Item Harvesting and Disaggregation: An Overlooked Step in Biofilm Methods Research(MyJove Corporation, 2022-04) Buckingham-Meyer, Kelli; Miller, Lindsey A.; Parker, Albert E.; Walker, Diane K.; Sturman, Paul; Novak, Ian; Goeres, Darla M.Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the surface and disaggregating the clumps, and analyzing the sample. Of the four steps, harvesting and disaggregation are the least studied but nonetheless critical when considering the potential for test bias. This article demonstrates commonly used harvesting and disaggregation techniques for biofilm grown on three different surfaces. The three biofilm harvesting and disaggregation techniques, gleaned from an extensive literature review, include vortexing and sonication, scraping and homogenization, and scraping, vortexing and sonication. Two surface types are considered: hard non-porous (polycarbonate and borosilicate glass) and porous (silicone). Additionally, we provide recommendations for the minimum information that should be included when reporting the harvesting technique followed and an accompanying method to check for bias.Item Harvesting and Disaggregation: An Overlooked Step in Biofilm Methods Research(MyJove Corporation, 2022-04) Buckingham-Meyer, Kelli; Miller, Lindsey A.; Parker, Albert E.; Walker, Diane K.; Sturman, Paul; Novak, Ian; Goeres, Darla M.Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the surface and disaggregating the clumps, and analyzing the sample. Of the four steps, harvesting and disaggregation are the least studied but nonetheless critical when considering the potential for test bias. This article demonstrates commonly used harvesting and disaggregation techniques for biofilm grown on three different surfaces. The three biofilm harvesting and disaggregation techniques, gleaned from an extensive literature review, include vortexing and sonication, scraping and homogenization, and scraping, vortexing and sonication. Two surface types are considered: hard non-porous (polycarbonate and borosilicate glass) and porous (silicone). Additionally, we provide recommendations for the minimum information that should be included when reporting the harvesting technique followed and an accompanying method to check for bias.Item Imaging and plate counting to quantify the effect of an antimicrobial: A case study of a photo-activated chlorine dioxide treatment(Oxford University Press, 2022-12) Parker, Albert E.; Miller, Lindsey; Adams, Jacob; Pettigrew, Charles; Buckingham-Meyer, Kelli; Summers, Summers; Christen, Andres; Goeres, DarlaAim. To assess removal versus kill efficacies of antimicrobial treatments against thick biofilms with statistical confidence. Methods and results. A photo‐activated chlorine dioxide treatment (Photo ClO2) was tested in two independent experiments against thick (>100 μm) Pseudomonas aeruginosa biofilms. Kill efficacy was assessed by viable plate counts. Removal efficacy was assessed by 3D confocal scanning laser microscope imaging (CSLM). Biovolumes were calculated using an image analysis approach that models the penetration limitation of the laser into thick biofilms using Beer's Law. Error bars are provided that account for the spatial correlation of the biofilm's surface. The responsiveness of the biovolumes and plate counts to the increasing contact time of Photo ClO2 were quite different, with a massive 7 log reduction in viable cells (95% confidence interval [CI]: 6.2, 7.9) but a more moderate 73% reduction in biovolume (95% CI: [60%, 100%]). Results are leveraged to quantitatively assess candidate CSLM experimental designs of thick biofilms. Conclusions. Photo ClO2 kills biofilm bacteria but only partially removes the biofilm from the surface. To maximize statistical confidence in assessing removal, imaging experiments should use fewer pixels in each z‐slice, and more importantly, at least two independent experiments even if there is only a single field of view in each experiment. Significance and impact of study. There is limited penetration depth when collecting 3D confocal images of thick biofilms. Removal can be assessed by optimally fitting Beer's Law to all of the intensities in a 3D image and by accounting for the spatial correlation of the biofilm's surface. For thick biofilms, other image analysis approaches are biased or do not provide error bars. We generate unbiased estimates of removal and assess candidate CSLM experimental designs of thick biofilms with different pixilations, numbers of fields of view and number of experiments using the included design tool.Item Harvesting and Disaggregation: An Overlooked Step in Biofilm Methods Research(MyJove Corporation, 2022-04) Buckingham-Meyer, Kelli; Miller, Lindsey A.; Parker, Albert E.; Walker, Diane K.; Sturman, Paul; Novak, Ian; Goeres, Darla M.Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the surface and disaggregating the clumps, and analyzing the sample. Of the four steps, harvesting and disaggregation are the least studied but nonetheless critical when considering the potential for test bias. This article demonstrates commonly used harvesting and disaggregation techniques for biofilm grown on three different surfaces. The three biofilm harvesting and disaggregation techniques, gleaned from an extensive literature review, include vortexing and sonication, scraping and homogenization, and scraping, vortexing and sonication. Two surface types are considered: hard non-porous (polycarbonate and borosilicate glass) and porous (silicone). Additionally, we provide recommendations for the minimum information that should be included when reporting the harvesting technique followed and an accompanying method to check for bias.Item Drip flow reactor method exhibits excellent reproducibility based on a 10-laboratory collaborative study(Elsevier BV, 2020) Goeres, Darla M.; Parker, Albert E.; Walker, Diane K.; Meier, Kelsey; Lorenz, Lindsey A.; Buckingham-Meyer, KelliA standard method for growing Pseudomonas aeruginosa biofilm in the Drip Flow Biofilm Reactor was assessed in a 10-laboratory study. The mean log density was 9.29 Log10(CFU/cm2). The repeatability and reproducibility SDs were equal to 0.22 and 0.24, respectively, providing statistical confidence in data generated by the method.Item Development, standardization, and validation of a biofilm efficacy test: The single tube method(2019-10) Goeres, Darla M.; Walker, Diane K.; Buckingham-Meyer, Kelli; Lorenz, Lindsey A.; Summers, Jennifer; Fritz, Blaine; Goveia, Danielle; Dickerman, Grace; Schultz, Johanna M.; Parker, Albert E.Methods validated by a standard setting organization enable public, industry and regulatory stakeholders to make decisions on the acceptability of products, devices and processes. This is because standard methods are demonstrably reproducible when performed in different laboratories by different researchers, responsive to different products, and rugged when small (usually inadvertent) variations from the standard procedure occur. The Single Tube Method (ASTM E2871) is a standard method that measures the efficacy of antimicrobials against biofilm bacteria that has been shown to be reproducible, responsive and rugged. In support of the reproducibility assessment, a six-laboratory study was performed using three antimicrobials: a sodium hypochlorite, a phenolic and a quaternary/alcohol blend, each tested at low and high efficacy levels. The mean log reduction in viable bacteria in this study ranged from 2.32 to 4.58 and the associated reproducibility standard deviations ranged from 0.89 to 1.67. Independent follow-up testing showed that the method was rugged with respect to deviations in sonication duration and sonication power but slightly sensitive to sonicator reservoir degassing and tube location within the sonicator bath. It was also demonstrated that when a coupon was dropped into a test tube, bacteria can splash out of reach of the applied antimicrobials, resulting in substantial bias when estimating log reductions for the products tested. Bias can also result when testing products that hinder the harvesting of microbes from test surfaces. The culmination of this work provided recommended changes to the early version of the standard method E2871-13 (ASTM, 2013b) including use of splashguards and microscopy checks. These changes have been incorporated into a revised ASTM method E2871-19 (ASTM 2019) that is the basis for the first regulatory method (ATMP-MB-20) to substantiate “kills biofilm” claims for antimicrobials registered and sold in the US.Item Holistic Management of Textile Odor Using Novel Silver-Polymeric Complexes(2018-08) Frattarelli, Dave; Powers, Lisa; Doshi, Deepack; Vargo, Kevin; Patel, Bhavin; Liboon, Jennifer; Gallagher, Michelle; Monticello, Robert; Goeres, Darla M.; Lorenz, Lindsey A.; Buckingham-Meyer, KelliOdor poses a growing concern in clothing and apparel applications due to laundering limitations at managing odor-causing microorganisms. Herein, a novel silver-polymer complex was applied to textile materials and studied using quantitative antimicrobial assays, gas chromatography techniques, and odor panel sensory tests to ascertain odor control function and effectiveness. A known chemical odor pathway involving leucine conversion to isovaleric acid was studied and found to be disrupted in silver-treated fabrics. Furthermore, its odor absorption function was confirmed with up to 90% retention of select thiol and fatty acid odors at body temperature in a model odor bouquet. Lastly, human sensory studies were used to support laboratory odor measurements using seven-day wear trials and milk odor generation techniques after 50 launderings.Item Development of a laboratory model to assess the removal of biofilm from interproximal spaces by powered tooth brushing(2002-11) Adams, Heather; Winston, Matthew T.; Heersink, Joanna; Buckingham-Meyer, Kelli; Costerton, J. William; Stoodley, PaulPurpose: To develop an interproximal laboratory model to compare the potential effectiveness of powered brushing to remove biofilm plaque from interproximal spaces beyond the reach of bristles. Materials and Methods: Streptococcus mutans biofilms were first grown onglass microscope slides in a drip-flow reactor. The slides were removed and positioned in the interproximal model. Each slide was exposed to 15 seconds powered brushing with either the Sonicare® Elite or the Braun Oral-B 3D Excel. The thickness of the biofilm was measured with confocal microscopy at various distances from the bristle tips. Results: The Sonicare® Elite reduced the thickness of biofilm by 57% at a distance of 0-5 mm from the bristle tips, 46% at 5-10 mm and 43% at 10-15 mm, relative to biofilm in areas unexposed to brushing. All reductions in thickness were statistically significant (P< 0.01). The Braun Oral-B 3D reduced the biofilm thickness by 16%, 13%, and 19% at the same distances respectively, but the thickness reductions were not statistically significant from those in the unexposed areas (P> 0.1).Item Monitoring of microbial souring in chemically treated, produced-water biofilm systems using molecular techniques(2005-04) Kjellerup, B. V.; Veeh, Richard Harold; Sumithrarathne, P.; Thomsen, T. R.; Buckingham-Meyer, Kelli; Frolund, B.; Sturman, Paul J.The identification of bacteria in oil production facilities has previously been based on culture techniques. However, cultivation of bacteria from these often-extreme environments can lead to errors in identifying the microbial community members. In this study, molecular techniques including fluorescence in situ hybridization, PCR, denaturing gradient gel electrophoresis, and sequencing were used to track changes in bacterial biofilm populations treated with nitrate, nitrite, or nitrate+molybdate as agents for the control of sulfide production. Results indicated that nitrite and nitrate+molybdate reduced sulfide production, while nitrate alone had no effect on sulfide generation. No long-term effect on sulfide production was observed. Initial sulfate-reducing bacterial numbers were not influenced by the chemical treatments, although a significant increase in sulfate-reducing bacteria was observed after termination of the treatments. Molecular analysis showed a diverse bacterial population, but no major shifts in the population due to treatment effects were observed.