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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 Design and fabrication of biofilm reactors(2020) Goeres, Darla M.; Pedersen, Stephen; Warwood, B. K.; Walker, Diane K.; Parker, Albert E.; Mettler, Madelyn; Sturman, Paul J.Laboratory biofilm reactors are tools that researchers use to grow biofilms that exhibit characteristics sufficiently similar to the environment of interest. Numerous biofilm reactors that model various fluid dynamics are described in scientific literature, each with its associated list of advantages and limitations. This chapter focuses on the process used to design and fabricate biofilm reactors with the stated goal of generating a commercial product. The process begins with identifying the environment of interest and key attributes the reactor should include or model. A prototype is then designed, built, and tested in the laboratory. Modifications are made based upon laboratory performance until a design is achieved that is affordable, practical, operationally simple, and relevant and that provides repeatable, convincing results. This process was used to design the industrial surfaces biofilm reactor, developed to model cooling tower biofilms but suitable to study biofilms grown under low shear, high gas transfer, and intermittently wet conditions.Item Interlaboratory study for the evaluation of three microtiter plate-based biofilm quantification methods(2021-07) Allkja, Jontana; van Charante, Frits; Aizawa, Juliana; Reigada, Ines; Guarch-Perez, Clara; Vazquez-Rodriguez, Jesus Augusto; Cos, Paul; Coenye, Tom; Fallarero, Adyary; Zaat, Sebastian A. J.; Felici, Antonio; Ferrari, Livia; Azevado, Nuno F.; Parker, Albert E.; Goeres, Darla M.Microtiter plate methods are commonly used for biofilm assessment. However, results obtained with these methods have often been difficult to reproduce. Hence, it is important to obtain a better understanding of the repeatability and reproducibility of these methods. An interlaboratory study was performed in five different laboratories to evaluate the reproducibility and responsiveness of three methods to quantify Staphylococcus aureus biofilm formation in 96-well microtiter plates: crystal violet, resazurin, and plate counts. An inter-lab protocol was developed for the study. The protocol was separated into three steps: biofilm growth, biofilm challenge, biofilm assessment. For control experiments participants performed the growth and assessment steps only. For treatment experiments, all three steps were performed and the efficacy of sodium hypochlorite (NaOCl) in killing S. aureus biofilms was evaluated. In control experiments, on the log10-scale, the reproducibility SD (SR) was 0.44 for crystal violet, 0.53 for resazurin, and 0.92 for the plate counts. In the treatment experiments, plate counts had the best responsiveness to different levels of efficacy and also the best reproducibility with respect to responsiveness (Slope/SR = 1.02), making it the more reliable method to use in an antimicrobial efficacy test. This study showed that the microtiter plate is a versatile and easy-to-use biofilm reactor, which exhibits good repeatability and reproducibility for different types of assessment methods, as long as a suitable experimental design and statistical analysis is applied.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 Guidelines for the statistical analysis of a collaborative study of a laboratory method for testing disinfectant product performance(2013-09) Hamilton, Martin A.; Hamilton, G. C.; Goeres, Darla M.; Parker, Albert E.This paper presents statistical techniques suitable for analyzing a collaborative study (multilaboratory study or ring trial) of a laboratory disinfectant product performance test (DPPT) method. Emphasis is on the assessment of the repeatability, reproducibility, resemblance, and responsiveness of the DPPT method. The suggested statistical techniques are easily modified for application to a single laboratory study. The presentation includes descriptions of the plots and tables that should be constructed during initial examination of the data, including a discussion of outliers and QA checks. The statistical recommendations deal with evaluations of prevailing types of DPPTs, including both quantitative and semiquantitative tests. The presentation emphasizes tests in which the disinfectant treatment is applied to surface-associated microbes and the outcome is a viable cell count; however, the statistical guidelines are appropriate for suspension tests and other test systems. The recommendations also are suitable for disinfectant tests using any microbe (vegetative bacteria, virus, spores, etc.) or any disinfectant treatment. The descriptions of the statistical techniques include either examples of calculations based on published data or citations to published calculations. Computer code is provided in an appendix.Item Evaluation of Petrifilm™ Aerobic Count Plates as an Equivalent Alternative to Drop Plating on R2A Agar Plates in a Biofilm Disinfectant Efficacy Test(2014-12) Fritz, Blaine; Walker, Diane K.; Goveia, Dani; Parker, Albert E.; Goeres, Darla M.This paper compares Petrifilm™ aerobic count (AC) plates to drop plating on R2A agar plates as an alternative method for biofilm bacteria enumeration after application of a disinfectant. A Pseudomonas aeruginosa biofilm was grown in a Centers for Disease Control and Prevention biofilm reactor (ASTM E2562) and treated with 123 ppm sodium hypochlorite (as free chlorine) according to the Single Tube Method (ASTM E2871). Aliquots from the same dilution tubes were plated on Petrifilm™ AC plates and drop plated on R2A agar plates. The Petrifilm™ AC and R2A plates were incubated for 48 and 24 h, respectively, at 36 ± 1 °C. After nine experimental runs performed by two technicians, the mean difference in biofilm log densities [log biofilm density (LD) = log10(CFU/cm2)] between the two methods for control coupons, treated coupons, and log reduction (LR) was 0.052 (p = 0.451), −0.102 (p = 0.303), and 0.152 (p = 0.313). Equivalence testing was used to assess equivalence of the two plating methods. The 90 % confidence intervals for the difference in control and treated mean LDs between methods were (−0.065, 0.170) and (−0.270, 0.064), both of which fall within a (−0.5, +0.5) equivalence criterion. The 90 % confidence interval for the mean LR difference (−0.113, 0.420) also falls within this equivalence criterion. Thus, Petrifilm™ AC plates were shown to be statistically equivalent to drop plating on R2A agar for the determination of control LDs, treated LDs, and LR values in an anti-biofilm efficacy test. These are the first published results that establish equivalency to a traditional plate counting technique for biofilms and for a disinfectant assay.