Browsing by Author "Fritz, Blaine"

Now showing 1 - 3 of 3

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.
Evaluation of 3M Petrifilm as an equivalent alternative to drop-plating on agar plates in a biofilm system
(2013-03) Fritz, Blaine; Goeres, Darla
This project evaluated 3M Petrifilm as an alternative, more efficient method for bacterial enumeration. Using Petrifilm allows the researcher to avoid preparing agar plates for bacterial enumeration. Currently, the majority of scientific literature concerning enumeration of bacteria on Petrifilm is from the food industry. There are no published studies examining the use of Petrifilm for enumeration of biofilm bacteria. A Pseudomonas aeruginosa biofilm was grown in a CDC reactor according to ASTM Method E2562. The mature biofilm was exposed to chlorine (buffered water for controls) and neutralized. The biofilm was removed from the surface, disaggregated, and serially diluted. Samples from the dilution tubes were plated in duplicate on Petrifilm Aerobic Count plates and drop plated on R2A plates. The Petrifilm and R2A plates were incubated at 36oC and colonies enumerated after 24 and 48 hours. The experiment was replicated three times by two technicians. The time required for both plating methods was recorded to help assess the efficiency of both methods. The results from this study may demonstrate that Petrifilm could replace drop plating as a more efficient and cost effective method for bacterial enumeration.
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.