Browsing by Author "Pines, R. M."

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Determining the efficacy of liquid sporicides against spores of Bacillus subtilis on a hard nonporous surface using the quantitative three step method: Collaborative study
(2008-07) Tomasino, S. F.; Pines, R. M.; Cottrill, M. P.; Hamilton, Martin A.
A collaborative study was conducted to validate the quantitative Three Step Method (TSM), a method designed to measure the performance of liquid sporicides on a hard nonporous surface. Ten laboratories agreed to participate in the collaborative study; data from 8 of 10 participating laboratories were used in the final statistical analysis. The TSM uses 5 x 5 x 1 mm glass coupons (carriers) upon which spores have been inoculated and which are introduced into liquid sporicidal agent contained in a microcentrifuge tube. Following exposure to a test chemical and a neutralization agent, spores are removed from carriers in 3 fractions: passive removal (Fraction A), sonication (Fraction B), and gentle agitation (Fraction C). Liquid from each fraction is serially diluted and plated on a recovery medium for spore enumeration. Control counts are compared to the treated counts, and the level of efficacy is determined by calculating the log10 reduction (LR) of spores. The main statistical goals were to evaluate the repeatability and reproducibility of the LR values, to estimate the components of variance for LR, and to assess method responsiveness. AOAC Method 966.04â€“Method II was used as a reference method. The scope of the validation was limited to testing liquid formulations against spores of Bacillus subtilis, a surrogate for virulent strains of B. anthracis, on a hard nonporous surface (glass). The test chemicals used in the study were sodium hypochlorite, a combination of peracetic acid and hydrogen peroxide, and glutaraldehyde. Each test chemical was evaluated at 3 levels of presumed efficacy: high, medium, and low. Three replications were required. The TSM was validated as it successfully met the statistical parameters for quantitative test methods. Satisfactory validation parameters, such as the repeatability standard deviation (Sr) and reproducibility standard deviation (SR), were obtained for control carrier counts and LR values. Both the TSM and the reference method were responsive to the efficacy levels of the test chemicals. For the 72 total TSM tests conducted, the mean (Â± standard error of the mean) log density of spores per control carrier was 6.86 (Â± 0.08); the Sr and SR were low at 0.15 and 0.27, respectively. Across the range of test chemicals, the Sr and SR estimates associated with LR were also acceptably low. The Sr ranged from 0.17 to 0.72 and the SR ranged from 0.34 to 1.43. Overall, the Sr and SR estimates associated with the efficacy data were within the ranges published for other quantitative methods and meet the performance characteristics necessary for validation. Collaborators: Alvey K; Buen M; Chan-Myers H; Chang G; Dellâ€™Aringa B; Gonzales E; Hitchins V; Hollingsworth A; Jeske A; Kingma D; Kitchen nee Dormstetter K; Klein D; Lappalainen S; Lawrence J; Lehman L; Malulla K; Michler T; Paulson D; Regan P; Rodriguez A; Rottjakob D; Sathe M; Steinagel S; Suchmann D; Tester J; To T; Wieland D; Zhang Q
Improving the AOAC use-dilution method by establishing a minimum log density value for test microbes on inoculated carriers
(2009-09) Tomasino, S. F.; Pines, R. M.; Hamilton, Martin A.
The AOAC Use-Dilution methods, 955.14 (Salmonella enterica), 955.15 (Staphylococcus aureus), and 964.02 (Pseudomonas aeruginosa), are used to measure the efficacy of disinfectants on hard inanimate surfaces. The methods do not provide procedures to assess log density of the test microbe on inoculated penicylinders (carrier counts). Without a method to measure and monitor carrier counts, the associated efficacy data may not be reliable and repeatable. This report provides a standardized procedure to address this method deficiency. Based on carrier count data collected by four laboratories over an 8 year period, a minimum log density value is proposed to qualify the test results. Carrier count data were collected concurrently with 242 Use-Dilution tests. The tests were conducted on products bearing claims against P. aeruginosa and S. aureus with and without an organic soil load (OSL) added to the inoculum (as specified on the product label claim). Six carriers were assayed per test for a total of 1452 carriers. All 242 mean log densities were at least 6.0 (geometric mean of 1.0 Â´ 106 CFU/carrier). The mean log densities did not exceed 7.5 (geometric mean of 3.2 Â´ 107 CFU/carrier). For all microbes and OSL treatments, the mean log density (Â± SEM) was 6.7 (Â± 0.07) per carrier (a geometric mean of 5.39 Â´ 106 CFU/carrier). The mean log density for six carriers per test showed good repeatability (0.29) and reproducibility (0.32). A minimum mean log density of 6.0 is proposed as a validity requirement for S. aureus and P. aeruginosa. The minimum level provides for the potential inherent variability that may be experienced by a wide range of laboratories and the slight effect due to the addition of an OSL. A follow-up report is planned to present data to support the carrier count procedure and carrier counts for S. enterica.
Procedural revision to the AOAC germicidal spray products as disinfectants test method: Establishment of minimum and maximum log density values for test microbes on inoculated carriers
(2013-06) Pines, R. M.; Tomasino, S. F.; Cottrill, M. P.; Hamilton, G. C.; Parker, Albert E.
The AOAC Germicidal Spray Products as Disinfectants test method (AOAC Official Method 961.02) is used to measure the efficacy of spray products on hard inanimate surfaces; however, the method does not provide procedures to determine the population of the test microbe on inoculated glass slide carriers (e.g., carrier counts reported as CFU/carrier). Without a method to measure and monitor carrier counts, the associated efficacy data may not be reliable and repeatable. This report provides a standardized procedure to address this issue and, based on carrier count data collected by four laboratories from 2000 to 2010, proposes a specific range for the mean log density per carrier as a requirement. Laboratory-based carrier count data were collected concurrently with 116 Method 961.02 efficacy tests conducted on spray products bearing claims against Pseudomonas aeruginosa and Staphylococcus aureus. For many of the tests a soil load (SL) was added to the inoculum (as specified on the product label claim). Six carriers were assayed per test for a total of 696 carriers. All but two of the 116 mean log densities were at least 5.0 (a geometric mean of 1.0 Ã— 105 CFU/carrier). Across the four combinations of microbes and SL treatments, the mean TestLD (mean log density across all enumerated carriers in a test) ranged from approximately 6.0 (a geometric mean of 0.9 Ã— 106 CFU/carrier) to 6.3 (a geometric mean of 2.0 Ã— 106 CFU/carrier). Across all microbes and SL treatments, the mean log density (Â±SEM) was 6.2 (Â±0.07) per carrier (a geometric mean of 1.5 Ã— 106 CFU/carrier). The mean log density for six carriers per test showed good repeatability (0.32) and reproducibility (0.34). The proposed requirement for S. aureus tests and P. aeruginosa tests is a mean log density (across six carriers) between 5.0 and 6.5. A separate 2009 study at three laboratories was conducted to evaluate the persistence of P. aeruginosa, S. aureus, and Salmonella enterica on glass carriers. Based on the persistence data, a 2 h use period is proposed for using the inoculated carriers post drying. The persistence data set was also used to assess the carrier counts for S. enterica. The carrier counts were approximately one log lower for S. enterica compared to S. aureus and P. aeruginosa; a range of 4.0 to 5.5 logs is proposed as a requirement for S. enterica tests.
Use of Alternative Carrier Materials in AOAC Official Method SM2008.05, Efficacy of Liquid Sporicides Against Spores of Bacillus subtilis on a Hard, Nonporous Surface, Quantitative Three-Step Method
(2010-01) Tomasino, S. F.; Rastogi, Vipin K.; Wallace, Lalena; Smith, Lisa S.; Hamilton, Martin A.; Pines, R. M.
The quantitative Three-Step Method (TSM) for testing the efficacy of liquid sporicides against spores of Bacillus subtilis on a hard, nonporous surface (glass) was adopted as AOAC Official MethodSM 2008.05 in May 2008. The TSM uses 5x5x1 mm coupons (carriers) upon which spores have been inoculated and which are introduced into liquid sporicidal agent contained in a microcentrifuge tube. Following exposure of inoculated carriers and neutralization, spores are removed from carriers in three fractions (gentle washing, fraction A; sonication, fraction B; and gentle agitation, fraction C). Liquid from each fraction is serially diluted and plated on a recovery medium for spore enumeration. The counts are summed over the three fractions to provide the density (viable spores per carrier), which is log10-transformed to arrive at the log density. The log reduction is calculated by subtracting the mean log density for treated carriers from the mean log density for control carriers. This paper presents a single-laboratory investigation conducted to evaluate the applicability of using two porous carrier materials (ceramic tile and untreated pine wood) and one alternative nonporous material (stainless steel). Glass carriers were included in the study as the reference material. Inoculated carriers were evaluated against three commercially available liquid sporicides (sodium hypochlorite, a combination of peracetic acid and hydrogen peroxide, and glutaraldehyde), each at two levels of presumed efficacy (medium and high) to provide data for assessing the responsiveness of the TSM. Three coupons of each material were evaluated across three replications at each level; three replications of a control were required. Even though all carriers were inoculated with approximately the same number of spores, the observed counts of recovered spores were consistently higher for the nonporous carriers. For control carriers, the mean log densities for the four materials ranged from 6.63 for wood to 7.14 for steel. The pairwise differences between mean log densities, except for glass minus steel, were statistically significant (P < 0.001). The repeatability standard deviations (Sr) for the mean control log density per test were similar for the four materials, ranging from 0.08 for wood to 0.13 for tile. Spore recovery from the carrier materials ranged from approximately 20 to 70%: 20% (pine wood), 40% (ceramic tile), 55% (glass), and 70% (steel). Although the percent spore recovery from pine wood was significantly lower than that from other materials, the performance data indicate that the TSM provides a repeatable and responsive test for determining the efficacy of liquid sporicides on both porous and nonporous materials.