Use of Alternative Carrier Materials in AOAC Official MethodSM 2008.05, Efficacy of Liquid Sporicides Against Spores of Bacillus subtilis on a Hard, Nonporous Surface, Quantitative Three-Step Method

Abstract 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 5 5 1 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.

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Modified AOAC Three Step Method (Official Method 2008.05): Consolidation of Fractions B and C

Journal of AOAC International ◽

10.5740/jaoacint.13-031 ◽

2013 ◽

Vol 96 (5) ◽

pp. 947-950

Author(s):

Vipin K Rastogi ◽

Lisa S Smith ◽

Lalena Wallace ◽

Stephen F Tomasino

Keyword(s):

Official Method ◽

Step Method ◽

Log Reduction ◽

Microcentrifuge Tube ◽

Gentle Agitation ◽

Plate Counts ◽

The Mean ◽

Procedural Modification ◽

The Comparative Study ◽

Subsequent Neutralization

Abstract The AOAC Quantitative Three Step Method (TSM; AOAC Official MethodSM 2008.05) is validated for testing the efficacy of liquid sporicides against spores of Bacillus subtilis and Bacillus anthracis on selected hard, nonporous, and porous surfaces. The TSM uses 5 × 5 × 1 mm inoculated coupons (carriers), which are placed in 400 μL liquid sporicidal agent contained in a microcentrifuge tube. Following exposure of inoculated carriers to the test chemical and subsequent neutralization, viable spores are recovered in three fractions: A (gentle tapping), B (sonication), and C (gentle agitation). The spores in suspension are serially diluted and plated on a recovery medium for enumeration. The plate counts are summed over the three fractions to provide the number of viable spores per carrier, which is log10-transformed to generate a mean log density (LD) value across carriers. As a measure of product efficacy, a log reduction (LR) value is calculated by subtracting the mean LD for treated carriers from the mean LD for control carriers. This paper reports on the comparative evaluation of the current and modified versions of the TSM in order to support a modification to simplify the procedure. The proposed modified TSM (mTSM) consolidates fractions B and C in the same tube. Thus, the sonication (fraction B) and gentle agitation (fraction C) steps are carried out in the same tube, thereby reducing the number of tubes and associated resources and time necessary to complete the test. Glass, steel, pine wood, and ceramic tile carriers were included in the comparative study. Inoculated carriers were evaluated against two preparations of sodium hypochlorite to generate two presumed levels of efficacy (intermediate and high); the control LD and LR values associated with testing each carrier type for the TSM and the mTSM were compared. For control carriers, the mean log densities per carrier (for each carrier material) were not significantly different based on the TSM compared to the mTSM. Furthermore, the treated carrier data showed comparable LR values for the TSM and mTSM. The data provided in this report demonstrate equivalency between the TSM and mTSM and support the proposed procedural modification to consolidate fractions B and C.

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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

Journal of AOAC International ◽

10.1093/jaoac/91.4.833 ◽

2008 ◽

Vol 91 (4) ◽

pp. 833-852 ◽

Cited By ~ 15

Author(s):

Stephen F Tomasino ◽

Rebecca M Pines ◽

Michele P Cottrill ◽

Martin A Hamilton ◽

K Alvey ◽

...

Keyword(s):

Bacillus Subtilis ◽

Standard Deviation ◽

Quantitative Methods ◽

Collaborative Study ◽

Reference Method ◽

Step Method ◽

Components Of Variance ◽

Test Chemical ◽

Validation Parameters ◽

The Mean

Abstract 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 5 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 966.04Method 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 ( standard error of the mean) log density of spores per control carrier was 6.86 ( 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 rangedfrom 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.

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Report of Ad Hoc Committee on Vitamin D Methodology, AOAC

Journal of AOAC INTERNATIONAL ◽

10.1093/jaoac/58.2.330 ◽

1975 ◽

Vol 58 (2) ◽

pp. 330-332

Author(s):

Forrest W Quackenbush ◽

Daniel Banes ◽

Philip H Derse

Keyword(s):

Maleic Anhydride ◽

Chemical Method ◽

Ad Hoc ◽

Collaborative Study ◽

Mean Values ◽

Biological Tests ◽

Aoac Official ◽

The Mean ◽

Biological Methods ◽

Good Agreement

Abstract In a collaborative study 6 samples of vitamin D3 resins were analyzed chemically by 9 laboratories and tested biologically by 3 laboratories to determine whether there exists a discrepancy between chemical and biological methods of measuring potency of these materials. The chemical methods employed saponification with and without subsequent maleic anhydride treatment. The biological method was the AOAC official final action rat assay, 39.149–39.162. The results showed good agreement between laboratories in the chemical analysis and relatively poor agreement in the biological tests. However, based upon the mean values as reported by the different laboratories, the chemical method employing saponification alone consistently overestimated biopotency, whereas the chemical method including maleic anhydride treatment gave results which averaged much closer to the mean biopotency values. There was no evidence that maleic anhydride treatment reduced values significantly below biopotency values.

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The Validation of the Dai Nippon Medi·Ca SA Method for AOAC Research Institute Performance Tested MethodsSM Certification

Journal of AOAC International ◽

10.5740/jaoacint.17-0487 ◽

2018 ◽

Vol 101 (5) ◽

pp. 1522-1533

Author(s):

Mai Shimizu ◽

Kentaro Takenaka ◽

Takeo Suzuki ◽

Aya Miyasaka ◽

Taiki Matsuda ◽

...

Keyword(s):

Reference Method ◽

Sample Volume ◽

Contamination Level ◽

Stability Studies ◽

Cooked Ham ◽

Aoac Official ◽

The Mean ◽

Food Matrices ◽

Mean Differences ◽

Medium Method

Abstract A ready-made dry medium method for Staphylococcus aureus count, the Medi·Ca SA method incubated at 35 or 37°C, was compared with the Baird-Parker method (AOAC Official MethodSM975.55) for 11 food matrices: raw beef, raw ground beef, raw lamb, cooked ham, raw salmon, frozen prawn, fresh chilled pasta, pasteurized milk, natural cheese, cream puff, and potato salad. The mean difference between the two methods at each contamination level for each matrix was <0.5 log10, and the 95% confidence intervals on the mean differences fell within the range of −0.50 to 0.50. Standard deviation of repeatability and RSDr values of the Medi·Ca SA method were generally the same level as those of the Baird-Parker method, and r2 ranged from 0.98 to 1.00. Product consistency and stability studies showed little variability between productions lots and a shelf-life of 16 months. Incubation time within the range of 22–26 h and variations to the sample volume did not adversely affect the results. These results showed that the Medi·Ca SA method is a reasonable alternative to the reference method for selected food matrices and makes it possible to simultaneously detect and enumerate S. aureus in only 24�h.

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Brechung und Reflexion schneller Elektronen durch dünne Folien

Zeitschrift für Naturforschung A ◽

10.1515/zna-1968-1215 ◽

1968 ◽

Vol 23 (12) ◽

pp. 1988-1994

Author(s):

H. Zeidl ◽

H. Baier

Keyword(s):

Energy Loss ◽

Secondary Electron Emission ◽

Experimental Methods ◽

Relativistic Electrons ◽

Angle Of Incidence ◽

Step Method ◽

Computational Program ◽

Thin Foils ◽

The Monte Carlo Method ◽

The Mean

The Monte Carlo method is applied to investigate the penetration of fast electron through thin foils of matter. The “step by step method” is used. Energy loss and secondary electron emission are taken into account. As an example for the possible application of the computational program we calculated “refraction” and “reflection” of relativistic electrons on thin Al-foils. Scattering and reflection depends significantly on the energy loss of the electrons in the foil. The “mean scattering angle” of the electron beam (with respect to the foil normal) has been shown to be smaller than the angle of incidence (with respect to the foil normal). Possible experimental methods to test the predictions on mean scattering and reflection angles (as defined in this paper) are discussed.

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Disinfection efficacy studies on three different disinfection methods in health care facilities by ATP method

BCIT Environmental Public Health Journal ◽

10.47339/ephj.2015.118 ◽

2015 ◽

Author(s):

Harry Zhuang ◽

BCIT School of Health Sciences, Environmental Health ◽

Helen Heacock ◽

Fred Shaw

Keyword(s):

Health Care ◽

Medical Equipment ◽

Health Care Facilities ◽

Relative Light Unit ◽

Monitoring Methods ◽

Log Reduction ◽

Disinfection Methods ◽

Care Facilities ◽

The Mean ◽

High Level

Objectives: Nosocomial infection has always been a significant topic in the field of public health. The disinfection procedures involved in health care facilities are extremely important to prevent potential transmission of diseases. Therefore, this study was performed to compare the disinfection efficacy between three different disinfection methods (Accel wipes, Hubscrub industrial washer, and Steam vapor) on three pieces of non-critical medical equipment: wheelchairs, mattresses and bath chairs. Methods: The method used to evaluate the disinfection efficacy compared the reduction of contaminants count in the relative light unit using ATP monitoring methods. 30 samples of each of the three types of medical equipment were swabbed pre-disinfection and post-disinfection using the three disinfection methods. The recorded reduction number was then converted using log transformation. Statistical analysis was conducted using NCSS to assess differences between the disinfection methods. Results: The mean log-reduction of disinfection for Accel wipes, Hubscrub, and steam vapor were 1.067, 1.490, and 1.485 respectively. Steam vapor and Hubscrub displayed statistically significantly better disinfection efficacy compared to Accel wipes in terms of log reduction (overall p=0.000002). Conclusion: Hubscrub and steam vapor are better disinfectants compared to Accel wipes in terms of mean log reduction values; however, all three disinfection methods demonstrated effectiveness when cleaning and disinfecting non-critical medical equipment. For critical medical equipment, steam vapor and Hubscrub industrial washing are effective while Accel wipes do not meet the standards of high-level disinfection. As a result, combination usages of all three disinfection methods are recommended at health care facilities based on the categories of the medical equipment.

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Simple, Rapid and on Spot Dye-based Sensor for the Detection of Vibrio Load in Shrimp Culture Farms

10.21203/rs.3.rs-265744/v1 ◽

2021 ◽

Author(s):

Sweta Binod Kumar ◽

Ambika Hemant Shinde ◽

Maheshawari Jagadishbhai Behere ◽

Dhruvi Italiya ◽

Soumya Haldar

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Water Sample ◽

Pond Water ◽

Negative Control ◽

Second Step ◽

Step Method ◽

Shrimp Culture ◽

Aquaculture Pond ◽

Aquaculture Ponds

Abstract For the detection of Vibrio bacteria, a kit involving two-step method was developed. In the in first step a specific media was added in the water sample which selectively promote the growth of vibrios and inhibit the growth of other bacteria. The second step involved addition of dye-based sensor (already developed in our previous work) in the sample which detect the active Vibrio and changed the colour of the sample to red/pink. The vibrio detection kit was optimized on five different species of Vibrio (V. cholerae and V. parahaemolyticus, V. campbellii, V. harveyi & V. proteolyticus) and two negative control bacteria (Escherichia coli and Bacillus subtilis). The kit was further evaluated on aquaculture pond water and probiotics used in aquaculture farms. It successfully estimated Vibrio concentration of all the five strains and in aquaculture ponds. The negative control bacteria and probiotics were not sensed by the kit. Hence, the kit developed here is perfect for the detection of Vibrio, especially in aquaculture farms.

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Sporicidal Activity of Chemical Germicides on Bacillus subtilis Spores Under Various Conditions

Journal of AOAC International ◽

10.1093/jaoac/77.1.53 ◽

1994 ◽

Vol 77 (1) ◽

pp. 53-57 ◽

Cited By ~ 1

Author(s):

James W Danielson ◽

James T Peeler ◽

Gordon S Oxborrow

Keyword(s):

Bacillus Subtilis ◽

Chlorine Dioxide ◽

Peracetic Acid ◽

Bovine Serum ◽

Hard Water ◽

Soft Water ◽

Initial Concentration ◽

Log Reduction ◽

Time Required ◽

Intended Use

Abstract Sporicidal tests were conducted on chemical germicides used for reprocessing hemodialyzers. The germicides that were tested for sporicidal activity contain some of the same active ingredients as many other commercial germicides, although instructions for dilution and exposure time may vary according to intended use. Cidex-Dialyzer™ (glu-taraldehyde), formaldehyde, Renalin™ (peracetic acid), and RenNew-D™ (chlorine dioxide) were each tested under the following conditions: at 50, 100, and 150% of the recommended concentration in soft water at 22°C; and at 1× the recommended concentration in soft water at 26.7°C, in hard water at 22°C, and in hard water containing 5% bovine serum at 22°C. Sporicidin-HD™ (glutaraldehyde–phenol) was tested only at the recommended concentration in soft water at 22°C. The time required for a 2-log reduction in the initial concentration of spores ranged from 1.8 min for Renalin at 150% of the recommended concentration to 26.4 h for formaldehyde in hard water containing 5% bovine serum. Cidex-Dialyzer at 50% of the recommended concentration and Sporicidin-HD at the recommended concentration were ineffective against Bacillus subtilis var. niger spores. The sporicidal activities of RenNew-D and formaldehyde were greatly reduced when bovine serum was added to hard water. Renalin had the highest sporicidal activity and was the least affected by each of the conditions tested.

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Bacillus stearothermophilus Disk Assay for Determining Ampicillin Residues in Fish Muscle

Journal of AOAC INTERNATIONAL ◽

10.1093/jaoac/74.6.910 ◽

1991 ◽

Vol 74 (6) ◽

pp. 910-912 ◽

Cited By ~ 1

Author(s):

Steven M Plakas ◽

Angelo DePaola ◽

Michael B Mox

Keyword(s):

Striped Bass ◽

Bacillus Stearothermophilus ◽

Fish Muscle ◽

Control Fish ◽

Official Method ◽

Limit Of Determination ◽

Cultured Fish ◽

Aoac Official ◽

The Mean

Abstract The Bacillus stearothermophilus disk assay for penicillin in milk (AOAC official method) was adapted for the determination of ampicillin in fish muscle. The method was evaluated in 2 species of cultured fish: channel catfish and striped bass. Recoveries of ampicillin ranged from 99 to 104% when muscle specimens from both species were spiked at concentrations of 0.025-1.00 μg/g. The lower limit of determination (LOD) was 0.025 μg/g. The assay was applied to monitor the elimination of ampicillin from the muscle of striped bass after intravascular administration (dosage of 10 mg/kg body weight). The mean concentrations in the muscle declined from 1.160 μg/g at 2 h to 0.063 μg/g at 18 h. The half-life of ampicillin in the muscle was 3.6 h. Ampicillin concentrations were below LOD at 24 h. No inhibitory activity was observed in the muscle of control fish.

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An Oral Formulation of the Probiotic, Bacillus subtilis HU58, Was Safe and Well Tolerated in a Pilot Study of Patients with Hepatic Encephalopathy

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2020/1463108 ◽

2020 ◽

Vol 2020 ◽

pp. 1-7

Author(s):

Sayed Yossef ◽

Frances Clark ◽

Sarah S. Bubeck ◽

John Abernethy ◽

Thomas Bayne ◽

...

Keyword(s):

Bacillus Subtilis ◽

Hepatic Encephalopathy ◽

Pilot Study ◽

Oral Formulation ◽

Ammonia Level ◽

Blood Ammonia ◽

Probiotic Group ◽

Blood Ammonia Level ◽

Study Product ◽

The Mean

Background. Hepatic encephalopathy often results in high blood ammonia levels because of inefficient ammonia processing by the liver. Lactulose treatment promotes the growth of urease-producing gut bacteria and a reduced colon pH, thus reducing blood ammonia absorption. It is thought that probiotics as an add-on therapy may be beneficial. Patients and Methods. Bacillus subtilis HU58 was tested for safety and tolerability in patients with hepatic encephalopathy taking lactulose in this double-bind, placebo-controlled, 4-week pilot study. Study participants received one dose of B. subtilis HU58 or placebo (orally) for the first five days and two daily doses thereafter. Participants were monitored for safety and blood ammonia levels. Results. Forty patients participated (placebo, 11; probiotic, 29). Baseline characteristics were generally comparable; the mean baseline blood ammonia level was somewhat higher in the probiotic group. Mild or moderate treatment-emergent adverse events (TEAEs) were reported in 27.3% and 17.2% of patients in the placebo and probiotic groups, respectively; no severe TEAEs were reported. One patient (9.1%) taking placebo and two (6.9%) taking the probiotic experienced serious TEAEs (SAEs); none resulted in study discontinuation and all were considered to have no/unlikely relationship to the study product. There were no significant differences in the mean percent change (MPC) of blood ammonia levels between groups, though the probiotic group exhibited a trend toward a milder increase. Stratification of the probiotic group by baseline blood ammonia level (>60 μg/dL and ≤60 μg/dL) resulted in a significantly reduced MPC in the >60 μg/dL subgroup (MPC (SD); ≤60 μg/dL (n = 14), 35.3% (73.3); >60 μg/dL (n = 14), −26.5% (24.4); p=0.0087). Conclusions. Daily treatment with oral B. subtilis HU58 was safe and well tolerated over a 4-week period in patients with hepatic encephalopathy, and a significantly reduced MPC of blood ammonia level was observed in patients with a baseline level >60 µg/dL.

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