The scope for using the volatile profiles of Pinus caribaea var. bahamensis as indicators of susceptibility to pine tortoise scale and as predictors of environmental stresses.

Climate change, unseasonal fire and urbanization are contributing to the decline of Pinus caribaea var. bahamensis populations in the Turks and Caicos Islands (TCI). Infestation of pines with the invasive pine tortoise scale (PTS, Toumeyella parvicornis) is accelerating this decline. Pine trees in the Bahamas are larger and healthier and are not infested with PTS although they are subject to some of the same environmental pressures as the trees in TCI. Volatile compounds were collected from wild and nursery-reared P. caribaea var. bahamensis from TCI and the Bahamas and characterized using GC/MS analysis, to look for differences between the compounds detected in insect-infested pines of TCI and the healthy pines of the Bahamas. Ten compounds contributing at least 1% of the total detected peak areas in any one of the samples were selected for further study. Eight of these compounds were identified using authentic standards and mass spectral libraries. The main constituents in the samples were α- and ß-pinene as well as ß-phellandrene, and, together with ß-myrcene, their contents varied the most between samples collected at different locations. Principal-component analysis showed that the two structural isomers of pinene, together with ß-myrcene and ß-phellandrene, contributed 98.4% of the variance between samples. There was a positive relationship between the concentrations of the two structural isomers of pinene and between levels of ß-myrcene and ß-phellandrene. The results are discussed in relation to the biology and adaptations of invasive scale insects, the importance of monoterpenes in pine as a defense against insect predation, whether these compounds can be used as indicators of tree health, and future directions for research into conserving the Caicos pine.

A statistical model for assessing performance standards for quantitative and semiquantitative disinfectant test methods.

A performance standard for a disinfectant test method can be evaluated by quantifying the (Type I) pass-error rate for ineffective products and the (Type II) fail-error rate for highly effective products. This paper shows how to calculate these error rates for test methods where the log reduction in a microbial population is used as a measure of antimicrobial efficacy. The calculations can be used to assess performance standards that may require multiple tests of multiple microbes at multiple laboratories. Notably, the error rates account for among-laboratory variance of the log reductions estimated from a multilaboratory data set and the correlation among tests of different microbes conducted in the same laboratory. Performance standards that require that a disinfectant product pass all tests or multiple tests on average, are considered. The proposed statistical methodology is flexible and allows for a different acceptable outcome for each microbe tested, since, for example, variability may be different for different microbes. The approach can also be applied to semiquantitative methods for which product efficacy is reported as the number of positive carriers out of a treated set and the density of the microbes on control carriers is quantified, thereby allowing a log reduction to be calculated. Therefore, using the approach described in this paper, the error rates can also be calculated for semiquantitative method performance standards specified solely in terms of the maximum allowable number of positive carriers per test. The calculations are demonstrated in a case study of the current performance standard for the semiquantitative AOAC Use-Dilution Methods for Pseudomonas aeruginosa (964.02) and Staphylococcus aureus (955.15), which allow up to one positive carrier out of a set of 60 inoculated and treated carriers in each test. A simulation study was also conducted to verify the validity of the model's assumptions and accuracy. Our approach, easily implemented using the computer code provided, offers a quantitative decision-making tool for assessing a performance standard for any disinfectant test method for which log reductions can be calculated.

Use of statistical modeling to reassess the performance standard for the AOAC use-dilution methods (955.15 and 964.02).

The AOAC Use-dilution methods (UDM) 955.15 (Staphylococcus aureus) and 964.02 (Pseudomonas aeruginosa) are laboratory methods used to substantiate antimicrobial efficacy claims for liquid disinfectants on inanimate surfaces. The UDM is accepted by the U.S. Environmental Protection Agency for the purpose of product registration. To attain a hospital-level claim, testing against S. aureus and P. aeruginosa is required, and the product must pass against both microbes. Currently, the UDM's performance standard for a single 60-carrier test is the same for both microbes, and allows up to one positive carrier for the product to be considered as a pass. In this paper, the performance standards for these methods are reassessed using data from a 2009 five-laboratory collaborative study and a recently published statistical model. The reassessment focuses on the pass-error rate for ineffective products and the fail-error rate for highly effective products. The calculations indicate that the pass-error rate is between 9 and 24% and the fail-error rate between 18 and 23% when the current performance standard is used for a single test. For product registration, a smaller pass-error rate (1%) historically has been maintained by requiring that a disinfectant pass three UDM tests for each of the two microbes; however, the calculations also indicate that the fail-error rate is between 42 and 45%. This large fail-error rate is a compelling reason to consider a new performance standard for the two UDM methods, 955.15 (S. aureus) and 964.02 (P. aeruginosa). One alternative performance standard allows no more than six positive carriers out of 60 as a pass when using P. aeruginosa and no more than three positive carriers out of 60 when using S. aureus. In addition, the new performance standard requires that three UDM tests be performed with each of the two microbes, and the disinfectant must pass all six tests to be considered efficacious. The statistical calculations for this alternative performance standard indicate that the pass-error rate is no more than 3%, while the fail-error rate is as small as 5%. Based on these error rate calculations, proposed revisions to the performance standards for AOAC Methods 955.15 and 964.02 are provided.

Guidelines for the statistical analysis of a collaborative study of a laboratory method for testing disinfectant product performance.

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.

Calculating the limit of detection for a dilution series.

AIMS: Microbial samples are often serially diluted to estimate the number of microbes in a sample, whether as colony-forming units of bacteria or algae, plaque forming units of viruses, or cells under a microscope. There are at least three possible definitions for the limit of detection (LOD) for dilution series counts in microbiology. The statistical definition that we explore is that the LOD is the number of microbes in a sample that can be detected with high probability (commonly 0.95). METHODS AND RESULTS: Our approach extends results from the field of chemistry using the negative binomial distribution that overcomes the simplistic assumption that counts are Poisson. The LOD is a function of statistical power (one minus the rate of false negatives), the amount of overdispersion compared to Poisson counts, the lowest countable dilution, the volume plated, and the number of independent samples. We illustrate our methods using a data set from Pseudomonas aeruginosa biofilms. CONCLUSIONS: The techniques presented here can be applied to determine the LOD for any counting process in any field of science whenever only zero counts are observed. SIGNIFICANCE AND IMPACT OF STUDY: We define the LOD when counting microbes from dilution experiments. The practical and accessible calculation of the LOD will allow for a more confident accounting of how many microbes can be detected in a sample.

Performance of the AOAC use-dilution method with targeted modifications: collaborative study.

The U.S. Environmental Protection Agency (EPA), in collaboration with an industry work group, spearheaded a collaborative study designed to further enhance the AOAC use-dilution method (UDM). Based on feedback from laboratories that routinely conduct the UDM, improvements to the test culture preparation steps were prioritized. A set of modifications, largely based on culturing the test microbes on agar as specified in the AOAC hard surface carrier test method, were evaluated in a five-laboratory trial. The modifications targeted the preparation of the Pseudomonas aeruginosa test culture due to the difficulty in separating the pellicle from the broth in the current UDM. The proposed modifications (i.e., the modified UDM) were compared to the current UDM methodology for P. aeruginosa and Staphylococcus aureus. Salmonella choleraesuis was not included in the study. The goal was to determine if the modifications reduced method variability. Three efficacy response variables were statistically analyzed: the number of positive carriers, the log reduction, and the pass/fail outcome. The scope of the collaborative study was limited to testing one liquid disinfectant (an EPA-registered quaternary ammonium product) at two levels of presumed product efficacies, high and low. Test conditions included use of 400 ppm hard water as the product diluent and a 5% organic soil load (horse serum) added to the inoculum. Unfortunately, the study failed to support the adoption of the major modification (use of an agar-based approach to grow the test cultures) based on an analysis of method's variability. The repeatability and reproducibility standard deviations for the modified method were equal to or greater than those for the current method across the various test variables. However, the authors propose retaining the frozen stock preparation step of the modified method, and based on the statistical equivalency of the control log densities, support its adoption as a procedural change to the current UDM. The current UDM displayed acceptable responsiveness to changes in product efficacy; acceptable repeatability across multiple tests in each laboratory for the control counts and log reductions; and acceptable reproducibility across multiple laboratories for the control log density values and log reductions. Although the data do not support the adoption of all modifications, the UDM collaborative study data are valuable for assessing sources of method variability and a reassessment of the performance standard for the UDM.

Conservation status of native plant hybrids in the British Virgin Islands.

BACKGROUND: Hybridization is an evolutionary event present in the natural world. Several studies suggest that natural hybridization is an important process in plant evolution, creating new genetic combinations which can play a vital role in speciation (Soltis and Soltis 2009, Soltis 2013, Neri et al. 2017, Taylor and Larson 2019). Therefore, it is important to understand and protect naturally occurring hybrids, conserving their ecological novelties and new traits, such as the ability to explore new niches, different from those of the parental species (Soltis 2013, Supple and Shapiro 2018).The British Virgin Islands (BVI) is a UK Overseas Territory situated in the Caribbean biodiversity hotspot (Myers et al. 2000). To date, three natural hybrids are known to occur within this territory: Tillandsia × lineatispica Mez, Anthurium × selloanum K.Koch and Coccoloba krugii × C. uvifera R.A.Howard (Howard 1957, Acevedo-Rodriguez and Strong 2005, Acevedo-Rodriguez and Strong 2012).Tillandsia × lineatispica is endemic to the Puerto Rican Bank, occurring in Puerto Rico, the US Virgin Islands (USVI) and the British Virgin Islands with an extent of occurrence estimated to be 3,390 km2 and a limited number of locations. The suitable habitat for this hybrid is declining mainly due to the negative impacts of feral ungulates, development for tourism and residential infrastructure and the impact of human-induced wildfires. In addition, it is suspected that the global population does not exceed 10,000 individuals with the largest subpopulation on Beef Island in the BVI thought to have no more than 1,000 mature individuals. This hybrid is therefore evaluated as Vulnerable, based on IUCN Red List Criteria, B1a(iii)+2b(iii) + C2a(i).Anthurium × selloanum is an endemic hybrid to BVI and USVI with a very restricted extent of occurrence which was estimated to range between 103 km2 and 207 km2 and an area of occupancy which was estimated to range between 56 km2 and 188 km2 and a limited number of locations. The suitable habitat of this species is declining mainly due to the negative impacts of feral ungulates, development for tourism and residential infrastructure and the negative impact of recreation activities in protected areas. This species is therefore evaluated as Endangered, based on IUCN Red List Criteria B1a+ b(iii) + B2a+b(iii).Coccoloba krugii × C. uvifera is native to the BVI, USVI, Puerto Rico, Dominican Republic, Haiti and Anguilla. It is estimated to have an extent of occurrence of 89,412 km2. This value exceeds the threshold for any threatened category. Despite an observed continuing decline of suitable habitat for this species, which is being degraded mainly through ongoing development pressures, this species occurs in more than 10 locations. It is therefore assessed as Least Concern (LC). NEW INFORMATION: In this paper, we discuss the conservation status of all the known, naturally occurring, native hybrids in the the British Virgin Islands and we provide distribution data, including new records, from across these hybrid species ranges. Although conservation assessments of hybrids are out of the scope of the published IUCN Red List of Threatened Species (IUCN Standards and Petitions Committee 2019), we use the IUCN Red List Criteria and Categories (version 3.1) to establish an equivalent conservation status of these hybrids and discuss conservation action due to the potential evolutionary importance of these naturally occurring hybrids. These assessments provide the necessary baseline information for prioritising species conservation and making informed management decisions, such as establishing the BVI's Tropical Important Plant Areas (TIPAS) network (Sanchez et al. 2019).

Reassessment of Varronia bellonis - a threatened, endemic plant from Puerto Rico.

BACKGROUND: Varronia bellonis (Urb.) Britton is a lianescent or recumbent shrub that is endemic to Puerto Rico where it is restricted to specific geology types with a limited extent on the western half of the Island. The species occurs on serpentinite geology covered by serpentine-derived soils in the west-central mountains and on limestone geology in the the northern karst region. The species area of occupancy is estimated to range between 108 km2 and 268 km2 and its extent of occurrence to be between 644 km2 and 852 km2. The number of locations are estimated to be four. There are 418 known mature individuals in the wild (Hamilton 2020a). The species was previously assessed as Critically Endangered (Linsky and Sustache 2014), based on available information. However, an international team have been collaborating to conserve the species and, based on new information derived from this work, the species is reassessed as Endangered (EN), based on Criteria B1ab(i,ii,iii,iv,v)+2ab(i,ii,iii,iv,v), according to the IUCN Red List Categories and Criteria (version 3.1) and guidelines (IUCN Standards and Petitions Committee 2019). NEW INFORMATION: Areas of suitable habitat across the native range of the threatened plant, V. bellonis, were surveyed by a team of experts between 2016 and 2019 to determine the species habitat preferences, identify threats to the species survival and provide an up-to-date meta-population status. The new information enabled members of the international team to reassess the species status and will enable sound and scientifically-based recovery actions to be recommended that can secure Varronia bellonis populations for the future. Parallel efforts are ongoing to explore the species population genetics and reproductive biology.

Range extension and conservation status of the rare Solanaceae shrub, Solanum conocarpum.

BACKGROUND: The British Virgin Islands and the US Virgin Islands, two island groups located in the Caribbean archipelago, hold unique plant diversity and high endemism. Until recently, Solanum conocarpum was considered a rare plant species endemic to the island of St. John in the US Virgin Islands. Ongoing botanical surveys in this region are revealing new populations and refining our understanding of the distribution of these narrow endemic plant species. The objective of this paper is to assess the conservation status of S. conocarpum, including a review of its geographic range, population numbers, threats and conservation actions needed for its long-term survival. NEW INFORMATION: In this paper, we present new occurrences for S. conocarpum, extending its geographic range to a new island, Tortola and new territory, the British Virgin Islands. Despite this range expansion, this species is evaluated as Endangered (EN), based on Criteria B1b(iii,v)+2b(iii,v)+C2a(i), according to the IUCN Red List Categories and Criteria. The extent of occurrence (EOO = 46 km2) and area of occupancy (AOO = 20 km2) are highly restricted. On St. John (US Virgin Islands), the historically recorded individuals at Reef Bay, Europa Ridge and Sabbat Point are now considered extirpated due to disturbance from development compounded by invasive species, as well as the impact of feral ungulates and drought stress. These threats are impacting the species across the whole island of St. John and contributing to a continuing decline of suitable habitat, despite the island being a National Park. On the island of Tortola, the species occurs on unprotected lands subject to development and habitat modification and decline by feral ungulates. Based on these threats acting separately across the two islands, two locations were defined. The estimated total number of mature individuals ranges between 150 and 250, with the largest subpopulation at Nanny Point in the US Virgin Islands, containing 108 mature individuals. Conservation action, focused on protecting this species' habitat, is urgently needed.

Use of alternative carrier materials in AOAC Official Method 2008.05, efficacy of liquid sporicides against spores of Bacillus subtilis on a hard, nonporous surface, quantitative three-step method.

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 Method 2008.05 in May 2008. The TSM uses 5 x 5 x 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.

New island record and conservation status of Puerto Rican Bank endemic plant species, Ruehssia woodburyana (Acev.-Rodr.) Goyder, comb. nov., formally transferred from Marsdenia.

BACKGROUND: Thought to be endemic to the Commonwealth of Puerto Rico, Ruehssia woodburyana (Apocynaceae) was recently discovered at a single location on Norman Island in the British Virgin Islands. Despite an increase in the extent of occurrence and area of occupancy, this species meta-population is very limited with a total of 37 individuals known in the wild. The largest subpopulation, on Mona Island, has only 26 individuals. The species suitable habitat is experiencing a continuing decline due to urban development, grazing by feral ungulates and human-induced forest fires. Conservation action is urgently needed and should be directed towards establishing genetically representative ex situ collections, such as seed for long term storage and live material for propagation. This species is evaluated as Critically Endangered (CR), based on Criteria C2a(i)+D, according to the IUCN Red List Categories and Criteria (version 3.1) and guidelines (Subcommittee IUCN Standards and Petitions 2016). NEW INFORMATION: Extensive and regular surveys to the region enable the discovery of new plant records for different countries and islands. In this paper, we record a new island record for Ruehssia woodburyana on Norman Island, in the British Virgin Islands and discuss the species conservation status. Marsdenia woodburyana is transferred to the genus Ruehssia to reflect the resurrection of that genus for species of Marsdenia native to the New World.

Checking the validity of the harvesting and disaggregating steps in laboratory tests of surface disinfectants.

A chemical disinfectant against surface-associated bacteria typically uses carriers (e.g., glass disks) that are purposely contaminated with bacteria prior to disinfection. After disinfection, the bacteria are harvested by mechanically separating them from the carrier surface to form a suspension of cells in a dilution tube. Bacterial clumps in the tube are disaggregated using mechanical or chemical techniques, thereby creating a well-mixed suspension of single cells suitable for enumeration. Efficacy is quantified by comparing the viable cell count for a disinfected carrier to the viable cell count for sham-disinfected (control) carrier. A test is said to be biased (invalid) if the observed efficacy measure is systematically higher or lower than the true efficacy. It is shown here for the first time that the bias attributable to the harvesting and disaggregating steps of a disinfectant test can be measured. For some conventional biofilm harvesting and disaggregating techniques, laboratory checks showed either negligible bias or important bias, depending on the disinfectant. Quantitative bias checks on the harvesting and disaggregating steps are prudent for each combination of carrier material, microorganism, and disinfectant. The quantitative results should be augmented by microscopic examination of harvested disinfected and control carriers and of the disaggregated suspensions.

Improving the AOAC use-dilution method by establishing a minimum log density value for test microbes on inoculated carriers.

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 x 10(6) CFU/carrier). The mean log densities did not exceed 7.5 (geometric mean of 3.2 x 10(7) CFU/carrier). For all microbes and OSL treatments, the mean log density (+/- SEM) was 6.7 (+/- 0.07) per carrier (a geometric mean of 5.39 x 10(6) 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.

Determining the efficacy of liquid sporicides against spores of Bacillus subtilis on a hard nonporous surface using the quantitative three step method: collaborative study.

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 966.04-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 (+/- 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 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.

Reproducibility of antimicrobial test methods.

We review reproducibility results for methods that test antimicrobial efficacy against biofilms, spores and bacteria dried onto a surface. Our review, that included test results for Pseudomonas aeruginosa, Salmonella choleraesuis and Bacillus subtilis, suggests that the level of reproducibility depends on the efficacy of the antimicrobial agent being tested for each microbe and microbial environment. To determine the reproducibility of a method, several laboratories must independently test the same antimicrobial agent using the method. Little variability among the efficacy results suggests good reproducibility. Such reproducibility assessments currently are hampered by the absence of an objective process for deciding whether the variability is sufficiently small. We present a quantitative decision process that objectively determines whether any method that assesses antimicrobial efficacy is reproducible. Because the perception of acceptable reproducibility may differ among stakeholders, the decision process is governed by a stakeholder's specifications that necessarily includes the efficacy of the agents to be tested.

Comparative evaluation of biofilm disinfectant efficacy tests.

Regulatory agencies are receiving registration applications for unprecedented, antibiofilm label claims for disinfectants. Reliable, practical, and relevant laboratory biofilm test methods are required to support such claims. This investigation describes the influence of fluid dynamics on the relevancy of a laboratory test. Several disinfectant formulations were tested using three different biofilm testing systems run side-by-side: the CDC biofilm reactor system that created turbulent flow (Reynolds number between 800 and 1850), the drip flow biofilm reactor system that created slow laminar flow (Reynolds number between 12 and 20), and the static biofilm system that involved no fluid flow. Each comparative experiment also included a dried surface carrier test and a dried biofilm test. All five disinfectant tests used glass coupons and followed the same steps for treatment, neutralization, viable cell counting, and calculating the log reduction (LR). Three different disinfectants, chlorine, a quaternary ammonium compound, and a phenolic, were each applied at two concentrations. Experiments were conducted separately with Pseudomonas aeruginosa and Staphylococcus aureus and every experiment was independently repeated. The results showed that biofilm grown in the CDC reactor produced the smallest LR, the static biofilm produced the largest LR, and biofilm grown in the drip flow reactor produced an intermediate LR. The differences were large enough to be of practical importance. The dried surface test often produced a significantly higher LR than the tests against hydrated or dried biofilm. The dried biofilm test produced LR values similar to those for the corresponding hydrated biofilm test. These results show that the efficacy of a disinfectant must be measured by using a laboratory method where biofilm is grown under fluid flow conditions similar to the environment where the disinfectant will be applied.

A laboratory hot tub model for disinfectant efficacy evaluation.

This paper describes a novel laboratory hot tub (LHT) apparatus and associated standard operating procedure (SOP) designed to reproduce the key biological, chemical, and engineering parameters associated with recreational and therapeutic hot tubs. Efficacy, as measured quantitatively by log reduction values, was determined against both biofilm and planktonic bacteria. When the LHT was run according to the SOP, with no antimicrobial treatment, a consistent level of bacterial contamination occurred. The means of log10 viable cell densities (+/- the repeatability standard deviation of log densities) were 7.2 (+/-0.31) for the bulk water (density in units of cfu ml-1), 5.3 (+/-0.56) for the coupons (density in units of cfu cm-2), and 6.6 (+/-0.50) for the filters (density in units of cfu cm-2). When control and chlorine treated LHTs were run in parallel, the log reduction increased significantly with chlorine concentration for samples of planktonic bacteria in the bulk water (p=0.016), biofilm bacteria on the coupons (p=0.09) and biofilm bacteria on the filter (p=0.005), indicating that the method was sensitive to chlorine concentration. The method also displayed sensitivity by differentiating between chlorine and bromine treatments; in every case, chlorine produced a greater log reduction than did the same concentration of bromine. The model and SOP were shown to be rugged with respect to slight changes in fluid mixing intensity, water chemistry (saturation index), inoculum size, and organic loading. The LHT and associated SOP provide a reliable second tier in a three-tiered testing process, in which the first tier is a suspension test and the final tier is a field test.

Comparative evaluation of two quantitative test methods for determining the efficacy of liquid sporicides and sterilants on a hard surface: a precollaborative study.

Two quantitative carrier-based test methods for determining the efficacy of liquid sporicides and sterilants on a hard surface, the Standard Quantitative Carrier Test Method-ASTM E 2111-00 and an adaptation of a quantitative micro-method as reported by Sagripanti and Bonifacino, were compared in this study. The methods were selected based on their desirable characteristics (e.g., well-developed protocol, previous use with spores, fully quantitative, and use of readily available equipment) for testing liquid sporicides and sterilants on a hard surface. In this paper, the Sagripanti-Bonifacino procedure is referred to as the Three Step Method (TSM). AOAC Official Method 966.04 was included in this study as a reference method. Three laboratories participated in the evaluation. Three chemical treatments were tested: (1) 3000 ppm sodium hypochlorite with pH adjusted to 7.0, (2) a hydrogen peroxide/peroxyacetic acid product, and (3) 3000 ppm sodium hypochlorite with pH unadjusted (pH of approximately 10.0). A fourth treatment, 6000 ppm sodium hypochlorite solution with pH adjusted to 7.0, was included only for Method 966.04 as a positive control (high level of efficacy). The contact time was 10 min for all chemical treatments except the 6000 ppm sodium hypochlorite treatment which was tested at 30 min. Each chemical treatment was tested 3 times using each of the methods. Only 2 of the laboratories performed the AOAC method. Method performance was assessed by the within-laboratory variance, between-laboratory variance, and total variance associated with the log reduction (LR) estimates generated by each quantitative method. The quantitative methods performed similarly, and the LR values generated by each method were not statistically different for the 3 treatments evaluated. Based on feedback from the participating laboratories, compared to the TSM, ASTM E 2111-00 was more resource demanding and required more set-up time. The logistical and resource concerns identified for ASTM E 2111-00 were largely associated with the filtration process and counting bacterial colonies on filters. Thus, the TSM was determined to be the most suitable method.

Modification to the AOAC Sporicidal Activity of Disinfectants Test (Method 966.04): collaborative study.

In an effort to improve AOAC Method 966.04, the Sporicidal Activity of Disinfectants Test, selected modifications to the procedure were evaluated in a collaborative study. Method 966.04 is used to generate efficacy data to support the product registration of sporicides and sterilants. The method is a carrier-based test that provides a qualitative measure of product efficacy against spores of Bacillus subtilis and Clostridium sporogenes. The use of garden soil extract and the lack of standard procedures for the enumeration of spores and neutralization of the test chemicals have been considered problematic for many years. The proposed modifications were limited to the B. subtilis and hard surface carrier (porcelain penicylinder) components of the method. The study included the evaluation of a replacement for soil extract nutrient broth and an establishment of a minimum spore titer per carrier, both considered crucial for the improvement and utilization of the method. Additionally, an alternative hard surface material and a neutralization confirmation procedure were evaluated. To determine the equivalence of the proposed alternatives to the standard method, 3 medium/carrier combinations, (1) soil extract nutrient broth/porcelain carrier (current method), (2) nutrient agar amended with 5 microg/mL manganese sulfate/porcelain carrier, and (3) nutrient agar amended with 5 microg/mL manganese sulfate/stainless steel carrier were analyzed for carrier counts, HCI resistance, efficacy, quantitative efficacy, and spore wash-off. The test chemicals used in the study represent 3 chemical classes and are commercially available antimicrobial liquid products: sodium hypochlorite (bleach), glutaraldehyde, and a combination of peracetic acid and hydrogen peroxide. Four laboratories participated in the study. The results of the spore titer per carrier, HCI resistance, efficacy, and wash-off studies demonstrate that amended nutrient agar in conjunction with the porcelain is comparable to the current method, soil extract nutrient broth/porcelain. The nutrient agar method is simple, inexpensive, reproducible, and provides an ample supply of high quality spores. Due to the current use of porcelain carriers for testing C. sporogenes, it is advisable to retain the use of porcelain carriers until stainless steel can be evaluated as a replacement carrier material for Clostridium. The evaluation of stainless steel for Clostridium has been initiated by the Study Director. Study Director recommendations for First Action revisions are provided in a modified method.

A microtiter-plate screening method for biofilm disinfection and removal.

A quantitative spectrophotometric method was developed to measure the removal and killing efficacy of antibiofilm agents. Biofilms of Pseudomonas aeruginosa or Staphylococcus epidermidis were grown in 96-well plates, treated with an agent, then stained with either the biomass indicator crystal violet or the respiratory indicator 5-cyano-2,3-ditolyl tetrazolium chloride. This rapid screening method is sensitive enough to elucidate concentration-response relationships as well as differences between species responses to treatments. Using these assays, agents can be ranked by their ability to remove or kill biofilm.