Clinical Characterisation of Caudal Traumatic Malocclusions and Treatment Outcomes in Cats (2018-2022).

Caudal malocclusions in cats may result in a variety of traumatic lesions affecting the soft tissues of the ipsilateral mandible such as fovea, gingival cleft, and proliferative lesions. Fifty-one cats diagnosed with a traumatic caudal malocclusion were compared with a control hospital population and evaluated for prevalence with respect to breed and sex. Twenty-two cats that were treated had radiographic, clinical findings, and the outcome of treatment (extraction or odontoplasty) recorded. Maine Coon, Persian, and male neutered cats were overrepresented while Domestic Shorthairs were underrepresented within the study population. Radiographically, 50% of the fovea lesions had an area of decreased bone density in the region of the lesion and none of these had evidence of periodontal disease. All gingival cleft lesions had radiographic changes consistent with periodontal disease. 15.4% of proliferative lesions presented with radiographic changes, with only half of those presenting with both radiographic and clinical evidence of periodontal disease. Eleven cats were treated by odontoplasty and eleven by extraction. One cat treated by odontoplasty developed new lesions caudally, and another had persistence of the initial lesions. Two cats in the extraction group developed new lesions rostral to the extracted teeth. In most instances, odontoplasty or extraction resulted in successful soft tissue lesion resolution. In rare cases, additional treatment was necessary due to either persistence or development of new lesions.

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.

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.

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.

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).

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.

Conserving and restoring the Caicos pine forests: The first decade.

The severe and rapid attack on the Caicos pine Pinus caribaea var. bahamensis (Pinaceae) by the non-native invasive pine tortoise scale, Toumeyella parvicornis, has resulted in the death of most of the trees in the Turks and Caicos Islands (TCI) in just over a decade. Local and international conservation efforts have enabled the necessary multi-disciplinary research, data gathering, and monitoring to develop and implement a restoration strategy for this endemic tree from the Bahaman archipelago. The native plant nursery established on North Caicos and horticultural expertise acquired throughout the years were crucial to the successful rescue of Caicos pine saplings from the wild populations and cultivation of new saplings grown from locally sourced seeds. These saplings have been used to establish six Restoration Trial Plots on Pine Cay and a seed orchard on North Caicos in TCI. Core Conservation Areas (CCAs) for the Caicos pine forests have been identified and mapped. To date, forest within the Pine Cay CCA has been supplemented by planting more than 450 pine trees, which have survived at a high (>80%) rate.

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.

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.

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.

The role of botanic gardens in the science and practice of ecological restoration.

Many of the skills and resources associated with botanic gardens and arboreta, including plant taxonomy, horticulture, and seed bank management, are fundamental to ecological restoration efforts, yet few of the world's botanic gardens are involved in the science or practice of restoration. Thus, we examined the potential role of botanic gardens in these emerging fields. We believe a reorientation of certain existing institutional strengths, such as plant-based research and knowledge transfer, would enable many more botanic gardens worldwide to provide effective science-based support to restoration efforts. We recommend botanic gardens widen research to include ecosystems as well as species, increase involvement in practical restoration projects and training practitioners, and serve as information hubs for data archiving and exchange.

Microtomographic porosity determination in alginate mixed with various methods.

PURPOSE: Mechanical spatulation of alginate impression materials reportedly produces fewer voids and superior casts than hand mixing. Two current methods of alginate mechanical preparation are a vacuum mixer Vac-U-Vestor, (Whip Mix Corp, Louisville, KY) and a semiautomated method that involves hand spatulation in a rotating bowl Alginator II (Cadco, Oxnard, CA). A new alginate-mixing machine has been introduced, TurboMax (Dentsply Raintree Essex, Sarasota, FL), with a centrifugal-spinning action that reportedly incorporates the alginate powder into the water more efficiently. The purpose of this study was to determine the number, percent, and volume distribution of porosities in alginate mixed with three mechanical-mixing methods using a nondestructive, microtomographic analysis method. MATERIALS AND METHODS: Alginate was mixed by each of the three mechanical methods per respective manufacturer's guidelines, with the set alginate analyzed using a microtomography unit and proprietary software. A mean and standard deviation was determined per group and analyzed with Kruskal-Wallis ANOVA/Mann-Whitney tests. RESULTS: Significant differences (p < 0.001) were found between groups per each of the three testing parameters (number, percent, volume distribution of porosities). The vacuum mixer produced significantly less percent porosity and number of porosities than the centrifugal mixer and semiautomated hand mixer. Both the vacuum mixer and centrifugal mixer produced porosities of significantly smaller volume than the semiautomated hand mixer. CONCLUSION: Of the three mechanical mixing methods, the vacuum mixer had the best performance overall in reducing the number, percent, and volume of porosities in the mixed alginate.

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.

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.

A method for growing a biofilm under low shear at the air-liquid interface using the drip flow biofilm reactor.

This protocol describes how to grow a Pseudomonas aeruginosa biofilm under low fluid shear close to the air-liquid interface using the drip flow reactor (DFR). The DFR can model environments such as food-processing conveyor belts, catheters, lungs with cystic fibrosis and the oral cavity. The biofilm is established by operating the reactor in batch mode for 6 h. A mature biofilm forms as the reactor operates for an additional 48 h with a continuous flow of nutrients. During continuous flow, the biofilm experiences a low shear as the media drips onto a surface set at a 10 degrees angle. At the end of 54 h, biofilm accumulation is quantified by removing coupons from the reactor channels, rinsing the coupons to remove planktonic cells, scraping the biofilm from the coupon surface, disaggregating the clumps, then diluting and plating for viable cell enumeration. The entire procedure takes 13 h of active time that is distributed over 5 d.

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.

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.