Efficacy of individual biocides and synergistic combinations

A method for quantitative estimation of the biocidal effect of compounds on microbial cells has been developed based on colony growth on solid nutrient media. The method allows for specifying a mode of action and for calculating the most effective ratio of biocides in synergistic blends. The test organism Serratia marcescens was grown in the presence of copper sulfate and kathon (2-N-octyl-4-isothiazolin-3-one) on agar medium supplemented with glucose and peptone. The approach made it possible to achieve respectively 96 and 67% decreases in effective concentrations of kathon and copper sulfate in the mixture.     

A rapid bactometer method for screening of biocides against sulfate-reducing bacteria

A rapid and efficient bactometer method was developed for screening biocides against sulfate-reducing bacteria. The method is based on impedance microbiology principles and uses double-layer API (American Petroleum Institute) agar medium supplemented with 0.1% sodium thioglycolate as a reducing agent. Compared to the conventional API procedure, which requires 28 days, the present technique takes only 1 day to obtain test results. Excellent linear correlation (r=–0.98) was found between the impedance detection time and log initial cell concentration. The results of the bactometer test were comparable to that of the API bottle test.     

A rapid technique for assessing the suitability of areas for invasive species applied to New Zealand's rivers

Early responses to incursions of non‐indigenous species (NIS) into new areas include modelling and surveillance to define the organisms’ potential and actual distributions. For well‐studied invasive species, predictive models can be developed based on quantitative data describing environmental tolerances. In late 2004, an invasive freshwater diatom Didymosphenia geminata, an NIS for which we had no such quantitative data, was detected in a New Zealand river. We describe a procedure used to rapidly develop a classification of suitability for all New Zealand's rivers, based on two sources of information. First, from a review of the limited available literature and unpublished data, we determined that temperature, hydrological and substrate stability, light availability, and water pH were the most important environmental gradients determining D. geminata's broad‐scale distribution and capacity for establishing and forming blooms in rivers. The second information source was a GIS‐based river network developed for a national classification of New Zealand's rivers, with associated data describing environmental characteristics of each section of the network. We used six variables that were available for every section of the network as surrogates for the environmental gradients that determine suitability. We then determined the environmental distance of all the river sections in the network from our assessment of the optimal conditions conducive to D. geminata blooms. The analysis suggested that > 70% of New Zealand's river sections (stream order > 3) fell into the two highest suitability categories (on a five‐point scale). At the time of writing, D. geminata had spread to 12 catchments, all of which were within these two categories. The technique is applicable in initial responses to incursions of NIS where quantitative information is limited, and makes optimal use of available qualitative information. Our assessment contributed to evaluations of the potential ecological, social, and economic impacts of D. geminata and is currently being used to stratify site selection for ongoing surveillance.     

A rapid method for assessing the resistance of polyurethanes to biodeterioration

A rapid soil burial method for assessing the susceptibility of polyurethanes to biodeterioration was developed. The time of the test was reduced by prestressing the polyurethanes. The degree of deterioration was measured by following changes in the appearance of the polyurethanes and in selected physical properties. It was found that pre-stressing produced significant reductions in the tensile strength of a known susceptible polyurethane after burial in soil for 2 weeks. The reduction was greater than that found with unstressed polyurethanes buried for 26 weeks in active soil. Changes in tensile strength were less after burial for 4 weeks in sterile soil than after burial in active soil for the same period. The results suggest that deterioration of polyurethane during soil burial is a result of both chemical and microbial action.     

A rapid method for assessing the RNA-binding potential of a protein

In recent years, evidence has emerged for the existence of many diverse types of RNA, which play roles in a wide range of biological processes in all kingdoms of life. These molecules generally do not, however, act in isolation, and identifying which proteins partner with RNA is a major challenge. Many methods, in vivo and in vitro, have been used to address this question, including combinatorial or high-throughput approaches, such as systematic evolution of ligands, cross-linking and immunoprecipitation and RNA immunoprecipitation combined with deep sequencing. However, most of these methods are not trivial to pursue and often require substantial optimization before results can be achieved. Here, we demonstrate a simple technique that allows one to screen proteins for RNA-binding properties in a gel-shift experiment and can be easily implemented in any laboratory. This assay should be a useful first-pass tool for assessing whether a protein has RNA- or DNA-binding properties, prior to committing resources to more complex procedures.     

Investigating the suitability of the Calgary Biofilm Device for assessing the antimicrobial efficacy of new agents

This study investigated the suitability of the Calgary Biofilm Device (CBD), originally designed as a test surrogate for indwelling medical devices, for assessing the efficacy of antimicrobials developed for food and food contact surface disinfection applications. The conditions for the development of uniform biofilms from pure and mixed bacterial cultures of wild type Escherichia coli and Listeria innocua were optimized. We were able to recover approximately 2 x 10(6) colony forming units (CFU) from the biofilms formed on the individual pegs of the device in 24 h. Further, the parameters for the consistent release of the cells from the biofilms were optimized; test showed that the number of cells released was uniform and reproducible. The consistency and reproducibility of the biofilms formed on the pegs was evaluated using scanning electron microscopy and by plate count method. The efficacies of disinfectants on cells residing in biofilms versus planktonic cells were compared. For both species, higher concentrations of disinfectants were needed to eliminate attached cells as compared with planktonic cells. This study establishes the value of the CBD for generating consistent biofilms from either pure or mixed cultures. These biofilms can be used to assess efficacies of disinfectants against cells that have colonized the surfaces of foods and food-processing equipment. Such a system could serve as a standard surrogate for evaluating new disinfectants designed to reduce or eliminate biofilms from food-contact surfaces.     

A rapid and sensitive method for the estimation of individual tRNA pools

A rapid and sensitive method is described to quantitatively compare tRNA pools for individual aminoacids in a single experiment. The procedure comprises of: charging of total tRNA with a mixture of radiolabeled aminoacids, deacylation of the esterified tRNA with a volatile base and the recovery of the labeled aminoacid, derivatisation of the aminoacid with phenylisothiocyanate after mixing with excess of nonradioactive aminoacids, baseline separation of the phenylthiocarbamyl aminoacids by reverse phase high performance liquid chromatography monitored by A254nm and quantitation of the radioactivity in individual aminoacid peaks. The radioactivity in the aminoacid peak corresponds to the quantity of the aminoacylated tRNA. The method has been successfully applied to quantitate the individual tRNA pools in the developing silk glands of Bombyx mori, a functionally adapted tissue which undergoes considerable variations in tRNA content.     

A rapid method for assessing the distribution of gold labeling on thin sections.

Particulate gold labeling on ultrathin sections is in widespread use for antigen localization at the EM level. To extend the usefulness of gold labeling technology, we are evaluating different methods for sampling and estimating quantities of gold labeling. Here we present a simple, rapid, and unbiased method for assessing the relative pool sizes of immunogold labeling distributed over different cell compartments. The method uses a sampling approach developed for stereology in which a regular array of microscopic fields or linear scans is positioned randomly on labeled sections. From these readouts, gold particles are counted and assigned to identifiable cell structures to construct a gold labeling frequency distribution of those labeled compartments. Here we use ultrathin cryosections labeled for a range of different proteins and for a signaling lipid. We show by scanning labeled sections at the electron microscope that counting 100-200 particles on each of two grids is sufficient to obtain a reproducible and rapid assessment of the pattern of labeling proportions over 10-16 compartments. If more precise estimates of labeling proportions over individual compartments are required (e.g., to achieve coefficients of error of 10-20%), then 100-200 particles need to be counted over each compartment of interest.     

Nanocomplexes on the basis of Taunit associated with biocides as effective anti-cyanobacterial agents

Cyanobacteria are photoautotrophic bacteria that are known also as blue-green algae. They accumulate on different surfaces and objects and contribute to their biodegradation. Moreover, cyanobacteria produce toxins, which lead to harmful environmental and human health impacts. Hence, cyanobacterial growth control problem is very vital. The goal of the study was to obtain new nanocomplexes on the basis of a modern nanomaterial Taunit associated with antibiotic chloramphenicol and herbicide diuron and to test their antimicrobial effect against a model organism such as the unicellular cyanobacterium Synechocystis sp. PCC 6803. A nanomaterial made of multiwalled carbon nanotubes (MWCNTs) called Taunit was used for the first time to obtain nanocomplexes coupled either with herbicide diuron (DCMU (3-(3,4-dichlorophenyl)- 1,1-dimethylurea) or with antibiotic chloramphenicol. A small amount of Taunit (~1 mg) was needed to adsorb micrograms of diuron or chloramphenicol. The new formed nanocomplexes differentiate in their antimicrobial activity, which could be explained by the difference in their chemical mechanism of action. Taunit ? diuron complex showed a higher biocide action against cyanobacterium than the Taunit ? chloramphenicol complex. The results allow to discuss the prospects of research on the use of Taunit ? diuron complex as a coating for various surfaces exposed to cyanobacteria fouling.     

A chemical quenching apparatus for studying rapid reactions.

A chemical quench-flow apparatus is described for studying enzymatic reactions with half-lives of 0.005 sec or longer. The syringe pistons are driven by a stepping motor which provides precise control over the volume and rate of flow of reactants. The drive mechanism also ensures a rapid approach to a steady flow velocity and thus minimizes the amount of material used per stroke. Improved mixing efficiency is accomplished by means of ball mixers which utilize the zone of turbulence in the wake of a sphere as the mixing mechanism. The instrument was used to follow the presteady state time course of phosphorylation and dephosphorylation of a microsomal preparation of (Na⁺ + K⁺)-stimulated ATPase.     

A simple method to evaluate the potential for degradation of antifouling biocides

A simple method to measure the degradation of antifouling biocides is described which measures the loss of biocidal activity from seawater by bioassay. The bioassay employs either the ship‐fouling diatom Amphora or the brine shrimp Anemia. Loss of bioaclivity from sterile seawater indicates abiotic degradation whilst loss of bioactivity from natural seawater indicates biodegradation. Results are presented for three biocides, viz. the trihalomethylthio compound, N‐dichlorofluoromethylthio‐N’,N'‐dimethyl‐N‐phenyl‐sulphamide (Preventol A4S), di‐n‐octylamine, and the isothiazolone compound 4,5‐dichloro‐2‐n‐octyl‐4‐isothiazolin‐3‐one (Sea‐Nine 211).     

A proposed metric for assessing the measurement quality of individual microarrays

Background  

High-density microarray technology is increasingly applied to study gene expression levels on a large scale. Microarray experiments rely on several critical steps that may introduce error and uncertainty in analyses. These steps include mRNA sample extraction, amplification and labeling, hybridization, and scanning. In some cases this may be manifested as systematic spatial variation on the surface of microarray in which expression measurements within an individual array may vary as a function of geographic position on the array surface.     

A simple and rapid protein array based method for the simultaneous detection of biowarfare agents

A protein chip has been developed that allows the simultaneous detection of a multitude of different biowarfare agents. The chip was developed for the ArrayTube platform providing a cheap and easy to handle technology solution that combines a microtube-integrated protein chip with the classical procedure of a sandwich-enzyme-linked immunosorbent assay and signal amplification by streptavidin-poly-horseradish peroxidase. Specific immunoassays for Staphylococcus enterotoxin B, ricin, Venezuelan equine encephalitis virus, St. Louis encephalitis virus, West Nile virus, Yellow fever virus, Orthopox virus species, Francisella tularensis, Yersinia pestis, Brucella melitensis, Burkholderia mallei and Escherichia coli EHEC O157:H7 were developed and optimized. All assays could be completed within 1 to 1 1/2 h and detection levels were demonstrated to be as low as in well established ELISAs. Most interesting, as a result of careful antibody screening and testing, it is currently possible to analyse at least five of the "dirty dozen" agents on one single protein chip in parallel.     

Evaluation of hydrogen peroxide-based disinfectants in a new resazurin microplate method for rapid efficacy testing of biocides

Aims:  Development of the resazurin microplate method (RMM) as a novel test system for the evaluation of the antimicrobial activity of antiseptics and disinfectants. The validated RMM was subsequently applied for the evaluation of hydrogen peroxide (H₂O₂) and stabilized H₂O₂ combination products.
Methods and Results:  The European Committee for Standardization prescribes the plate count challenge test (PCCT) for antiseptic and disinfectant efficacy testing. This protocol was adapted to a microplate-based assay, using resazurin as viability indicator. The RMM was as accurate as the PCCT, had an identical detection limit and showed high intermediate precision. Using the validated RMM, it was shown that H₂O₂ combined with silver possessed a higher bactericidal and fungicidal activity compared to native H₂O₂ with and without glycerol.
Conclusions:  Validation showed that the RMM may replace the PCCT. When applying the RMM, H₂O₂ combined with silver was clearly a more potent disinfectant compared to H₂O₂ in killing bacteria and fungi.
Significance and Impact of the Study:  The RMM is easier to use for antimicrobial efficacy testing of antiseptics and disinfectants. As the RMM is in accordance with the norms of the European Committee for Standardization, it may become a more cost-effective alternative to the more laborious PCCT reference method. H₂O₂ with silver may replace native H₂O₂ to increase overall disinfection efficiency.     

A method for assessing sustainability,with beef production as an example

A comprehensive approach to decisions about the use of land and other world resources, taking full account of biological and other scientific information, is crucial for good decisions to be made now and in future. The sustainability of systems for producing food and other products is sometimes assessed using too narrow a range of component factors. A production system might be unsustainable because of adverse effects on a wide range of aspects of human welfare, animal welfare, or the environment. All factors should be included in sustainability evaluation, otherwise products or actions might be avoided without adequate consideration of key factors or of the diversity of production systems. A scoring method that is based on scientific information and potentially of general relevance is presented here, using beef production as a example with a review of each of its sustainability components. This includes an overall combined score and specific factors that make the system unacceptable for some consumers. The results show that, in this example, the sustainability of the best systems is very much better than that of the worst systems. By taking account of scores for a wide range of components of sustainability in comparing beef-production systems, better quality policies about beef use can be formulated than when statements referring only to one system are considered. The least sustainable beef-production systems are extensive grazing that causes land degradation and the use of feedlots or indoor housing with grain feeding. Semi-intensive silvopastoral systems are the most sustainable beef-production systems, and well-managed pasture-fed beef from areas where crop production is uneconomic is also sustainable. This simple, scientifically based scoring system could be modified to use positive as well as negative scores and is of value for policy makers, researchers, producers, organisations aiming to improve sustainability, and the general public.     

Validation of respirometry as a short-term method to assess the efficacy of biocides

This study shows that a short-term respirometric measurement based on the rate of oxygen uptake needed to oxidize glucose is a reliable and fast method to assess biocide efficacy against P. fluorescens cells. Respiratory activity using oxygen consumption rate, the determination of viable and nonviable cells using Live/Dead BacLight kit and colony formation units (CFU), were compared as indicators of the biocidal efficacy of ortho-phthalaldehyde (OPA). The results showed that determining the effect of OPA against P. fluorescens using the different methods leads to different conclusions. The minimum bactericidal concentration (MBC) was 80 mgl(-1), 100 mgl(-1) and 65 mgl(-1) respectively, using respiratory activity, viability using BacLight counts and culturability. The plate count method was shown to underestimate the biocidal action of OPA, whilst data from respirometry and viability using Live/Dead BacLight kit correlated strongly and were not statistically different when yellow cells were considered nonviable. Respirometry therefore represents an expeditious, non-destructive and accurate method to determine the antimicrobial action of biocides against aerobic heterotrophic bacteria.     

A repeatable laboratory method for testing the efficacy of biocides against toilet bowl biofilms

AIMS: The purpose of this study was to develop a laboratory biofilm growth reactor system that simulated the toilet bowl environment and which could be used for biocide efficacy testing. METHODS AND RESULTS: A microbial biofilm reactor system incorporating intermittent flow and nutrient provision was designed. The reactor system was open to the air and was inoculated with organisms collected from toilet bowl biofilms. Once per hour, reactors were supplied with a nutrient solution for a period of 5 min, then flushed and refilled with tap water or tap water amended with chlorine. Quantitative measures of the rate and extent of biofilm accumulation were defined. Biofilm accumulated in untreated reactors to cell densities of 108 cfu cm-2 after approximately 1 week. Biofilm accumulation was also observed in reactors in the continuous presence of several milligrams per litre of free chlorine. Repeatability standard deviations for the selected efficacy measures were low, indicating high repeatability between experiments. Log reduction values of viable cell numbers were within ranges observed with standard suspension and hard surface disinfection tests. Biofilm accumulated in laboratory reactors approximately seven times faster than it did in actual toilet bowls. The same ranking was achieved in tests between laboratory biofilms and field-grown biofilms with three of the four measures, using three different concentrations of chlorine. CONCLUSION: This reactor system has been shown to simulate, in a repeatable way, the accumulation of bacterial biofilm that occurs in toilet bowls. The results demonstrate that this system can provide repeatable assays of the efficacy of chlorine against those biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: The laboratory biofilm reactor system described herein can be used to evaluate potential antimicrobial and antifouling treatments for control of biofilm formation in toilet bowls.