Characterization of methods for determining sterilization efficacy and reuse efficiency of oxygen biosensor multiwell plates

High-throughput screening (HTS) assays based upon fluorometric detection of oxygen consumption in microtiter plates were primarily developed for applications in drug discovery and ecotoxicology but have recently been adopted for use in microbial community-level physiological profiling assays (CLPP). The widespread use of oxygen biosensor systems for CLPP applications has, however, been hindered by the relatively high cost of oxygen biosensor reagent systems and limited access to microplate fluorometer instrumentation platforms. The ability to recycle and reuse oxygen biosensor system plates would expand their utilization for CLPP assays and other research applications in microbial ecology. Here, the efficacy and cost effectiveness of multiple procedures for sterilization of Oxygen Biosensor System (OBS; BD Biosciences) plates for reuse was evaluated. OBS plates were sterilized using ethylene oxide, ultraviolet radiation, and bleach treatments, then evaluated for biosensor response and plate life-cycle performance. Of the sterilization methods tested, ethylene oxide sterilization was most effective based on its low cost, high sterilization efficacy, and minimal impact upon OBS plate response.     

Characterization of methods for determining sterilization efficacy and reuse efficiency of oxygen biosensor multiwell plates

High-throughput screening (HTS) assays based upon fluorometric detection of oxygen consumption in microtiter plates were primarily developed for applications in drug discovery and ecotoxicology but have recently been adopted for use in microbial community-level physiological profiling assays (CLPP). The widespread use of oxygen biosensor systems for CLPP applications has, however, been hindered by the relatively high cost of oxygen biosensor reagent systems and limited access to microplate fluorometer instrumentation platforms. The ability to recycle and reuse oxygen biosensor system plates would expand their utilization for CLPP assays and other research applications in microbial ecology. Here, the efficacy and cost effectiveness of multiple procedures for sterilization of Oxygen Biosensor System™ (OBS; BD Biosciences) plates for reuse was evaluated. OBS plates were sterilized using ethylene oxide, ultraviolet radiation, and bleach treatments, then evaluated for biosensor response and plate life-cycle performance. Of the sterilization methods tested, ethylene oxide sterilization was most effective based on its low cost, high sterilization efficacy, and minimal impact upon OBS plate response.     

A comparison of the traditional method of counting viable cells and a quick microplate method for monitoring the growth characteristics of Listeria monocytogenes

AIMS: To determine: (i) the growth parameters (specific growth rate, lag time, asymptotic amount of growth, generation time and time for maximum growth rate) of Listeria monocytogenes in different broths by standard cultivation methods and (ii) whether a microplate method in conjunction with a standard nondedicated plate reader could be adapted to routine assay. METHODS AND RESULTS: Growth curves were determined from cell numbers in a standard tube method at 2 h intervals by serial dilution and plating, and in a microplate method by absorbance measurements. Growth curves were fitted with a modified Gompertz function. CONCLUSIONS: The microplate method was similar to the standard cultivation methods in accuracy, required less chemical reagents, and considerably reduced the time required for analyses. This work also illustrates that growth characteristics of bacteria are not necessarily constant, and depend on the methodology used. SIGNIFICANCE AND IMPACT OF THE STUDY: It is not the intended purpose of this paper to present all the data for the media tested but instead to illustrate the success of the microplate method for studying growth kinetics compared to a standard cultivation method and system precision. The method will be of considerable benefit to laboratories unable to afford dedicated workstations.     

Bacterial oxygenases: in vivo enzyme biosensors for organic pollutants

An in vivo enzyme-based biosensor platform was developed that uses specific oxygenase enzymes to detect aromatic compounds in water. Bacteria capable of degrading highly reduced hydrocarbons initiate substrate oxidation using well-characterised oxygenase enzymes, which due to their specificity, stability and high activity can be applied in vivo as biosensor components. Oxygenase enzyme activity was determined in vivo using BD Oxygen Biosensor plates to measure oxygenase-mediated oxygen depletion in the presence of specific aromatic analytes. The BTEX family of compounds (benzene, toluene, ethylbenzene and the three isomers of xylene) were used as model compounds. Detection limits and sensitivity achieved using this approach (microM detection range) was similar to levels achieved with oxygen electrode-based and some recombinant DNA-based approaches. No significant signals were detected with hydrocarbons that were not substrates of the initial oxygenases.     

Physiological Profiling of Soil Microbial Communities in a Florida Scrub-Oak Ecosystem: Spatial Distribution and Nutrient Limitations

Rapid physiological profiling of heterotrophic microbial communities enables intensive analysis of the factors affecting activity in aerobic habitats, such as soil. Previous methods for performing such profiling were severely limited due to enrichment bias and inflexibility in incubation conditions. We tested a new physiological profiling approach based on a microtiter plate oxygen sensor system (Becton Dickinson Oxygen Biosensor System (BDOBS)), which allows for testing of lower substrate addition (i.e., lower enrichment potential) and manipulation of physiochemical assay conditions, such as pH and nutrients. Soil microbial communities associated with a scrub-oak forest ecosystem on Merritt Island Wildlife Refuge in central Florida, USA, were studied in order to evaluate microbial activity in a nutrient poor soil and to provide baseline data on the site for subsequent evaluation of the effects of elevated CO₂ on ecosystem function. The spatial variation in physiological activity amongst different habitats (litter, bulk soil, and rhizosphere) was examined as a function of adaptation to local resources (i.e., water soluble extracts of roots and leaf litter) and the degree of N and P limitation. All the communities were primarily N-limited, with a secondary P limitation, which was greater in the rhizosphere and bulk soil. The litter community showed greater overall oxygen consumption when exposed to litter extracts relative to the rhizosphere or soil, suggesting acclimation toward greater use of the mixed substrates in the extract. Root extracts were readily used by communities from all the habitats with no habitat specific acclimation observed. A priming effect was detected in all habitats; addition of glucose caused a significant increase in the use of soil organic carbon. Response to added glucose was only observed with N and P addition, suggesting that C may be lost to the groundwater from these porous soils because nutrient limitation prevents C immobilization.     

ESTIMATION OF MICROBIOLOGICAL QUALITY OF CHILLED BEEF USING AUTOMATED TURBIDIMETRY

Total bacterial counts on chilled beef samples were estimated by the standard plate count method and by an automated turbidimetric system. The latter method is based on product-specific calibration curves constructed by correlating growth curve parameters calculated for the turbidimeter to the log CFU values obtained by plate counts. A total of 74 beef samples was used to construct the calibration curves. Correlation analysis between turbidimetric parameters and plate count values showed that detection time was the best predictor to estimate microbial loads on fresh (r=0.91) and aged beef (r=0.94). Microbial loads for a different set of aged beef samples (n = 37) refrigerated for 7, 9, 10, 17 and 45 days were compared by turbidimetric measurements and plate counts. Mean total viable counts were log 5.92 ± 1.17 and log 5.54 ± 1.28 CFU/mL, respectively. Results showed that total bacterial counts on chilled beef could be estimated accurately from turbidimetric parameters. Furthermore, setting a cut-off value of log 6 CFU/mL allowed to accepting/rejecting samples according to their microbial condition in shorter periods of time compared to the traditional plate count method.     

Impedance microbiology: quantification of bacterial content in milk by means of capacitance growth curves

The impedancimetric method is a technique for the rapid evaluation of milk bacterial content and also of its subproducts. Several authors have made use of culture conductance changes during bacterial growth for quantitative and qualitative assessments of microbial growth. However, interface capacitance curves, Ci, have not been used. In this paper, we quantify bacteria in cow raw milk by following their growth as the above-mentioned capacitance change time course event. With it, bigger growth variations, shorter detection times and a better coefficient of correlation with the plate count method were obtained than those yielded by conductance curves. Calibration was performed by plotting initial known concentrations, IC (CFU/ml), as a function of the time detection theshold (TDT).     

Protocols and Programs for High-Throughput Growth and Aging Phenotyping in Yeast

In microorganisms, and more particularly in yeasts, a standard phenotyping approach consists in the analysis of fitness by growth rate determination in different conditions. One growth assay that combines high throughput with high resolution involves the generation of growth curves from 96-well plate microcultivations in thermostated and shaking plate readers. To push the throughput of this method to the next level, we have adapted it in this study to the use of 384-well plates. The values of the extracted growth parameters (lag time, doubling time and yield of biomass) correlated well between experiments carried out in 384-well plates as compared to 96-well plates or batch cultures, validating the higher-throughput approach for phenotypic screens. The method is not restricted to the use of the budding yeast Saccharomyces cerevisiae, as shown by consistent results for other species selected from the Hemiascomycete class. Furthermore, we used the 384-well plate microcultivations to develop and validate a higher-throughput assay for yeast Chronological Life Span (CLS), a parameter that is still commonly determined by a cumbersome method based on counting “Colony Forming Units”. To accelerate analysis of the large datasets generated by the described growth and aging assays, we developed the freely available software tools GATHODE and CATHODE. These tools allow for semi-automatic determination of growth parameters and CLS behavior from typical plate reader output files. The described protocols and programs will increase the time- and cost-efficiency of a number of yeast-based systems genetics experiments as well as various types of screens.     

Growth Curves of Anaerobic Bacteria in Solid Media

Simple pour plate and spectrophotometric techniques for the evaluation of growth curves of several anaerobic bacteria on solid media are described. Three basic patterns of anaerobic growth were observed. The curves obtained were very reproducible when studied on separate occasions. The curves obtained by spectrophotometric measurement were comparable to those obtained by the pour plate method, especially when a large bacterial inoculum was used. Limitations in the interpretation of the results are discussed. The methods and principles reported could provide the basis for the determination of bacterial growth on solid media using other organisms and different experimental conditions.     

Use of impedance test for testing effectiveness of drug preservatives

Pharmacopoeias contain preservation efficacy test for estimating antimicrobial activity of chemical compounds added to pharmaceutical preparations in multidose containers in order to inhibit bioburden growth. This method involves the treating of preserved products with bacteria and yeast cells and monitoring the survival of microorganisms through the specified time periods up to 28 days. The last stage of assay--incubation and colony counting--is very time consuming. Recent advance in technology enables faster and more convenient detection in comparison to traditional methods. Impedance method is based on the principle that conductance and capacitance of cultivation medium increases when bacteria grow and metabolize, Impedance time detection is inversely proportional to initial bacterial population. Six different products were utilised throughout the study. The calibration curves were calculated for each of the tested strains by comparison between standard plate count method and detection time measured in Bactometer system. In our study log reduction calculated in alternative method were similar to those obtained in plate count assay. All of the tested preparations, except one, exhibited acceptable activity against bacteria and fungi and meet the pharmacopoeal requirements. The studies indicated a positive correlation between standard plate count results and impedance reading. The procedure with the usage of Bactometer, provides a rapid and accurate system for the determination of bacterial content.     

Age-, gender-, and species-dependent mutagenicity in T cells of mice and rats exposed by inhalation to 1,3-butadiene

Experiments were performed: (i) to investigate potential age- and gender-dependent differences in mutagenic responses in T cells following exposures of B6C3F1 mice and F344 rats by inhalation for 2 weeks to 0 or 1250 ppm butadiene (BD), and (ii) to determine if exposures for 2 weeks to 62.5 ppm BD produce a mutagenic effect in female rats. To evaluate the effect of age on mutagenic response, mutant manifestation curves for splenic T cells of female mice exposed at 8-9 weeks of age were defined by measuring Hprt mutant frequencies (MFs) at multiple time points after BD exposure using a T cell cloning assay and comparing the resulting mutagenic potency estimate (calculated as the difference of areas under the mutant manifestation curves of treated versus control animals) to that reported for female mice exposed to BD in the same fashion beginning at 4-5 weeks of age. The shapes of the mutant T cell manifestation curves for spleens were different [e.g., the maximum BD-induced MFs in older mice (8.0+/-1.0 [S.D.]x10(-6)) and younger mice (17.8+/-6.1 x 10(-6)) were observed at 8 and 5 weeks post-exposure, respectively], but the mutagenic burden was the same for both age groups. To assess the effect of gender on mutagenic response, female and male rodents were exposed to BD at 4-5 weeks of age and Hprt MFs were measured when maximum MFs are expected to occur post-exposure. The resulting data demonstrated that the pattern for mutagenic susceptibility from high-level BD exposure is female mice>male mice>female rats>male rats. Exposures of female rats to 62.5 ppm BD caused a minor but significant mutagenic response compared with controls (n=16/group; P=0.03). These results help explain part of the differing outcomes/interpretations of data in earlier Hprt mutation studies in BD-exposed rodents.     

Applications of a microplate reader in yeast physiology research

Microplate readers have been useful assistants of researchers for several decades. This work is focused on the applications of a simple absorbance microplate reader in yeast physiology research, and its advantages and limitations in comparison with alternative methods are discussed. The two main procedures involved are measuring growth curves and monitoring the pH changes of medium using two different pH indicators. We suggest mathematical formulas for converting absorbance data into pH values. With a microplate reader as many as 96 samples can be simultaneously analyzed, while medium consumption is minimized to 100 microL per sample. The results can be observed in 24-48 h (for growth curves) or in 1-3 h (for pH changes) with minimal hands-on time required.     

A Bayesian model for sparse functional data

Summary.   We propose a method for analyzing data which consist of curves on multiple individuals, i.e., longitudinal or functional data. We use a Bayesian model where curves are expressed as linear combinations of B-splines with random coefficients. The curves are estimated as posterior means obtained via Markov chain Monte Carlo (MCMC) methods, which automatically select the local level of smoothing. The method is applicable to situations where curves are sampled sparsely and/or at irregular time points. We construct posterior credible intervals for the mean curve and for the individual curves. This methodology provides unified, efficient, and flexible means for smoothing functional data.     

Identification and characterization of 4-hexylbenzoic acid and 4-nonyloxybenzoic acid as substrates of CYP102A1

CYP102A1 is an efficient medium- to long-chain fatty acid hydroxylase that is able to accept a wide range of non-natural substrates which bear no resemblance to the natural ones. 4-Hexylbenzoic acid (HBA) and 4-nonyloxybenzoic acid (NOBA) were identified as CYP102A1 substrates via screening studies using the BD Oxygen Biosensor System. Spectroscopic binding studies showed that these two substrates bind in the active site of CYP102A1 with K _d values of 2.6 ± 0.1 μM for HBA and 1.9 ± 0.2 μM for NOBA. NADPH consumption rates in the presence of HBA and NOBA were 45 ± 1 min⁻¹ and 61 ± 1 min⁻¹, respectively. The coupling efficiency for NADPH was 57% for NOBA, while it was 77% for HBA. During whole-cell biotransformations, HBA was converted into ω−1- and ω−2-hydroxyhexylbenzoic acid, whereas NOBA was oxidized to ω−2-hydroxynonyloxybenzoic acid and ω−2,ω−4-dihydroxynonyloxybenzoic acid. HBA was used as a fatty acid mimic to compare whole-cell biotransformations with cell-free extracts. Whole-cell biotransformations carried out in a biphasic system resulted in 86% conversion of 5 mM HBA, producing 3.8 mM ω−2- and 0.5 mM ω−1-hydroxyhexylbenzoic acid in 4 h with a turnover number of 4.1 min⁻¹, whereas 100% conversion of 5 mM HBA was obtained in 1 h with crude cell extracts and a cofactor regeneration system, giving a turnover number of 10.5 min⁻¹.     

The use of automated tubidimetric data for the construction of kinetic models

Summary An automated tubidimetric instrument (Bioscreen) was used to observe the growth response ofListeria monocytogenes to combinations of temperature (15–30°C), hydrogen-ion (0.1–21.9 m) (equivalent pH 4.66–7.0) and NaCl concentration (0.5–9.5% w/v). Compared to traditional plate count techniques, the technique allowed many more data points to be captured and replicates to be used, with less expenditure of effort. Optical density curves were filtered (smoothed) to minimize the effect of signal noise and the mean signal from uninoculated wells was subtracted to minimize the effect of signal draft. A novel procedure for fitting growth curves to optical density data has been developed. The procedure involves the use of the logistic function and a calibration equation for fitting, in a single step, in the dimension of optical density. This approach allowed the four parameters of the logistic equation to be derived at each set of experimental conditions. A quadratic response surface was then fitted to the curve parameters using temperature, NaCl and hydrogen-ion concentration as three independent variables. Predicted time to 1000-fold increase in cell numbers compared well to predictions from predictive microbial growth equations generated in other laboratories using traditional plate counting. We propose that this technique should be further evaluated as a method for generating data for modeling the kinetics of microbial growth.Mention of brand or firm names does not constitute an endorsement by the US Department of Agriculture over others of a similar nature not mentioned.     

Exopolysaccharide Distribution of and Bioemulsifier Production by Acinetobacter calcoaceticus BD4 and BD413

The heavily encapsulated Acinetobacter calcoaceticus BD4 and the “miniencapsulated” single-step mutant A. calcoaceticus BD413 produced extracellular polysaccharides in addition to the capsular material. The molar ratio of rhamnose to glucose (3:1) in the extracellular BD413 polysaccharide fraction was similar to the composition of the capsular material. In both strains, the increase in capsular polysaccharide was parallel to cell growth and remained constant in stationary phase. The extracellular polysaccharides were detected starting from mid-logarithmic phase and continued to accumulate in the growth medium for 5 to 8 h after the onset of stationary phase. Strain BD413 produced one-fourth the total rhamnose exopolysaccharide per cell that strain BD4 did. Depending on the growth medium, 32 to 63% of the rhamnose polysaccharide produced by strain BD413 was extracellular, whereas in strain BD4 only 7 to 14% was extracellular. In all cases, strain BD413 produced more extracellular rhamnose polysaccharide than strain BD4 did. In glucose medium, strain BD413 also produced approximately 10 times more extracellular emulsifying activity than strain BD4 did. The isolated capsular polysaccharide obtained after shearing of BD4 cells showed no emulsifying activity. Thus, strain BD413 either produces a modified extracellular polysaccharide or excretes an additional substance(s) that is responsible for the emulsifying activity. Emulsions induced by the ammonium sulfate-precipitated BD413 extracellular emulsifier require the presence of magnesium ion and a mixture of an aliphatic and an aromatic hydrocarbon.     

Automated measurements of antilisterial activities of lactate and diacetate in ready-to-eat meat

Standard procedures to enumerate Listeria organisms rely on plating food samples on selective agar media. The procedures are labor-intensive, and because of the limited sensitivity, pre-enrichment step is required for the detection of low numbers of the pathogen. In the present study, an automated rapid optic procedure and the standard procedure were used to determine the behavior of the pathogen in ready-to-eat (RTE) meat and to test the effect of antilisterial agents. Listeria monocytogenes strain Scott A or a six-strain mixture of Listeria was studied using lactate (2.5%), diacetate (0.2%) and their combination in beef bologna and in sterile beef emulsion. Samples stored for up to 60 days at 5 and 10 degrees C were tested at time intervals during storage. Using the plate count method, each of the salts caused a delay in growth of the pathogen, and the salt combination was most effective causing listeriostatic effects and decline in growth of the pathogen at 5 degrees C. High negative correlation (r), ranging from 0.92 to 0.99, was obtained between the detection time (DT) recorded by the optic procedure (BioSys instrument) and cell numbers determined by the plate count procedure. The rapid (< 24 h) optic procedure was reliable in assessing the efficacy of antimicrobials and in rapid detection of low levels of listeriae that are undetectable by direct plating procedure.     

Prey selection and efficiency of naïve and experienced juvenile sockeye salmon

Prey selection and growth efficiency of juvenile sockeye salmon Oncorhynchus nerka switched between live prey and pelleted diets were investigated. First feeding sockeye salmon fry were placed into one of three dietary treatments for 7 months prior to assessing potential differences with a growth and a behavioural assay. Dietary treatments were (1) adult Artemia franciscana for 1 month, followed by pelleted feed for an additional 6 months ( Art − BD), (2) pelleted feed from first feeding for 7 months (BD) and (3) adult A. franciscana for 1 month, and a combination of pelleted feed and live adult A. franciscana for 6 months ( Art + BD). Equal numbers from each treatment group were then tagged, pooled into replicate 'common garden' tanks and fed novel live prey items ( Daphnia sp. and mosquito Culex pipiens larvae) for an additional 3 weeks. No significant differences in the growth efficiency of sockeye salmon were found during the 3 week feeding trial on the novel prey items. Additional sockeye salmon from each dietary treatment were used in a behavioural assay to determine if the treatments had an impact on foraging efficiency (prey selection or time to capture prey). No significant differences in prey selection were found among treatment groups in time to capture pellets, A. franciscana or mosquito larvae. Also, no significant differences were found within treatment groups in time to capture different food sources. No substantive benefits in foraging efficiency of sockeye salmon associated with prior exposure to live prey were demonstrated. This suggests that altering existing hatchery practices for juvenile sockeye salmon by offering live food prior to release is unlikely to influence post-hatchery feeding behaviour or increase post-release survival.     

Mutagenicity of 1,3-butadiene at the Hprt locus of T-lymphocytes following inhalation exposures of female mice and rats.

The species specific response to 1,3-butadiene (BD), an important industrial chemical, was investigated by determining the influence of exposure duration and exposure concentration on the mutagenicity of BD in mice and rats and by defining the spectra of mutations in the Hprt gene T-cell mutants from control and BD-exposed mice. Female B6C3F1 mice and F344 rats (4-5 weeks old) were exposed by inhalation to 0, 20, 62.5, or 625 ppm of BD for up to 4 weeks (6 h/day, 5 days/week). Groups of control and exposed animals (n=4-12/group) were necropsied at multiple time points after exposure and the T-cell cloning assay was used to measure Hprt mutant frequencies in lymphocytes isolated from spleen. Mutant clones collected from control and BD-exposed mice were propagated and analyzed by RT-PCR to produce Hprt cDNA for sequencing. In animals necropsied 4 weeks after 2 or 4 weeks of BD exposure (0 or 625 ppm), the rate of accumulation of mutations was greater in mice than in rats. Supra-linear dose-response curves were observed in BD-exposed mice, indicating a higher efficiency of mutant induction at lower concentrations of BD. The mutagenic potency estimates (represented by the differences in the areas under the mutant T-cell 'manifestation' curves of treated vs. control animals) in mice were 11 and 61 following 4 weeks of exposures to 62.5 and 625 ppm of BD, respectively, while mutant frequencies (Mfs) in rats were significantly increased only at 625 ppm BD (mutagenic potency of 7). Molecular analysis of Hprt cDNA from expanded T-cell clones from control and BD-exposed mice demonstrated an increased frequency of mutants in exposed animals that likely contain large deletions in the Hprt gene (P=0.016). These data indicate that both exposure duration and exposure concentration are important in determining the magnitude of mutagenic response to BD, and that mutagenic and carcinogenic properties of BD in mice may be related more to the ability of its metabolites to cause chromosomal deletions than to produce point mutations.