Miniaturized kinetic growth inhibition assay with denitrifying bacteria Paracoccus denitrificans

Koutný, Marek; Zaorálková, Linda

doi:10.1016/j.chemosphere.2005.01.007

Cited by 10 publications

(5 citation statements)

References 8 publications

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“…A directly comparable, classical high cell density inhibition test on strain SV3 based on culture turbidity measurements also showed naphthalene-dependent growth rate reduction, but less severe than in the oligotrophic test (see Figure S7, Supporting Information). Similarly, population average growth reduction inferred for the other compounds tested here falls in the concentration range of what has been reported for other high cell density/high nutrient tests, although we realize that there are important strain to strain differences in sensitivity. − As an alternative to the population average growth reduction, one could deduce LC 50 values on the basis of increased proportions of cells in the membrane-compromised gate (Figure ).…”

Section: Discussionsupporting

confidence: 72%

“…Toxicant-induced reproduction inhibition tests are common in ecotoxicology using microplate or regular batch cultured microorganisms under standardized high nutrient conditions. ,,− Such tests measure average growth behavior for all individuals in the culture either as midexponential phase growth rates in the presence or absence of toxicants − or as the amount of biomass formed. , Here we demonstrate that average population growth rates upon toxicant exposure are a reflection of subpopulations that divide with possibly normal generation times and those with injured cells that may or may not reproduce at all. Moreover, we show that even in the absence of population growth rate effects there is a tendency for the proportions of subpopulations of injured cells to increase at higher toxicant dosages in the assay (Figure ).…”

Section: Discussionmentioning

confidence: 76%

“…Toxicity of chemical pollutants to organisms in aquatic systems is regularly assessed by a variety of different methods. − Such methods can involve physiological, behavioral, or molecular methods on multicellular organisms present or not at a site (“biomarkers”) , or, for instance, entail examination of enzymatic activity or de novo gene expression in test organisms under well-defined conditions (e.g., Microtox, umu-test). ,, Other methods directly interrogate toxicant-induced reproductive effects on microbial test species with rapid generation times, while scoring the decrease of population growth rate or biomass yield as a consequence of chemical exposure. ,− Such tests are typically carried out in replicate microplate or regular suspended batch cultures incubated under standardized nutrient conditions, mostly with high cell numbers and optimal high concentrations of nutrients. ,− Exposure effects are translated as the toxicant concentration at which the maximum specific growth rate of the population (i.e., the growth rate calculated during exponential phase) is 50% reduced compared to that in nonexposed cultures growing in the same medium conditions but without toxicants (e.g., EC 50 ). − …”

Section: Introductionmentioning

confidence: 99%

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A Flow Cytometry Based Oligotrophic Pollutant Exposure Test To Detect Bacterial Growth Inhibition and Cell Injury

Czechowska

Meer

2011

Environ. Sci. Technol.

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Toxicity of chemical pollutants in aquatic environments is often addressed by assays that inquire reproductive inhibition of test microorganisms, such as algae or bacteria. Those tests, however, assess growth of populations as a whole via macroscopic methods such as culture turbidity or colony-forming units. Here we use flow cytometry to interrogate the fate of individual cells in low-density populations of the bacterium Pseudomonas fluorescens SV3 exposed or not under oligotrophic conditions to a number of common pollutants, some of which derive from oil contamination. Cells were stained at regular time intervals during the exposure assay with fluorescent dyes that detect membrane injury (i.e., live-dead assay). Reduction of population growth rates was observed upon toxicant insult and depended on the type of toxicant. Modeling and cell staining indicate that population growth rate decrease is a combined effect of an increased number of injured cells that may or may not multiply, and live cells dividing at normal growth rates. The oligotrophic assay concept presented here could be a useful complement for existing biomarker assays in compliance with new regulations on chemical effect studies or, more specifically, for judging recovery after exposure to fluctuating toxicant conditions.

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Section: Discussionsupporting

confidence: 72%

Section: Discussionmentioning

confidence: 76%

Section: Introductionmentioning

confidence: 99%

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A Flow Cytometry Based Oligotrophic Pollutant Exposure Test To Detect Bacterial Growth Inhibition and Cell Injury

Czechowska

Meer

2011

Environ. Sci. Technol.

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“…Several approaches have been used to circumvent the low-throughput nature of anaerobe cultivation. One approach excluded oxygen by sealing lids onto microtiter plates and flushing the headspace with N 2 to form anaerobic conditions (Koutny and Zaoralkova 2005). A second approach enzymatically removed oxygen from the culture medium ahead of growth analysis (Lam et al 2018).…”

Section: Introductionmentioning

confidence: 99%

Cultivating efficiency: High-throughput growth analysis of anaerobic bacteria in compact microplate readers

Snoeyenbos-West,

Guerrero,

Valencia

et al. 2023

Preprint

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Anaerobic microbes play crucial roles in environmental processes, industry, and human health. Traditional methods for monitoring the growth of anaerobes, including plate counts or subsampling broth cultures for optical density measurements, are time and resource intensive. The advent of microplate readers revolutionized bacterial growth studies by enabling high-throughput and real-time monitoring of microbial growth kinetics but their use in anaerobic microbiology has remained limited. Here, we present a workflow for using small-footprint microplate readers and the Growthcurver R package to analyze the kinetic growth metrics of anaerobic bacteria. We benchmarked the small-footprint Cerillo Stratus microplate reader against a BioTek Synergy HTX microplate reader in aerobic conditions usingEscherichia coliDSM 28618 cultures. The growth rates and carrying capacities obtained from the two readers were statistically indistinguishable. However, the area under the logistic curve was significantly higher in cultures monitored by the Stratus reader. We used the Stratus to quantify the growth responses of anaerobically grownE. coliandClostridium bolteaeDSM 29485 to different doses of the toxin sodium arsenite. The growth ofE. coliandC. bolteaewas sensitive to arsenite doses of 1.3 μM and 0.4 μM, respectively. Complete inhibition of growth was achieved at 38 μM arsenite forC. bolteae, and 338 μM inE. coli. These results show that the Stratus performs similarly to a leading brand of microplate reader and can be reliably used in anaerobic conditions. We discuss the advantages of the small format microplate readers and our experiences with the Stratus.Importance statementWe present a workflow that facilitates the production and analysis of growth curves for anaerobic microbes using small-footprint microplate readers and an R script. This workflow is a cost and space-effective solution to most high-throughput solutions for collecting growth data from anaerobic microbes. This technology can be used for applications in which high-throughput would advance discovery, including microbial isolation, bioprospecting, co-culturing, host-microbe interactions, and drug/toxin-microbial interactions.

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“…In a study on growth inhibition, Salmonella enteriditis, Escherichia coli, and Paracoccus denitrificans were grown on plates in a variety of media under anaerobic conditions (Brewster, 2003;Koutny and Zaoralkova, 2005), The lids were sealed onto plates with silicone and then flushed with nitrogen to remove oxygen. Problems with condensation during incubation were overcome with the use of anti-fogging agents on the lids.…”

Section: Previous Work: Anaerobic Growth On Microtiter Platesmentioning

confidence: 99%

Sulfate-Reducing Bacteria

大毛宏喜¹,

竹末芳生²,

末田泰二郎³

et al.

SpringerReference

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Growing anaerobic microorganisms in phenotypic microarrays (PM) and 96-well microtiter plates is an emerging technology that allows high throughput survey of the growth and physiology and/or phenotype of cultivable microorganisms. For non-model bacteria, a method for phenotypic analysis is invaluable, not only to serve as a starting point for further evaluation, but also to provide a broad understanding of the physiology of an uncharacterized wild-type organism or the physiology/phenotype of a newly created mutant of that organism. Given recent advances in genetic characterization and targeted mutations to elucidate genetic networks and metabolic pathways, high-throughput methods for determining phenotypic differences are essential. Here we outline challenges presented in studying the physiology and phenotype of a sulfatereducing anaerobic delta proteobacterium, Desulfovibrio vulgaris Hildenborough. Modifications of the commercially available OmniLog™ system (Hayward, CA) for experimental setup, and configuration, as well as considerations in PM data analysis are presented. Also highlighted here is data viewing software that enables users to view and compare multiple PM data sets. The PM method promises to be a valuable strategy in our systems biology approach to D. vulgaris studies and is readily applicable to other anaerobic and aerobic bacteria.Published by Elsevier B.V.

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Miniaturized kinetic growth inhibition assay with denitrifying bacteria Paracoccus denitrificans

Cited by 10 publications

References 8 publications

A Flow Cytometry Based Oligotrophic Pollutant Exposure Test To Detect Bacterial Growth Inhibition and Cell Injury

A Flow Cytometry Based Oligotrophic Pollutant Exposure Test To Detect Bacterial Growth Inhibition and Cell Injury

Cultivating efficiency: High-throughput growth analysis of anaerobic bacteria in compact microplate readers

Sulfate-Reducing Bacteria

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