A flow cytometry method for bacterial quantification and biomass estimates in activated sludge

Brown, Mathew; Hands, Catherine Lauren; Coello-Garcia, T.; Sani, B. M.; Ott, Amelie; Smith, S.J.; Davenport, Russell J.

doi:10.1016/j.mimet.2019.03.022

Cited by 43 publications

(27 citation statements)

References 53 publications

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“…It was calculated using Eq. (1) 56 , assuming a bacterial biovolume (V) of 0.25 μm 3 cell −1 , a carbon content per unit of cell volume (Cs) of 310 fg C μm −3 and considering that only ~53% of a cell’s dry weight constitutes carbon. …”

Section: Methodsmentioning

confidence: 99%

Extended spectrum β-lactamase and carbapenemase genes are substantially and sequentially reduced during conveyance and treatment of urban sewage

Nesme

Quintela-Baluja

et al. 2020

Preprint

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Integrated and quantitative observations of antibiotic resistance genes (ARGs) in urban water systems (UWSs) are lacking. We sampled three UWSs for clinically important extended spectrum β-lactamase (ESBL) and carbapenemase (CP) genes, mobile genetic elements and microbial communities. Sewage – especially from hospitals – carried substantial loads of ESBL and CP genes (106 – 107 per person equivalent), but those loads progressively declined along the UWS, resulting in minimal emissions (101 – 104 copies per person equivalent). Removal was primarily during sewage conveyance (65% ± 36%) rather than within sewage treatment (34% ± 23%). The ARGs clustered in groups based on their persistence; less persistent groups were associated to putative host taxa (especially Enterobacteriaceae and Moraxellaceae), while more persistent groups appear horizontally transferred as they correlated with mobile genetic elements. This first documentation of a substantial ARG reduction during sewage conveyance provides opportunities for antibiotic resistance management and a caution for sewage-based ARG surveillance.

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Section: Methodsmentioning

confidence: 99%

Extended spectrum β-lactamase and carbapenemase genes are substantially and sequentially reduced during conveyance and treatment of urban sewage

Nesme

Quintela-Baluja

et al. 2020

Preprint

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“…However, for microbiological applications, it remains difficult in practice, and there is no consensus yet on how to best handle cell aggregate signals. We recommend the optimization of sample preparation protocols to reduce the percentage of cell aggregates; these can include the use of filtration, ultrasonication, surfactants (e.g., Tween, Triton X-100), complexing agents (e.g., EDTA, sodium pyrophosphate), and/or Ca 21 /Mg 21 -free buffers (4,40).…”

Section: Preprocessingmentioning

confidence: 99%

Computational Analysis of Microbial Flow Cytometry Data

Rubbens

Props

2021

mSystems

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Flow cytometry is an important technology for the study of microbial communities. It grants the ability to rapidly generate phenotypic single-cell data that are both quantitative, multivariate and of high temporal resolution. The complexity and amount of data necessitate an objective and streamlined data processing workflow that extends beyond commercial instrument software. No full overview of the necessary steps regarding the computational analysis of microbial flow cytometry data currently exists. In this review, we provide an overview of the full data analysis pipeline, ranging from measurement to data interpretation, tailored toward studies in microbial ecology. At every step, we highlight computational methods that are potentially useful, for which we provide a short nontechnical description. We place this overview in the context of a number of open challenges to the field and offer further motivation for the use of standardized flow cytometry in microbial ecology research.

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“…The nucleic acid dye SYTO9 (ThermoFisher Scientific), which has been shown to preferentially stain live and dead total bacteria cells, was tested at varying dilutions (1:80, 1:500, 1:1000 v/v of the dye's stock solution and dimethyl sulfoxide (DMSO)) 28,29 . All dilutions were tested using triplicate positive (stained) and negative controls (no stain).…”

Section: Flow Cytometry Staining Optimizationmentioning

confidence: 99%

“…All dilutions were tested using triplicate positive (stained) and negative controls (no stain). After stain optimisation using an analogue river water sample, all aliquots were subsequently stained using 0.2 µL of SYTO9 (1:1000 v/v dilution of commercial stock solution with DMSO) and incubated for 15 minutes in the dark at room temperature prior to analysis 28 . Note the differentiation and quantification of both live and dead cells, using Propidium Iodide, was purposely omitted, since sequencing typically incorporates both live and dead cells, and the results were aimed at determining the total number of bacteria cells (live and dead) present in the sample.…”

Section: Flow Cytometry Staining Optimizationmentioning

confidence: 99%

A novel real-world ecotoxicological dataset of pelagic microbial community responses to wastewater

et al. 2020

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Real-world observational datasets that record and quantify pressure-stressor-response linkages between effluent discharges and natural aquatic systems are rare. With global wastewater volumes increasing at unprecedented rates, it is urgent that the present dataset is available to provide the necessary information about microbial community structure and functioning. Field studies were performed at two time-points in the austral summer. Single-species and microbial community whole effluent toxicity (WET) testing was performed at a complete range of effluent concentrations and two salinities, with accompanying environmental data to provide new insights into nutrient and organic matter cycling, and to identify ecotoxicological tipping points. the two salinity regimes were chosen to investigate future scenarios based on a predicted salinity increase at the study site, typical of coastal regions with rising sea levels globally. Flow cytometry, amplicon sequencing of 16S and 18S rRNA genes and micro-fluidic quantitative polymerase-chain reactions (MFQPCR) were used to determine chlorophyll-a and total bacterial cell numbers and size, as well as taxonomic and functional diversity of pelagic microbial communities. this strong pilot dataset could be replicated in other regions globally and would be of high value to scientists and engineers to support the next advances in microbial ecotoxicology, environmental biomonitoring and estuarine water quality modelling.

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A flow cytometry method for bacterial quantification and biomass estimates in activated sludge

Cited by 43 publications

References 53 publications

Extended spectrum β-lactamase and carbapenemase genes are substantially and sequentially reduced during conveyance and treatment of urban sewage

Extended spectrum β-lactamase and carbapenemase genes are substantially and sequentially reduced during conveyance and treatment of urban sewage

Computational Analysis of Microbial Flow Cytometry Data

A novel real-world ecotoxicological dataset of pelagic microbial community responses to wastewater

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