Dataset Information

Evaluating the Effects of Disinfectants on Bacterial Biofilms Using a Microfluidics Flow Cell and Time-Lapse Fluorescence Microscopy.

ABSTRACT: A commercially available microfluidics flow cell was utilized together with widefield fluorescence microscopy to evaluate the effects of disinfectants on bacterial strains. The flow cell's inner surface supports the formation of biofilms of numerous bacterial species. The modular setup of the flow cell accessories allows connection to syringes, pumps and collection vials, facilitating aseptic experiments in a controlled fluidics environment which can be documented with precisely timed microscopy imaging. The flow cell is inoculated with a suspension of bacteria in a nutrient medium and incubated for several days allowing bacterial cells to form a biofilm. Shortly before performing an assay, the biofilm is labelled with a dual-fluorescent DNA probe which distinguishes unharmed and damaged bacteria. Then a disinfectant sample (or control) is gently injected and time-lapse imaging is used for quantifying the course of bacterial biomass response. We use a simplified widefield microscopy method that allows intensive recording and quantification of time series of two-dimensional frames for tracking the course of disinfectant action on a variety of microbial strains. This procedure has potential for the rapid evaluation of novel products.

SUBMITTER: Legner M

PROVIDER: S-EPMC7700140 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Evaluating the Effects of Disinfectants on Bacterial Biofilms Using a Microfluidics Flow Cell and Time-Lapse Fluorescence Microscopy.

Legner Milos M Jonkman James J Swift Dean D

Microorganisms 20201122 11

A commercially available microfluidics flow cell was utilized together with widefield fluorescence microscopy to evaluate the effects of disinfectants on bacterial strains. The flow cell's inner surface supports the formation of biofilms of numerous bacterial species. The modular setup of the flow cell accessories allows connection to syringes, pumps and collection vials, facilitating aseptic experiments in a controlled fluidics environment which can be documented with precisely timed microscopy ...[more]

PMID: 33266442

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Project description:BACKGROUND:Clonal microbial populations often harbor rare phenotypic variants that are typically hidden within the majority of the remaining cells, but are crucial for the population's resilience to external perturbations. Persister and viable but non-culturable (VBNC) cells are two important clonal bacterial subpopulations that can survive antibiotic treatment. Both persister and VBNC cells pose a serious threat to human health. However, unlike persister cells, which quickly resume growth following drug removal, VBNC cells can remain non-growing for prolonged periods of time, thus eluding detection via traditional microbiological assays. Therefore, understanding the molecular mechanisms underlying the formation of VBNC cells requires the characterization of the clonal population with single-cell resolution. A combination of microfluidics, time-lapse microscopy, and fluorescent reporter strains offers the perfect platform for investigating individual cells while manipulating their environment. METHODS:Here, we report a novel single-cell approach to investigate VBNC cells. We perform drug treatment, bacterial culturing, and live/dead staining in series by using transcriptional reporter strains and novel adaptations to the mother machine technology. Since we track each cell throughout the experiment, we are able to quantify the size, morphology and fluorescence that each VBNC cell displayed before, during and after drug treatment. RESULTS:We show that VBNC cells are not dead or dying cells but share similar phenotypic features with persister cells, suggesting a link between these two subpopulations, at least in the Escherichia coli strain under investigation. We strengthen this link by demonstrating that, before drug treatment, both persister and VBNC cells can be distinguished from the remainder of the population by their lower fluorescence when using a reporter strain for tnaC, encoding the leader peptide of the tnaCAB operon responsible for tryptophan metabolism. CONCLUSION:Our data demonstrates the suitability of our approach for studying the physiology of non-growing cells in response to external perturbations. Our approach will allow the identification of novel biomarkers for the isolation of VBNC and persister cells and will open new opportunities to map the detailed biochemical makeup of these clonal subpopulations.

Project description:BackgroundThe software available to date for analyzing image sequences from time-lapse microscopy works only for certain bacteria and under limited conditions. These programs, mostly MATLAB-based, fail for microbes with irregular shape, indistinct cell division sites, or that grow in closely packed microcolonies. Unfortunately, many organisms of interest have these characteristics, and analyzing their image sequences has been limited to time consuming manual processing.ResultsHere we describe BactImAS - a modular, multi-platform, open-source, Java-based software delivered both as a standalone program and as a plugin for Icy. The software is designed for extracting and visualizing quantitative data from bacterial time-lapse movies. BactImAS uses a semi-automated approach where the user defines initial cells, identifies cell division events, and, if necessary, manually corrects cell segmentation with the help of user-friendly GUI and incorporated ImageJ application. The program segments and tracks cells using a newly-developed algorithm designed for movies with difficult-to-segment cells that exhibit small frame-to-frame differences. Measurements are extracted from images in a configurable, automated fashion and an SQLite database is used to store, retrieve, and exchange all acquired data. Finally, the BactImAS can generate configurable lineage tree visualizations and export data as CSV files. We tested BactImAS on time-lapse movies of Mycobacterium smegmatis and achieved at least 10-fold reduction of processing time compared to manual analysis. We illustrate the power of the visualization tool by showing heterogeneity of both icl expression and cell growth atop of a lineage tree.ConclusionsThe presented software simplifies quantitative analysis of time-lapse movies overall and is currently the only available software for the analysis of mycobacteria-like cells. It will be of interest to the community of both end-users and developers of time-lapse microscopy software.

Dataset Information

Evaluating the Effects of Disinfectants on Bacterial Biofilms Using a Microfluidics Flow Cell and Time-Lapse Fluorescence Microscopy.

Publications

Evaluating the Effects of Disinfectants on Bacterial Biofilms Using a Microfluidics Flow Cell and Time-Lapse Fluorescence Microscopy.

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