2018
DOI: 10.1371/journal.pone.0193267
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Fast and simple tool for the quantification of biofilm-embedded cells sub-populations from fluorescent microscopic images

Abstract: Fluorescent staining is a common tool for both quantitative and qualitative assessment of pro- and eukaryotic cells sub-population fractions by using microscopy and flow cytometry. However, direct cell counting by flow cytometry is often limited, for example when working with cells rigidly adhered either to each other or to external surfaces like bacterial biofilms or adherent cell lines and tissue samples. An alternative approach is provided by using fluorescent microscopy and confocal laser scanning microsco… Show more

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Cited by 84 publications
(39 citation statements)
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“…The biofilms were imaged with confocal laser scanning microscopy (CLSM; A1R, Nikon, Tokyo, Japan) and biofilm thickness quantified using ImageJ (NIH, Bethesda, MD) by referencing at least 4 different images per condition. The proportions of live and dead bacteria were also quantified using BioFilmAnalyzer version 1.0 (https://bitbucket.org/rogex/biofilmanalyzer/downloads/) by counting fluorescence‐specific pixels in digital fluorescent images. Five different images were selected for analysis per condition.…”
Section: Methodsmentioning
confidence: 99%
“…The biofilms were imaged with confocal laser scanning microscopy (CLSM; A1R, Nikon, Tokyo, Japan) and biofilm thickness quantified using ImageJ (NIH, Bethesda, MD) by referencing at least 4 different images per condition. The proportions of live and dead bacteria were also quantified using BioFilmAnalyzer version 1.0 (https://bitbucket.org/rogex/biofilmanalyzer/downloads/) by counting fluorescence‐specific pixels in digital fluorescent images. Five different images were selected for analysis per condition.…”
Section: Methodsmentioning
confidence: 99%
“…The analysis of TEM images was based on the shape-based algorithm of globular vs. brillar structures selection 55 with subsequent sub-populations quantication using an in-house developed algorithm and soware. 56…”
Section: Discussionmentioning
confidence: 99%
“…Experiments were carried out in six biological repeats with newly prepated cultures and medium in each of them. The fraction of non-viable cells in microscopic images was estimated as the relative fraction of the red cells among all cells in the combined images obtained by overlaying of the green and the red fluorescence microphotographs (10 images per each sample) by using BioFilmAnalyzer software 92 .The statistical significance of the discrepancy between monoculture and mixed biofilms treatment efficacy was determined using the Kruskal-Wallis statistical test with significance threshold at p < 0.05.…”
Section: Methodsmentioning
confidence: 99%