Characterizing Microbiome Dynamics – Flow Cytometry Based Workflows from Pure Cultures to Natural Communities

ABSTRACT The distribution of DNA among bacterioplankton and bacterial isolates was determined by flow cytometry of DAPI (4′,6′-diamidino-2-phenylindole)-stained organisms. Conditions were optimized to minimize error from nonspecific staining, AT bias, DNA packing, changes in ionic strength, and differences in cell permeability. The sensitivity was sufficient to characterize the small 1- to 2-Mb-genome organisms in freshwater and seawater, as well as low-DNA cells (“dims”). The dims could be formed from laboratory cultivars; their apparent DNA content was 0.1 Mb and similar to that of many particles in seawater. Preservation with formaldehyde stabilized samples until analysis. Further permeabilization with Triton X-100 facilitated the penetration of stain into stain-resistant lithotrophs. The amount of DNA per cell determined by flow cytometry agreed with mean values obtained from spectrophotometric analyses of cultures. Correction for the DNA AT bias of the stain was made for bacterial isolates with known G+C contents. The number of chromosome copies per cell was determined with pure cultures, which allowed growth rate analyses based on cell cycle theory. The chromosome ratio was empirically related to the rate of growth, and the rate of growth was related to nutrient concentration through specific affinity theory to obtain a probe for nutrient kinetics. The chromosome size of aMarinobacter arcticus isolate was determined to be 3.0 Mb by this method. In a typical seawater sample the distribution of bacterial DNA revealed two major populations based on DNA content that were not necessarily similar to populations determined by using other stains or protocols. A mean value of 2.5 fg of DNA cell−1was obtained for a typical seawater sample, and 90% of the population contained more than 1.1 fg of DNA cell−1.

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Automated flow cytometry as a flexible tool for comparing disinfection characteristics of indigenous bacterial communities and pure cultures

Ecotoxicology and Environmental Safety ◽

10.1016/j.ecoenv.2021.112799 ◽

2021 ◽

Vol 225 ◽

pp. 112799

Author(s):

Guannan Mao ◽

Yingying Wang ◽

Frederik Hammes

Keyword(s):

Flow Cytometry ◽

Bacterial Communities ◽

Flexible Tool ◽

Pure Cultures

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Flow Cytometric Analysis of Freshwater Cyanobacteria: A Case Study

Water ◽

10.3390/w11071422 ◽

2019 ◽

Vol 11 (7) ◽

pp. 1422 ◽

Cited By ~ 1

Author(s):

Ruchit Patel ◽

Aline de Oliveira ◽

Robert Newby ◽

Tinchun Chu

Keyword(s):

Flow Cytometry ◽

Harmful Algal Blooms ◽

Algal Blooms ◽

Flow Cytometric Analysis ◽

Anthropogenic Sources ◽

Nitrogen And Phosphorus ◽

Flow Cytometric ◽

Freshwater Bodies ◽

Pure Cultures ◽

Cyanobacterial Harmful Algal Blooms

Eutrophication is a process that occurs due to the excessive accumulation of nutrients, primarily nitrogen and phosphorus, from natural and anthropogenic sources. This phenomenon causes cyanobacterial overgrowth, which over time leads to cyanobacterial harmful algal blooms (CHABs) that affect public drinking water sources and water sites with recreational usage. The rapid detection of bloom-forming cyanobacteria in freshwater bodies is critical in order to implement prevention strategies. Cyanobacteria contain phycobiliproteins such as phycoerythrin and allophycocyanin as part of the phycobilisome that allows autofluorescence. In this study, samples from 36 freshwater bodies in 14 New Jersey counties were collected and analyzed using flow cytometry with forward-scatter phycoerythrin and allophycocyanin parameters. Pure cultures of Synechococcus sp. IU 625, Cylindrospermum spp. and Microcystis aeruginosa were used as references. The results revealed that 17 out of the 36 analyzed sites contained all three references and related species. Seven sites showed Microcystis and Cylindrospermum-like species, while four sites indicated Microcystis and Cylindrospermum-like species. Six water bodies showed Cylindrospermum-like species, and two sites showed Microcystis-like species. Polymerase chain reaction (PCR)-based assays further confirmed the flow cytometric results. The findings from this study suggest that flow cytometry could potentially serve as a rapid method for freshwater cyanobacteria detection and screening.

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Use of Flow Cytometry To Follow the Physiological States of Microorganisms in Cider Fermentation Processes

Applied and Environmental Microbiology ◽

10.1128/aem.01183-06 ◽

2006 ◽

Vol 72 (10) ◽

pp. 6725-6733 ◽

Cited By ~ 35

Author(s):

Mónica Herrero ◽

Covadonga Quirós ◽

Luis A. García ◽

Mario Díaz

Keyword(s):

Flow Cytometry ◽

Fluorescent Dyes ◽

Malolactic Fermentation ◽

Stress Conditions ◽

Mixed Fermentation ◽

Fermentation Processes ◽

Sequential Inoculation ◽

Pure Cultures ◽

Synthetic Media ◽

Over Time

ABSTRACT The flow cytometry (FC) technique used with certain fluorescent dyes (ChemChrome V6 [CV6], DRAQ5, and PI) has proven useful to label and to detect different physiological states of yeast and malolactic bacterium starters conducting cider fermentation over time (by performing sequential inoculation of microorganisms). First, the technique was tested with pure cultures of both types of microorganisms grown in synthetic media under different induced stress conditions. Metabolically active cells detected by FC and by the standard plate-counting method for both types of microorganisms in fresh overnight pure cultures gave good correlations between the two techniques in samples taken at this stage. Otherwise, combining the results obtained by FC and plating during alcoholic and malolactic fermentation over time in the cider-making process, different subpopulations were detected, showing significant differences between the methods. A small number of studies have applied the FC technique to analyze fermentation processes and mixed cultures over time. The results were used to postulate equations explaining the different physiological states in cell populations taken from fresh, pure overnight cultures under nonstress conditions or cells subjected to stress conditions over time, either under a pure-culture fermentation process (in this work, corresponding to alcoholic fermentation) or under mixed-fermentation conditions (for the malolactic-fermentation phase), that could be useful to improve the control of the processes.

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