A simple micro-growth assay for enumerating bacteria

Brewster, Jeffrey D.

doi:10.1016/s0167-7012(02)00226-9

Cited by 94 publications

(93 citation statements)

References 9 publications

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“…The inoculated plates were then incubated in the Synergy H1 hybrid reader at 37°C with a 1-min period of shaking every 10 min. The OD 660 of each well was recorded every 10 min for a period of 40 h. To prevent condensation, the lids of the microtiter plates were precoated with 10 ml of 0.05% Triton X-100 in 20% ethanol for 30 min and air dried (39).…”

Section: Methodsmentioning

confidence: 99%

Secreted Pyomelanin of Legionella pneumophila Promotes Bacterial Iron Uptake and Growth under Iron-Limiting Conditions

et al. 2013

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Iron acquisition is critical to the growth and virulence of Legionella pneumophila. Previously, we found that L. pneumophila uses both a ferrisiderophore pathway and ferrous iron transport to obtain iron. We now report that two molecules secreted by L. pneumophila, homogentisic acid (HGA) and its polymerized variant (HGA-melanin, a pyomelanin), are able to directly mediate the reduction of various ferric iron salts. Furthermore, HGA, synthetic HGA-melanin, and HGA-melanin derived from bacterial supernatants enhanced the ability of L. pneumophila and other species of Legionella to take up radiolabeled iron. Enhanced iron uptake was not observed with a ferrous iron transport mutant. Thus, HGA and HGA-melanin mediate ferric iron reduction, with the resulting ferrous iron being available to the bacterium for uptake. Upon further testing of L. pneumophila culture supernatants, we found that significant amounts of ferric and ferrous iron were associated with secreted HGA-melanin. Importantly, a pyomelanin-containing fraction obtained from a wild-type culture supernatant was able to stimulate the growth of iron-starved legionellae. That the corresponding supernatant fraction obtained from a nonpigmented mutant culture did not stimulate growth demonstrated that HGA-melanin is able to both promote iron uptake and enhance growth under iron-limiting conditions. Indicative of a complementary role in iron acquisition, HGA-melanin levels were inversely related to the levels of siderophore activity. Compatible with a role in the ecology and pathogenesis of L. pneumophila, HGA and HGA-melanin were effective at reducing and releasing iron from both insoluble ferric hydroxide and the mammalian iron chelates ferritin and transferrin.

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Section: Methodsmentioning

confidence: 99%

Secreted Pyomelanin of Legionella pneumophila Promotes Bacterial Iron Uptake and Growth under Iron-Limiting Conditions

et al. 2013

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“…Growth rates of duplicate cultures of each strain were compared using the microgrowth assay developed by Brewster (2003). The cell density of each strain was adjusted to the same starting concentration, and cell density measurements (optical density at 595 nm [OD 595 ]) were taken every hour using a Wallac 1420 Victor 2 spectrophotometer (PerkinElmer Life Sciences, USA).…”

Section: Gfp Labeling Of Vibrio Coralliilyticusmentioning

confidence: 99%

Visualization of coral host–pathogen interactions using a stable GFP-labeled Vibrio coralliilyticus strain

et al. 2015

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The bacterium Vibrio coralliilyticus has been implicated as the causative agent of coral tissue loss diseases (collectively known as white syndromes) at sites across the Indo-Pacific and represents an emerging model pathogen for understanding the mechanisms linking bacterial infection and coral disease. In this study, we used a mini-Tn7 transposon delivery system to chromosomally label a strain of V. coralliilyticus isolated from a white syndrome disease lesion with a green fluorescent protein gene (GFP). We then tested the utility of this modified strain as a research tool for studies of coral host-pathogen interactions. A suite of biochemical assays and experimental infection trials in a range of model organisms confirmed that insertion of the GFP gene did not interfere with the labeled strain's virulence. Using epifluorescence video microscopy, the GFP-labeled strain could be reliably distinguished from non-labeled bacteria present in the coral holobiont, and the pathogen's interactions with the coral host could be visualized in real time. This study demonstrates that chromosomal GFP labeling is a useful technique for visualization and tracking of coral pathogens and provides a novel tool to investigate the role of V. coralliilyticus in coral disease pathogenesis.

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“…Therefore, the method presented in this study is faster, as it requires about 30 min of work for analysis of a 96-well plate plus the incubation of the dye, and is comparable in terms of sensitivity to flow cytometry (13) or microgrowth assays (7). Since it requires only small sample volumes, the remaining sample volume can be used for subculturing or chemical or molecular analysis.…”

Section: Vol 72 2006 Nucleic Acid Staining For Microbial Growth Detmentioning

confidence: 99%

Sensitive Determination of Microbial Growth by Nucleic Acid Staining in Aqueous Suspension

Martens‐Habbena

Sass

2006

Appl Environ Microbiol

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The determination of cell numbers or biomass in laboratory cultures or environmental samples is usually based on turbidity measurements, viable counts, biochemical determinations (e.g., protein and lipid measurements), microscopic counting, or recently, flow cytometric analysis. In the present study, we developed a novel procedure for the sensitive quantification of microbial cells in cultures and most-probable-number series. The assay combines fluorescent nucleic acid staining and subsequent fluorescence measurement in suspension. Six different fluorescent dyes (acridine orange, DAPI [4,6-diamidino-2-phenylindole], ethidium bromide, PicoGreen, and SYBR green I and II) were evaluated. SYBR green I was found to be the most sensitive dye and allowed the quantification of 50,000 to up to 1.5 ؋ 10 8 Escherichia coli cells per ml sample. The rapid staining procedure was robust against interference from rRNA, sample fixation by the addition of glutaric dialdehyde, and reducing agents such as sodium dithionite, sodium sulfide, and ferrous sulfide. It worked well with phylogenetically distant bacterial and archaeal strains. Excellent agreement with optical density measurements of cell increases was achieved during growth experiments performed with aerobic and sulfate-reducing bacteria. The assay offers a time-saving, more sensitive alternative to epifluorescence microscopy analysis of most-probable-number dilution series. This method simplifies the quantification of microbial cells in pure cultures as well as enrichments and is particularly suited for low cell densities.

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A simple micro-growth assay for enumerating bacteria

Cited by 94 publications

References 9 publications

Secreted Pyomelanin of Legionella pneumophila Promotes Bacterial Iron Uptake and Growth under Iron-Limiting Conditions

Secreted Pyomelanin of Legionella pneumophila Promotes Bacterial Iron Uptake and Growth under Iron-Limiting Conditions

Visualization of coral host–pathogen interactions using a stable GFP-labeled Vibrio coralliilyticus strain

Sensitive Determination of Microbial Growth by Nucleic Acid Staining in Aqueous Suspension

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