Application of Flow Cytometry for Measuring Changes in Intracellular RNA Contents of Marine Bacteria.

MASAHIKO NISHIMURA; ATSUSHI TSUDA; KAZUHIRO KOGURE; KOUICHI OHWADA

doi:10.1264/microbes1986.10.73

Application of Flow Cytometry for Measuring Changes in Intracellular RNA Contents of Marine Bacteria.

Bulletin of Japanese Society of Microbial Ecology ◽

10.1264/microbes1986.10.73 ◽

1995 ◽

Vol 10 (2) ◽

pp. 73-79 ◽

Cited By ~ 2

Author(s):

MASAHIKO NISHIMURA ◽

ATSUSHI TSUDA ◽

KAZUHIRO KOGURE ◽

KOUICHI OHWADA

Keyword(s):

Flow Cytometry ◽

Marine Bacteria

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Use of the 5-cyano-2,3-ditolyl tetrazolium chloride reduction test to assess respiring marine bacteria and grazing effects by flow cytometry during linear alkylbenzene sulfonate degradation

FEMS Microbiology Ecology ◽

10.1111/j.1574-6941.1998.tb00523.x ◽

1998 ◽

Vol 27 (1) ◽

pp. 33-42 ◽

Cited By ~ 5

Author(s):

Ricard LÃ³pez-AmorÃ³s ◽

Jaume Comas ◽

MarÄ±Ìa Teresa GarcÄ±Ìa ◽

Josep Vives-Rego

Keyword(s):

Flow Cytometry ◽

Marine Bacteria ◽

Linear Alkylbenzene Sulfonate ◽

Tetrazolium Chloride ◽

Linear Alkylbenzene ◽

Reduction Test ◽

Grazing Effects ◽

Alkylbenzene Sulfonate

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Use of the 5-cyano-2,3-ditolyl tetrazolium chloride reduction test to assess respiring marine bacteria and grazing effects by flow cytometry during linear alkylbenzene sulfonate degradation

FEMS Microbiology Ecology ◽

10.1016/s0168-6496(98)00053-1 ◽

1998 ◽

Vol 27 (1) ◽

pp. 33-42 ◽

Cited By ~ 7

Author(s):

R López-Amorós

Keyword(s):

Flow Cytometry ◽

Marine Bacteria ◽

Linear Alkylbenzene Sulfonate ◽

Tetrazolium Chloride ◽

Linear Alkylbenzene ◽

Reduction Test ◽

Grazing Effects ◽

Alkylbenzene Sulfonate

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Flow Cytometric Analysis of 5-Cyano-2,3-Ditolyl Tetrazolium Chloride Activity of Marine Bacterioplankton in Dilution Cultures

Applied and Environmental Microbiology ◽

10.1128/aem.65.6.2409-2417.1999 ◽

1999 ◽

Vol 65 (6) ◽

pp. 2409-2417 ◽

Cited By ~ 51

Author(s):

Michael E. Sieracki ◽

Terry L. Cucci ◽

Justyna Nicinski

Keyword(s):

Flow Cytometry ◽

Growth Rate ◽

Marine Bacteria ◽

Flow Cytometric Analysis ◽

Reducing Power ◽

Automated Analysis ◽

Significant Loss ◽

Epifluorescence Microscopy ◽

Tetrazolium Chloride ◽

Marine Bacterioplankton

ABSTRACT The respiratory activity of marine bacteria is an important indication of the ecological functioning of these organisms in marine ecosystems. The redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) is reduced intracellularly in respiring cells to an insoluble, fluorescent precipitate. This product is detectable and quantifiable by flow cytometry in individual cells. We describe here an evaluation of flow cytometry for measuring CTC activity in natural assemblages of marine bacteria growing in dilution cultures. We found that more CTC-positive cells are detected by flow cytometry than by visual epifluorescence microscopy. Samples can be stored refrigerated or frozen in liquid nitrogen for at least 4 weeks without a significant loss of total cells, CTC-positive cells, or CTC fluorescence. Cytometry still may not detect all active cells, however, since the dimmest fluorescing cells are not clearly separated from background noise. Reduction of CTC is very fast in most active cells, and the number of active cells reaches 80% of the maximum number within 2 to 10 min. The proportion of active cells is correlated with the growth rate, while the amount of fluorescence per cell varies inversely with the growth rate. The CTC reduction kinetics in assemblages bubbled with nitrogen and in assemblages bubbled with air to vary the oxygen availability were the same, suggesting that CTC can effectively compete with oxygen for reducing power. A nonbubbled control, however, contained more CTC-positive cells, and the amount of fluorescence per cell was greater. Activity may have been reduced by bubble-induced turbulence. Addition of an artificial reducing agent, sodium dithionite, after CTC incubation and fixation resulted in a greater number of positive cells but did not “activate” a majority of the cells. This indicated that some of the negative cells actually transported CTC across their cell membranes but did not reduce it to a detectable level. Automated analysis by flow cytometry allows workers to study single-cell variability in marine bacterioplankton activity and changes in activity on a small temporal or spatial scale.

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