Detection, enumeration, and sizing of planktonic bacteria by image-analyzed epifluorescence microscopy

Epifluorescence microscopy is now being widely used to characterize planktonic procaryote populations. The tedium and subjectivity of visual enumeration and sizing have been largely alleviated by our use of an image analysis system consisting of a modified Artek 810 image analyzer and an Olympus BHT-F epifluorescence microscope. This system digitizes the video image of autofluorescing or fluorochrome-stained cells in a microscope field. The digitized image can then be stored, edited, and analyzed for total count or individual cell size and shape parameters. Results can be printed as raw data, statistical summaries, or histograms. By using a stain concentration of 5 micrograms of 4'6-diamidino-2-phenylindole per ml of sample and the optimal sensitivity level and mode, counts by image analysis of natural bacterial populations from a variety of habitats were found to be statistically equal to standard visual counts. Although the time required to prepare slides, focus, and change fields is the same for visual and image analysis methods, the time and effort required for counting is eliminated since image analysis is instantaneous. The system has been satisfactorily tested at sea. Histograms of cell silhouette areas indicate that rapid and accurate estimates of bacterial biovolume and biomass will be possible with this system.

[1]  J. Sieburth,et al.  Metabolic and ultrastructural response to glucose of two eurytrophic bacteria isolated from seawater at different enriching concentrations , 1984, Applied and environmental microbiology.

[2]  E. N. Buckley,et al.  Response of marine bacterioplankton to differential filtration and confinement , 1984, Applied and environmental microbiology.

[3]  J. Fuhrman,et al.  Bacterioplankton growth in seawater: I.Growth kinetics and cellular characteristics in seawater cultures , 1984 .

[4]  Louis Legendre,et al.  Flow cytometry and cell sorting: A technique for analysis and sorting of aquatic particles1 , 1983 .

[5]  E. Carpenter,et al.  Identification and Enumeration of Marine Chroococcoid Cyanobacteria by Immunofluorescence , 1983, Applied and environmental microbiology.

[6]  D. Caron.  Technique for Enumeration of Heterotrophic and Phototrophic Nanoplankton, Using Epifluorescence Microscopy, and Comparison with Other Procedures , 1983, Applied and environmental microbiology.

[7]  T. Platt,et al.  Autotrophic Picoplankton in the Tropical Ocean , 1983, Science.

[8]  E. Sherr,et al.  Enumeration of Heterotrophic microprotozoa by epifluorescence microscopy , 1983 .

[9]  E. Carpenter.  Identification andEnumeration ofMarine Chroococcoid Cyanobacteria byImmunofluorescence , 1983 .

[10]  D. Kirchman,et al.  Statistical analysis of the direct count method for enumerating bacteria , 1982, Applied and environmental microbiology.

[11]  K. Johnson,et al.  Diel relationships of microbial trophic groups and in situ dissolved carbohydrate dynamics in the caribbean sea , 1982 .

[12]  R. Christian,et al.  Comparison of methods for measurement of bacterial growth rates in mixed batch cultures , 1982, Applied and environmental microbiology.

[13]  J. Paul.  Use of Hoechst Dyes 33258 and 33342 for Enumeration of Attached and Planktonic Bacteria , 1982, Applied and environmental microbiology.

[14]  J. Fuhrman,et al.  Thymidine incorporation as a measure of heterotrophic bacterioplankton production in marine surface waters: Evaluation and field results , 1982 .

[15]  M. Laake,et al.  Diversity Dynamics of Marine Bacteria Studied by Immunofluorescent Staining on Membrane Filters , 1982, Applied and environmental microbiology.

[16]  C. Sullivan,et al.  The seasonal abundance, vertical distribution, and relative microbial biomass of chroococcoid cyanobacteria at a station in southern California coastal waters , 1981 .

[17]  Christiane Krambeck,et al.  Microcomputer-Assisted Biomass Determination of Plankton Bacteria on Scanning Electron Micrographs , 1981, Applied and environmental microbiology.

[18]  S. Y. Newell,et al.  Frequency of Dividing Cells as an Estimator of Bacterial Productivity , 1981, Applied and environmental microbiology.

[19]  J. Fuhrman.  Influence of Method on the Apparent Size Distribution of Bacterioplankton Cells: Epifluorescence Microscopy Compared to Scanning Electron Microscopy , 1981 .

[20]  K. Porter,et al.  The use of DAPI for identifying and counting aquatic microflora1 , 1980 .

[21]  M. Perry,et al.  Immunofluorescent Assay for the Marine Ammonium-Oxidizing Bacterium Nitrosococcus oceanus , 1980, Applied and environmental microbiology.

[22]  J. Waterbury,et al.  Widespread occurrence of a unicellular, marine, planktonic, cyanobacterium , 1979, Nature.

[23]  P. Dugan,et al.  Study of Developmental Stages of Methylosinus trichosporium with the Aid of Fluorescent-Antibody Staining Techniques , 1979, Applied and environmental microbiology.

[24]  Paul W. Johnson,et al.  Chroococcoid cyanobacteria in the sea: A ubiquitous and diverse phototrophic biomass1 , 1979 .

[25]  U. Larsson,et al.  Frequency of Dividing Cells, a New Approach to the Determination of Bacterial Growth Rates in Aquatic Environments , 1979, Applied and environmental microbiology.

[26]  Victor Smetacek,et al.  Pelagic ecosystem structure: Heterotrophic compartments of the plankton and their relationship to plankton size fractions 1 , 1978 .

[27]  J. Hobbie,et al.  Use of nuclepore filters for counting bacteria by fluorescence microscopy , 1977, Applied and environmental microbiology.

[28]  Woods,et al.  Determination of bacterial number and biomass in the marine environment , 1977, Applied and environmental microbiology.

[29]  R. Zimmermann.  Estimation of Bacterial Number and Biomass by Epifluorescence Microscopy and Scanning Electron Microscopy , 1977 .

[30]  R. Ferguson,et al.  Contribution of bacteria to standing crop of coastal plankton1 , 1976 .

[31]  W. Walton.  Automatic Counting of Microscopic Particles , 1952, Nature.