Dilution culture, a method for growing the typical small bacteria from natural aquatic assemblages, has been developed. Each of 11 experimental trials of the technique was successful. Populations are measured, diluted to a small and known number of cells, inoculated into unamended sterilized seawater, and examined three times for the presence of 104 or more cells per ml over a 9-week interval. Mean viability for assemblage members is obtained from the frequency of growth, and many of the cultures produced are pure. Statistical formulations for determining viability and the frequency of pure culture production are derived. Formulations for associated errors are derived as well. Computer simulations of experiments agreed with computed values within the expected error, which verified the formulations. These led to strategies for optimizing viability determinations and pure culture production. Viabilities were usually between 2 and 60%o and decreased with >5 mg of amino acids per liter as carbon. In view of difficulties in growing marine oligobacteria, these high values are noteworthy. Significant differences in population characteristics during growth, observed by high-resolution flow cytometry, suggested substantial population diversity. Growth of total populations as well as of cytometry-resolved subpopulations sometimes were truncated at levels of near 104 cells per ml, showing that viable cells could escape detection. Viability is therefore defined as the ability to grow to that population; true viabilities could be even higher. Doubling times, based on whole populations as well as individual subpopulations, were in the 1-day to 1-week range. Data were examined for changes in viability with dilution suggesting cell-cell interactions, but none could be confirmed. The frequency of pure culture production can be adjusted by inoculum size if the viability is known. These apparently pure cultures produced retained the size and apparent DNA-content characteristic of the bulk of the organisms in the parent seawater. Three cultures are now available, two of which have been carried for 3 years. The method is thus seen as a useful step for improving our understanding of typical aquatic organisms.

Marine bacteria in Resurrection Bay near Seward, Alaska, and in the central North Sea off the Dutch coast were cultured in filtered autoclaved seawater following dilution to extinction. The populations present before dilution varied from 0.11 x 109 to 1.07 x 109 cells per liter. The mean cell volume varied between 0.042 and 0.074 ,um3, and the mean apparent DNA content of the cells ranged from 2.5 to 4.7 fg of DNA per cell. All three parameters were determined by high-resolution flow cytometry. All 37 strains that were obtained from very high dilutions of Resurrection Bay and North Sea samples represented facultatively oligotrophic bacteria.

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Flow cytometry offers a rapid method for characterizing aquatic populations according to the properties of individual cells. This technology has been extended to aquatic bacteria by using high-intensity UV excitation, condensing the laser beam onto a small area, using blemish-free flow cells, optimizing organism staining protocol, segregating the optical signal produced with high-transmittance optical filters, collecting the signal with sensitive photomultipliers, and expanding the range of data displayed from individual samples with calibrated circuitry. Bacteria could be counted according to event frequency, and populations agreed with direct counts by epifluorescence microscopy. Forward scatter intensity was a linear function of volume for bacterial cells between 1.3 and 0.25 pm3 as calibrated by Coulter impedance. Plastic spheres down to 0.014 pm3, 0.3 pm in diameter, were resolved. Aquatic bacteria 0.05 pm3 in volume were clearly resolved according to DNA content by staining with DAPI. The observed signal was DNA-dependent because DNase treatment eliminated most fluorescence.These procedures are suitable for direct analysis of the bacteria in marine and freshwater samples without interference from algae, sediment, or most DNA-free organic particles. Cytograms indicated one or more clearly resolved subpopulations of bacteria of substantially smaller size and DNA content than the laboratory organisms typically classified.Key terms: Marine bacteria, size distribution, DAPI, small particles, light scatter Speed and sensitivity for describing the physical and biochemical aspects of individual cells in mixed populations have been improved by the use of flow cytometers (13,211. While attempts have been made to analyze bacterial populations in this way, the focus has been on large and easily cultured organisms (12,16,25,26,29). Tyndall et al. (27) were the first to report resolution of smaller aquatic bacteria by flow cytometry when they detected Legionella sp. in cooling tower waters. Unable to observe the organisms by light scatter when using an unmodified Cytofluorograf (Ortho Diagnostic Instruments, Westwood, MA), resolution was achieved by using a combination of the red fluorescence from propidium iodide-stained DNA and the green fluorescence from FITC-labeled antibodies.Bacteria are ubiquitous in oceans, lakes, and rivers. They process most marine primary productivity and comprise a major component of aquatic biomass (28). However, most are very small and resist growth to high populations in the laboratory (23), characteristics which have impeded the usual taxonomic, genetic, and biochemical studies. Current investigations of aquatic bacteria rely on metabolic measurements of whole water samples (€9, and electron (6,11,31) or epifluorescence microscopy (6,7,11,18,24,31) of collected organisms.Because aquatic bacteria are environmentally important, inconvenient to measure, and present a t about lo6 cells/mL, which is ideal for flow cytometry, we set out to improve the potential of a commercial instrument to ...