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The concentration of aquatic bacteria is basic information required to evaluate the status of environments and to assess bacterial contribution to material cycles. However, the standard direct counting method using epifluorescence microscopy (EFM) is tedious and there is variation in the counts among workers. Here an automatic counting system that consists of Bioplorer (BP) and image analysis has been applied to marine bacteria. BP is composed of a light-emitting diode (LED) illuminant, an optical unit, a driving stage and a charge-coupled device camera. In combination with fluorescent labeling and simplified membrane filtration, bacteria are enumerated automatically. The reproducibility, sensitivity and accuracy of the system were tested for natural marine bacteria, in comparison with EFM and flow cytometry (FCM). The counts obtained by BP showed good correlation with those obtained by EFM and FCM methods. The counts were significantly higher in inshore and oceanic samples, indicating high sensitivity with low background noise. Considering its reproducibility, objectivity, ease of use and compact size, BP can be used as a routine tool for counting aquatic bacteria in substitution for EFM or FCM.

We developed a method for the separate and simultaneous analysis of the community structure of heterotrophic nanopkankton (HNP) and autotrophic nanoplankton (ANP). This method consists of three steps. First, nanoplankton cells were concentrated using a cross-flow filtration system because cell densities in natural seawater are usually too low for genetic studies. Second, HNP and ANP were separated by flow cytometric sorting ("flow sorting") on the basis of the presence or absence of chlorophyll. Finally, the community structure was analyzed using denaturing gradient gel electrophoresis targeting 18S rRNA gene. The newly developed method was applied to the coastal surface water of Aburatsubo Inlet, Japan, in July 2008. The separation of nanoplankton into HNP and ANP was validated by phylogenetic analysis, and the trophic mode of uncultured nanoplankton was confirmed (e.g. Marine Alveolata group II [MALV II] and Marine Stramenopile clade-2 [MAST-2]). This new method involving cell concentration, flow sorting and phylogenetic analysis is a potentially powerful tool for evaluating the population dynamics and ecology of marine protozoa.

The co-existence of physiologically different cells in bacterial cultures is a general phenomenon. We have examined the applicability of the density dependent cell sorting (DDCS) method to separate subpopulations from a long-term starvation culture of Vibrio parahaemolyticus. The cells were subjected to Percoll density gradient and separated into 12 fractions of different buoyant densities, followed by measuring the cell numbers, culturability, respiratory activity and leucine incorporation activity. While more than 78% of cells were in lighter fractions, about 95% of culturable cells were present in heavier fractions. The high-density subpopulations also had high proportion of cells capable of forming formazan granules. Although this was accompanied by the cell specific INT-reduction rate, both leucine incorporation rates and INT-reduction rates per cell had a peak at mid-density fraction. The present results indicated that DDCS could be used to separate subpopulations of different physiological conditions.