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The relationship between flow cytometry data and epifluorescence microscopy measurements was assessed in bacterioplankton samples from 80 lakes to estimate bacterial biovolume and cell size distribution. The total counts of 4,6-diamidino-2-phenylindole-stained cells estimated by both methods were significantly related, and the slope of their linear regression was not significantly different from 1, indicating that both methods produce very similar estimates of bacterial abundance. The relationships between side scatter (SSC) and 4,6-diamidino-2-phenylindole fluorescence and cell volume (microscopy values) were improved by binning of the data in three frequency classes for each, but further increases in the number of classes did not improve these relationships. Side scatter was the best cell volume predictor, and significant relationships were observed between the SSC classes and the smallest (R 2 ‫؍‬ 0.545, P < 0.001, n ‫؍‬ 80) and the largest (R 2 ‫؍‬ 0.544, P < 0.001, n ‫؍‬ 80) microscopy bacterial-size classes. Based on these relationships, a reliable bacterial biomass estimation was obtained from the SSC frequency classes. Our study indicates that flow cytometry can be used to properly estimate bacterioplankton biovolume, with an accuracy similar to those of more time-consuming microscopy methods.Planktonic bacteria are important members of aquatic ecosystems, and the calculation of their biovolume is relevant to our understanding of their roles within the microbial food web and in the cycling of organic matter and nutrients. Direct epifluorescence microscopy (EFM) counts of samples stained with nucleic acid fluorochromes such as acridine orange or 4Ј,6Ј-diamidino-2-phenylindole (DAPI) has for the last few decades been the standard method for determining bacterial abundance and biovolume in plankton samples (16,26,39). Based on these measurements, bacterial carbon biomass can be estimated by applying a general conversion factor (24, 28). However, despite improvements through the application of automated image analysis systems (29), microscopy counts are still time-consuming and require a considerable effort to obtain accurate measurements of bacterial cell volumes. In the early 1990s, flow cytometry (FC) was introduced as an alternative to EFM for estimating bacterial abundance in mixed natural assemblages (see references 13 and 30). FC has become a key tool in aquatic microbial ecology because it constitutes a rapid cell counting method and also makes it possible to process a high number of samples in a short time (33). Besides estimates of bacterial abundance, FC also provides information on single-cell parameters (e.g., light scatter values and specific channels of fluorescence) that can be useful for further discriminating distinct fractions of bacteria within mixed assemblages and thus for analyzing the heterogeneity of bacterial communities (3,11,38). The increase in commercially available fluorochromes, the use of molecular techniques, the application of cell sorting, and technological progress have i...

A B S T R A C TThree aspects of size selective feeding by the scuticociliate Cyclidium glaucoma were studied in continuous cultivation systems. Firstly, grazing-induced changes in abundance, biomass, and size structure of a bacterial community were investigated. Secondly, we studied possible grazingprotection mechanisms of bacteria as a response to permanent presence of the predator. And finally, we were looking for potential feedback mechanisms within this predator-prey relationship, i.e., how the ciliate population reacted to a changed, more grazing-protected bacterial community. The first stage of the cultivation system consisted of the alga Cryptomonas sp. and the accompanying mixed bacterial community. These organisms were transferred to two second stage vessels, a control stage without ciliates and a second one inoculated with C. glaucoma. After the first week, the abundance of bacteria in the latter decreased by 60% and remained stable until the end of the experiment (65 d), whereas bacterial biomass was less affected (393 µg C L −1 during days 0-7, 281 µg C L −1 afterwards). The mean bacterial cell volume doubled from 0.089 µm 3 to 0.167 µm 3 , which was mainly due to increasing cell widths. During the whole investigation period formation of colonies or filaments was not observed, but we found a clear feedback of ciliates on bacterial size. An increase in bacterial cell volume was always followed by a decline of the predator population, resulting in a yet undescribed type of microbial predator-prey relation. Literature and our own data on the optimal food size range grazed by C. glaucoma showed that bacterial cell width rather than length was responsible for that observed phenomenon. Finally, we suggest that uptake rates of spherical latex beads give only limited information on truly ingestible prey volumes and that prey geometry should be considered in future studies on size selective feeding of protists.

We describe a procedure to measure the cell sizes of pelagic bacteria after determinative hybridization with rRNA-targeted fluorescently labeled oligonucleotide probes. Our approach is based on established image analysis techniques modified for objects simultaneously stained with two fluorescent dyes. It allows the estimation of biomass and cell size distribution and the morphological characterization of different bacterial taxa in plankton samples. The protocol was tested in a study of the bacterioplankton community of a high mountain lake during and after the ice break period. Cells that hybridized with a probe for the domain Bacteria accounted for 70% of the bacterial abundance (range, 49 to 83%) as determined by 4,6-diamidino-2-phenylindole staining (K. G. Porter and Y. S. Feig, Limnol. Oceanogr. 25:943-948, 1980), but for >85% of the total biomass (range, 78 to 99%). The size distribution for members of the beta subclass of the Proteobacteria shifted toward larger cells and clearly distinguished this group from the total bacterial assemblage. In the surface water layer beneath the winter cover, bacteria belonging to the beta 1 subgroup constituted about one-half of the beta subclass abundance. The mean cell volume of the beta 1 subgroup bacteria was significantly less than that of the beta subclass proteobacteria, and the beta 1 subgroup accounted for less than 30% of the total beta subclass biovolume. Two weeks later, the biovolume of the beta Proteobacteria had decreased to the level of the beta 1 subgroup, and both the biovolume size distributions and cell morphologies of the beta Proteobacteria and the beta 1 subgroup were very similar. We could thus quantify the disappearance of large, morphologically distinct beta subclass proteobacteria which were not members of the beta 1 subgroup during the ice break period. Our results demonstrate that changes in biovolumes and cell size distributions of different bacterial taxa, and eventually of individual populations, reveal hitherto unknown processes within aquatic bacterial assemblages and may open new perspectives for the study of microbial food webs.

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