In situ classification and image cytometry of pelagic bacteria from a high mountain lake (gossenkollesee, austria)

Abstract

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.

A transplant experiment to identify the factors controlling bacterial abundance, activity, production, and community composition in a eutrophic canyon‐shaped reservoir

“…They are based on splitting the community into a few subgroups consisting of an unknown number of bacterial phylotypes. As an example of the problems associated to this approach, our proportions of EUB-detectable cells were rather low (40%-70%) but were similar to those of most other freshwater studies Glöckner et al 1996;Weiss et al 1996;Pernthaler et al 1997;Glöckner et al 1999;Jürgens et al 1999;Š imek et al 1999;Langenheder and Jürgens 2001). A more serious problem could be that a large number of EUB-detectable bacteria remained unaffiliated with the very small set of probes that we used, and this could be related to the presence in these environments of organisms that would not be targeted by our probes (Grampositive, as shown in Fig.…”

“…During the past few years, we have come to learn quite a lot about BCC in freshwater environments with in situ hybridization with oligonucleotide rRNA-targeted probes. Data are now available from oligotrophic Pernthaler et al 1997), oligomesoeutrophic (Glöckner et al 1996Methé et al 1998), and eutrophic lakes (Casamayor et al 2000) and reservoirs (Š imek et al 1999). And although experimental evidence of the role of in situ manipulating predation pressure on BCC exists (Jürgens et al 1999;Š imek et al 1999), nutrient availability is not commonly manipulated (but see Fisher et al 2000, for an exception).…”

Morphological and Compositional Changes in a Planktonic Bacterial Community in Response to Enhanced Protozoan Grazing

“…Image analysis. For selected time points we measured the size distribution of hybridized bacterial cells (Ͻ10 m in length) with an automated image analysis system as described by Pernthaler et al (29). Briefly, the method consisted of recording images of the hybridized filter sections under UV and under green light excitation with a charge-coupled device camera.…”

“…Therefore, such a system allows us to examine the potential of a bacterial community to develop grazing resistance and to analyze possible underlying mechanisms and resulting changes in bacterial taxonomic composition. Fluorescent in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes enables a rapid and cultivationindependent analysis of the bacterial community structure (reviewed in reference 2) and has been used to examine the composition of freshwater bacterioplankton (1,6,29,30,42). Here, oligonucleotide probes for the major phylogenetic groups of planktonic bacteria were applied to study the bacterial composition during a predation-induced shift towards a community with a high proportion of grazing-resistant cells.…”

Quantification of pelagic filamentous microorganisms in aquatic environments using the line-intercept method

“…We used the object-measurement method (based on measurement of individual cells, for details see [16]), to size the non-¢lamentous bacteria ( 6 5 Wm) in all samples and, for comparison with the line-intercept method, also the ¢laments in selected samples containing only short ¢laments (915 Wm).…”