Automated image analysis and in situ hybridization as tools to study bacterial populations in food resources, gut and cast of Lumbricus terrestris L.

Document Type

Article

Publication Date

1-1-2002

Abstract

An image analysis procedure was developed for bacterial cells after staining with the DNA-intercalating dye 4/-6-diamidino-2-phenylindole (DAPI), and after in situ hybridization with Cy3-labeled, rRNA-targeted oligonucleotide probes. DAPI- and Cy3-images were captured separately from the same scenery with a cooled digital video camera with three CCD chips for the basic colors red (R), green (G) and blue (B). Using the appropriate filter sets, images of DAPI-stained cells were captured with the red channel shut down, while Cy3-stained cells were captured with the green and blue channels shut down. DAPI images and Cy3 images were subsequently merged to produce virtual color (RGB)-images. Processing of all color channels allowed to specifically enumerate DAPI-stained and hybridized bacteria, to measure their cell sizes, to subsequently calculate their biovolumes and to estimate their biomass. Using this procedure, significant differences were detected in bacterial populations in food resources, digestive tract and cast of the earthworm L. terrestris L. In leaves, bacteria were on average ten times more abundant and two times larger than in soil. In the digestive tract of L. terrestris, however, numbers and volumes of bacteria were comparable to those in soil indicating the disruption of cells originating from leaves before arriving in the foregut. Passage through the digestive tract of L. terrestris significantly reduced bacterial populations belonging to the α-, β- and γ-subdivisions of Proteobacteria. While these populations did not recover during incubation of cast, populations of the δ-subdivision of Proteobacteria and the Cytophaga-Flavobacterium cluster of the CFB phylum increased in cast. These results suggest a large impact of passage through the digestive tract of L. terrestris on bacterial community structure and demonstrate the usefulness of our image analysis procedure for the determination of cell sizes and biovolumes and thus biomass of specific bacterial populations in different terrestrial habitats. © 2002 Elsevier Science B.V. All rights reserved.

Identifier

0036132828 (Scopus)

Publication Title

Journal of Microbiological Methods

External Full Text Location

https://doi.org/10.1016/S0167-7012(01)00345-1

ISSN

01677012

PubMed ID

11733082

First Page

53

Last Page

68

Issue

1

Volume

48

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