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In situ detection of bacteria in continuous-flow cultures of seawater sediment suspensions with f luorescently labelled rRNA-directed oligonucleotide probes

Fachbereich Biologie, C.v.0. Universitat Oldenburg, PO Box 2503, D-26111 Oldenburg, Germany

¹ Author for correspondence: Luise Berthe-Corti. Tel: + 441 7983290. Fax: + 441 7983250.

ABSTRACT

SUMMARY: RNA-targeted and f luorescently labelled oligonucleotide probes were used to study the composition of natural bacterial populations in continuous-f low cultures of seawater sediment suspensions. The cultures were run as enrichment cultures with increasing dilution rates, and hexadecane as the sole carbon source. Total cell numbers were analysed by counting DAPI (4',6- diamidino-2-phenylindole)-stained cells. To differentiate the population composition, oligonucleotide probes for eubacteria, for Cytophagd Flavobacteria, and for four subclasses of the Proteobacteria (a, b, y and 6) were used. About 4&80°/o of the DAPI-stained cells could be detected with the EUB338 probe. Moreover, it was possible to detect a shift in the composition of the natural bacterial population with increasing dilution rate of the continuous culture, from large amounts of CytophagdFlavobacteria to large numbers of members of the pProteobacteria. The cell recovery rate for bacteria labelled with specific oligonucleotide probes was analysed with defined cell numbers of Rhodospirillum nrbnrm, Cornamonas testosteroni and Desulfowibrio vulgaris subsp. vulgaris introduced into the seawater sediment suspension, and was determined to be 13*%33*5 %. The standard deviation determined for this method applied to sediment suspensions was 28.3 %. The results suggest that the application of the in situ hybridization technique allows a good insight into the structure of populations growing in sediment suspensions.

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