A morphologically conspicuous bacterium that constituted a very small fraction (< 0.01%) of the total microbial community of activated sludge was enriched and analysed phylogenetically by a combination of cultivation‐independent molecular and physical methods. The large, corkscrew‐shaped, filamentous bacteria were first detected in municipal activated sludge by light microscopy owing to their unusual rotating gliding motility. Various attempts at microbiological enrichment and pure culture isolation with traditional techniques failed, as did attempts to retrieve the morphotype of interest by micromanipulation.
In situ
hybridization with the group‐specific, rRNA‐targeted oligonucleotide probe CF319a indicated a phylogenetic affiliation to the
Cytophaga–Flexibacter
group of the
Cytophaga
–
Flavobacterium–Bacteroides
phylum. Based on strong morphological resemblance to members of the genus
Saprospira
, additional 16S rRNA‐targeted oligonucleotides with more narrow specificity were designed and evaluated for
in situ
hybridization to the morphotype of interest. Flow cytometric cell sorting based on the fluorescence conferred by probe SGR1425 and forward scatter enabled a physical enrichment of the helical coiled cells. Subsequent polymerase chain reaction (PCR) amplification of 16S rDNA fragments from whole fixed sorted cells with a primer pair based on probes CF319a and SGR1425 resulted in the retrieval of 12 almost identical partial 16S rDNA fragments with sequence similarities among each other of more than 99.2%.
In situ
hybridizations proved that the sequences that showed the highest similarity (88.4%) to the 16S rRNA of
Saprospira grandis
were indeed retrieved from the corkscrew‐shaped filaments. The bacterium is likely to be a member of a genus of which no species has been cultured hitherto. It was consequently tentatively named ‘Magnospira bakii’ and has the taxonomic rank of
Candidatus
Magnospira bakii, as the ultimate taxonomic placement has to await its cultivation. In this study, it was demonstrated that even bacteria occurring at very low frequencies in highly complex environmental samples can be retrieved selectively without cultivation for further molecular analysis.