FEMS Microbiology Letters 100 (1992)45-50 © 1992 Federation of European MicrobiologicalSocieties0378-1097/92/$05.00 Published by Elsevier 45 FEMSLE 80023 Identification and in situ detection of individual bacterial cells R. Amann, W. Ludwig and K.-H. Schleifer Lehrstuhl fiir Mikrobiologie, Technische Universitiit Miinchen, Miinchen, FRG Received 12 May 1992 Accepted 13 May 1992 Key words: In situ identification; Oligonucleotide probe; Non-culturable bacteria 1. SUMMARY Microbial ecology has long been hampered by the fact that most microorganisms cannot be identified in situ because of the lack of morpho- logical diversity. The immunofluorescence ap- proach has yielded important insights into the spatial distribution of microorganisms but has its severe limitations. The recently introduced fluo- rescently labelled, ribosomal RNA-targeted oligonucleotide probes have successfully been ap- plied for the detection and identification in situ of individual microbial ceils and evade some of the principal problems of fluorescent antibodies. The design and synthesis of these phylogeneti- cally nested probes does not require cultivation and isolation of the target organism and can therefore be used to monitor the population dis- tribution and dynamics of hitherto uncultured microorganisms. Correspondence to: K.-H. Schleifer, Lehrstuhl fiir Mikrobiolo- gie, Technische Universit~itMiinchen, Arcisstrasse 21, D-8000 Miinchen 2, FRG. 2. INTRODUCTION The morphological diversity within the do- mains Bacteria and Archaea [1] is rather re- stricted. Whereas higher plants and animals can readily be identified in their natural habitats based on morphology, even the best light micro- scopes do not reveal enough characteristic details to allow the reliable identification of individual microbial cells in situ. The classic approach to enumerate bacteria in environmental samples has been the plating technique combined with a si- multaneous or subsequent differentiation of the isolates based on physiological and biochemical characteristics. Besides only offering very limited insight into the spatial distribution of microorgan- isms plating has other severe limitations: (1) all techniques relying on cultivation are quite time consuming as are the physiological and biochemi- cal differentiation tests; (2) often the number of colony forming units is only a minor fraction (less than 1%) of the cell counts determined by direct microscopic techniques. These significant differ- ences cannot only be attributed to non-viable cells but are in part caused by viable cells con-