231 Fluorescence in situ hybridisation (FISH) with rRNA-targeted oligonucleotide probes facilitates the rapid and specific identification of individual microbial cells in their natural environments. Over the past year there have been a number of methodological developments in this area and new applications of FISH in microbial ecology and biotechnology have been reported. Addresses Molecular Ecology Group, Max Planck Institute for Marine Microbiology, Celsiusstraße 1, D-28359 Bremen, Germany *e-mail: ramann@mpi-bremen.de † e-mail: bfuchs@mpi-bremen.de ‡ e-mail: sbehrens@mpi-bremen.de Current Opinion in Biotechnology 2001, 12:231–236 0958-1669/01/$ —see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations DGGE denaturing gradient gel electrophoresis FISH fluorescence in situ hybridisation PCR polymerase chain reaction PNA peptide nucleic acid rRNA ribosomal RNA SRB sulphate-reducing bacteria Introduction A method for the rapid and specific identification of individual microbial cells within their natural environments has been long awaited. Most microorganisms have very limited mor- phological detail, preventing the visual identification possible with higher animals and plants. Traditional cultivation methods are time-consuming and frequently only work for a minority of the bacterial species present in a sample. In recent years, molecular biological methods have extended our view to those microorganisms that have proved impossible to culture. Techniques based on the polymerase chain reaction (PCR) now facilitate the rapid and sensitive detection of bacteria independent of whether or not they can be cultured; however, these techniques provide only limited information on the number and spatial distribution of microorganisms. There was therefore a need for a microscopy technique simi- lar to that of the famous Gram-staining method. The test needed to be as sensitive as the well-established immunoflu- orescence techniques [1], but instead of targeting antigens would be based on nucleic acids. The comparative analysis of homologous nucleic acid sequences, most notably of ribosomal RNA (rRNA) molecules and the genes encoding them, has over the past 25 years profoundly changed our view of micro- bial systematics [2]. Large databases of sequence information exist, and rRNA gene fragments are today routinely retrieved without prior cultivation. Since their first application as ‘phylogenetic stains’ in 1989 [3], fluorescently labelled, rRNA-targeted oligonucleotide probes have become a com- mon tool for the direct, cultivation-independent identification of individual bacterial cells. Fluorescence in situ hybridisation (FISH) with rRNA-targeted oligonucleotide probes has been developed for the in situ identification of individual microbial cells and is now a well-established technique. A detailed account of the development of FISH methods in microbial ecology has been published previously [4]. This review will focus on methodological improvements and applications of FISH in microbial ecology and biotech- nology over the past year. Updated information on medical applications is available in a recent review by Moter et al. [5 • ]. More information on in situ nucleic acid amplification and flow cytometric analysis can be found elsewhere [6]. Methodological aspects of FISH rRNAs are the main target molecules for FISH for several reasons: they can be found in all living organisms; they are relatively stable and occur in high copy numbers (usually several thousand per cell); and they include both variable and highly conserved sequence domains [4,7]. Signature sequences unique to a chosen group of microorganisms, ranging from whole phyla to individual species, can there- fore be identified by comparative sequence analysis. Bacteria and archaea contain 5S, 16S, and 23S rRNAs with lengths of approximately 120, 1500 and 3000 nucleotides, respectively. In the vast majority of applications FISH probes target 16S rRNA. The public databases now include 16S rRNA sequences for most cultured microbial species, as well as numerous sequences directly retrieved from the environment [8 •• ,9 •• ]. Probes are designed using sequence information from these databases and program packages such as ARB [10 • ,11 • ]. A typical FISH protocol includes four steps: the fixation and permeabilisation of the sample; hybridisation; washing steps to remove unbound probe; and the detection of labelled cells by microscopy or flow cytometry. Detailed descriptions of this procedure, which can be completed within a few hours, are available elsewhere [12]. FISH is fully compatible with direct count methods [13,14]. The oligonucleotide probes used in FISH are generally between 15 and 30 nucleotides long and covalently linked at the 5′-end to a single fluorescent dye molecule. Common fluorophors include fluorescein, tetramethylrhodamine, Texas red and, increasingly, carbocyanine dyes like Cy3 or Cy5 [15]. The carbocyanine dyes have greatly increased the sensitivity of FISH [13], but further improvements are still needed. The microorganisms living in oligotrophic environ- ments, such as the open ocean, are typically small with low The identification of microorganisms by fluorescence in situ hybridisation Rudolf Amann*, Bernhard M Fuchs † and Sebastian Behrens ‡