Fluorescent In Situ Hybridization and the Analysis of the Single Cell

Abstract

The discovery that prokaryotic and eukaryotic cells could be made permeable to fluorescently labeled, sequence specific oligonucleotides makes possible the determinative probing of intact microbial cells (1). Thus, individual target cells can be identified and enumerated in heterogeneous populations (or even when present as endosymbionts of other organisms (2) without the need for direct isolation and culture of the organisms of interest. In microbial ecology, the primary targets for such procedures, referred to collectively as fluorescent in situ hybridization (FISH) techniques, have been the ribosomal RNAs (rRNAs). The rRNAs have proved exceptionally good targets for determinative probes for several reasons. First of all, despite being highly conserved biopolymers owing to their role in protein synthesis, they also exhibit regions of marked sequence variability. Thus, the rRNAs can be considered as mosaics of highly conserved and highly variable sequence. Regions of highly conserved sequence have remained virtually unchanged throughout evolution and provide ideal targets for so-called universal or consensus probes and for probes directed at higher levels of taxonomic rank. The variable regions, on the other hand, have evolved more rapidly and can be used to differentiate among species or even subspecies of bacteria. A second advantage is that they are present in high copy numbers in active cells (1000–10,000 ribosomes per cell) (3), thereby increasing the sensitivity of direct determinative examinations.