In situ identification of cyanobacteria with horseradish peroxidase-labeled, rRNA-targeted oligonucleotide probes

Individual cyanobacterial cells are normally identified in environmental sa mples only on the basis of their pigmentation and morphology. However, thes e criteria are often insufficient for the differentiation of species. Here, a whole-cell hybridization technique is presented that uses horseradish pe roxidase (HRP)-labeled, rRNA-targeted oligonucleotides for in situ identifi cation of cyanobacteria. This indirect method, in which the probe-conferred enzyme has to be visualized in an additional step, was necessary since flu orescently monolabeled oligonucleotides were insufficient to overstain the autofluorescence of the target cells. Initially, a nonfluorescent detection assay was developed and successfully applied to cyanobacterial mats. Later , it was demonstrated that tyramide signal amplification (TSA) resulted in fluorescent signals far above the level of autofluorescence. Furthermore, T SA-based detection of HRP was more sensitive than that based on nonfluoresc ent substrates. Critical points of the assay, such as cell fixation and per meabilization, specificity, and sensitivity, were systematically investigat ed by using four oligonucleotides newly designed to target groups of cyanob acteria.