Use of an alternative Archaea-specific probe for methanogen detection.

An alternative 16S rRNA-targeted oligonucleotide probe specific for Archaea was developed and used for detection of methanogens in anaerobic reactors. The designed probe was checked for its specificity by computer-aided comparative sequence analysis. For in situ application, optimal stringency conditions were adjusted by performing whole cell hybridization using target and nontarget organisms. Anaerobic sludge samples were examined by in situ hybridization for methanogenic populations. The relative abundance of methanogens was monitored with epifluorescence microscopy. Individual cells could be visualized with strong fluorescence signals after hybridization with the newly developed probe.

Use of fluorochrome-labeled rRNA targeted oligonucleotide probe and tyramide signal amplification to improve sensitivity of fluorescence in situ hybridization.

A tyramide signal amplification (TSA) system was used in combination with a conventional fluorochrome-labeled 16S rRNA oligonucleotide probe to increase the sensitivity of fluorescence in situ hybridization. TSA was performed after hybridization resulted in a low fluorescence signal intensity. In contrast to the horseradish peroxidase-tyramide signal amplification (HRP-TSA) system and biotin-tyramide signal amplification (biotin-TSA) system, no additional expensive probe labeling was required. A whole cell hybridization technique was used to compare the fluorescence signal obtained using a monolabeled probe with that obtained using the TSA system. The fluorescence signal of the probe obtained using the TSA system was much higher than that obtained using the monolabeled probe. The technique was successfully applied to the in situ detection of microbial communities in anaerobic sludge. It was demonstrated that TSA resulted in an increased in sensitivity, as the fluorescence signal intensity was much higher than that obtained using a conventional probe.