Comprehensive Analysis of Cell Wall-Permeabilizing Conditions for Highly Sensitive Fluorescence in Situ Hybridization

The simultaneous detection of different bacterial strains carrying a certain functional gene is absolutely imperative in order to detect bacteria on the basis of functional gene expression products (mRNA) in situ. Functional genes are possessed by bacterial strains with different types of cell walls; therefore, the optimization of conditions for digesting different types of cell walls is significant. In this study, we defined the optimal conditions for permeabilization in eight bacterial strains belonging to different phylogenetic divisions using solutions containing different concentrations of lysozyme and/or achromopeptidase, for conventional fluorescence in situ hybridization (FISH), digoxigenin (DIG)-FISH, and catalyzed reporter deposition (CARD)-FISH with a rRNA-targeting oligonucleotide probe. Most bacterial strains were successfully detected using CARD-FISH with lysozyme 10 mg/ml pretreatment. Additionally, achromopeptidase pretreatment combined with lysozyme pretreatment was a highly effective means of permeabilizing bacterial strains that were unable to be detected using lysozyme pretreatment. However, this additional pretreatment resulted in a loss of cell morphology in some bacterial strains due to excessive permeabilization. Consequently, permeabilizing conditions for applying highly sensitive FISH to the well-defined target bacterial strains used in this study were optimized. The results of this study will contribute to the optimization of permeabilizing conditions, which is one of the most important factors for the successful application of highly sensitive FISH.