Catalyzed reporter deposition-fluorescence in situ hybridization allows for enrichment-independent detection of microcolony-forming soil bacteria

Abstract

Advances in the growth of hitherto unculturable soil bacteria have emphasized the requirement for rapid bacterial identification methods. Due to the slow-growing strategy of microcolony-forming soil bacteria, successful fluorescence in situ hybridization (FISH) requires an rRNA enrichment step for visualization. In this study, catalyzed reporter deposition (CARD)-FISH was employed as an alternative method to rRNA enhancement and was found to be superior to conventional FISH for the detection of microcolonies that are cultivated by using the soil substrate membrane system. CARD-FISH enabled real-time identification of oligophilic microcolony-forming soil bacteria without the requirement for enrichment on complex media and the associated shifts in community composition.

FIG. 1.

Comparison of conventional FISH and CARD-FISH for the detection of bacterial microcolonies grown on standard or enriched PC membranes using the SSMS. Oligophilic bacteria grown in diluted nutrient media following SSMS cultivation were also analyzed using both detection methods.

FIG. 2.

The total numbers of bacterial microcolonies detected per field of view following SSMS microcultivation on standard and enriched PC membranes, determined by SYBR green staining.

FIG. 3.

Confocal images of standard and enriched membranes from three soil samples following hybridization (red-orange) and total bacterial staining (green) at a magnification of ×600. The left panels refer to standard membranes, and the right panels represent enriched membranes. MQA (A) soil was stained with only SYBR green. MQB (B) soil and RH (C) soil were dual; stained with CARD-FISH (red) and SYBR green. Marked differences in microcolony morphology following rRNA enrichment were observed.

FIG. 4.

Dendrogram generated by cluster analysis of 16S rRNA gene DGGE profiles indicating similarity of SSMS-cultivated bacterial communities from MQB soil grown on standard (S) or enriched (E) polycarbonate membranes.