rRNA Sequence-Based Scanning Electron Microscopic Detection of Bacteria

• Published in: Applied and Environmental Microbiology Vol. 71; no. 9; pp. 5523 - 5531
• Main Authors: Kenzaka, Takehiko, Ishidoshiro, Ai, Yamaguchi, Nobuyasu, Tani, Katsuji, Nasu, Masao
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.09.2005
• Subjects: Bacteria; Bacteria - genetics; Bacteria - isolation & purification; Bacteria - ultrastructure; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Cells; Colony Count, Microbial - methods; Electron microscopes; Fluorescence in situ hybridization; Fundamental and applied biological sciences. Psychology; Geologic Sediments - microbiology; Gold; Methods; Microbiology; Microscopy, Electron, Scanning - methods; Oligonucleotide Probes; Ribonucleic acid; Rivers - microbiology; RNA; RNA, Ribosomal - genetics; Sequence Analysis, DNA; Sonication; Molecular hybridization; Scanning electron microscopy; Bacteria; Method; Detection; Ribosomal RNA
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.71.9.5523-5531.2005

A new scanning electron microscopic method was developed for gaining both phylogenetic and morphological information about target microbes using in situ hybridization with rRNA-targeted oligonucleotide probes (SEM-ISH). Target cells were hybridized with oligonucleotide probes after gold labeling. Gold enhancement was used for amplification of probe signals from hybridized cells. The hybridized cells released a strong backscatter electron signal due to accumulation of gold atoms inside cells. SEM-ISH was applied to analyze bacterial community composition in freshwater samples, and bacterial cell counts determined by SEM-ISH with rRNA-targeted probes for major phyla within the domain Bacteria were highly correlated to those by fluorescent in situ hybridization (FISH). The bacterial composition on surface of river sediment particles before and after cell dispersion treatment by sonication was successfully revealed by SEM-ISH. Direct enumeration of bacterial cells on the surface of sonicated sediment particles by SEM-ISH demonstrated that members of Cytophaga-Flavobacterium existed tightly on the surface of particles. SEM-ISH allows defining the number and distribution of phylogenetically defined cells adherent to material surfaces, which is difficult in FISH, and it gives new insight into electron microscopic studies of microorganisms in their natural environment.