Chapter 43 - In situ techniques to study noncultivable microbial symbionts

Abstract

To study the microbial communities from various environments, in situ methods or culture-independent methods have been developed. Most of the microbes in the symbiotic association are noncultivable in laboratory conditions. In the past 20 years, various methods or techniques have been developed like small subunit ribosomal RNA gene analysis and direct amplification to study microbial communities from living environments. Such methods include single-strand-conformation polymorphism, (SSCP), temperature gradient/denaturing gradient gel electrophoresis (DGGE/TGGE), RFLP, terminal-RFLP, q-PCR, etc. The non-PCR-based molecular techniques have also been implicated like fluorescence in situ hybridization (FISH), flow cytometry, cell sorting, and microarray. Recently, several novel techniques which are largely propelled by the innovation and next-generation sequencing (NGS)-based methods have been developed such as metagenomics, metaproteomics, metatranscriptomics (omics approach), single-cell-based technologies like Raman microspectroscopy and nano-scale secondary ion mass spectrometry (NanoSIMS). These techniques are used in the field of environmental microbiology and microbial ecology. Applications of such methods allow scientists to analyze microbial communities in situ including proteins, genes, transcripts, and metabolites and their interactions' impact on the distribution patterns. This book chapter, therefore, discusses the different in situ methods and their applications in the microbial community, diversity analysis, and to study the noncultivable symbionts.