Table 1.

List of microbiological, biochemical and molecular techniques applied to analyze the microbiome components of an environment

Sl. No	Technique	Principle	References
Microbiological and Biochemical Techniques
1	Plate counts	Direct cultivable bacterial count upon growth on media plates based on morphological differences	Kirk et al. 2004
2	Community Level Physiological Profiling (CLPP)	Physiological profiling based on sole carbon source utilization properties of microbial communities helpful in detecting copiotrophic organisms	Lladó and Baldrian 2017
3	Fatty Acid Methyl Ester analysis (FAME)	Gas chromatographic analysis of cellular fatty acids through	Ghosh et al. 2020
4	Guanine plus Cytosine (GC)	GC content of the genomic DNA, a taxon level characteristic feature, is analyzed through melting temperature curve of DNA renaturation studies	Kirk et al. 2004
Molecular Techniques
1	16S rDNA sequencing approach	a. 16S rRNA gene amplification, preparation of clone libraries (for metagenomic samples), forming operational taxonomic units (OTUs) upon Amplified Ribosomal DNA Restriction Analysis (ARDRA), selection of representative members and sequencing b. 16S rRNA gene amplicon library preparation, next generation high-throughput sequencing, OTU formation and bioinformatic analysis for phylogenetic classification	Ghosh and Sar 2013 Dutta et al. 2018
2	Polymorphism based techniques	a. Denaturing Gradient Gel Electrophoresis (DGGE): 16S rRNA gene amplicon from bacterial genomic DNA differing in sequence composition is resolved electrophoretically based on their difference in denaturation at increasing concentrations of the denaturant in the gel b. Temperature Gradient Gel Electrophoresis (TGGE): Electrophoretic separation of 16S rRNA gene amplicon based on the varying melting temperature on a temperature gradient gel c. Amplified ribosomal DNA restriction analysis (ARDRA) or restriction fragment length polymorphism (RFLP) Terminal restriction fragment length polymorphism (T- RFLP): Polymorphism of restriction sites of 16S rRNA gene for a particular restriction enzyme is utilized to differentiate the microbial communities d. Single strand conformation polymorphism (SSCP): Separation based on the electrophoretic mobility of secondary structures formed out of DNA single strands under non-denaturing conditions	Kirk et al. 2004 Theron and Cloete 2000 Schwieger et al., 1998
3	Nucleic acid reassociation and hybridization techniques	a. DNA-DNA hybridization: Genomic DNA hybridization with known microbial taxa to analyze distinctness of the taxon to species level with 70% as cornerstone b. DNA microarray: Microscopic DNA slides with spots of known DNA sequences to which the unknown DNA sequences are hybridised and fluorescence measured c. DNA Reassociation: Lower the DNA reassociation kinetics of a microbial community, higher is the diversity d. Reciprocal Hybridization of Community DNA: Reciprocal hybridization of total community DNA indicates the presence of same kinds of organisms in two samples based on the idea that only identical or very closely related species would show significant cross-hybridization of pure culture DNA	Theron and Cloete 2000 Cho and Tiedje 2001
4	Ribosomal Intergenic Spacer Analysis (RISA)/Automated Ribosomal Itergenic Spacer Analysis (ARISA)	DNA fingerprinting technique based on the amplification of the intergenic region between 16 and 23S rRNA genes in the rRNA operon. Different microbial taxon has characteristic and significant variability in the length and nucleotide sequence of this region. ARISA is an updated and more efficient technique to get high-resolution data involving a fluorescence-tagged oligonucleotide primer for PCR amplification and subsequent electrophoresis in an automated system	Ciesielski et al. 2013
5	Flow cytometry	Flow cytometry conjugated with fluorescence-activated cell sorting (FACS) relying on fluorescent dyes for detection helps quantify and fractionate complex bacterial communities	Park et al. 2005
6	Fluorescence In Situ Hybridization (FISH)	Fluorescently labelled DNA probes are used to target rRNA of defined taxonomic or phylogenetic groups for microbial identification. Recently, an updated technique named live-FISH combined with FACS has been developed to sort specific taxonomic groups of bacteria and culture them for their further taxon level identification	Batani, et al. 2019