Techniques for Microbial Taxonomy and Phylogeny

Techniques for Determining Microbial Taxonomy and Phylogeny

Technique	Description	Applications
Morphological Analysis	Involves studying the physical characteristics of microorganisms, such as shape, size, color, and arrangement.	Preliminary classification based on basic visible traits (e.g., cocci, bacilli, spirilla).
Gram Staining	A differential staining method that classifies bacteria into Gram-positive (purple) or Gram-negative (pink).	Initial identification and classification based on cell wall composition.
Biochemical Tests	Tests that identify microbial enzymes, fermentation patterns, and metabolic products.	Differentiation of bacteria based on metabolic activities (e.g., lactose fermentation, catalase test).
Fatty Acid Profiling	Analysis of the fatty acid composition of the bacterial cell membrane.	Chemotaxonomic classification, especially useful for identifying species in environmental samples.
Protein Profiling (e.g., MALDI-TOF)	Mass spectrometry-based method that identifies microorganisms by analyzing their protein patterns.	Fast identification of bacterial species, particularly in clinical microbiology.
DNA-DNA Hybridization (DDH)	Measures the genetic similarity between two DNA samples by hybridizing them and assessing the degree of pairing.	Determining relatedness between bacterial species or strains; used to confirm species designation.
16S rRNA Gene Sequencing	Sequencing of the 16S ribosomal RNA gene to identify and classify bacteria based on evolutionary relationships.	Highly accurate identification of prokaryotes; a cornerstone in bacterial phylogeny and taxonomy.
Whole Genome Sequencing (WGS)	Sequencing the entire genome of a microorganism to obtain comprehensive genetic data.	Provides in-depth phylogenetic and taxonomic resolution; helps define novel species.
Multilocus Sequence Typing (MLST)	Sequencing several "housekeeping" genes to create a sequence type for comparison between strains.	Useful for in-depth strain comparison and epidemiological tracking.
Polymerase Chain Reaction (PCR)	Amplifies specific regions of microbial DNA to identify species or strains.	Used for species identification, detection of specific genes (e.g., virulence, resistance genes).
Restriction Fragment Length Polymorphism (RFLP)	DNA fragments are generated by digesting DNA with restriction enzymes, and the fragment patterns are analyzed.	Helps identify genetic diversity within microbial populations; used for species or strain differentiation.
Phylogenetic Tree Construction	Based on sequence data (e.g., 16S rRNA or whole genome) to create a tree showing evolutionary relationships.	Used for understanding the evolutionary history and relationships of microbial species.
DNA Microarray	A tool that detects the expression of thousands of genes simultaneously by hybridizing DNA or RNA samples.	For functional analysis, identification of specific microbial strains, and analysis of gene presence.
Fluorescent in situ Hybridization (FISH)	Uses fluorescent probes that bind to specific nucleic acid sequences to identify microorganisms directly in samples.	Identification and quantification of specific microorganisms in complex environmental or clinical samples.
Comparative Genomics	Involves comparing the entire genome of different strains to study genetic similarities and differences.	Used to examine evolutionary relationships, functional genomics, and strain-level diversity.