Classification of Microorganisms
Bergey' s Manual of Determinative Bacteriology is the standard reference for laboratory identification of bacteria.
Morphological characteristics are useful in identifying microorganisms, especially when aided by differential staining techniques (Gram staining and acid fast stains can suggest a course of treatment before the organism is positively identified).
Differential staining for cell wall characteristics is also used as part of a classification scheme, so this particular method is used for both identification and classification. (See Table 10.5)
Biochemical testing to determine the presence of various enzymes is used in identifying microorganisms.
Rapid identification can be accomplished with specific sets of biochemical tests.
Serological tests, involving the reactions of microorganisms with specific antibodies, are useful in determining the identity of strains and species, as well as relationships among organisms. Slide agglutination, ELISA, and Western blotting are examples of serological tests. I would argue that flow cytometry also employs serological methods, but doesn't have to.
Phage typing is the identification of bacterial species and strains by determining their susceptibility to various phages.
Fatty acid profiles can be used identify some organisms. Examination of esterification patterns (ester vs. ether linkages in lipids) can also indicate phylogenetic relatedness on a broad scale.
Flow cytometry measures physical and chemical characteristics of cells.
The DNA and RNA based methods (including % G+C comparisons, PCR, rRNA sequencing, DNA fingerprinting by restriction fragment length polymorphism, or RFLP, nucleic acid hybridization) are used primarily for classification rather than identification, although PCR, nucleic acid hybridization, and DNA fingerprinting can be used for either depending on the application of the methodology.
The percentage of G-C base pairs in the nucleic acid of cells can be used in the classification of organisms. closely related organisms should have similar %G-C numbers, those that vary considerably are not closely related. Note that having a similiar %G-C doesn't necessarily mean organisms are related, but different means different.
The polymerase chain reaction (PCR) can be used to detect small amounts of microbial DNA in a sample. Amplification by PCR may be used for DNA fingerprinting, sequencing, and detection of specific nucleotide sequences characteristic of specific organisms.
rRNA sequencing: The sequence of bases in ribosomal RNA can be used in the classification of organisms. Be careful here. It might not be the end-all be-all that many think, depending on how the sequencing data is used.
Prokaryotes have 70S ribosomes, each consisting of a small (30S) and a large (50S) subunit.
Their large 50S subunit is composed of a 5S RNA subunit (consisting of 120 nucleotides), a 23S RNA subunit (2900 nucleotides) and 34 proteins.
The small 30S subunit has a 1540 nucleotide RNA subunit (16S) bound to 21 proteins.
Eukaryotes have 80S ribosomes, each consisting of a small (40S) and large (60S) subunit.
Their large 60S subunit is composed of a 5S RNA (120 nucleotides), a 28S RNA (4700 nucleotides), a 5.8S subunit (160 nucleotides) and ~49 proteins.
The small 40S subunit has a 1900 nucleotide (18S) RNA and ~33 proteins.
DNA Fingerprinting by RFLP
Nucleic acid hybridization may be done with large or small DNA fragments. Single strands of DNA and RNA from related organisms will hydrogen-bond to form a double-stranded molecule; the percent hybridization may indicate relatedness between organisms.
Colony hybridization, Southern blotting, DNA chips (microarrays), and FISH (fluorescent in-situ hybridization) are also examples of hybridization techniques using smaller DNA probes.
Fluorescence In-Situ Hybridization (FISH)
Dichotomous keys are used for the identification of organisms. A dichotomous key may incorporate information from a variety of identification methods to identify organisms. The dichotomous key illustrated below uses both morphological and biochemical data for identification.
Cladograms show phylogenetic relationships among organisms. A cladogram can be constructed using data from different methods but typically uses one type of information.