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Methods of Classifying and Identifying Microorganisms

Methods of Classifying and Identifying Microorganisms. Identifying: determining what species it is for research or disease treatment

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Methods of Classifying and Identifying Microorganisms

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  1. Methods of Classifying and Identifying Microorganisms • Identifying: determining what species it is for research or disease treatment • Classifying: to discover which group the species is most closely related to (evolutionary relatedness); how similar (closely related or how different (distantly related)

  2. Morphological Characteristics • Size, shape, • Presence of endospores, flagella, capsules, pili, fimbriae • ID

  3. Differential Staining • Gram positive or gram negative (gram stain divides bacteria into 2 large groups • Acid-fast stain: usually used just to identify Mycobacterium • ID

  4. Biochemical Tests • Fermentation of carbohydrates • Fermentation end products • Hydrolysis reactions • Oxidation reactions • Rapid identification methods: several tests performed simultaneously; each test result (+ or -) is given a coded value • (Enterotubes, OxyFerm tubes)

  5. Serological Testing • Reaction of bacteria to antibodies that are specific for that organism’s antigens • i.e. Agglutination tests: if antibody is specific for a certain bacterium, it will cause agglutination (clumping) of the organisms

  6. Phage Typing • Bacteriophages are specific to the bacterial species that they infect (similar idea to antigens/antibodies) • If a phage that is specific for a bacterium is placed on agar with bacterial growth, it will destroy cells where is is placed creating a plaque

  7. Fatty Acid Profiles • Separate and identify fatty acids and compare to the list (profile) of fatty acids that the suspected bacterium can synthesize

  8. Flow Cytometry • Bacteria flows through a tube and size/shape can be determined by how much light is scattered • Can also detect fluorescent cells (naturally or dyes) • Can be done without staining

  9. DNA Base Composition • Determine percentage of G-C pairs in DNA • Difference in more than 10% between two species: they are not closely related • Very close percentages: might not be closely related since percentages could be similar but arrangements in genes could be quite different

  10. DNA fingerprinting • Cut DNA with restriction enzymes; run electrophoresis to separate bands of DNA fragments • Compare bands of different bacteria: more bands similar - more closely related; exactly same bands - same species

  11. Ribosomal RNA Sequencing • Determine sequence of nucleotide bases on the ribosomal RNA in the small subunit of the ribosome • More similar sequences - more closely related • Used to organize classification groups around this characteristic

  12. Nucleic Acid Hybridization • Separate the double stranded DNA of two organisms; mix the strands together and see how much DNA from one organism base pairs with the DNA from the other organism (hybridization) • Higher percentage of DNAs hybridizing - more closely related • Can also hybridize DNA with mRNA

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