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)
Morphological Characteristics
• Size, shape,
• Presence of endospores, flagella, capsules,
pili, fimbriae
• ID
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
Biochemical Tests
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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)
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
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
Fatty Acid Profiles
• Separate and identify fatty acids and
compare to the list (profile) of fatty acids
that the suspected bacterium can synthesize
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
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
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
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
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