Transcript Bacterial Identification

Bacterial Identification
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• History- bacterial ID methods have changed
– Early methods have not been so much “replaced”
as “added to”.
– Methods used for ID not always adequate for
classifying
• Taxonomies depend on species relatedness
– Species concept in bacteria is difficult; e.g. no sex
• Newer methods are molecular, so more basic
– Sometimes you want to know what the bacterium is
more than who it is related to.
Advances in taxonomy
• Basic: morphological tests
– Gram reaction, size, shape, motility, pigments, etc.
– Provide foundational information
• But many unrelated bacteria appear similar
• Physiological tests- reflect biochemistry
– Enzymes specified by genes, so more basic
– Use of different compounds, production of others
• The molecular age: use of DNA/RNA
– G + C content: if different, then 2 species unrelated
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More molecular methods
• DNA-DNA hybridization
– 2 strands of DNA bind together only if 2 organisms
are closely related; good for species level.
• rRNA – revolutionizing taxonomy
– Ribosomal genes slowly mutate
– Sequence analysis reveals differences over all
living things: responsible for the 3 Domain view
– Can be used to group bacteria into genera and
species; now a major taxonomic tool.
• PCR: highly sensitive method for ID by looking
for specific DNA sequences.
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Technology/specificity
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• Fatty acid profile analysis
– ID microbes by particular fatty acids in lipids of
microbe and the relative abundances.
• Antibody (Aby) and bacteriophage methods
– Highly specific, rely on interactions between Aby or
phage and specific molecules on bacterium
– Used for distinguishing between strains of a species
• Multi-test methods (see Lab Manual)
– API strip, Enterotube combine many physiological
tests into one test package for easy ID
– Biolog: carbon source utilization patterns for ID
Unknown #1
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• Collect morphological data
– Gram reaction, size, shape, etc.
• Collect physiological data: does it ferment?
• Follow flow chart
– Gram reaction will determine if you should use
Biolog for ID.
– Gram positive, spore-forming rods are difficult to ID
using Biolog
• Separate database of results will be used for
spore-formers
Flow chart
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Flow chart comments
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• Separate handouts will explain the principle and
procedures behind the Biolog method.
• Oxidase test (see Lab Manual)
– Easy test for presence of cytochrome C
– Pull up glob of cells from slant w/ cotton swab,
moisten swab w/ reagent, watch for color change
dark red.
• If Biolog is inconclusive,
– tests tailored to info that you already have (Gram
reaction, fermentation) will be done. Examples in
Lab Manual.
• Large G+ rods usually endospore-formers
– NA is the best medium to get them to make spores