1. Bacterial Identification • 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.

2. 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

3. 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.

4. Technology/specificity • 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

5. Unknown #1 • 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

6. Flow chart

7. Flow chart comments • 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