1. 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. 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. 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. 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. 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. 6 Flow chart

7. 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