Bacterial Identification 1 History bacterial ID methods have
Bacterial Identification 1 • 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 2
More molecular methods 3 • DNA-DNA hybridization – 2 strands of DNA bind together only if 2 organisms are closely related; good for species level. • r. RNA – 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.
Technology/specificity 4 • 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 • 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 5
Flow chart 6
Flow chart comments 7 • 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
- Slides: 7