Chapter 10: Classification of Microorganisms 1. The Taxonomic Hierarchy 2. Methods of Identification 1. The Taxonomic Hierarchy Phylogenetic Tree of the 3 Domains Taxonomic Hierarchy • 8 successive taxa are used to classify each species: Domain Kingdom Phylum Class Order Family Genus **species can also contain different strains** Species Scientific Nomenclature To avoid confusion, every type of organism must be referred to in a consistent way. The current system of nomenclature (naming) has been in use since the 18th century: • every type of organism is referred by its genus name followed by its specific epithet (i.e., species name) Homo sapiens (H. sapiens) Escherichia coli (E. coli) • name should be in italics and only the genus is capitalized which can also be abbreviated • names are Latin (or “Latinized” Greek) with the genus being a noun and the specific epithet an adjective **strain info can be listed after the specific epithet (e.g., E. coli DH5α)** 2. Methods of Identification Biochemical Testing In addition to morphological (i.e., appearance under the microscope) and differential staining characteristics, microorganisms can also be identified by their biochemical “signatures”: • the nutrient requirements and metabolic “by-products” of of a particular microorganism • different growth media can be used to test the physiological characteristics of a microorganism • e.g., medium with lactose only as energy source • e.g., medium that reveals H2S production **appearance on test medium reveals + or – result!** Commercial devices for rapid Identification Perform multiple tests simultaneously Enterotube II Such devices involve the simultaneous inoculation of various test media: • ~24 hrs later the panel of results reveals ID of organism! Use of Dichotomous Keys Series of “yes/no” biochemical tests to ID organism. • tests done in a logical order, each test result indicates next test to be done • collective results of multiple tests create a profile allowing ID of microorganism Serology (i.e., antibodies) Specific antibodies can be used to ID bacteria: • antibodies are produced by animals to anything “foreign” • animals (rabbits, goats…) are routinely injected with biological material for which antibodies are needed • antibodies present in the animal serum can then be used in various ID tests: e.g., the agglutination test differences in antibody reactivity can reveal different bacterial strains or serovars Phage (virus) Typing Bacteriophages (viruses that infect bacteria) have very specific hosts and can be use to ID bacteria: • grow a “lawn” of bacteria to be tested on agar plate • “dot” different test phage samples on surface • after ~24 hr, clear zones appear where bacteria have been infected & killed • profile of phage sensitivity can reveal ID of bacteria DNA Base Composition Members of the same genera or species have nearly identical DNA sequences, and hence the same proportions of G/C base pairs & A/T base pairs: • because they base pair, G = C and A = T • G/C + A/T = 100% (e.g., if G/C = 40% then A/T = 60%) Determining the G/C content of the DNA from a test organism and comparing to known values is a quick way to eliminate possible identities: • if %G/C is different, cannot be a match! • if %G/C is same, might be a match but additional testing is necessary to confirm The Use of DNA Hybridization With enough heat, DNA strands will separate. Cooling allows complementary strands to base pair. • this technique is used in a variety of ways to see if DNA from two different sources are similar • usually the DNA from one source is immobilized, the other is labeled to allow detection “FISH” Fluorescent in situ hybridization: 1) label DNA “probe” (fr. species of interest) w/fluorescent tag 2) chemically treat cells to allow DNA to enter, hybridize 3) wash & view with fluorescence microscopy **cells w/DNA complementary to probe will fluoresce!** PCR Polymerase Chain Reaction • selectively amplifies only desired DNA (if present) • e.g., DNA from suspected pathogen PCR is a technique that involves manipulating DNA replication in vitro… Overview of PCR Technique Every PCR reaction requires the following: • DNA source to be tested (or amplified) • artificial primers specific for DNA of interest • heat-stable DNA polymerase • free nucleotides (dNTP’s) Plus an automated thermocycler to facilitate repeated cycles of: 1) denaturation of DNA (separation of strands) @ ~95o C 2) hybridization of primers to template @ ~50-60o C 3) DNA synthesis @ ~72o C Ribosomal RNA (rRNA) Comparison Prokaryotic ribosomes contain 3 different rRNA mol.: • large subunit contains 23S (2900 nt) & 5S (120 nt) rRNA • small subunit contains 16S (1500 nt) 16S rRNA sequence is typically used for ribotyping: • sequence is highly conserved (varies little) • degree of difference reflects “evolutionary distance” **primary method for classifying prokaryotic species** Key Terms for Chapter 10 • dichotomous key • serology • phage typing • hybridization • FISH • PCR, thermocycler • ribotyping Relevant Chapter Questions rvw: 4-10, 13, 14 MC: 2-8 Chapter 3: Microscopy 1. Types of Microscopy 2. Staining 1. Types of Microscopya Scale of Magnification Light microscopy • limit of resolution* ~0.2 μm • sufficient to see most organelles, bacteria Electron microscopy (TEM & SEM): • limit of resolution ~2.5-20 nm • sufficient to see subcellular detail, large molecular complexes *resolution = ability to distinguish objects close to each other Light Microscopy Most common type is the Compound Light 3 Microscope: 1) condenser lens focuses light source on sample 2 2) objective lens magnifies 1 the image 3) ocular lens further magnifies image Oil Immersion & Light Refraction Different media (air, water, glass, oil…) bend light to different degrees. • i.e., have different refractive indexes • the oil immersion lens is too small to capture all light refracted by air • immersion oil has refraction index similar to glass, allows more light to enter the lens Bright & Dark Field Microscopy Bright Field Microscopy Dark Field Microscopy • standard or “default” • barrier in condenser type of light microscopy eliminates all direct light • only light reflected by specimen enters the objective lens Phase Contrast & DIC Microscopy Phase-Contrast Differential Interference Microscopy Contrast (DIC) Microscopy • provides internal detail, • variation on phase-contrast contrast, w/o staining with a 2nd light source • useful for live specimens • greater detail, contrast Fluorescence Microscopy Fluorescent dyes or antibodies with a fluorescent tag stick to specific targets. Under UV light, dye fluoresces, only labeled cells or structures are seen. standard confocal Confocal Microscopy Only light from a given depth or plane is transmitted, “out of focus” light excluded Electron Microscopy Electromagnetic lenses focus electron beam onto metal-stained specimen. • electron beams have very short wavelengths • allows far greater resolution than with light microscopy Transmission EM (TEM) • thin sections of specimen, highest resolution Scanning EM (SEM) • reveals surface features 2. Staining Why the Need for Stains? Because, no matter how high the magnification or resolution, you need contrast to be able to see anything. If contrast is not sufficient in the sample or the microscopic method used, staining can provide the necessary contrast: • stains used for viewing bacteria via light microscopy are typically positively charged chromophores (basic dyes) • chromophore = “color-bearing” ion of a salt • bacteria have a net negative charge (i.e., bind positive ions) General Types of Stains Simple stain • dye that non-specifically stains all organisms, features Differential stain • dye that binds various structures or organisms differently Counter stain • a 2nd dye added that is a different color than original dye Negative stain • dye that stains background, not specimen Special stain • dye that specifically stains certain subcellular structures **a mordant is any chemical added to enhance a stain** Gram Staining A very common stain to distinguish 2 bacterial types: 1 23*4 Gram positive • retain primary stain • thick cell wall, NO outer membrane Process: Gram negative 1) primary stain • retain only the counter stain 2) add mordant • thin cell wall, have 3) decolorize* outer membrane * key step 4) counter stain Acid-Fast Staining Most bacteria are not “acid-fast” (i.e., don’t retain stain after acid wash). • only stains species in the genera: Mycobacterium Nocardia • “non-acid-fast” cells revealed by counter acid fast non-acid fast stain Other Types of Staining Negative Staining • e.g., capsule stain Spore Staining • specific for endospores Flagella Staining Key Terms for Chapter 3 • resolution • fluorescent, confocal microscopy • transmission vs scanning EM • refraction & oil immersion • bright & dark field, phase contrast microscopy • simple, differential, counter, negative stains • gram & acid-fast stains, mordant, chromophore Relevant Chapter Questions rvw: 1-13 MC: 10.