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UIUC MCB 100 - Ch.4: Microscopy, Staining, and Classification (cont.)

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MCB 100 1st Edition Lecture 12Outline of Last Lecture I. Stains for Microscopy A. Simple stains B. Negative stains C. Differential stains i. The Gram stain ii. Schaeffer-Fulton Endospore Stain Outline of Current Lecture I. The Gram StainII. Schaeffer-Fulton endospore stain III. Ziehl-Neelsen acid-fast stainIV. Classification of living creaturesV. Modern molecular phylogeny VI. Indentification of bacteriaVII. Media for culturing microorganisms VIII. Cell theory of life Current LectureI. The Gram Stain A. Used to divide bacteria into 2 groups based on the thickness of the cell wallB. Bacteria with THICK cell walls: purple (Gram positive) C. Bacteria with THIN cell walls: pink (Gram negative) II. Schaeffer-Fulton Endospore StainA. Used to identify spore forming bacteria in the Gram positive genera Bacillus and Clostridium B. Especially useful for identifying: Clostridium botulinum, Clostridium tetani, C. perfringens and Bacillus anthracis C. Endospore cell wall is mineralized with dipicolinic acid and calcium. D. Endospore can be stained by the dyeMalachite Green. Most bacterial cells don’t stain well with Malachite Green. The red dye Safranin is used as a counterstain to seethe vegetative (non-spore) cells II. Ziehl-Neelsen Acid-Fast StainA. Used to identify bacteria in the Genera Mycobacteria and Nocardia, including the clinically very significant bacterium Mycobacterium tuberculosis These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.B. Basis of this differential stain is that cell walls of Mycobacteria contain a lot of hydrophobic waxy mycolic acids. The dye carbol fuchsin sticks tightly to the waxy mycolic acids. Decolorization with acidified alcohol removes the Carbol Fuchsin from other types of bacteria. The counterstain is typically methylene blue. II. Classification of Living Creatures A. Linnean System (1735): Two kingdoms: plants and animals i. Seven layers: (from most general to most specific) kingdom, phylum, class, order, family, genus, species ii. Species: a group of organisms that are capable of mating and producing viable offspringiii. Genus: a group of related species, several distinct species that share many common traits and from an evolutionary standpoint are closely related with a relatively recent common ancestor iv. Family: a group of closely related genera v. Binomial nomenclature (two names): Genus, specific epithet vi. Fungi are not plants. They don’t use photosynthesis, they need organic nutrients, cell walls are differentvii. Euglena are hard to classify. They use photosynthesis, but don’t have cell walls and they are motileviii. Bacterial cells are very different from plant cells. They don’t have chloroplasts nor even a proper nucleus B. Modern molecular phylogenyi. Taxonomy: a taxa is a group of organisms lumped together because they share certain morphological traits ii. Phylogeny: the grouping of organisms is based on their genetic relationships iii. Woese (1978): the three domains of life: archaea, Eucarya, Bacteria (based on 16S rRNA sequence comparisons) B. Identification and classification of bacteria i. Physical characteristics (useful, but not definitive; for identifying broad groups of bacteria)1. Shape, size, arrangement2. Staining characteristics (cell wall structure and composition) ii. Biochemical tests (classical way of differentiating bacterial species) 1. Check for presence of specific enzymes and matabolic pathways2. Carbohydrate fermentation (If bacteria can ferment carbohydrates, they make acid and the medium turns yellow. If can't ferment, medium will remain red)3. Indole test: looks for presence of enzyme tryptophanase, which catalyzes the conversion of tryptophan to indole. Red layer forms at the top if the bacterium is able to make indole 4. utilization of specific amino acids/citric acid5. production of specific waste productsii. Serological tests (strain specific identification)1. Recognition of bacteria by specific antibodiesii. Phage typing (strain specific identification)1. Differentiating strains on basic of bacteriophage host rage2. A bacterial lawn inoculated with a selection of bacteriophages- bacteria are spread over the surface of an agar medium and a range of bacteriophage strains are then inoculated onto the lawn and the plate is incubated. Some phages have no effect on the bacteria, others can attack cells causing formation of plaques 3. Different bacterial strains within a species show different bacteriophage susceptibilities ii. Molecular analysis 1. Uses computer programs to calculate phylogenetic relationships2. Ribosomal RNA sequence comparisons3. Other gene sequences II. Media for culturing microorganisms A. General purpose media- used to grow wide variety of microorganisms, RICH B. Selective media- some species of microorganisms grow, others don’t C. Differential media- used to reveal metabolic difference between species D. Enrichment media- used to enhance growth of some species over others II. Cell theory of life: A. A single cell is the smallest unit of life and is capable of carrying out all of basic processes of a living organismB. 5 important traits of all living organisms:i. Ability to reproduceii. Ability to take in food, produce energy and growiii. Ability to excrete wastesiv. Ability to respond to environmentv.Susceptible to


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UIUC MCB 100 - Ch.4: Microscopy, Staining, and Classification (cont.)

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