Microscopy, Staining and Classification - Chapter 4 Most of these sections will be covered in lab

1. Microscopy, Staining and Classification - Chapter 4 Most of these sections will be covered in lab -- Principles of Staining -- Taxonomic and Identifying Characteristics Relevant metric units mm = 10-3 meter μm (micrometer) nm (nanometer) Resolution vs Magnification Microscopy and Classification

2. Microscopy How does a lens work? Optical properties refraction magnification resolution distortion, e.g., - spherical - chromatic Limitations of a single lens magnification illumination aberration Wikipedia link http://en.wikipedia.org/wiki/Lens_%28optics%29 Microscopy and Classification

3. Some principles of a compound microscope Ocular lens Objective lens Condenser lens How is magnification calculated? Limitations to a compound microscope Microscopy and Classification

4. What were the key advances • in light microscopy? • Oil immersion lens • Fused lenses • Advanced optics • How do oil immersion and • fused lenses improve resolution? • Refractive index = • Borosilicate glass = 1.52 • Air = 1.00 • Water = 1.33 • Immersion oil = 1.52 • Fused lenses combine different types of glass • -- compensate for aberrations Speed of light in vacuum Speed of light in medium Microscopy and Classification

5. Advanced optics only improve contrast Theoretical limit of resolution = 0.2 uM Dark field manipulates the path of light Phase contrast and DIC manipulate phase of light Dark field Bright field DIC Microscopy and Classification

6. Fluorescence Microscopy allows precise staining of cells and cellular components Standard fluorescence manipulates the path of light Confocal -- puts everything in focus Standard Fluorescence vs Confocal image of Tetrahymena Microscopy and Classification

7. How does electron microscopy yield higher resolution? Resolution - Eye ~ 0.2 mm - LM ~ 0.2 uM - EM ~ 0.2 nM (0.0002 uM) Wavelengths of … of electrons ~ 0.005 nm … visible light ~ 550 nm Microscopy and Classification

8. What are types of electron microscopy? Scanning vs Transmission STM is different Driscoll et al. 1990. Nature, 346, 6281 Microscopy and Classification

9. Biological classification Biological taxa -- Muddled in the middle -- Blurry at the bottom Binomial naming Writing conventions Staphylococcus aureus Staphylococcusaureus S. aureus Common names and strains Pneumoncoccus E. coli O157 H7 Domain: Bacteria Kingdom: Eubacteria Phylum: Firmicutes Class: Bacilli Order: Bacillales Family: Staphylococcaceae Genus: Staphylococcus Species: aureus The Antiquity of Microbes Adam Had ‘em -- Strickland Gillilan Microscopy and Classification

10. What are the 3 domains of life? Eukarya – Archaea – Bacteria What traits do Archaea and Bacteria share? -- prokaryotic cell structure -- circular chromosome What traits do Archaea and Eukarya share? -- Nucleosomes -- Multiple RNA Polymerases -- Ribosome structure Some Archaea have unique properties -- Genetically distinctive -- Cell membrane -- Cell wall structure New 2016 Tree of Life Hug, et al. 2016. Nature Microbiology Article #16048 Microscopy and Classification

11. What are the characteristics of Eukaryotes vs Prokaryotes? -- all “cellular organisms” Basic similarities? membrane genetic information biochemistry Distinctive properties of eukaryotes • ‘ eu’ = true ‘karyo’ = nucleus • size • organelles mitochondria, chloroplasts, etc • chromosomes …vs prokaryotes ‘pre-nucleus’ Microscopy and Classification

12. Archaea Extremophiles -- some notable ones… Pyrolobus fumarii -- “Fire lobe of the chimney” Hyper-thermophile -- 90OC – 113OC (106OC) hydrothermal vents Strain 121: http://www.genomenewsnetwork.org/articles/08_03/hottest.shtml Picrophilus spp. Extreme acidophiles -- pH <4 (opt 0.7) Volcanic steam vents Halobacterium spp. Extreme halophiles -- 3 – 5 M salt Dead sea; great salt lake; Salted foods Microscopy and Classification