C H 4 M ICROSCOPY

M EASUREMENTS - METRIC UNITS 1. Meter 2. Decimeter 3. Centimeter 4. Millimeter(mm)- bacterial colony 5. Micrometer(um)- blood cells 6. Nanometer (nm)- poliovirus

M ICROSCOPY - USE OF LIGHT OR ELECTRONS TO MAGNIFY OBJECTS -we see visible light, ROYGBV -differ in wavelengths-the distance b/w 2 corresponding parts of the wave -magnification-make object appear larger, but doesn’t actually change size -resolution (resolving power)-ability to distinguish b/w objects that are close together, limited by wavelength of light *today's microscopes are greater b/c use shorter wavelength radiation- blue light or electron beams -contrast-intensity b/w 2 objects or background, use stains Oil Immersion- helps magnification & resolution

L IGHT M ICROSCOPES 1. Bright field -simple-one lense -compound-series of lenses 2. Dark field- for pale objects -stop in the condenser to prevent light from directly entering the objective lens 3. Phase-living or specimens that would get damaged from stains -phase-contrast-sharply defined images, living cells -differential interference contrast (Nomarski)-3D 4. Fluorescent-use UV light, show against black -emits light of longer wavelength 5. Confocal-ultraviolet lasers, biofilms

E LECTRON M ICROSCOPES 1. TEM- uses very short electron wavelength, much greater magnification, very thin specimens -use diamond edge knife, stain w/ heavy metals 2. SEM- electrons don't penetrate specimen, are bombarded and reflected at different angles -use for 3-D, whole specimens

S TAINING - COLORING SPECIMENS 1. make a smear-film of organisms 2. heat fixation (flame)/ chemical fixation (methyl alcohol) -kills them and preserves shape and size -chromophore- colored portion of a dye acidic dyes- (-, anionic), alkaline structures in low PH basic dyes- (+, cationic), acidic structures in high PH

1. Simple stains- single basic dye: crystal violet, safranin, methylene blue 2. Differential stains- more than one dye A. Gram stain- (+ = purple) (- = pink) 1. Flood smear with primary stain, crystal violet, rinse 2. Flood with mordant(bind dyes), iodine, rinse 3. Flood with decolorizing agent, ethanol and acetone, rinse 4. Flood with counterstain, safranin, rinse

B. Acid-fast- differentiate cells with waxy cell walls (those that cause many human diseases) C. Endospore (Schaeffer-Fulton)- bacteria walls impermeable to all chemicals -uses heat to drive malachite green into the endospore

Special Stains D. Negative/Capsule- stain background & leave cells colorless E. Flagellar- stain flagella, increase the diameter

C LASSIFICATION & I DENTIFICATION Taxonomy- science of classification 1. Classification-assigning organisms to a taxa based on similarities 2. Nomenclature- rules of naming 3. Identification- determining belonging Important for: sense of order, enhance communication, make predictions about similar organisms

C AROLUS L INNAEUS Species-similar organisms that can interbreed Didn’t work well for asexual organisms- called strains Linnaeus divided into plantae & animalia only Kingdom, Phylum, Class, Order, Family, Genus, Species Binomial Nomenclature- use Genus & species Latin names Genus-noun, written first, capitalized Species- adjective, lowercase *both written in italics or underlined Diplococcus pneumoniae or Homo sapiens

R OBERT W HITTAKER - 5 KINGDOM SYSTEM Plantae, Animalia, Fungi, Protista, Prokaryotes Did not address viruses Today Prokaryotes> Eubacteria & Archabacteria

W OESE - 3 D OMAINS Determined by rRNA sequencing Eukarya, Bacteria, Archae

I DENTIFYING C HARACTERISTICS 1. Physical Characteristics Morphology (shape) Stains to view features such as flagella 2. Biochemical Tests Ability to utilize or produce certain chemicals

3. Seriological Tests Agglutination test- antiserum mixed with a sample that may be antigenic (triggers immune response) able to find antigens that cause disease 4. Phage typing (bacteriophages)- viruses that infect bacteria Produces a plaque- clear area

5. Nucleic Acid sequences

Taxonomic/Dichotomous Keys Dichotomous Key Video