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LAB PRACTICAL 1 - LAB PRACTICAL 1 Lab 1 Phase Contrast...

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LAB PRACTICAL 1 Lab 1: Phase Contrast Microscopy I can explain how phase microscopy works -uses an optical system to exploit the relatively high refractive index of cells relative to water in wet mount of live cells -undiffracted background light passes through portion of phase plate that is thinner and lighter in color. -specimen diffracted light passes thru thicker, darker portion of phase plate and is therefore slowed. -cells have denser material, thus light is slowed relative to background light. -:LIVE CELLS I can set up the microscope for phase contrast I can make wet mount 1. clean slide 2. pipet 10 ul of broth culture or water onto a cover sli Motility Brownian Movement (fake): this is due to random molecular bombardment of tiny bacterial cells by the molecules of the solvent **”shaking” I can estimate size of cells Lab 2: Gram Stain and Bright Field Microscopy SCAGS : used for GRAM STAIN slides Shape, Color, Arrangement, Gram, size I can explain how brightfield microscopy works -no auxillary condensor lens used -path of light is focused on specimen and goes straight into your eyes
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-uses different stains to stain cells -cells fized by heat, so DEAD I can explain how gram stain differentiates between cell types -Gram +: thick peptidoglycan -Gram -: thin peptidoglycan, so ethanol can extract lipid and violet-I leak out I can set up bright field microscopy 1. Kohler illuminate 2. use H for bright field on new microscope I can do Gram stain 1.Fixation: place loopful of cells from a 24hr culture on each slide air dry, then pass thru flame 3 times 2. Initial staining: flood with crystal violet, then rinse with water flood with iodine, then rinse with water 3. Decolorizing step: angle slide and drop EtOH until no color, quickly rinse with water 4. Counterstaining: flood with safranin, rinse with water 5. USE BRIGHTFIELD TO SEE! Lab 3: Purifying mixed culture CHESST -to observe culture plate -Color: -Height: flat, raised, convex, umbonate -Edge: entire (smooth circle), undulate, lobate, filamentous -Size: measure with ruler -Shape: punctiform, circular, irregular, filamentous -Texture : smooth, rough, wrinkled, dry I can streak plate -used to dilute down and isolate loading zone 1 3
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