Lab 3 - Second Microscopy Lab

Lab 3 - Second Microscopy Lab - Anil Kanungo BIO 206L...

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5/8/2009 Anil Kanungo BIO 206L Thursday 6-10pm Lab #3: Fluorescence Microscopy Introduction: This laboratory session involved learning to use fluorescence in microscopy. Different methods were used and the advantages of each method were illustrated. Other videos and demonstrations in the lab also contributed to our first experience with fluorescence microscopy. Materials: Slides (one composed of 4-micron beads and one composed of .1-micron beads) Coverslips Fluorescence Microscope (with UV, green, and blue filter sets) Phase Contrast Microscope Onion Root Tip Porcelain Spot Plate Bottle of 1N HCl Bottle of PBS (Phosphate Buffered Saline) Bottle of DAPI Solution Bottle of FITC Solution Toothpick Bottle of H 2 O Bottle of Methylene Blue Bottle of Propidium Iodide Kimwipes (tissue paper) Mounting Medium 6 Small Petri Dishes (each containing a coverslip with growing mammalian cells) Paraformaldehyde Cooled Absolute Methanol Pasteur Pipette Procedures: The second microscopy lab focused on developing experience in fluorescence. New staining procedures involving DAPI, methylene blue, propidium iodide and methanol were used. Some of the staining processes involved more extensive preparation involving incubation. A time lapse video was shown on our station’s monitor to demonstrate mitosis and our TA demonstrated fluorescent beads with a fluorescence microscope. Results and Analysis: Exercise B: Observations of Mitosis in Onion Root Tip Made Using Fluorescence Microscopy For this exercise, about 2mm of an onion root tip was cut and placed in a depression of a spot plate to help break down cell walls. It was briefly rinsed with PBS and allowed to soak. The tip was then placed in a spot containing DAPI which is a fluorochrome that emits blue light when excited by UV light. Next the tip was rinsed and soaked in more PBS before finally placing it on a slide and coverslip. The tip was squashed and placed on the fluorescence microscope with the UV excitation filter set. Observing the tip showed some parts to be more visible than others but the nuclei was uniquely bright blue.
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  • Spring '08
  • Unknown
  • Fluorescence Microscopy, Fluorescence microscope

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