Microscopy and Staining

Microscopy and Staining

1. Microscopes

   • Compound Microscope
     • Can be monocular or binocular
     • Has objective lenses
     • Light based
       • Blue light can be used to improve resolution
   • Magnification vs Resolution
     • Magnification is the amount zoomed in
     • Resolution is the amount of detail that can be seen
       • Distinguish two adjacent points as separete entities
       • Resolving Power
         • Shorter wavelength = better resolution
         • Oil Immersion Lense
           • Works on the concept of refractive indicies
             • Glass and air have different values
             • Oil has the same RI as glass, therefore can channel the light more directly into the lense whihc improves resolution

2. Light Microscopes

   • Bright Field
   • Dark Field
   • Phase Contrast
   • Flurescence
   • Confocal
   • Can magnify up to 1000 times
   • Max resolution of 0.2um

3. Electron Microscopes

   • Transmission
     • Specimens must be thin
     • Specimens must be treated by staining to enhance contrast
   • Scanning
     • 3D image produced
     • Specimens may be coated with a think layer metal
     • Good for surface structure

4. Specimen Preparation

   • Wet and Hanging drop mounts
     • Live cells to be examined for motility, shape and arrangements
   • Fixed Mounts
     • Specimen is gently heated to fix it to the slide. Can also be stained to improved visualization of cells
   • Staining
     • Cationic Dyes
       • Basic dyes with positive charges
       • Bind to negatively charged molecules such as Nucleic acids
     • Anionic Dyes
       • Acidic Dyes with a negative charge
       • Bind to positively charged structures
     • Dyes have a chromophore
       • Ring structure with an unbonded electron. Gives the dye it's colour
     • Simple stain
       • Only one dye is used
     • Differential Stains
       • 2 Dyes are used
         • Stain and Counterstain
           • Gram stain
             • Distinguishes between Gram positive and negative
               • Based on differences on the cell wall
             • Cells are fixed and treated with primary stain crystal violet
               • Washed with Iodine. Is a mordant that helps CV to fix into cell walls
                 • Cells are treated with acetone to remove some of the primary stain
                   • Counter-stained with basic fuchsin or safranin