Microscopy 01/10/18

Microscopy 01/10/18

1. Common Units

   • 1 micrometer (um) is 1x10-6m
   • 1 nanometer (nm) is 1x10-9m
   • 1 milimeter (mm) is 1x10-3

2. Refractive Index is how much a substance slows the velocity of light passing through it

   • Short focal length means larger magnification

3. Resolution is the ability to distinguish two adjacent objects as separate and distinct

   • Magnification is the ability to make an object as separate and distinct

4. Working Distance

   • Distance between the front surface lens and surface of the coverslip when it is in sharp focus
     • Oil can be used to increase the resolution of images when using very short working distances

5. Types of Microscope

   • Bright Field
     • Produces dark image on light background
     • Produce of ocular lens and object lens gives total magnification
   • Dark Field
     • Bright image on a dark background
     • Image formed by light reflected or refracted by specimen
     • Used to view eukaryote organelles and certain bacteria
   • Phase contract microscopy
   • Differential Interference Contrast Microscope
   • Fluoresence Microscope
     • Stained with fluorochromes and exposed to UV light
   • Confocal Scanning Laser Microscopy
     • Computerised microscope and a laser source to generate 3D images

6. Fixation preserves internal and external structures and holds them in place

   • Heat fixation used on Archaea and Bacteria preserves external structure but damages internal
   • Chemical Fixation is used with larger more delicate organisms and preserves internal features and morphology

7. Dyes are also used to stain cells and provide a better contrast to improve the final image

   • Gram Staining
     • 1) Crystal violet stain applied for 1 minute then rinsed in water, specimen is now dyed purple
       • 2) Iodine Mordant applied for 1 min then rinsed
         • 3) Alcohol decolourizer for 10-30 seconds then rinsed, gram positive cells remain purple, Gram Negative cells become colourless
           • 4) Safranin counter stain for 30-60 seconds and rinse with water and blot to dry, Gram negative cells will turn red.
   • Acid Fast Staining
   • Endospore Staining
   • Capsule staining
   • Flagella staining

8. Electrion Microscopy

   • Higher resolution due to electrons having a shorter wavelength than light
   • Transmission Electron Microscope
     • Electrons are transmitted in a vacuum
   • Scanning Electron Microscope
     • Heavy Metal Dyes
     • Produces a 3D image of the cells exterior

9. Cell Size

   • Very large cells would limit nutrient uptake and inhibit metabolism reducing their ability to compete with smaller cells
   • Smaller cells have a smaller surface area to volume ratio