TEM Visualization of Microtubules

LEVEL II

Materials

• Coated grid for TEM
  

• 0.1 M ammonium acetate
  

• 5% ethanol saturated uranyl acetate
  

• Transmission electron microscope

Procedure

1. At the conclusion of a 37° C incubation from Exercise 9.6, remove one drop (approximately 20 microliters) of the extract, place it on a collodion or formvar and carbon-coated EM grid, and allow it to remain for about 20-30 seconds. 6

   
   

2. Rinse the grid carefully with 0.1 M ammonium acetate, and float the grid upside down on a drop of 5% uranyl acetate for 15 minutes. Rinse the grid with water, blot all excess fluids from the grid, and allow it to air dry.

   
   

3. Examine the stained grid in a transmission electron microscope.

   
   

4. Draw or photograph your observations -- calculate all appropriate dimensions (length, diameter) of the formed tubules.