Colony staining

This procedure can be used to permanently stain adherent colonies of tissue culture cells on 10 cm tissue culture plates. Colonies are first fixed in methanol, then stained with diluted GiemsaStain. When performed correctly, the colonies will stain dark blue against a clear and colorless background.

1. Wait until the colonies on a plate are macroscopic in size (at least 0.5 mm diameter).
   
   • Do not rush this step. Give the colonies long enough to be easily visible to the naked eye.
     
2. Gently decant the medium from the plate, and add back 2 ml of PBS taking care not to mechanically disturb the colonies.
   
   • if the colonies are very loosely attached to the plate, skip this step and proceed to add methanol directly to the medium as described immediately below
     
3. Tilt the plate to pool the PBS on one side and gently add 2 ml of 75% methanol into the PBS with very gentle mixing.
   
   • As the methanol dissolves into the PBS there can be a strong roiling action as the solutions dissolve into each other which can dislodge the colonies if care is not taken to add the methanol to a sufficiently deep region of the PBS.
     
4. Add 2 ml of 75% methanol three more times with gentle mixing for a final mix of 2 ml PBS and 8 ml 75% methanol.
   
5. Incubate at least five minutes at room temperature until the colonies start to take on a whitish appearance.
   
   • incubating longer is fine
     
6. Decant the PBS/methanol mixture
   
7. Gently add 5 ml of 100% methanol to the plate by slowly dripping the menthanol down the plate's side. Then gently tilt the plate until the methanol is homogeneously spread over the plate.
   
   • Do not drop the methanol directly onto the colonies as this may dislodge them.
     
8. Incubate the plate with 100% methanol at room temperature at least 10 minutes. The colonies should appear pure white and there may be a fine silty white precipitate in the plate.
   
   • incubating longer is ok
     
9. Decant the methanol and add 5ml of 5% GiemsaStain. Gently tilt the plate until the stain is homogeneously spread over the plate.
   
   • dilute the 100% GiemsaStain stock solution with 19 volumes of water to make a 5% solution
     
10. Stain the plates until the colonies are uniformly dark blue throughout.
    
11. Swirl gently, aspirate the stain, gently add back 5 ml water and tilt the plate until the water is homogeneously spread over the plate.
    
12. Repeat the previous step: aspirate, add 5 ml water and swirl homogeneously over the plate.
    
13. Aspirate the water and stand the stained plates at an angle to allow residual water to pool, then aspirate the residual water.
    

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Comments

• The stained colonies can be kept at room temperature indefinitely without loss of contrast.
  
• using a 50 ml Finnpipette stepper (ThermoLabsystems) makes this protocol much less tedious