Protocol For Spore Staining

Protocol For Spore Staining 

(Schaeffer-Fulton method for staining endospores)

Ø  Air dry and heat fix the organism on a glass slide

Ø  cover the slide with a blotting paper or toweling cut to fit the slide.

Ø  Saturate the blotting paper with malachite green stain solution and steam for 5 minutes

Ø  Keep the paper moist by adding more dye as required.  Alternatively, the slide may be steamed over a container of boiling water.

Ø  Wash the slide in tap water.

Ø  Counterstain with safranin for 30 seconds. 

Ø  Wash with tap water; blot dry.

Ø  Examine the slide under the oil immersion lens (1,000X) for the presence of endospores. 

Endospores are bright green and vegetative cells are brownish red to pink.  

Spore staining for bacterial cells

Reagents For Spore Staining:

Malachite green stain  (0.5% (wt/vol) aqueous solution)

0.5 g of malachite green
100 ml of distilled water

Decolorizing agent

Tap water

Safranin counterstain

  Stock solution (2.5% (wt/vol) alcoholic solution)
    2.5 g of safranin O
    100 ml of 95% ethanol

   Working solution
   10 ml of stock solution
   90 ml of distilled water