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
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
