ENDOSPORE STAINING

ENDOSPORE STAINING (Schaeffer-Fulton method)

Aim:  

To determine the spore forming (Endospore) bacteria by Schaeffer-Fulton method

Principle :

Endospore staining is used to visualize specialized cell structures. The endospore stain is used to determine the highly resistant spores of certain microorganisms within their vegetative cells. The multiple thick coats of the spore made the endospore resistant to stain with most dyes. In Schaeffer-Fulton method, the primary stain, Malachite Green, is added over the heat fixed bacterial smear and heated over a steam bath for few minutes. This will soften the hard outer coverings of the spore and the primary stain gets stick to the spore. When taken from the steam bath followed by further cooling hardens the outer layer of the spore. During this stage both the spore and vegetative cells appear as green in color. But later the thick outer layer makes the spore resistant to the action of decolorizing agent (water), but however, water can easily decolorize the vegetative cells.  When counterstained with Safranin, vegetative cells are easily stained with Safranin, and the cells appear in red or pink color.

Materials required

Clean glass slide in box

Inoculation loop

Test organism

Absorbent paper

Boiling water in water bath

Water in wash bottle

Microscope

Primary Stain – Malachite green

Counter stain - Safranin

Procedure

1. Aseptically transfer the bacterium with an inoculating loop to a clean glass slide and prepare a thin smear of the bacterium.

2. Air dry and heat-fix the bacterial smear.

3. Cover the bacterial smear with a piece of absorbent paper cut to fit the smear and slide.

Place the slide over a container of boiling water.

4. Saturate the absorbent paper with malachite green stain solution and steam for 5 minutes.  Keep the paper moist by adding more stain as required.

5. Remove the absorbent paper using forceps, allow the slide to cool, and rinse the slide with water for 30 seconds.

6. Wash the slide with water.

7. Counter stain with safranin for 30 seconds.

8. Wash the slide with water and blot dry the slide.

9. Examine the slide under the oil immersion lens for the presence of endospores.

Result: 

 On microscopic observation endospores appear in green color and the vegetatives cells as pink.