SPORE STAINING

 

SPORE STAINING

INTRODUCTION:

In 1922, Dorner published a method for staining endospores. Shaeffer and Fulton modified Dorner’s method in 1933 to make the process faster The endospore stain is a differential stain which selectively stains bacterial endospores. The main purpose of endospore staining is to differentiate bacterial spores from other vegetative cells and to differentiate spore formers from non-spore formers.

AIM:

To perform spore Staining technique to demonstrate the Spore in a culture.

PRINCIPLE:

Bacterial endospores are metabolically inactive, highly resistant structures produced by some bacteria as a defensive strategy against unfavorable environmental conditions. The bacteria can remain in this suspended state until conditions become favorable and they can germinate and return to their vegetative state.

In the Schaeffer-Fulton`s method, a primary stain-malachite green is forced into the spore by steaming the bacterial emulsion. Malachite green is water soluble and has a low affinity for cellular material, so vegetative cells may be decolorized with water. Safranin is then applied to counterstain any cells which have been decolorized. At the end of the staining process, vegetative cells will be pink, and endospores will be dark green.Spores may be located in the middle of the cell, at the end of the cell, or between the end andmiddle of the cell. Spore shape may also be of diagnostic use. Spores may be spherical or elliptical.

MATERIAL’S REQUIRED:

1)Primary Stain: Malachite green (0.5% (wt/vol) aqueous solution)0.5 gm of malachite green,100 ml of distilled water

2)Decolorizing agent:Tap water or Distilled Water

3)Counter Stain: Safranin solution (2.5% (wt/vol) alcoholic solution)

2.5 gm of safranin O,100 ml of 95% ethanol

PROCEDURE;

1)Take a clean grease free slide and make smear using sterile technique

2)Air dry and heat fix the organism on a glass slide and cover with a square of blotting paper or

 toweling cut to fit the slide.

3)Saturate the blotting paper with malachite green stain solution and steam for 5 minutes,

 keeping the paper moist and adding more dye as required. Alternatively, the slide may be

steamed over a container of boiling water.

4)Wash the slide in tap water.

5)Counterstain with 0.5% safranin for 30 seconds. Wash with tap water; blot dry.

6)Examine the slide under microscope for the presence of endospores. Endospores are bright

green and vegetative cells are brownish red to pink.

RESULT:

Endospores: Endospores are bright green.

Vegetative Cells: Vegetative cells are brownish red to pink.Spores may be located in the middle of the cell, at the end of the cell, or between the end and middle of the cell. Spore shape may also be of diagnostic use. Spores may be spherical or elliptical.