Endospore Staining : Principle, Procedure and Result

            ENDOSPORE STAINING

Endospore staining is a differential staining technique that selectively stains the spores and makes them distinguishable from the vegetative part of the cells. Endospores are produced by a few genera of Gram Positive bacilli such as Bacillus and Clostridium, in response to adverse environmental conditions.

Endospores are highly resistant to environmental conditions such as heat, chemicals and therefore require special technique for staining.

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 and 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 and middle of the cell. Spores may be spherical or elliptical.

Reagents Used : 

Primary stain : Malachite green

0.5 gm of malachite green

100ml of distilled water

Decolorizing agent 

Tap water or Distilled water

Counter stain : Safranin

2.5gm of safranin

100ml of 95% ethanol

Procedure : 

Preparation of microscope slide

1. Clean the glass slide, with alcohol to remove any stains.
2. Using a sterile inoculation loop, put two small drops of water in each circle.
3. Aseptically, open the tube with bacteria culture and flame it at the top and collect a loopful of bacterial culture from the tube, flame the tube again and close.
4. Smear the bacterial culture in the drop of water on the slide.
5. Air dry till its completely dry.
6. Heat fix the slide with smear facing up, by running it over the blue flame 3-4 times.
7. Leave to cool and then start to stain.

Staining procedure : 

1. Cover the smear with a piece of absorbent paper.
2. Place the slide over a staining rack, that has a beaker/water bath of steaming water.
3. Flood the absorbent paper with malachite green and let it steam for 3-5 minutes.
4. Remove the stained absorbent paper carefully and discard and allow it to cool for 1-2 minutes.
5. Gently rinse the slide with tap water by tilting the slide to allow the water to flow over the smeared stain. This is to remove the extra dye present on the slide on both slides and to also remove extra dye staining any vegetative forms in the heat fixed smear.
6. Add the counterstain, saffarnin for 1 minute.
7. Rinse the slide with water, on both sides to remove the safranin reagent.
8. Ensure the bottom of the slide is dry before placing it on the stage of the Microscope to view with the oil immersion lens, at 1000x for. Maximum magnification

Result : 

The vegetative forms will take up the pink / red stain. from safranin while the endospore will stain strat green.

Interpretation of results : 

The vegetative forms stain pink / red because they take up the counterstain while the endospores take up the green from the malachite green.

This is because during smearing and heat fixing, the malachite green penetrates into the endospores with the help of the heat from the steam, and during the water- rinse, the dye is not easily washed away.

And for the vegetative forms, the dye is easily washed away because of their fragile outer covering, hence they talk up the last stain which is the counterstain, hence they appear pink-red.