Spore staining (Schaeffer-Fulton method)

·        Take a Malachite Green 1% w/v is used as staining solution in spore staining and    simple staining.

·         Check the compositions.

Ingredients

Malachite green--------------- 1.000 gm

Distilled water------------------ 100.000 ml

·         Prepare a smear on a clear, dry glass slide.

·         Allow it to air dry and fix it with gentle heat.

·         Flood the slide with 1% w/v Malachite Green.

·        Allow the stain to be in contact with the smear for 2-3 minutes and heat the preparation for 3-6 minutes and then allow to cool.

·         Wash in slow-running tap water.

·         Counter stain with safranin for 30 seconds.

·         Wash with water, blot dry and examine under oil immersion objective.

Note:
       Endo spore stains are performed on smear that have been heat –fixed.

Stain applied both vegetative and endospore cell

Heat is applied to help the primary stain (malachite green ) penetrate the endospores.

      

     Primary stain           :   Malachite Green (Aqueous solution or water soluble) 

     Decolonizing agent :   Tap water (raw water)

    Counter Stain           :   safranin

Results:

1. Examine the slide under the oil immersion lens (1000 X) for the presence of      endospores. 

2. Endo spores are bright green and vegetative cell are brownish red to pink.

          Vegetative cell show- pink color (red color)

     3. Endo spore cell – Red colour cells containing green spores can be seen

Malachite Green

1. Aqueous solution(water soluble).
2. Weakly binding to the cell wall and spore cell. (low affinity for cellular membrane) Bind     to the peptidoglycan.

3. Washed the tap water then dye comes right out of the cell wall not from the spore wall. 
4. Vegetative cells may be decolonized with water. That’s why that do not needed to                 decolonizer in this staining.
5. Vegetative cells are then counter stained with Safranin. 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.
6. Spores may be spherical or elliptical.