Question

Describe a spore forming bacterium stained with the endospore stain and magnified 1000X.

Describe the spore and vegetative cell and note the colors you would expect to see using the stains we performed. What would you expect the Gram reaction of this cell to be?

Answer 1

Bacteria such as Bacillus spp and Clostridium spp produce metabolically inactive or dormant form-endospore under environmental stresses such as nutrient deprivation. Formation of endospore circumvent the problems associated with environmental stress and helps them to survive. Endospores can form within different areas of the vegetative cell. They can be central, subterminal, or terminal. Central endospores are located within the middle of the vegetative cell. Terminal endospores are located at the end of the vegetative cell. Sub-terminal endospores are located between the middle and the end of the cell.

vegetative cell is normal bacterial cell and can be stained directly by simple staining techniques like Gram staining but for endospore staining differential staining technique is used

Principle of Endospore Staining:

A differential staining technique (the Schaeffer-Fulton method) is used to distinguish between the vegetative cells and the endospores. A primary stain (malachite green) is used to stain the endospores. Because endospores resist staining, the malachite green will be forced into (i.e, malachite green permeate the spore wall) the endospores by heating. In this technique heating acts as a mordant.

There is no need of using any decolorizer in this spore staining as the primary dye malachite green bind relatively weakly to the cell wall and spore wall .In fact If washed well with water the dye come right out of cell wall however not from spore wall once the dye is locked in. Water is used to decolorize the vegetative cells.

( Note: In Gram Staining and AFB Staining we use Alcohol or Acid Acohol or Acid as a decolorizer but in spore staining water is sufficient ( to be used as decolorizer) because:

• malachite green dye is water-soluble and does not adhere well to the cell wall
• vegetative cells have been disrupted by heat,
  because of these reasons, the malachite green rinses easily from the vegetative cells. )

As the endospores are resistant to staining, the endospores are equally resistant to de-staining and will retain the primary dye while the vegetative cells will lose the stain. The addition of a counterstain or secondary stain (safranin) is used to stain the decolorized vegetative cells.

RESULT OF STAINING: differentially stained endospore producing bacteria at 1000X magnification looks like-

1. the vegetative cells should appear pink/red (i.e. color of counter stain),
2. the vegetative cells that contain endospores should stain pink while the spores should be seen as green ellipses within the cells.
3. Mature, free endospores should not be associated with the vegetative bacteria and should be seen as green ellipses

Gram staining result is different in different bacteria as is it positive for Bacillus and Clostridium and hence looks voilet colored