Endospore Staining Practical- Aim, Introduction, Principle, Procedure, Results, Conclusions


Due to the highly resistant nature of endospores,they are not easily penetrated by stains. Thus, it is necessary to steam the stain into an endospore.

Dorner’s endospore staining is the first endospore staining method, which was published in 1922.The Schaeffer-Fulton method is the most commonly used endospore staining technique.Schaeffer-Fulton endospore staining procedure is a faster endospore staining method,modified from Dorner’s procedure in 1933.

The Schaeffer-Fulton method uses malachite green as the primary stain.Once the endospore has absorbed the stain, it is resistant to de-colorization, but the vegetative cell is easily de-colourized with water (leave the vegetative cells colorless). Finally, the vegetative cells are counter stained with Safranin to aid in their visualization.When viewed under a microscope, the endospores appear green, while the vegetative cells are red or pink.

This article of Schaeffer-Fulton method of endospore staining practical covers aim, theory, principle, procedure, possible results, conclusions.


Aim

The main objectives of endospore staining is to stain and observe the shape, size and locations of the bacterial endospore by Schaeffer Fulton’s method and to differentiate between bacterial spore and vegetative cell forms and to differentiate spore formers from non-spore formers.

  • Observe the shape, size and locations of spore
  • Differentiate between bacterial spore & vegetative cell (green vs red)
  • Differentiate spore formers & non-spore formers.

Principle

Endospore Staining Practical- Aim , Introduction, Principle , Procedure, Results , Conclusions

Endospore staining is one type of differential staining which selectively stains bacterial endospores through which we can observe the endospors. With the help of this staining technique we can distinguish two kind of cells like endospore former and non-endospore former by the use of two stain like malachite green and safranin.

Endospore are extremely resistant due to thick wall which is known as sporecoat. Spore does not take any kind of stain easily but only malachite green penetrates the coat of endospore after considerable heating. Once stained, endospore does not decolourized easily. But during water flow the vegetative cells decolourized. So after treating with safranin, the vegetative cells are stained with it but not the endospores. That is why we can observe the endospore green and vegetative cells red or pink.

Theory

Membrane of the anaerobic genera Clostridium and the aerobic genus Bacillus are examples of organisms that have the capacity to exist either as metabolically active vegetative cells or as highly resistance metabolically inactive cell types called Spores. When environmental condition became unfavourable for the continuing vegetative cellular activities particularly with the exhaustion of a nutritional carbon source, these cells have the capacity to undergo sporogenesis ( vegetative cell to spore formation) and give rise to a new intracellular structure called endospore which is surrounded by the impervious layer called sporecoats. As conditions continue to worsen, the endospore is released from the degenerating vegetative cell and becomes an independent cell called spore. Because of the chemical composition of spore layers, the spore is resistant to the deleterious effects of excessive heat, freezing, radiation, desiccation and chemical agents as well as to the commonly employed microbiological stains. With the return of favourable environmental conditions, the free spore may refer to be metabolically active and less resistant vegetative cell through germination (spore to normal vegetative cell) are not mean of reproduction but merely mechanisms that ensures cell survival under all environmental conditions

Note – Bacteria can form endospores in approximately 6 to 8 hours after being exposed to adverse conditions.The normally-growing cell forms vegetative cell. In growing stage number of endospore will be very less.
So to see endospore first you have to take any known spore forming bacterial culture and second you have to sure about the present stage of the culture.

Reagents

In practice of the endospore staining procedure uses two different agents-

Endospore Staining Practical- Aim , Introduction, Principle , Procedure, Results , Conclusions

Malachite green

Unlike most vegetative cell types that stain by common procedures, the spore because of its impervious coats it will not accept the primary stain easily. To further penetration, the application of heat is required. After the primary stain is applied and the smear is heated, both the vegetative cell and the spot will appear green.

  • Malachite green – 5 g
  • Distilled water – 1L or 1000 ml

Function – Primary stain – Malachite green is accepted by the spore only and appear as green.

Tap water

Once the spore accepts the malachite green it cannot be de-clolurized, but the tap water which will remove the excess primary stain. The spore will remain stained. On the other hand the malachite green stain does not demonstrate a strong affinity for vegetative cell components; the water removes it and this cell will be colorless.

Function – Washing agent – wash excess green color from vegetative cell

Safranin

This constructing red stain is used as the second reagent to colour the decolorized vegetative cells, which will absorb the counter-stain and appear red. The spores retain the green of the primary stain.

  • Safranin O – 25g
  • Ethyal alcohol – 1000ml

Function – Counter stain – Applied after primary stain and is accepted by vegetative cell ,appear as red

Requirements

  • Bacterial CultureBacillus sp culture
  • Reagents (malachite green, distilled water , safranin )
  • Glass slide(grease free)
  • Inoculating loop
  • Standing tray
  • Water bath
  • Bunsen burner
  • Microscope
  • Blotting paper

Procedure

Pre Endospore Staining (Smear Preparation)

  1. A clean grease free glass slide was taken for staining purpose.
  2. A loopful of bacterial i.e.bacillus culture was taken by inculating loop on the middle of glass slide and culture was plugged in as before without any contamination.
  3. With 2 drop of distilled water a bacterial smear was prepared in the middle of slide by the loop.
  4. Bacterial mear was air dried and gently heat fixed .

Main Endospore Staining

  1. The heat fixed slide was kept over boiling water-bath with a piece of filter paper towel covering the smear,then it was flooded with solution of malachite green
  2. The stained slide was steamed for 5 minutes and case was taken to see that the solution did not dry up.
  3. Then the malachite green flooded slide was washed gently with tap water
  4. Again it was flooded with counter-stain with safranin for 30 seconds.
  5. The counter-stained smear was washed gently with tap water and dried
  6. The prepared final slide was observed under microscope

Observation and Results

By performing this Schaeffer-Fulton endospore staining procedure we can observe that vegetative cells appear pink and the spore appear green in color. Spore under microscope may locate in middle ,end of the cell and the size of spore will also vary in size.Some may be spherical or others in shape

Conclusions

The endospore are extremely resistant due to their thick wall,for which it does not take any kind of stain easily but only malachite green can penetrate the wall after considerable heating.That’s why once endospores are vegetative cells can take the color of safranin and become pink or red in colour

Some Endospore Forming Bacteria

Bacillus cereus , Bacillus anthracis , Clostridium botulinum , Clostridium tetani

References and Source


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