Endospore Staining by Dorner’s Method

Endospore Staining by Dorner’s Method

Introduction

  • Endospore is a resistant refractile structure formed inside the cell.
  • Endospore is generally formed by some gram positive bacteria.
  • When a bacteria come across some unfavorable conditions it gives rise to resistant structure called as endospore.
  • These endospore is metabolically inactive,it has low moisture content and it is made up of several layers.
  • Endospore of bacterial is different from fungal spores.
  • It is resistant to heat, freezing, desiccation, radiation and chemical agents.
  • So staining of such a resistant structure is not a easy job.
  • The endospore structure doesn’t get stain easily by a simple staining procedure it requires a technique.
  • Here we have two staining techniques and they are
  1. Dorner’s staining technique.
  2. Bartholomew and Mittwer’s staining technique.

Dorner’s method

Aim

To stain endospore of bacterial cell by Dorner’s method.

Requirement

  1. Cell suspension of sporulating bacteria.
  2. ZNCF stain.
  3. 10 % Nigrosine stain.

Procedure

  1. Take a thick cell suspension and add two drops of distill water.
  2. Further add equal amount of ZNCF stain and keep this tube containing mixture in boiling water bath for 15 minutes.
  3. After 15 minutes take a loopful of suspension from the tube and make a smear on a clean grease free slide.
  4. Air dry and heat fix the slide.
  5. Further take a drop of nigrosine on one end of a slide and make a thin film of a stain all over the smear with the help of other slide.
  6. Allow the film of  Nigrosin to air dry.
  7. After air drying observe the slide under oil immersion.

Flow chart of Endospore staining procedure

Endospore staining by Dorner's method

Mechanism

  1. Here we use a physical and chemical method to increase the penetration of stain in endospore.
  2. First of all we take a thick suspension plus distill water and add equal amount of ZNCF stain.
  3. Firstly ZNCF stain is prepared in phenolic solution and phenol helps in penetration of stain in endospore.
  4. Secondly we place this mixture in boiling water bath for 15 minutes so here also heating increases penetration of stain.
  5. Now once the endospore is stained it doesn’t get decolourise easily by water wash treatment.
  6. Further when we apply 10 % Nigrosine it acts as a decolourising agent as well as stain the background of a cell.
  7. After Nigrosine treatment it creates a clear contrast by removing the stain from cytoplasm.

Observation

Here we can observe red colour endospores in colourless cytoplasm against black background.

 

 

 

 

 

 

 

 

 

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