Endospore Staining by Dorner’s Method
- 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
- Dorner’s staining technique.
- Bartholomew and Mittwer’s staining technique.
To stain endospore of bacterial cell by Dorner’s method.
- Cell suspension of sporulating bacteria.
- ZNCF stain.
- 10 % Nigrosine stain.
- Take a thick cell suspension and add two drops of distill water.
- Further add equal amount of ZNCF stain and keep this tube containing mixture in boiling water bath for 15 minutes.
- After 15 minutes take a loopful of suspension from the tube and make a smear on a clean grease free slide.
- Air dry and heat fix the slide.
- 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.
- Allow the film of Nigrosin to air dry.
- After air drying observe the slide under oil immersion.
Flow chart of Endospore staining procedure
- Here we use a physical and chemical method to increase the penetration of stain in endospore.
- First of all we take a thick suspension plus distill water and add equal amount of ZNCF stain.
- Firstly ZNCF stain is prepared in phenolic solution and phenol helps in penetration of stain in endospore.
- Secondly we place this mixture in boiling water bath for 15 minutes so here also heating increases penetration of stain.
- Now once the endospore is stained it doesn’t get decolourise easily by water wash treatment.
- Further when we apply 10 % Nigrosine it acts as a decolourising agent as well as stain the background of a cell.
- After Nigrosine treatment it creates a clear contrast by removing the stain from cytoplasm.
Here we can observe red colour endospores in colourless cytoplasm against black background.