Endospore staining by Bartholomew and Mittwer’s Method

Endospore staining by Bartholomew and Mittwer’s Method

Aim

To stain Endospore of a bacterial cell by Bartholomew and Mittwer’s Method.

Requirement

  1. Cell suspension of endospore producing bacteria.
  2. Malachite green stain.
  3. Saffranin stain.

Procedure

  1. Take a clean grease free slide and prepare a thick smear on a slide.
  2. The smear is heat fixed by passing the slide from the flame for about 25 times.
  3. The slide is allowed to cool.
  4. Further the slide is treated with Malachite green stain and allowed it to react for about 10 minutes.
  5. After 10 minutes slide is given a water wash treatment.
  6. Further the slide is treated with counter stain that is saffranin for about 30 seconds.
  7. After 30 seconds the slide is water washed, air dried and observed under oil immersion.

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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.

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