Endospore staining by Bartholomew and Mittwer’s Method
To stain Endospore of a bacterial cell by Bartholomew and Mittwer’s Method.
- Cell suspension of endospore producing bacteria.
- Malachite green stain.
- Saffranin stain.
- Take a clean grease free slide and prepare a thick smear on a slide.
- The smear is heat fixed by passing the slide from the flame for about 25 times.
- The slide is allowed to cool.
- Further the slide is treated with Malachite green stain and allowed it to react for about 10 minutes.
- After 10 minutes slide is given a water wash treatment.
- Further the slide is treated with counter stain that is saffranin for about 30 seconds.
- After 30 seconds the slide is water washed, air dried and observed under oil immersion.
Flow chart of Endospore staining by Bartholimew and Mittwer’s Method.
- Here in these staining technique we apply a longer heat treatment and prolonged staining technique.
- Endospore get stained due to longer heat treatment,prolonged staining and heavy concentration of stain.
- here we pass he slide from flame for about 25 times in addition we use concentrated stain that is 7.6 % Malachite green for about 10 minutes.
- This technique stains the cell as well as the endospore.
- When we give water wash treatment the water acts as a weak decolourising agent and decolourises cytoplasm and not endospore.
- So here further we apply a counter stain that is Saffranin .
- Due to application of saffranin the cytoplasm gets stain in pink colour.
After staining procedure the endospore appear green in colour as well as cytoplasm appear pink in colour.