Endospore staining and capsule staining of the bacteria

Aim

To study and differentiate bacterial species from other negative cells using endospore staining technique.
To observe capsule staining of capsulated bacteria by Maneval’s method.

Bacterial endospore are metabolically inactive and highly resistant structures that are produced by some bacteria to defend the unfavorable condition.
The bacteria have the ability to remain in the suspended state till the conditions are favorable enough to germinate and return to their vegetative state.
During unfavorable condition, endospores can form within different areas of vegetative cell. The regions of endospore are central, terminal, sub-terminal and the shape may differ from elliptical to spherical.
The Schaeffer- Fulton stain is the most common endospore staining technique, which differentiates vegetative cell and the endospore.
We will use malachite green, a primary water-soluble stain which is forced into the spore. To counterstain those cells which have been decolorized, a secondary stain safranin is applied.
Therefore, at the end of staining, endospores will appears dark green, whereas, vegetative cells will appears pink.

It is a method in which acidic and basic dyes are used for staining the background and bacterial cells respectively so that the capsule can be visualized easily.
Capsule is usually synthesized in the cytoplasm and secreted outside of the cell to surround the bacterium.
Capsules are usually made of polysaccharides but some capsules are made of polypeptides too.
The main principal of Maneval’s method is to stain the background using acidic solutions (congo red stain) and stain the bacterial cell using Maneval stain.
Acid fushin being the main component of Maneval’s stain penetrates the exopolysaccharide layer with the help of phenol component.

For Endospore staining

For Capsule staining

Take a clean, grease free slide.
Air dry this particular smear and then heat fix it.
Place the slide over steam bath and put a strip of blotting paper over the smear and stain it with malachite green stain for 3 minutes while this is in steam bath.
Wash the slide and air dry.
After air drying, add drops of safranin and let it stain for 5 minutes.
Wash the excess stain and again let it air dry.
Observe the slide under 100X objective lens after adding a drop of immersion oil.

Take a clean, grease free slide.
Add a few drops of Congo red stain on its terminal and add few drops of bacterial suspension using micropipette.
Spread the smear evenly throughout the slide at an angle of 45 degrees and let it air dry.
Flood the slide using Maneval’s stain and let it rest for 5-6 minutes.
Drain the excess stain and dry the stain gently suing blotting paper air dry it.
After adding a drop of immersion oil, Observe the slide under 100X objective lens.

Https://id.scribd.com/doc/126340469/Microbiology-Lab-Manual-2
Https://biologyreader.com/capsule-staining.html
Https://microbiologyinfo.com/endospore-staining-principle-reagents-procedure-and-result/
Https://www.scienceprofonline.org/vmc/vmc-lab-new-13/lab-3-differential-stains-specializedmedia/Differential-Stain-Microbiology-Lab-3-INSTRUCTIONS.pdf
Https://askinglot.com/why-is-capsule-staining-called-negative-staining-method