Spore Staining – A Basic Overview

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What is endospore?

Functions of endospore

How to stain endospore

Endospores – Some bacteria in the vegetative form such as Bacillus spp. and Clostridium spp. When subjected in a adverse condition of nutrient depletion, they produce endospores which are metabolically inactive. This endospore formation helps them to survive in adverse condition. When conditions become favourable they get germinated and forms vegetative cell of pathogenic bacteria. Endospores can form in any location in the vegetative cell like they can be terminal central or sub terminal.

Central endospore- forms in the centre of cell

Terminal endospore- forms in the end of cell

Sub terminal endospore- forms between the middle and end of cell.

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Functions of endospore – They are highly resistant , metabolically inactive forms in adverse condition. They are highly resistant to – heat , dehydration , radiation and chemicals. They are resistant because they contains –

1) Dipicolinic acid , a calcium ion chelator.

2) Keratin spore coat.

– Mature endospore contains DNA from vegetative cell , ribosomes and specialized enzymes.

– They vary in shape

– they can be spindle shaped , club shaped , tennis racket shaped and also  can appear as a drumstick.

Principle of spore staining – Primary stain malachite green is used in this staining. It is used to stain the endospore and endospore  cant  get stain easily so malachite green is forced to stain to spore by heating. This malachite green can be easily washed off from the vegetative cell because it is weakly bind to the cell but cant be easily washed off from the spores. So here water is used as a decolouriser so there is no need of another decolouriser and heat is acts as a mordant. And also because of heating cells get lysed so malachite can get easily washed off from the cell and cell will get red colour of saffranine.

Procedure of spore staining –  1) prepare a smear of organism which is to be test for the presence of endospore on clean Greece free glass slide. 2) Then heat fix. 3) Then place a small piece blotting paper on the smear and keep the slide on wire gauze heating on burner. 4) Heat the slide gently till it starts to evaporate. 5) Add 1-2 drops of malachite green on it for keeping it moist. Don’t heat too much ,  this process is to steaming not baking. 6) After 5 min. remove the slide from wire gauze.7) Remove the blotting paper and let the slide cool to room temperature for 2min. 8) Rinse the slide thoroughly with tap water. 9) Then add saffranine on it and keep it for 2min.10) rinse the slide with tap water to remove secondary stain and air dry and observe under oil immersion lens.

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Observation of endospore- spores will shows green colour and vegetative cell shows red colour. This is all about the endospore staining

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