Spore stain

 

Some Gram-positive bacteria produce endospores which are highly resistant to heating and a variety of chemicals. These spores are found in the genera Bacillus and Clostridium. The spores are difficult to stain using normal techniques, but it is possible to drive the stain into them using heat. Once the stain has been taken up by the spores, it is extremely difficult to decolourise them, although the vegetative portion of the cell can be decolourised easily. Several versions of the spore stain are used.

It is advisable to use old cultures grown on nutrient agar, as spore production is a survival mechanism found in ageing cultures. A smear should be made and fixed in the normal manner. The slide should be placed on the rim of a beaker half full of boiling water, with the bacterial film uppermost, and then the following procedure used.

(a) Flood the slide with 5% aqueous malachite green and leave for 2-3 minutes. Do not allow the stain to dry; if the stain starts to dry, add more.

(b) Remove the slide from the heat and wash well with cold water. Blot dry.

(c) Stain with 0.5% safranin for 30 seconds.

(d) Rinse quickly in cold water, blot and examine under an oil immersion lens.

The spores will stain green and vegetative portions of the cell will stain red/ pink. If a very old culture was used, it may be difficult to find vegetative material.

The size and position of the spores should be noted, as this may be of diagnostic value, especially when examining members of the genus Clostridium. They may be polar or central, and may be the same diameter as the cell or larger than it, causing it to bulge.