PRACTICAL 4: The Staining of Microorganisms – Capsule Staining and Spore

Staining

1.0 INTRODUCTION

When vegetative cells of certain bacteria such as Bacillus spp and Clostridium spp

are subjected to environmental stresses such as nutrient deprivation, they produce
metabolically inactive or dormant form-endospore. Formation of endospore
circumvent the problems associated with environmental stress and helps them
to survive. During unfavorable conditions (especially when carbon and nitrogen
become unavailable) endospores can form within different areas of the vegetative
cell. They can be central, subterminal, or terminal.  Central endospores are located
within the middle of the vegetative cell.  Terminal endospores are located at the end
of the vegetative cell.  Sub-terminal endospores are located between the middle and
the end of the cell.

A differential staining technique (the Schaeffer-Fulton method) is used to distinguish

between the vegetative cells and the endospores.  A primary stain (malachite
green) is used to stain the endospores.  Because endospores resist staining, the
malachite green will be forced into (i.e, malachite green permeate the spore wall) the
endospores by heating.  In this technique heating acts as a mordant. There is no
need of using any decolorizer in this spore staining as the primary dye malachite
green bind relatively weakly to the cell wall and spore wall .In fact If washed well with
water the dye come right out of cell wall however not from spore wall once the dye is
locked in. Water is used to decolorize the vegetative cells.

 Capsule stain is a type of differential stain which uses acidic and basic dyes to stain
background & bacterial cells respectively so that presence of capsule is easily
visualized.  Capsule is synthesized in the cytoplasm and secreted to the outside of
the cell where it surrounds the bacterium.  Most of the capsulated bacteria have a
capsule made up of polysaccharide layer but some bacteria have capsule made up
of polypeptide, or glycoprotein. Bacterial capsules are non-ionic, so neither acidic nor
basic stains will adhere to their surfaces. Therefore, the best way to visualize them is
to stain the background using an acidic stain (e.g., Nigrosine, congo red) and to stain
the cell itself using a basic stain (e.g.,crystal violet, safranin, basic fuchsin and
methylene blue). Crystal violet and india ink are used. The capsule is seen as a clear
halo around the microorganism against the black background. This method is used
for demonstrating Cryptococcus. The background will be dark (color of india ink).The
bacterial cells will be stained purple (bacterial cells takes crystal violet-basic dyes as
they are negatively charged). The capsule (if present) will appear clear against the
dark background (capsule does not take any stain).