Differential Staining Techniques:
The Spore Stain and the Acid-Fast Stain
Two major genera of bacteria, Bacillus and Clostridium, produce
structures called endospores
which serve as resting structures,
enabling cells to overcome harsh environmental changes such as drying.
These structures are resistant to heat, osmotic changes, radiation, and
pH, and can enable a cell to remain viable for months or even years until
conditions again become conducive for vegetative growth. Click on
the images to get a larger view.
is an image of a simple stained sample of Bacillus subtilis.
Note the clear areas within the cells. Endospores do not take up
the crystal violet-iodine mordant complex, nor the safranin counterstain
during gram staining, so they must be made visible by utilizing another
differential staining technique.
This is an image of a Bacillus subtilis culture which is over 72
hours old. It was prepared with the Schaeffer-Fulton endospore stain.
The oval green structures are endospores, and the red rods are young vegetative
The Acid-Fast Stain
Mycobacterium smegmatis (1000X).Acid-fast
bacteria, including the genera Mycobacterium and Nocardia
have cell wall structure similar to that of the gram positive cell, but
these also contain mycolic acids (waxy lipids). These organisms stain
gram variable and are best visuallized utilizing techniques such as the
Zeihl-Neelsen acid-fast stain, which utilizes a carbolfuschin primary stain,
followed by a 1% hydrochloric acid/aclcohol destain to make the cells appear
pink in color.