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.

This 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 cells.

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.