Fundamentals of Microbiology (2+2)

Background

 Bacilli belonging the genus Bacillus or Clostridium, and a few cocci, form resting structures known as endospores. Sporulation appears to be a natural part of the life cycle of these organisms when vegetative growth ceases. Sporulation is most often triggered by depletion of an essential nutrient. The endospores may occupy space within the confines of the cell or be greater in width than the cells and occur in central, sub-terminal, or terminal positions. Ultimately, endospores are released by autolysis of the parent cell.

Alterations occurring during sporulation cause endospores to have a refractile quality when observed with a bright-field microscope. The refractile cells are not only resistant to ordinary staining methods, but resistant to heat, cold, ultraviolet, drying, and the action of disinfectants. The structural compactness of endospores makes it necessary to use heat to permit dyes to permeate the cells. In the Schaeffer-Fulton technique, an aqueous solution of malachite green is applied to a smear and heated in a manner similar to that used in the Ziehl-Neelsen acid-fast staining method. Water is used to decolorize vegetative cells. Endospores are relatively resistant to the washing procedure because of their impermeable nature. The application of a counterstain of safranin causes vegetative cells to stain red, whereas endospores retain the green color of the primary dye.

This exercise has been designed to introduce the Schaeffer ­Fulton endospore-staining technique and to stress observation of endospores for shape and position. You should be able to see endospores still encased within the parent cell as well as free spores which have been released by autolysis.