Spore staining by Schafflor fulton method

SUITABLE  ORGANISMS : Bacillus subtilis

PRINCIPLE : Endospore staining is a differential staining technique where the spore is stained in a manner so that it can be distinguished from the vegetative part of the cell. Spores are structures remarkably resistant to heat, radiation, chemicals and other agents that are typically lethal to the organism. The heat resistence of spores has been linked to their high content of calcium and dipicolinic acid. Although sporulation is genetically regulated, the event is initiated when the organism senses depletion of nutrients or during unfavorable environmental conditions. During sporulation, a vegetative cell gives rise to a new intracellular structure termed as endospore that is surrounded by an impermeable layer called sporecoat. Complete transformation of a vegetative cell into a sporangium and then into a spore requires 6-8 hours in most spore-forming species. An endospore develops in a characteristics position within a cell, i.e. either central ,subterminal or terminal .Once an endospore is formed in a cell, the cell wall disintegrates ,releases the endospore that becomes an independent spore. Endospores may remain dormant for long period of time. However, a free spore may return to its vegetative or growing state with the return of favourable conditions. The spores are differentially stained by using special procedures that help dyes penetrate the spore wall. An aqueous primary stain (Malachite Green) is applied and steamed to enhance penetration of the impermeable spore cores. Once stained, the endospores do not readily decolonize and appear green within red cells.