Endospore Staining by Schaeffer –Fulton Method.
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 cellwall 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 decolourize and appear green within red
cells.
Suitable organism: Bacillus subtilis
Procedure: 1. Make a smear of the organisms on separate grease-free slides
2 Air dry and heat fix the smear.
3. Flood the smears with malachite green solution (stain for the spore).
3. Heat the slide to steaming and continue steaming for 5 minutes adding
more stains to the smear from time to time. (a set of slides may be steamed
for 10 and 15 minutes as well)
4. Wash under gentle stream of tap water.
5. Counter stain with safranin for 30 secs.
6. Wash smear with distilled water.
7. Air dry and examine under microscope.
Dorner’s Method:
Procedure: 1) 1ml Carbol fuchsin + 1 ml culture
2) boiling for 5, 10 & 15 min.
3) one drop placed in a grease free slide with inoculating needle .
4) The drop mixed with nigrosin.
5) Smear made very quickly.
6) observed under microscope.
