Make a bacterial smear. Dry and heat-fix the slide then cover it with malachite green solution. Heat the slide for 3 minutes; avoid boiling.
(If not using steam method then apply stain while keeping the slide wet for 10 minutes; add more stain to keep it wet if necessary).
Wash in running tap water. Cover the slide with Safranin solution and wait 1 minute. Remove solution, wash and dry.
Schaeffer & Fulton 1933
Endospores are non-reproductive structures produced by some Gram-positive bacteria, being a form of resistance in unfavorable
conditions, unlike the eukaryotic spores wich have a reproductive role. Bacterial endospores exists in a dormant state waiting for
reactivation when conditions become favorable.
Staining technique consists in dyeing the smear with malachite green. Only the
endospore retain the stain. Vegetative cell is decolorized when washed with water.
The counterstain safranin is then used to color vegetative cell.
Malachite green solution 5%,
Safranin solution 0.5% or diluted fuchsin.
Use a culture about one week old; it will contain both vegetative cells and
endospores. An older than two weeks culture may have few if any vegetative cells. A
younger than a few days culture may have less or no endospores.
|Malachite-green-stained, central, non-deforming
spores (Bacillus anthracis)
Light-green inclusions (endospores) in pink-to-red vegetative cells.
Excesive heating may deform the cells.
Short time malachite green staining may result in weak coloration of the spores.
A demonstration of staining procedure is available in video section.
(c) Costin Stoica