ENDOSPORE STAINING

Introduction
Members of the anaerobic genera Clostridium and Desulfotomaculum and the aerobic genus
Bacillus are examples of organisms that have the capacity to exist either as metabolically active
vegetative cells or as highly resistant, metabolically inactive cell types called spores. When
environmental conditions become unfavorable for continuing vegetative cellular activities,
particularly with the exhaustion of a nutritional carbon source, these cells have the capacity to
undergo sporogenesis and give rise to a new intracellular structure called the endospore, which
is surrounded by impervious layers called spore coats. As conditions continue to worsen, the
endospore is released from the degenerating vegetative cell and becomes an independent cell
called a free spore. Because of the chemical composition of spore layers, the spore is resistant
to the damaging effects of excessive heat, freezing, radiation, desiccation, and chemical agents,
as well as to the commonly employed microbiological stains. With the return of favorable
environmental conditions, the free spore may revert to a metabolically active and less resistant
vegetative cell through germination. It should be emphasized that sporogenesis and
germination are not means of reproduction but merely mechanisms that ensure cell survival
under all environmental conditions.

In practice, the spore stain uses two different reagents. An alternative method known as the
Dorner method is widely published and utilizes nigrosin as the counterstain.

Objectives of Spore Stain

1. To prepare an endospore stain of bacterial cells and demonstrate endospores in the
stained preparation.
2. To differentiate between vegetative cells and endospores.

Principle of Endospore Staining

Bacterial endospores are metabolically inactive, highly resistant structures produced by some
bacteria as a defensive strategy against unfavorable environmental conditions. The bacteria can
remain in this suspended state until conditions become favorable and they can germinate and
return to their vegetative state.
In the Schaeffer-Fulton`s method, a primary stain-malachite green is forced into the spore
by steaming the bacterial emulsion. Malachite green is water soluble and has a low affinity
for cellular material, so vegetative cells may be decolourized with water. Safranin is then
applied to counterstain any cells which have been decolorized. At the end of the staining
process, vegetative cells will be pink, and endospores will be dark green.

Spores may be located in the middle of the cell, at the end of the cell, or between the end
and middle of the cell. Spore shape may also be of diagnostic use. Spores may be spherical or
elliptical.

Reagents used for Endospore Staining (Schaeffer-Fulton’s Method)

Primary Stain: Malachite green (0.5% (wt/vol) aqueous solution)
0.5 gm of malachite green
100 ml of distilled water
(Malachite Green: Unlike most vegetative cell types that stain by common procedures, the
free spore, because of its impervious coats, will not accept the primary stain easily. For further
penetration, the application of heat is required. After the primary stain is applied and the smear
is heated, both the vegetative cell and spore will appear green).

Decolorizing agent:
Tap water or Distilled Water
(Decolorizing Agent: Water Once the spore accepts the malachite green, it cannot be
decolorized by tap water, which removes only the excess primary stain. The spore remains
green. On the other hand, the stain does not demonstrate a strong affinity for vegetative cell
components; the water removes it, and these cells will be colorless).

Counter Stain:
Safranin
Stock solution (2.5% (wt/vol) alcoholic solution)
2.5 gm of safranin O
100 ml of 95% ethanol
(Counterstain (Safranin): This contrasting red stain is used as the second reagent to color the
decolorized vegetative cells, which will absorb the counterstain and appear red. The spores
retain the green of the primary stain).

Procedure of Endospore Staining

1. Take a clean grease free slide and make smear using sterile technique.
2. Air dry and heat fix the organism on a glass slide and cover with a square of blotting
paper or toweling cut to fit the slide.
3. Saturate the blotting paper with malachite green stain solution and steam for 5
minutes, keeping the paper moist and adding more dye as required. Alternatively, the
slide may be steamed over a container of boiling water.
4. Wash the slide in tap water.
5. Counterstain with 0.5% safranin for 30 seconds. Wash with tap water; blot dry.
6. Examine the slide under microscope for the presence of endospores. Endospores are
bright green and vegetative cells are brownish red to pink.
Result of Endospore Staining

Endospores: Endospores are bright green.

Vegetative Cells: Vegetative cells are brownish red to pink.

Spores may be located in the middle of the cell, at the end of the cell, or between the end and
middle of the cell. Spore shape may also be of diagnostic use. Spores may be spherical or
elliptical.
Endospore Staining by Dorner’s Method
Carbolfuchsin stain
0.3 gm of basic fuchsin
10 ml of ethanol, 95% (vol/vol)
5 ml of phenol, heat-melted crystals
95 ml of distilled water
Dissolve the basic fuchsin in the ethanol; then add the phenol dissolved in the water.
Mix and let stand for several days. Filter before use.

Decolorizing solvent (acid-alcohol)

97 ml of ethanol, 95% (vol/vol)
3 ml of hydrochloric acid (concentrated)

Counterstain (Nigrosin solution)

10 gm of nigrosin
100 ml of distilled water

Procedure
1. Take a clean grease free slide and make smear using sterile technique.
2. Air dry and heat fix the organism on a glass slide and cover with a square of blotting
paper or toweling cut to fit the slide.
3. Saturate the blotting paper with carbolfuchsin and steam for 5 to 10 minutes, keeping
the paper moist and adding more dye as required. Alternatively, the slides may be
steamed over a container of boiling water.
4. Remove the blotting paper and decolorize the film with acid-alcohol for 1 minute;
rinse with tap water and blot dry.
5. Further take a drop of nigrosine on one end of a slide and make a thin film of a stain
all over the smear with the help of other slide.
6. Allow the film of Nigrosin to air dry.
7. After air drying observe the slide under oil immersion.
Vegetative cells are colorless, endospores are red, and the background is black.
Examples of Endospore Staining
Positive
Clostridium perfringens, C. botulinum, C. tetani, Bacillus anthracis, Bacillus
cereus, Desulfotomaculum spp, Sporolactobacillus spp, Sporosarcina spp, etc.

Negative
E. coli, Salmonella spp, etc.

Quality control of Endospore Staining

Positive control: Clostridium perfringens (ATCC 13124)
Negative control: Escherichia coli (ATCC 25922)