Due to the highly resistant nature of endospores, they
are not easily penetrated
by stains. Thus, it is necessary to steam the
stain into an endospore. The
Schaeffer-Fulton method is the most commonly used
endospore staining technique,
and it uses Malachite green as the primary stain.
Once the endospore has absorbed
the stain, it is resistant to decolorization, but
the vegetative cell is easily decolorized
with water (leaving the vegetative cells colorless).
Finally, the vegetative cells are
counterstained with Safranin to aid in their visualization.
When viewed under a
microscope, the endospores appear green, while the
vegetative cells are red or pink.
The steps in the endospore staining technique are
listed below.
01. Using aseptic technique, prepare a bacterial smear on a clean
slide,
air dry and gently heat fix.
02. Prepare a boiling water bath.
03. Cover the slide with a piece of paper towel, and place on a staining
rack over the water bath.
04. Flood the paper towel on the slide with Malachite green (primary
stain).
05. Steam the slide for five minutes.
06. Remove the slide from the water bath, and remove the paper towel
from the slide.
07. Allow the slide to cool, and then rinse with deionized water
until the
water runs clear.
08. Pour off any excess water and apply Safranin (counterstain) for
two
minutes.
09. Rinse excess Safranin off with deionized water, and blot the
slide dry
with bibulous paper.
10. Examine the slide with a light microscope under oil immersion.
The picture below shows the outcome of an endospore
stain. The vegetative cells
are pink/red and the endospores are green.
Results achieved by endospore staining. The green structures
are endospores,
and the pink structures are vegetative cells.
http://news.cn.edu/karrst/endospor.htm
• Introduction
•
Discovery of Endospores • Endospore
Structure •
Endospore Formation
•
• Staining Endospores •
Miscellaneous
Facts •
References •
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