Transcript Spore stain

Spore stain
(Schaeffer-Fulton )
Principle, Procedure
and Results
MSc. Sarah Ahmed
Spore stain
(Schaeffer-Fulton method)
• Some gram positive bacteria especially members of the
genera clostridium and bacillus have the ability to form
spore(endospore) when exposed to drastic unfavorable
conditions such as drying, heating, lack of nutrients and so
on.
• Spores formed by these bacteria are highly resistant to
unfavorable conditions and can remain viable for several
years, once the conditions become favorable the spore
germinates.
Principle of Spore Staining:
A differential staining technique (the Schaeffer-Fulton method) is
used to distinguish between the vegetative cells and the
endospores.
A primary stain (malachite green) is used to stain the
endospores. Because the malachite green permeate the spore
wall by heating. In this technique heating acts as a mordant.
There is no need of using any decolorizer in this spore staining as
the primary dye malachite green bind relatively weakly to the cell
wall and spore wall .In fact If washed well with water the dye come
right out of cell wall however not from spore wall once the dye is
locked in. Water is used to decolorize the vegetative cells.
The addition of a counterstain or secondary stain (safranin) is
used to stain the decolorized vegetative cells.
Spore stain(cont..)
,
• Spores don t accept the stain easily due to their thick
impervious coat and require the use of heating during
staining.
Procedure:
1. Prepare smear of the microorganism, allow to dry and the
heat-fix to a slide.
2. Place the slide over a steam bath, flood the smear with
malachite green dye and keep it for 3-5 mins, replenish
smear with malachite green dye if some evaporates and don't
allow the stain to boil.
Spore stain(cont..)
3. Allow the slide to cool and wash with tap water to remove
excess stain.
4. Counterstain with safranin for 2 min.
5. Rinse the slide with running tap water to remove excess stain.
6. Blot dry with bibulous paper and examine under oil
immersion.
• Results:
The vegetative cells will appear pink/red
• and the spores will appear green.
THE END