In Experiment 4, you may well have seen bacterial spores. They are
refractile to most staining procedures and appear as clear areas in the Gram
stain. Poly-beta hydroxybutyrate (bacterial fat) and other intracellular
inclusions can also not stain in the Gram stain and appear as clear areas. The
spore stain allows you to clearly demonstrate that the clear area in a Gram
stained or simple stained cell is a spore. Since spores are refractile to
stains, the Malachite green stain must be "driven" into the spore by
heat. That is, during the spore stain, you will heat the slide being stained to
steaming....this causes breaks in the spore such that the stain can penetrate
the spore coat, cortex and spore wall. After cooling the slide is rinsed in
water and counterstained with safranin. The cells stain red and the spores
stain green.
MATERIALS
1.
Two Nutrient Agar slant
cultures of Bacillus species. One grown for 48 hours and another grown
for 24 hours.
2.
Shaeffer-Fulton spore stain:
0.5% Malachite Green.
3.
Safranin.
4.
Slides and slide holders.
PROCEDURE
1.
Prepare two slides with the
two cultures of Bacillus as heat fixed smears.
2.
Simple stain one with
Crystal Violet.
3.
Spore Stain the other as
follows:
a. Drop Spore stain onto the smear and cover with a piece of paper
towel. The paper towel must be cut so that it does not extend beyond the sides
of the slide (Figure 1.)
b. Heat
the slide to steaming for 5 minutes. Do not let the stain dry on the slide, to
prevent this add stain when it gets close to drying out.
c. Allow
the slide to cool (place it on the cool bench). Remove the paper towel and put
it in the waste bucket (not the sink!).
d. Rinse
with water.
e. Counterstain
with safranin for 1 minute.
f. Rinse
with water, dry and observe with the oil immersion Lens.
Figure 1
