THE SPORE STAIN

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