7.3: Endspore Procedures and Results

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1. Heat-fix a smear of Bacillus megaterium as follows:

a. Using the dropper bottle of deionized water found in your staining rack, place 1/2 of a normal sized drop of water on a clean slide by touching the dropper to the slide. Alternately, use your sterilized inoculating loop to place a drop of deionized water on the slide.

b. Using your sterile inoculating loop, aseptically remove a small amount of the culture from the edge of the growth on the agar surface and generously mix it with the drop of water until the water becomes visibly cloudy .

c. Incinerate the remaining bacteria on the inoculating loop.

d. After the inoculating loop cools, spread the suspension over approximately half of the slide to form a thin film.

e. Allow this thin suspension to completely air dry.

f. To heat-fix the bacteria to the slide, pick up the air-dried slide with coverslip forceps and hold the bottom of the slide opposite the smear against the opening of the bacto-incinerator for 10 seconds as demonstrated by your instructor. If the slide is not heated enough, all the bacteria will wash off. If it is overheated, the bacteria structural integrity can be damaged.

2. Place a piece of blotting paper over the smear and saturate with malachite green.

3. Let the malachite green sit on the slide for one minute and proceed to the next step.

4. Fill a glass beaker approximately one-fourth full with tap water, place it on a hot plate, and bring the water to a boil. Reduce the heat so the water simmers and place your slide on top of the beaker. Your slide will get hot so be sure to handle the slide with a test tube holder. Steam the slide for 5 minutes. As the malachite green evaporates, continually add more. Do not let the paper dry out!

5. After five minutes of steaming, wash the excess stain and blotting paper off the slide with water. Don't forget to wash of any dye that got onto the bottom of the slide.

6. Blot the slide dry.

7. Now flood the smear with safranin and stain for one minute.

8. Wash off the excess safranin with water, blot dry, and observe using oil immersion microscopy. With this endospore staining procedure, endospores will stain green while vegetative bacteria will stain red. (See Fig. \(\PageIndex{1}\).)

Photomicrograph of an endospore stain of <i>Bacillus megaterium</i> showing green endospores inside red vegetative cells. — Figure \(\PageIndex{1}\): Endospore stain of *Bacillus megaterium*. Note green endospores within pink bacilli. (Copyright; Gary E. Kaiser, Ph.D. The Community College of Baltimore County, Catonsville Campus CC-BY-3.0)

9. Make sure you carefully pour the used dye in your staining tray into the waste dye collection container, not down the sink.

10. Observe the demonstration slide of Bacillus anthracis. (See \(\PageIndex{2}\).) With this staining procedure, the vegetative bacteria stain blue and the endospores are colorless. Note the long chains of rod-shaped, endospore-containing bacteria.

Photomicrograph of an endospore stain of <i>Bacillus anthracis</i> showing streptobacilli with colorless endospores inside streptobacilli. Vegetative cells are stained blue. — Figure \(\PageIndex{2}\): **Endospore stain of Bacillus anthracis**. Note the endospores within the streptobacillus. (Copyright; Gary E. Kaiser, Ph.D. The Community College of Baltimore County, Catonsville Campus CC-BY-3.0)

11. Observe the demonstration slide of Clostridium tetani (See \(\PageIndex{3}\).) With this staining procedure, the vegetative bacteria stain blue and the endospores are colorless. Note the "tennis racket" appearance of the endospore-containing Clostridium.