Endospore Staining and Bacterial Motility

Endospore Staining
Introduction
A few genera of bacteria, such as Bacillus and Clostridium, have the ability to produce resistant survival forms termed endospores. Unlike the reproductive spores of fungi and plants, these endospores are resistant to heat, drying, radiation, and various chemical disinfectants. Endospore formation (sporulation) occurs through a complex series of events. One is produced within each vegetative bacterium. Once the endospore is formed, the vegetative portion of the bacterium is degraded and the dormant endospore is released.

First, the DNA replicates and a cytoplasmic membrane septum forms at one end of the cell. A second layer of cytoplasmic membrane then forms around one of the DNA molecules (the one that will become part of the endospore) to form a forespore. Both of these membrane layers then synthesize peptidoglycan in the space between them to form the first protective coat, the cortex.

Calcium dipocolinate is also incorporated into the forming endospore. A spore coat composed of a keratin-like protein then forms around the cortex. Sometimes an outer membrane composed of lipid and protein, called an exosporium, is also seen.

Finally, the remainder of the bacterium is degraded and the endospore is released. Sporulation generally takes around 15 hours. The endospore is able to survive for long periods of time until environmental conditions again become favorable for growth. The endospore then germinates, producing a single vegetative bacterium.

Bacterial endospores are resistant to antibiotics, most disinfectants, and physical agents such as radiation, boiling, and drying. The impermeability of the spore coat is thought to be responsible for the endospore’s resistance to chemicals. The heat resistance of endospores is due to a variety of factors:

• Calcium-dipicolinate, abundant within the endospore, may stabilize and protect the endospore’s DNA.
• Specialized DNA-binding proteins saturate the endospore’s DNA and protect it from heat, drying, chemicals, and radiation.
• The cortex may osmotically remove water from the interior of the endospore, and the dehydration that results is thought to be very important in the endospore’s resistance to heat and radiation.
• Finally, DNA repair enzymes contained within the endospore are able to repair damaged DNA during germination.

Due to the resistant nature of the endospore coats, endospores are difficult to stain. Strong dyes and vigorous staining conditions, such as heat, are needed. Once stained, however, endospores are equally hard to decolorize. Since few bacterial genera produce endospores, the endospore stain is a good diagnostic

Organisms
Trypticase soy agar plate cultures of Bacillus megaterium.

Procedure

1. Heat-fix a smear of Bacillus megaterium as follows:
   
   1. Using the dropper bottle of distilled water found in your staining rack, place a small drop of water on a clean slide by touching the dropper to the slide.
   2. 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 turns cloudy.
   3. Burn the remaining bacteria off of the loop.
   4. Using the loop, spread the suspension over the entire slide to form a thin film.
   5. Allow this thin suspension to completely air dry.
   6. Pass the slide (film-side up) through the flame of the Bunsen burner 3 or 4 times to heat-fix.
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 1 minute and proceed to the next step.
4. Holding the slide with forceps, carefully heat the slide in the flame of a Bunsen burner until the stain begins to steam. Remove the slide from the heat until steaming stops; then gently reheat. Continue steaming the smear in this manner for 5 minutes. As the malachite green evaporates, continually add more. Do not let the paper dry out.
5. After 5 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 1 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.
9. Observe the demonstration slide of Bacillus anthracis. With this staining procedure, the vegetative bacteria stain blue and the endospores are colorless. Note the long chains of rod-shaped, endospore-containing bacteria.
10. Observe the demonstration slide of Clostridium tetani. With this staining procedure, the vegetative bacteria stain blue and the endospores are colorless. Note the “tennis racquet” appearance of the endospore-containing Clostridium.

Bacterial Motility
Introduction
Many bacteria are capable of motility (the ability to move under their own power). Most motile bacteria propel themselves by special organelles termed flagella. The bacterial flagellum is a noncontractile, semi-rigid, helical tube composed of protein and anchors to the bacterial cytoplasmic membrane and cell wall by means of disk-like structures. The rotation of the inner disk causes the flagellum to act much like a propeller.

Bacterial motility constitutes unicellular behavior. In other words, motile bacteria are capable of a behavior called taxis. Taxis is a motile response to an environmental stimulus, and functions to keep bacteria in an optimum environment.

The arrangement of the flagella about the bacterium is of use in classification and identification. The following flagellar arrangements may be found:

1. Monotrichous, a single flagellum at 1 pole.
2. Amphitrichous, a single flagella at both poles.
3. Lophotrichous, 2 or more flagella at 1 or both poles of the cell.
4. Peritrichous, completely surrounded by flagella.

One group of bacteria, the spirochetes, has internally located axial filaments or endoflagella. Axial filaments wrap around the spirochete towards the middle from both ends. They are located above the peptidoglycan cell wall, but underneath the outer membrane or sheath.

To detect bacterial motility, we can use any of the following 3 methods:

• direct observation by means of special-purpose microscopes (phase-contrast and dark-field),
• motility media, and
• flagella staining.

Direct observation of motility using special-purpose microscopes

1. Phase-contrast microscopy. A phase-contrast microscope uses special phasecontrast objectives and a condenser assembly to control illumination and produce an optical effect of direct staining. The special optics convert slight variations in specimen thickness into corresponding visible variation in brightness. Thus, the bacterium and its structures appear darker than the background.
2. Dark-field microscopy. A dark-field microscope uses a special condenser to direct light away from the objective lens. However, bacteria (or other objects) lying in the transparent medium will scatter light so that it enters the objective. This produces the optical effect of an indirect stain. The organism will appear bright against the dark background. Dark-field microscopy is especially valuable in observing the very thin spirochetes.

A semi-solid motility test medium may also be used to detect motility. The agar concentration (0.3%) is sufficient to form a soft gel without hindering motility. When a nonmotile organism is stabbed into a motility test medium, growth occurs only along the line of inoculation. Growth along the stab line is very sharp and defined. When motile organisms are stabbed into the soft agar, they swim away from the stab line. Growth occurs throughout the tube, rather than being concentrated along the line of inoculation. Growth along the stab line appears much more cloudlike as it moves away from the stab. A tetrazolium salt (TTC) is incorporated into the medium. Bacterial metabolism reduces the TTC producing formazan, which is red. The more bacteria present at any location, the darker red the growth appears.

Flagella staining
If we assume that bacterial flagella confer motility, flagella staining can then be used indirectly to denote bacterial motility. Since flagella are very thin (20–28 nm in diameter), they are below the resolution limits of a normal light microscope and cannot be seen unless one first treats them with special dyes and mordants, which build up as layers of precipitate along the length of the flagella, making them microscopically visible. This is a delicate staining procedure and will not be attempted here. We will, however look at several demonstration flagella stains.

Organisms
Trypticase soy broth cultures of Pseudomonas aeruginosa and Staphylococcus aureus.

Caution: Handle these organisms as pathogens.
Medium: Motility test medium (2 tubes).

Procedure

1. Observe the phase-contrast video microscopy demonstration of motile Pseudomonas aeruginosa.
2. Observe the dark-field microscopy demonstration of motile Pseudomonas aeruginosa.
3. Take 2 tubes of motility test medium per pair. Stab one with Pseudomonas aeruginosa and the other with Staphylococcus aureus. Incubate at 37°C until the next lab period.
4. Observe the flagella stain demonstrations of Pseudomonas aeruginosa (monotrichous), Proteus vulgaris (peritrichous), and Spirillum undula (lophotrichous), as well as the dark-field photomicrograph of the spirochete. When observing flagella stain slides, keep in mind that flagella often break off during the staining procedure, so you have to look carefully to observe the true flagellar arrangement.

Results
Endospore Stain:
Make drawings of the various endospore stain preparations.

Bacterial Motility:

1. Observe the phase—contrast and dark-field microscopy demonstrations of bacterial motility.
2. Observe the 2 tubes of motility test medium.
3. Make drawings of the flagella stain demonstrations.

Performance Objectives
Endospore Stain

Discussion

1. Name 2 endospore-producing genera of bacteria.
2. Describe the function of bacterial endospores.

Results

1. Recognize endospores as the structures observed in an endospore stain preparation.
2. Identify a bacterium as an endospore-containing Clostridium by its “tennis racquet” appearance.

Bacterial Motility
Discussion

1. Define the following flagellar arrangements: monotrichous, lophotrichous, amphitrichous, peritrichous, and axial filaments.
2. Describe the chemical nature and function of bacterial flagella.
3. Describe 3 methods of testing for bacterial motility and indicate how to interpret the results.

Results

1. Recognize bacterial motility when using phase-contrast or dark-field microscopy.
2. Interpret the results of the motility test medium.
3. Recognize monotrichous, lophotrichous, amphitrichous, and peritrichous flagellar arrangements.