A few genera of bacteria, such as Bacillus and Clostridium, have the ability to
produce resistant survival forms
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
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
Both of these membrane layers then synthesize peptidoglycan in
the space between them to form the first protective coat, the cortex.
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
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.
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
Trypticase soy agar plate cultures of Bacillus megaterium
- Heat-fix a smear of Bacillus megaterium as follows:
- 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.
- 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.
- Burn the remaining bacteria off of the loop.
- Using the loop, spread the suspension over the entire slide to form a
- Allow this thin suspension to completely air dry.
- Pass the slide (film-side up) through the flame of the Bunsen burner 3
or 4 times to heat-fix.
- Place a piece of blotting paper over the smear and saturate with malachite
- Let the malachite green sit on the slide for 1 minute and proceed to the
- 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.
- 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.
- Blot the slide dry.
- Now flood the smear with safranin and stain for 1 minute.
- 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.
- 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.
- 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.
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
The arrangement of the flagella about the bacterium is of use in classification
and identification. The following flagellar arrangements may be found:
- Monotrichous, a single flagellum at 1 pole.
- Amphitrichous, a single flagella at both poles.
- Lophotrichous, 2 or more flagella at 1 or both poles of the cell.
- 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
- motility media, and
- flagella staining.
Direct observation of motility using special-purpose microscopes
- 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
- 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.
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
Trypticase soy broth cultures of Pseudomonas aeruginosa and Staphylococcus aureus.
Caution: Handle these organisms as pathogens.
Medium: Motility test medium (2 tubes).
- Observe the phase-contrast video microscopy demonstration of motile Pseudomonas aeruginosa.
- Observe the dark-field microscopy demonstration of motile Pseudomonas
- 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.
- 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.
Make drawings of the various endospore stain preparations.
- Observe the phase—contrast and dark-field microscopy demonstrations of
- Observe the 2 tubes of motility test medium.
- Make drawings of the flagella stain demonstrations.
- Name 2 endospore-producing genera of bacteria.
- Describe the function of bacterial endospores.
- Recognize endospores as the structures observed in an endospore stain
- Identify a bacterium as an endospore-containing Clostridium by its “tennis
- Define the following flagellar arrangements: monotrichous, lophotrichous,
amphitrichous, peritrichous, and axial filaments.
- Describe the chemical nature and function of bacterial flagella.
- Describe 3 methods of testing for bacterial motility and indicate how to
interpret the results.
- Recognize bacterial motility when using phase-contrast or dark-field
- Interpret the results of the motility test medium.
- Recognize monotrichous, lophotrichous, amphitrichous, and peritrichous