LAB 7: ENDOSPORE STAIN AND BACTERIAL MOTILITY

 

A. ENDOSPORE STAIN

DISCUSSION

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 (see Labs 19 and 20)

To view an electron micrograph of an endospore of Bacillus stearothermophilus, see the Microbe Zoo web page of Michigan State University.

Although harmless themselves until they germinate, bacterial endospores are involved in the transmission of some diseases to humans. Infections transmitted to humans by endospores include:

a. Anthrax, caused by Bacillus anthracis.

Endospores can be inhaled, ingested, or enter wounds where they germinate and the vegetative bacteria subsequently replicate and produde exotoxins. In the case of the two anthrax exotoxins, two different A-components known as lethal factor (LF) and edema factor (EF) share a common B-component known as protective antigen (PA). Protective antigen, the B-component, first binds to receptors on host cells and is cleaved by a protease creating a binding site for either lethal factor or edema factor. At low levels, LF inhibits the release of proinflammatory cytokines such as interleukin-1 (IL-1), tumor necrosis factor-alpha, (TNF-alpha), and NO. This may initially reduce immune responses against the organism and its toxins. But at high levels, LF is cytolytic for macrophages, causing release of high levels of interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and NO. Excessive release of these cytokines can lead to a massive inflammatory response and the shock cascade, similar to septic shock. Edema factor impairs phagocytosis, and inhibits production of TNF and interleukin-6 (IL-6) by monocytes. This most likely impairs host defenses.

Scanning electron micrograph of Bacillus anthracis endospores; courtesy of CDC.

b. Tetanus, caused by Clostridium tetani.

Endospores enter anaerobic wounds where they germinate and the vegetative bacteria subsequently replicate and release exotoxin. Tetanus exotoxin (tetanospasmin), produced by Clostridium tetani is a neurotoxin that binds to inhibitory interneurons of the spinal cord and blocks their release of inhibitor molecules. It is these inhibitor molecules from the inhibitory interneurons that eventually allow contracted muscles to relax by stopping excitatory neurons from releasing the acetylcholine that is responsible for muscle contraction. The toxin, by blocking the release of inhibitors, keeps the involved muscles in a state of contraction and leads to spastic paralysis, a condition where opposing flexor and extensor muscles simultaneously contract. Death is usually from respiratory failure.

c. Botulism, caused by Clostridium botulinum.

Endospores enter the anaerobic environment of improperly canned food where they germinate and subsequently replicate and at a neutral pH, secrete botulinal exotoxin. This is a neurotoxin that acts peripherally on the autonomic nervous system. For muscle stimulation, acetylcholine must be released from the neural motor end plate of the neuron at the synapse between the neuron and the muscle to be stimulated. The acetylcholine then induces contraction of the muscle fibers. The botulism exotoxin binds to and enters the presynaptic neuron and blocks its release of acetylcholine. This causes a flaccid paralysis, a weakening of the involved muscles. Death is usually from respiratory failure.

Scanning electron micrograph of Clostridium botulinum with endospore; courtesy of Dennis Kunkel's Microscopy.

d. Gas gangrene, caused by Clostridium perfringens.

Endospores enter anaerobic wounds where they germinate and the vegetative bacteria subsequently replicate and produce a variety of exotoxins. This bacterium produces at least 20 exotoxins that play a role in the pathogenesis of gas gangrene and producing expanding zones of dead tissue (necrosis) surrounding the bacteria. Toxins include: Alpha toxin (lecithinase) that increases the permeability of capillaries and muscle cells by breaking down lecithin in cytoplasmic membranes resulting in the gross edema associated with gas gangrene as well as being necrotizing, hemolytic, and cardiotoxic; Kappa toxin (collagenase) breaks down supportive connective tissue resulting in the mushy lesions of gas gangrene and is also necrotizing; Mu toxin (hyaluronidase) breaks down the tissue cement that holds cells together in tissue; and epsilon toxin Increases vascular permeability and causes edema and congestion in various organs including lungs and kidneys. Additional necrotizing toxins include beta toxin, iota toxin, and nu toxin. A major characteristic of gas gangrene is the ability of C. perfringens to very rapidly spread from the initial wound site leaving behind an expanding zone of dead tissue. This organism spreads as a result of the pressure from fluid accumulation (due to increased capillary permeability from alpha toxin) and gas production (anaerobic fermentation of glucose by the organisms produces hydrogen and carbon dioxide), coupled with the breakdown of surrounding connective tissue (kappa toxin) and tissue cement (mu toxin).

e. Antibiotic-associated pseudomembranous colitis, caused by Clostridium difficile.

Clostridium difficile causes severe antibiotic-associated colitis and is an opportunistic Gram-positive, endospore-producing bacillus transmitted by the fecal-oral route. C. difficile is a common healthcare-associated infection (HAIs) and is the most frequent cause of health-care-associated diarrhea. C. difficile infection often recurs and can progress to sepsis and death. CDC has estimated that there are about 500,000 C. difficile infections (CDI) in health-care associated patients each year and is linked to 15,000 American deaths each year. Antibiotic-associated colitis is especially common in older adults. It is thought that C. difficile survives the exposure to the antibiotic by sporulation. After the antibiotic is no longer in the body, the endospores germinate and C. difficile overgrows the intestinal tract and secretes toxin A and toxin B that have a cytotoxic effect on the epithelial cells of the colon. C.difficile has become increasingly resistant to antibiotics in recent years making treatment often difficult. There has been a great deal of success in treating the infection with fecal transplants.

For further information on bacterial endospores, see the following in your Softchalk Lectures:

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 test for species of Bacillus and Clostridium.

2. Place a piece of blotting paper over the smear and saturate with malachite green (see Fig. 19).

Videos reviewing techniques used in this lab:
Video review - Focusing Using Oil Immersion (1000X) Microscopy
Video review - Aseptic Technique: Inoculation of broth tubes, slant tubes, and stab tubes

 

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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.

 

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ORGANISMS

Trypticase Soy broth cultures of Pseudomonas aeruginosa and Staphylococcus aureus. Caution: handle these organisms as pathogens.

 

MEDIUM

Motility Test medium (2 tubes)

PROCEDURE (to be done individually and in pairs)

1. Observe the phase-contrast microscopy demonstration of motile Pseudomonas aeruginos.

Movie of motile Pseudomonas from YouTube.

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. Stab the bacterium about 1/2 - 3/4 of an inch into the agar, taking care not to tilt or twist the loop so that the loop comes up through the same cut as it went down. Incubate the tubes in your test tube rack at 37°C until the next lab period.

4. Observe the flagella stain demonstrations of a Vibrio species (monotrichous), Proteus vulgaris (peritrichous) and Spirillum undula (amphitrichous) as well as the dark-field photomicrograph of the spirochete Leptospira. 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.

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RESULTS

A. Endospore Stain

Make drawings of the various endospore stain preparations.

 

 


Endospore stain of Bacillus megaterium

 


Endospore stain of Bacillus anthracis


Endospore stain of Clostridium tetani

B. Bacterial Motility

1. Observe the phase contrast and dark-field microscopy demonstrations of bacterial motility.

2. Observe the two tubes of Motility Test medium.

Conclusion:


Conclusion:

 

 

3. Make drawings of the flagella stain demonstrations.



Flagella stain of a
Vibrio
species


Flagella stain of
Proteus vulgaris


Flagella stain of
Spirillum undula

Arrangement =

 

Arrangement =

 

Arrangement =

 

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PERFORMANCE OBJECTIVES FOR LAB 7

After completing this lab, the student will be able to perform the following objectives:

A. ENDOSPORE STAIN

DISCUSSION

1. Name two endospore-producing genera of bacteria.

2. State 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.

B. BACTERIAL MOTILITY

DISCUSSION

1. Define the following flagellar arrangements: monotrichous, lophotrichous, amphitrichous, peritrichous, and axial filaments.

2. State the function of bacterial flagella.

3. Describe three 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 Motility Test Medium.

3. Recognize monotrichous, lophotrichous, amphitrichous, and peritrichous flagellar arrangements.

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SELF-QUIZ

Self-quiz

Answers

 

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Lab Manual Table of Contents


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Microbiology Laboratory Manual by Gary E. Kaiser, PhD, Professor of Microbiology
is licensed under a Creative Commons Attribution 4.0 International License.

Last updated: August, 2017
Please send comments and inquiries to Dr. Gary Kaiser