A. ENDOSPORE STAIN
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)
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 and called an exosporium is also seen (Fig. 1M).
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 (see Fig. 1N).
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:
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:
Trypticase Soy agar plate cultures of Bacillus megaterium.
PROCEDURE (to be done individually)
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 (see Fig. 13). Altenately, 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 (see Fig. 14) and generously mix it with the drop of water until the water becomes visibly cloudy (see Fig. 15).
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 (see Fig. 16).
e. Allow this thin suspension to completely air dry (see Fig. 17).
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 microincinerator for 10 seconds (see Fig. 18) as demonstrated by your instructor. If the slide is not heated enough, all of 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 (see Fig. 19).
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 (see Fig. 20). 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 (see Fig. 21).
8. Wash off the excess safranin with water (see Fig. 22), blot dry (see Fig. 23), and observe using oil immersion microscopy. With this endospore staining procedure, endospores will stain green while vegetative bacteria will stain red (see Fig. 2).
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 Fig. 3). With this staining procedure, the vegetative bacteria stain blue and the endospores are colorless. Note the long chains of rod-shaped, endospore-containing bacteria.
11. Observe the demonstration slide of Clostridium tetani (see Fig. 4). With this staining procedure, the vegetative bacteria stain blue and the endospores are colorless. Note the "tennis racquet" appearance of the endospore-containing Clostridium.
12. Endospore stain of Clostridium botulinum (see Fig. 13). Endospores stain green while vegetative bacteria stain red.
|Video review - Aseptic Technique: Inoculation of broth tubes, slant tubes, and stab tubes|
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B. BACTERIAL MOTILITY
A bacterial flagellum has 3 basic parts: a filament, a hook, and a basal body.
1) The filament is the rigid, helical structure that extends from the cell surface. It is composed of the protein flagellin arranged in helical chains so as to form a hollow core. During synthesis of the flagellar filament, flagellin molecules coming off of the ribosomes are transported through the hollow core of the filament where they attach to the growing tip of the filament causing it to lengthen. With the exception of a few bacteria, such as Bdellovibrio and Vibrio cholerae, the flagellar filament is not surrounded by a sheath (see Fig. 5).
2) The hook is a flexible coupling between the filament and the basal body (see Fig. 5).
3) The basal body consists of a rod and a series of rings that anchor the flagellum to the cell wall and the cytoplasmic membrane (see Fig. 5). Unlike eukaryotic flagella, the bacterial flagellum has no internal fibrils and does not flex. Instead, the basal body acts as a rotary molecular motor, enabling the flagellum to rotate and propell the bacterium through the surrounding fluid. In fact, the flagellar motor rotates very rapidly.
The MotA and MotB proteins form the stator of the flagellar motor and function to generate torque for rotation of the flagellum. The MS and C rings function as the rotor (see Fig. 5). Energy for rotation comes from the proton motive force provided by protons moving through the Mot proteins along a concentration gradient from the peptidoglycan and periplasm towards the cytoplasm.
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 (see Fig. 6):
- Scanning electron micrograph showing monotrichous flagellum of Vibrio; courtesy of CDC.
2. amphitrichous - a single flagellum at both poles. (see Fig. 8A)
3. lophotrichous - two or more flagella at one or both poles of the cell (see Fig. 8).
- Scanning electron micrograph of Helicobacter pylori showing lophotrichous arrangement of flagella ; from Science Photolab.com
4. peritrichous - completely surrounded by flagella (see Fig. 9).
One group of bacteria, the spirochetes, has internally-located axial filaments (see Fig. 10) 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.
Some bacteria use motility to contact host cells and disseminate within a host. The mucosal surfaces of the respiratory tract, the intestinal tract, and the genitourinary tract constantly flush bacteria away in order to prevent colonization of host mucous membranes. Motile bacteria can use their motility and chemotaxis to swim through mucus towards mucosal epithelial cells. Many bacteria that can colonize the mucous membranes of the bladder and the intestines, in fact, are motile. Motility probably helps these bacteria move through the mucus between the mucin strands or in places where the mucus is less viscous.
In addition, because of their thinness, their internal flagella (axial filaments), their corkscrew shape, and their motility, certain spirochetes are more readily able to penetrate host mucous membranes, skin abrasions, etc., and enter the body. Motility and penetration may also enable the spirochetes to penetrate deeper in tissue and enter the lymphatics and bloodstream and disseminate to other body sites. Spirochetes that infect humans include Treponema pallidum that causes syphilis, Leptospira that causes leptospirosis, and Borrelia burgdorferi that causes Lyme disease.
For further information on bacterial flagella and motility, see the following in your Softchalk Lectures:
To detect bacterial motility, we can use any of the following three methods: 1) direct observation by means of special-purpose microscopes (phase-contrast and dark-field), 2) motility media, and, indirectly, 3) flagella staining.
1. Direct observation of motility using special-purpose microscopes.Movies of bacterial motility:
a. Phase-contrast microscopy
A phase-contrast microscope uses special phase-contrast objectives and a condenser assembly to control illumination and give an optical effect of direct staining. The special optics will convert slight variations in specimen thickness into corresponding visible variation in brightness. Thus, the bacterium and its structures appear darker than the background.
Phase contrast microscopy of motile Pseudomonas from YouTube.
b. 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 gives 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 (see Fig. 11 and Fig. 14).
- Movie of motile Escherichia coli with fluorescent labelled-flagella #1 Courtesy of Dr. Howard C. Berg from the Roland Institute at Harvard.
- Movie of motile Escherichia coli with fluorescent labelled-flagella #2 Courtesy of Dr. Howard C. Berg from the Roland Institute at Harvard.
- Movie of swimming Escherichia coli as seen with phase contrast microscopy Courtesy of Dr. Howard C. Berg from the Roland Institute at Harvard.
- Movie of tethered Escherichia coli showing that the bacterial flagella rotate Courtesy of Dr. Howard C. Berg from the Roland Institute at Harvard.
- Movie of swarming motility of Escherichia coli Courtesy of Dr. Howard C. Berg from the Roland Institute at Harvard.
- Movie of motile Pseudomonas from YouTube.
- Movie of motile Rhodobacter spheroides with fluorescent labelled-flagella Courtesy of Dr. Howard C. Berg from the Roland Institute at Harvard.
- Movie of motile Borrelia bergdorferi, the spirochete that causes Lyme disease. From You Tube, courtesy of CytoVivo.
- Movie of motile Borrelia bergdorferi, the spirochete that causes Lyme disease.
- Movie of Biofilm formation by Borrelia bergdorferi, the spirochete that causes Lyme disease.
2. Motility Test medium
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 non-motile organism is stabbed into Motility Test medium, growth occurs only along the line of inoculation. Growth along the stab line is very sharp and defined (see Fig. 12A). 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 cloud-like as it moves away from the stab (see Fig. 12B). A tetrazolium salt (TTC) is incorporated into the medium. Bacterial metabolism reduces the TTC producing formazan which is red in color. The more bacteria present at any location, the darker red the growth appears.
3. 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 that 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.
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Trypticase Soy broth cultures of Pseudomonas aeruginosa and Staphylococcus aureus. Caution: handle these organisms as pathogens.
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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A. Endospore Stain
Make drawings of the various endospore stain preparations.
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.
3. Make drawings of the flagella stain demonstrations.
Flagella stain of
Flagella stain of
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After completing this lab, the student will be able to perform the following objectives:
A. ENDOSPORE STAIN
1. Name two endospore-producing genera of bacteria.
2. State the function of bacterial endospores.
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
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.
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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Lab Manual Table of Contents
Last updated: August, 2017
Please send comments and inquiries to Dr. Gary Kaiser