8.1: Spore Stain
- Execute the spore staining procedure accurately, distinguishing between spores and vegetative cells under the microscope.
- Understand the significance of spore formation in bacterial survival.
- Identify/give examples of environmental conditions that can stimulate spore formation.
- Interpret staining results accurately to determine the presence or absence of spores in bacterial cultures.
- Differentiate between endo and exospores.
- Identify when endospores are terminal, subterminal, and central in microscopic images, diagrams, and descriptions.
- Apply critical thinking skills to analyze the significance of spore formation in bacterial survival and pathogenicity.
- Discuss the clinical relevance of spore-forming bacteria in healthcare settings.
Spores
Bacterial spores are highly resistant, dormant structures formed by certain genera of bacteria in response to adverse environmental conditions. They can be formed within bacteria (i.e., endospores ) or on the bacterial surface (i.e., exospores ).These conditions may include nutrient limitation, high temperatures, desiccation, or exposure to toxic chemicals. Spores serve as a survival mechanism for the bacteria, allowing them to endure harsh conditions until more favorable conditions arise.
Spores are formed through a process called sporulation , during which a vegetative bacterial cell (i.e., active cell) undergoes a series of morphological changes to produce the spore (Fig. 1A). The spore is encased in a keratin-rich coat that protects against environmental stressors.
Spores can remain viable for extended periods, sometimes even thousands of years until conditions become suitable for germination. When conditions improve, the spore can undergo germination, leading to the emergence of a new vegetative bacterial cell capable of resuming normal metabolic activities and growth.
Not all bacteria have the ability to form spores, but those that do belong primarily to the genera Bacillus and Clostridium. These bacteria are commonly found in soil, water, and various other environments, including the human body, where they can be both beneficial and pathogenic. Bacillus and Clostridium species cause many dangerous and lethal diseases such as botulism, gangrene, tetanus, c-diff, and anthrax.
The location where an endospore is within a vegetative cell is also useful for distinguishing bacterial species. Endospores may be located in a terminal (end of the cell), subterminal (near the end of the cell), or central (middle of the cell) position.
Spore Stain
The spore stain is a differential staining technique used to visualize bacterial spores within a population of bacteria. This staining method utilizes heat to drive the primary stain, usually malachite green, into the spore structures, followed by a counterstain to color the vegetative cells.
First, the smear is flooded with malachite green, the primary stain for spores. Next, heat is applied to the slide using a steam bath. The heat helps drive the malachite green into the spore structures, where it binds to the keratin protein present in the spore coat. After the primary stain has been applied and heated, the smear is decolorized with water. Decolorization is crucial to remove the malachite green from the vegetative cells, leaving only the spores stained. Once decolorization is complete, a counterstain, commonly safranin, is applied to the smear. The counterstain stains the decolorized vegetative cells, differentiating them from the green-stained spores (Fig.2).
The keratin forming the outer portion of the spore wall resists stain. Heating the cells will make the spore wall more permeable to the malachite green so it can then attach. Once in, the malachite green will not come out because the overlying spore wall becomes less permeable when the smear cools.
Clostridium difficile
Clostridium difficile ( C. difficile ), is a spore-forming bacterium responsible for severe gastrointestinal infections, particularly in healthcare settings (e.g., hospitals, nursing homes, etc.). The use of broad-spectrum antibiotics among patients disrupts the normal gut flora, allowing C. difficile , a bacterium commonly found in the large intestine, to grow. This disruption increases the risk of developing a C. difficile infection (i.e., antibiotic-associated colitis), which is often difficult to treat and can lead to recurrent episodes. Symptoms include severe diarrhea, abdominal cramps, fever, nausea, and loss of appetite. Consequently, managing C. difficile infections is a critical concern among infection control programs in hospitals and long-term care facilities.
In healthcare settings, C. difficile is particularly concerning because its spores can persist on surfaces for long periods, resisting common disinfectants and spreading easily between patients and staff. The ability of the endospore stain to identify C. difficile endospores is critical for implementing proper infection control measures. Once detected, specialized cleaning protocols that target spore-forming bacteria, such as bleach or spore-specific disinfectants, are necessary to prevent further spread within healthcare facilities.
Most patients respond well to antibiotic therapy (e.g. Metronidazole or Vancomycin). However, due to damage to the normal gut flora, recurrent infections are common. Fecal microbiota transplantation (FMT) is an innovative treatment option for recurrent C . difficile infections. This procedure involves transferring fecal matter from a healthy donor into the gastrointestinal tract of the infected patient, effectively restoring a balanced gut microbiome. By reintroducing beneficial bacteria, FMT helps to outcompete C. difficile and reduces the likelihood of recurrence.
Laboratory Instructions
Prepare a Bacterial Smear
You may find it helpful to draw a circle (wax pencil is best) on the opposite side of the slide where you will spread your smear. This will help you later in locating the smear. The wax pencil is better than a marker because it will not wash off easily from the glass.
If you are using a liquid culture, gently mix the culture until you get an even, cloudy mixture (it should look somewhat like skim milk). If you mix too aggressively, you will lose the bacterial morphology.
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Prepare a bacterial smear on the slide:
- If you are taking bacteria from a solid culture (slant or Petri plate), place a loopful of saline, deionized (DI) water, or distilled water onto a microscope slide and use a loop to aseptically add bacteria to the water (avoid taking a large chunk of bacteria since the cells will be too dense to view individual cells). Use the loop to spread the bacteria in the water and spread the water out to make it thinner (it will dry faster).
- If you are taking bacteria from a liquid culture (broth), place 1 or 2 loopful(s) of bacteria directly onto a microscope slide (no saline or water added).
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Allow the slide(s) to air dry on the slide warmer (or air dry if a slide warmer is not available).
Heat Fix the Bacterial Smear
Once the liquid has completely evaporated on the surface of the slide, heat fix the slide:
Attach a wooden clip to the microscope slide to hold it.
- Pass the underside of the microscope slide through a flame three times. If using a hotplate, place the slide in the center of the hotplate for 30 seconds.
- Allow the slide to cool and then continue with your staining protocol.
If you heat fix too little, the bacteria will wash off the slide. If you heat fix too much, you will cook the bacteria and denature them.
Spore Stain
- Under the fume hood, put a beaker of water on the hot plate and boil until steam is coming up from the water.
- Turn the hot plate heat down so that the water is barely boiling.
- Place a wire staining rack over the beaker. Steam should be coming up through the wire rack.
- Cut a small piece of bibulous paper (or paper towel) and place it on top of the smear on the slide. This will keep the dye from evaporating too quickly, thereby giving more contact time between the dye and the bacteria.
- Flood the smear with the primary dye, malachite green, and leave for 5 minutes. Keep the paper moist with the malachite green. DO NOT let the dye dry on the towel. Add additional drops of malachite green as needed.
- Remove and discard the paper piece in the trash bin.
- Take the slide back to your lab table.
- Wash the slide with water.
- Sit the slide on your staining rack.
- Apply safranin to the smear and leave for 1 minute.
- Wash the slide with water.
- Blot the slide dry (do not wipe) with bibulous paper.
- Place the stained slide under a microscope for examination. Be sure to begin at the lowest power (look for the pink and green colors of the stains). Focus at the lowest power and then increase one objective magnification at a time until you reach oil immersion.
Attributions
- Bacillus subtilis Spore.jpg by Y tambe is licensed under CC BY-SA 3.0
- Chapter Image: OSC Microbio 02 04 Endospores.jpg by CNX OpenStax is licensed under CC BY 4.0
- Endospore Bazillus.jpg by Geoman3 is licensed under CC BY-SA 3.0
- Endospore Formation.png by Farah, Sophia, Alex is licensed under CC BY-SA 4.0
- Microbiology Labs I by Delmar Larsen and Jackie Reynolds is licensed under an undeclared license .
Citations
- Johns Hopkins Medicine. (n.d.). Fecal transplant . Retrieved November 7, 2024, from https://www.hopkinsmedicine.org/health/treatment-tests-and-therapies/fecal-transplant
- Centers for Disease Control and Prevention. (2023, September 19). Clostridioides difficile infection . U.S. Department of Health & Human Services. Retrieved November 7, 2024, from https://www.cdc.gov/c-diff/index.html