27: ENDOSPORE STAIN
- Page ID
- 157096
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)- Accurately identify endospores and vegetative cells.
- Differentiate between primary stains and counterstains based on their purpose in the staining procedure.
BACKGROUND
Endospores are unique survival structures produced only by bacteria. Unlike the true spores of fungi or plants—which serve as reproductive units—endospores are not reproductive. They do not result in the production of new bacterial cells, but instead help a single bacterium survive harsh or unfavorable environmental conditions. In this way, endospores function more like cysts or resting forms seen in other organisms. They represent one of the most resilient forms of life known and can withstand extreme heat, cold, dryness, and even radiation.
Endospore formation is triggered when environmental conditions become unfavorable for growth. The specific trigger can vary between bacterial species. Common triggers include nutrient depletion, toxic waste accumulation, unfavorable pH, or extreme salt or water levels. However, the most common cause is starvation. In response, some bacteria begin to form highly durable structures capable of surviving extreme stress.
Endospores are formed inside the bacterial cell, typically around the nuclear material. They are highly desiccated (very low in water content) and surrounded by a thick, protein-rich outer layer called the endospore coat. This coat is nearly crystalline in structure, offering strong protection. Once the endospore is fully developed, the remainder of the bacterial cell breaks down, leaving the endospore as a free-floating, dormant structure. At this stage, it is extremely resistant to damage and death.
Free endospores can survive boiling, freezing, drying, and radiation. They are unaffected by most disinfectants and antiseptics, and because they are not metabolically active, they are resistant to antibiotics. This resilience makes endospores a serious public health concern. For example, the food canning industry measures the effectiveness of sterilization by its ability to destroy endospores from Clostridium botulinum, the bacterium responsible for botulism. If the sterilization process successfully prevents endospore growth, the food is considered safe and sterile, since endospores are the hardest form of life to kill.
Only five genera of bacteria can form endospores. Four of these are rod-shaped: Bacillus, Sporolactobacillus, Clostridium, and Desulfotomaculum. The fifth, Sporosarcina, is spherical. Among these, Clostridium species are commonly found in soil and are medically significant. They are responsible for diseases such as tetanus, gas gangrene, Buruli ulcer, botulism, and possibly some cases of sudden infant death syndrome (SIDS).
It’s important to note that even endospore-forming bacteria cannot always make endospores on demand. If the cell lacks the necessary nutrients to form the endospore coat, it cannot complete the process. In addition, the bacterium must have time to complete endospore development. For example, if a culture containing only vegetative cells (non-spore forms) is suddenly exposed to boiling, the cells will die before they can form protective endospores. However, if the culture already contains mature endospores, it can survive boiling for up to four hours.
Endospores are barely visible under a light microscope, but they are notoriously difficult to stain. Their tough outer coat protects them from common dyes, and they do not stain during a typical Gram stain. Instead, they appear as clear, glassy bodies inside stained bacterial cells. These may be in different positions within the cell—central (middle), terminal (end), or sub-terminal (off-center). Free endospores can be difficult to see unless viewed with a phase contrast microscope, where they appear as bright, glowing structures.
To effectively visualize endospores, microbiologists use a special structural staining technique called the Schaeffer-Fulton method. This method uses malachite green, a powerful dye that can be forced into the endospore coat by applying heat. Heat allows the dye to penetrate the tough structure and stain the endospore green.
Use caution: Malachite green is a permanent dye. If the dye contacts clothing, it cannot be removed. If it contacts skin, it may take several days to fade. After the staining procedure discard the run off in the hazardous waste.
After staining the endospore green, a counterstain (usually safranin) is used to color the surrounding vegetative cells red or pink. The result is a striking image:
- Green free endospores
- Red or pink vegetative cells
- Red or pink cells with green endospores inside
This technique produces a vivid contrast that makes it easy to identify both the presence and location of endospores within a bacterial sample. It remains a classic and powerful example of a differential stain used to study bacterial structure and survival strategies.
MATERIALS (Per Group of 2)
1 Culture with endospores
1 Hot plate
1 Small glass beaker
Strips of paper towel
2 Microscope slides
Malachite green stain
Safranin stain
METHODS/PROCEDURES
1. Ensure your slides are very clean before beginning. Any residual soap or oil from your fingers can affect stain absorption and alter the final colors. If
needed, wipe the slide gently with a lens wipe and 95% ethanol. Allow it to dry completely before use.
2. Prepare a bacterial smear of Bacillus on the slide. Label the frosted edge of the slide in pencil. Allow the smear to air dry completely, then heat-fix the
slide by quickly passing it through a flame.
3. Place a small beaker filled with approximately 100 mL of water on a hot plate and bring it to a gentle boil. Monitor the water level carefully—do not let
it boil dry. Once boiling, lay the heat-fixed slide across the top of the beaker. Two slides may fit across one beaker if needed.
4. Tear a strip of paper towel slightly smaller than the slide in both length and width. This prevents stain from dripping over the edges. Place the paper
strip directly over the bacterial smear.
5. Completely saturate the paper towel with malachite green stain. As the stain begins to steam and evaporate, add more malachite green as needed—
just a few drops at a time. Avoid spilling stain onto the beaker or hot plate. The paper towel must remain moist and never be allowed to dry out.
Continue steaming the stain into the slide for 15 minutes.
6. After 15 minutes, carefully remove the slide using a clothespin and place it onto a staining rack over a stain tray. Hold a folded paper towel under the
slide as you move it to catch any drips. If any stain spills, clean it immediately with paper towels and alert your instructor.
7. Using forceps, carefully remove the saturated paper towel from the slide. Wrap the used paper in a dry paper towel and dispose of it in the regular
trash—not in the stain tray or chemical waste container.
8. Tilt the slide and gently rinse with distilled water until no more green stain runs off the surface.
9. Flood the slide with safranin and allow it to sit for 10 minutes to counterstain the vegetative cells.
10. Rinse briefly again with distilled water. Blot gently with bibulous paper or clean paper towels, and allow the slide to air dry completely.
11. Examine the slide under the microscope. Look for green endospores and pink/red vegetative cells. If endospores are still inside cells, you should see red
cells with green centers.
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Exercise #37 Endospore Stain
NAME ______________________
EXPECTATIONS
Describe what a correctly stained slide should look like under the microscope. What colors will indicate endospores versus vegetative cells?
RESULTS
Draw what you see on your slide under oil immersion. Identify endospores and vegetative cells.
CONCLUSIONS
1. Why is it important to use an older culture (24–48 hours) of Bacillus when staining for endospores?
2. Why are endospore-forming bacteria a concern in clinical or food safety settings?
3. Ultraviolet (UV) radiation is often used in hospitals and research labs to clean work surfaces. Do you think
this would be an effective method for killing Bacillus anthracis? Why or why not?