24: CAPSULE STAIN
- Page ID
- 157092
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)- Correctly prepare a bacterial smear for capsule staining.
- Use a microscope to focus, observe, and record capsule stain preparations at the appropriate magnification.
- Distinguish between capsule-producing and non–capsule-producing bacteria through careful microscopic observation.
BACKGROUND
Some bacterial cells produce an additional outer layer called a capsule or slime layer. Both of these structures are composed primarily of polysaccharides (complex carbohydrates made of long chains of sugar molecules), although in some species, small amounts of protein or other molecules may also be present. The difference between the two is structural: a capsule is firmly attached to the cell wall and is well-organized, while a slime layer is loosely attached and irregular in shape. Regardless of the type, both structures offer significant survival benefits for bacteria.
These polysaccharide coatings are not always produced. Instead, they are often made only when environmental conditions support or demand their formation. For example, when nutrients are plentiful and growth is rapid, many bacteria do not waste energy producing a capsule. But under stressful conditions—such as dehydration, chemical exposure, or host immune response—these coatings provide an important form of protection.
The presence of a capsule or slime layer alters the permeability (ability to allow substances to pass through) of the bacterial cell membrane. This can prevent antibiotics and chemical disinfectants from entering the cell, making these bacteria harder to kill. Capsules also help protect bacteria from desiccation (drying out) by retaining moisture around the cell.
Importantly, capsules are considered a major virulence factor (a characteristic that enhances a microorganism’s ability to cause disease). They can shield bacterial cells from detection and attack by the immune system. Under normal circumstances, the body’s phagocytes (white blood cells that engulf and destroy invaders) recognize and bind to antigens (specific molecules on the surface of pathogens that trigger an immune response). But when a capsule covers the cell, these antigens are hidden. As a result, phagocytes may not recognize the bacteria as harmful, allowing the pathogen to survive, multiply, and cause infection over time.
Bacteria that produce capsules can remain undetected in a host for an extended period. This "stealth mode" gives them time to grow in number and establish infection before the immune system can respond effectively. Some pathogens known for having prominent capsules include Streptococcus pneumoniae, Klebsiella pneumoniae, and Haemophilus influenzae, all of which are capable of causing serious illness, especially in vulnerable patients.
Another interesting benefit of capsules is their use as a backup energy source. When nutrients are scarce, bacteria can hydrolyze (break down with water) their own capsule into simpler sugar molecules, which can then be used for energy. This gives capsule-producing bacteria an edge in surviving longer between meals—or between hosts—compared to those without this feature.
Despite their importance, capsules are difficult to visualize using traditional microbiological staining techniques. The high water content and delicate structure of capsules mean they shrink or disappear when subjected to heat fixing. In addition, capsules do not take up most common stains like crystal violet or methylene blue. To detect capsules, microbiologists use a special method called negative staining.
Negative staining is a technique that allows capsules to be seen clearly by staining everything except the capsule itself. Two common dyes used in this technique are India ink and Congo Red. These stains are repelled by the capsule and do not penetrate it, which leaves a clear, unstained area around each capsule-producing cell.
To perform a negative stain, a small drop of dye is mixed with a sample of live bacteria on a glass slide. The mixture is spread out thinly and allowed to air dry—without applying heat. As the sample dries, the dye coats the background and outlines the cells, but it cannot enter the capsule. This results in a striking image under the microscope: cells are visible against a dark or colored background, and any capsule appears as a clear "halo" surrounding the cell.
In many cases, a counterstain (a secondary stain that does enter the bacterial cell, such as crystal violet or safranin) is applied after the background stain has dried. This colors the actual cell body, making it easier to distinguish the cell from its surroundings and to verify the presence or absence of a capsule.
When viewed under the microscope, the differences are clear:
- Cells without capsules are surrounded directly by the dark stain.
- Cells with capsules are surrounded by a clear, unstained zone between the cell and the background.
Since heat fixing can destroy capsules, it is important to avoid using this step during negative staining. By preserving the natural structure of the capsule, this method allows for accurate observation and identification.
Capsule staining is often used in clinical laboratories to help identify pathogenic bacteria. It’s a simple, rapid, and effective way to observe a structure that plays a critical role in bacterial survival and infection.
MATERIALS (Per Group of 4)
1 Bacillus culture grown for capsules
4 Clean slides (one per student)
Sheep serum (Instructors Desk)
1 Congo Red stain
1 Maneval’s stain
METHODS/PROCEDURES
1. Place a small drop of sheep serum near one end of a clean glass slide.
2. Add a small drop of Congo Red stain next to the serum.
3. Using aseptic technique, add a small amount of bacterial culture to the mixture and gently blend using a sterile loop or stick. Set the slide down on a
paper towel on the lab bench.
4. Prepare a smear using the slide spreader technique. Hold a second clean slide at a 30–45° angle. Touch it to the drop of stain until the liquid spreads
along the edge. Gently push or pull the spreader slide across the surface to evenly distribute the mixture in a thin smear.
5. Allow the smear to air dry completely.
**Do not heat fix the slide. Air drying sets the Congo Red in place around the cells without destroying the capsule.
6. Cover the dried smear with Maneval’s stain and let it sit for 1 minute. Maneval’s stain is a solution that contains acetic acid and acid fuchsin:
* The acetic acid lowers the pH and causes the background (containing Congo Red, a pH indicator) to shift from red to blue.
* The acid fuchsin is a basic dye that penetrates the bacterial cell wall and stains the cells red to pink.
* The capsules remain clear and unstained.
7. Rinse the slide briefly and gently with water. Tilt the slide and let water run gently over it to remove excess stain. Avoid spraying directly or too
forcefully.
8. Let the slide air dry completely.
9. View the slide under oil immersion using the 100x objective (final magnification 1000x).
- 2.4: Staining Microscopic Specimens by OpenStax is licensed CC BY 4.0. Original source: https://openstax.org/details/books/microbiology.
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Exercise #29 Capsule Stain
NAME ______________________
EXPECTATIONS
Describe what you expect to see under oil immersion if a bacterium produces a capsule.
RESULTS
Draw what you see on your capsule stain slide under oil immersion.
CONCLUSIONS
1. Describe what a capsule looks like under the microscope when using negative staining.
2. How can you tell the difference between a capsule-positive and capsule-negative bacterial cell on a stained slide?
3. Why might a bacterium that produces a capsule be more likely to cause a long-lasting infection?
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Attributions:
Chapter tile image: Bacteria with capsule.jpg by Microrao is licensed under CC BY-SA 4.0 |