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1.13: Capsule Stain

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    Learning Objectives
    • Describe what bacterial capsules are and where they are found in bacterial cells.
    • Tell how the capsule stain works.
    • Give at least three ways bacterial capsules benefit bacterial cells.
    • Successfully conduct a capsule stain.
    • Examine, illustrate, label, and interpret results of a capsule stain.


    Bacterial Capsules


    prokaryotic cell and its structures

    Figure 1: Diagram of a prokaryotic cell shows that capsules are a layer located outside of the cell wall.


    Bacterial capsules are typically composed of a polysaccharide layer which is thick, detectable, and discrete layer outside the cell wall. Capsules do not stain well for microscopic examination. Because of this, to visualize bacterial capsules, stains/reagents are used in a way that will stain the environment surrounding the bacterial cells and stains the bacterial cell itself, but not the capsule. This creates a non-stained area that is the capsule. Capsules appear as uncolored halos surrounding the bacterial cell.


    microscopic image of capsule stain with the capsule labeled

    Figure 2: Microscopic image of bacteria stained with the capsule stain. The environment surrounding bacterial cells and the bacterial cells are is stained pink/purple. The unstained region that appears white surrounding each bacterial cell is the capsule.


    The ability to produce a capsule is coded in the DNA of the bacteria and can therefore be species-dependent or even strain-dependent. The capsule is not an absolutely essential cellular component for bacteria and some species and strains do not form capsules. Capsules are often produced only under specific growth conditions. The thickness of the capsule can vary depending on the bacterial species, its age, and the medium in which the bacterium is growing. Even though not essential for life, capsules can help bacteria to survive.

    Capsules protect pathogenic bacteria from the phagocytic action of immune cells and allow pathogens to invade the body by enabling them to avoid being engulfed by white blood cells. If a pathogenic bacterium loses its ability to form capsules, it often ceases to be pathogenic. Some bacterial species of the microbiome also have capsules to protect them from phagocytosis. Environmental bacteria living in soil and water are protected by their capsules from being engulfed by free-living protozoa.

    In addition to protection from phagocytosis, the capsule protects cells against desiccation (drying out). Capsules also enhances the ability of cells to attach. For example, pathogenic bacterial cells with capsules aid in attachment to host cells to establish their growth/infection. Further, capsules enable bacterial cells can attach to other bacteria in biofilms. Bacteria that establish the first layer of a biofilm may utilize their capsules to adhere to a surface and enable attachment of other bacterial cells for biofilm development.


    the stages of biofilm development

    Figure 3: Process of biofilm development. Stage 1: initial attachment of bacteria to a surface. Stage 2: irreversible attachment. Stage 3: maturation I. Stage 4: maturation II. Stage 5: dispersion of cells out of the biofilm.


    Laboratory Instructions


    Capsule Stain (Anthony's Capsule Stain)

    1. Gently stir the skim milk broth culture with your loop and place 2-3 loops of Enterobacter aerogenes or Serratia marcescens (whichever is available) on a microscope slide.
    2. Using your inoculating loop, spread the sample out to cover about one inch of the slide.
    3. Let it completely air dry. Do not heat fix or use the slide warmer. Capsules stick well to glass, and heat may destroy the capsule.
    4. Stain with 1% crystal violet for two minutes (do not use the same crystal violet solution as the Gram stain).
    5. Gently wash off the excess crystal violet stain with 20% copper sulfate solution.
    6. Gently shake off the excess copper sulfate solution and let air dry (do not blot or use slide warmer).
    7. Examine the slide with the microscope. The bacterial cell and the milk dried on the slide will pick up the purple dye while the capsule will remain colorless.


    Results & Questions

    Microscopic illustrations.png

    1. Illustrate the bacterial cells viewed in the microscope. Label a bacterial cell and a capsule in your illustration.
    2. Where are bacterial capsules located in a bacterial cell?
    3. What type of molecule are bacterial capsules typically composed of?
    4. Why do bacterial capsules appear white/clear after staining?
    5. Give three different ways bacterial capsules give species/strains that have them an advantage.
    6. Do all bacteria have/need a capsule? Explain your answer.



    This page titled 1.13: Capsule Stain is shared under a CC BY-NC-SA 4.0 license and was authored, remixed, and/or curated by Rosanna Hartline.

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