This document discusses capsule staining, which is a technique used to identify the presence of bacterial capsules under a light microscope. It begins by defining bacterial capsules and explaining their functions, which include helping bacteria resist phagocytosis and providing protection. It then discusses the principle of capsule staining, which uses a negative stain to contrast the unstained capsule against stained bacterial cells. The procedure involves smearing a bacterial culture onto a slide with negative stain, staining with a counterstain like crystal violet, and examining under a microscope for unstained capsules surrounding stained cells. Examples of capsule-containing bacteria that can be identified this way include Klebsiella pneumoniae and Bacillus anthracis.

1. Presented by
Nagendra P
16PBT2004
Capsule Staining
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2. Know the bacterial cell
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3. Did you notice Capsule ?
• Some bacteria have a layer of material lying outside
the cell wall.
• When the layer is well organized and not easily
washed off, it is called a capsule.
• Capsules, usually are composed of polysaccharides,
known as Capsular Polysaccharides(CPS) but they
may be constructed of other materials. For example,
Bacillus anthracis has a capsule of poly-D-glutamic
acid.
• Capsules are clearly visible in the light microscope
when negative stains or special capsule stains are
employed.
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4. What does these capsules do ?
• Help bacteria resist phagocytosis.
• Protect bacteria against desiccation.
• Provide a food reserve when certain organic compounds are in excess.
• A virulence determinant of pathogenic microbes. E.g. Streptococcus pneumonia
• Prevention of complement-mediated bacterial cell lysis.
• Protection of anaerobes from oxygen toxicity.
• They exclude bacterial viruses and most hydrophobic toxic materials such as
detergents.
• It also aids bacterial attachment to surfaces of solid objects in aquatic
environments or to tissue surfaces in plant and animal hosts.
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5. Few Strains having Capsules
• Capsules are widely distributed and are found in such diverse pathogens as
• Escherichia coli,
• Neisseria meningitidis,
• Actinobacillus
• pleuropneumoniae,
• Sinorhizobium meliloti,
• as well as in important Gram-positive pathogens including
• Staphylococcus aureus and
• Streptococcus pneumoniae.
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6. Staining
• Staining -It’s a process of adding a dye to a microbial culture.
• Simple Staining (shapes and arrangements)
• Differential Staining (Gram reactions)
• Special Staining (Capsule, flagella, spores)
• Dye-Is a colored substance that has an affinity to the substrate to which it is
being applied.
• Basic dye—possess a positive charge (methylene blue, basic fuchsin, crystal
violet, safranin, malachite green)
• Acidic dye—possess a negative charge (eosin, rose bengal, and acid fuchsin)
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7. Why Capsule Staining ?
• Bacterial capsules are non-ionic, so neither acidic nor basic stains will adhere to
their surfaces.
• Because most capsule materials are water soluble, simple stains will not adhere to
them.
• Since the capsule is a major virulence factor in the major disease-causing bacteria,
it is essential to identify the strain.
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8. Principle of Capsule staining
• Negative staining methods contrast a translucent, darker colored, background
with stained cells but an unstained capsule. The background is formed with
• india ink or
• nigrosin or
• congo red.
• Next counterstaining with dyes like crystal violet or methylene blue, bacterial
cell wall takes up the dye.
• Capsules appear colourless with stained cells against dark background.
• Capsules are fragile and can be diminished, desiccated, distorted, or destroyed by
heating. A drop of serum can be used during smearing to enhance the size of the
capsule and make it more easily observed with a typical compound
light microscope.
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9. Procedure
• Place a small drop of a negative stain on the slide.
• Using sterile technique, add a loopful of bacterial culture to slide, smearing it in
the dye.
• With an other slide, drag the ink-cell mixture into a thin film along the first slide
and let stand for5-7 minutes.
• Allow to air dry (do not heat fix).
• Flood the smear with crystal violet stain (this will stain the cells but not the
capsules) for about 1 minutes.
• Drain the crystal violet by tilting the slide at a 45 degree angle and let stain run off
until it air dries .
• Examine the smear microscopically (100X) for the presence of encapsulated cells
as indicated by clear zones surrounding the cells. 9

10. Non Capsulated Bacterium
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11. Quality Control
• Positive control: Klebsiella pneumoniae (ATCC e13883)
• Negative control: Alacilgenes denitrificans (ATCC 15173)
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12. Uses of Capsule Staining
Detection of Anthrax
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13. References
1. http://homeinsurancequotations.com/post/bacterial-cell-diagram-and functions.html
2. http://www.microbiologyinfo.com/capsule-staining-principle-reagents-procedure-and-
result
3. http://microbeonline.com/bacterial-capsule-structure-and-importance-and-examples-
of-capsulated-bacteria
4. https://www.ncbi.nlm.nih.gov/books/NBK310485/#annex1.s68
5. http://textbookofbacteriology.net/Anthrax_3.html
6. Willis LM, Whitfield C. Structure, biosynthesis, and function of bacterial capsular
polysaccharides synthesized by ABC transporter-dependent pathways. Carbohydrate
research. 2013 Aug 30;378:35-44.
7. Rautemaa R, Meri S. Complement-resistance mechanisms of bacteria. Microbes and
infection. 1999 Aug 31;1(10):785-94.
8. Microbiology; 5th Edition; Lansing M. Prescott; October 2002. 13

14. Thank YOU
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