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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