Capsule staining: Principle, Requirements, Procedure and Microscopic Examination

  • Capsule staining is a differential technique commonly used in light microscopy.
  • A capsule is a gelatinous layer which lies immediately exterior to the cell wall and is often referred to as a slime layer.
  • It is secreted by the cell itself, and adheres to the cell wall.
  • Following are the examples of few capsulated organisms:
    • Gram negative and capsulated: Neisseria meningitides, Klebsiella pneumoniae, Haemophilus influenza type B,Pseudomonas aeruginosa,  Escherichia coli (few strains), Yersania pestis,
    • Gram positive and capsulated:Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogens, Bacillus megaterium, Bacillus anthracis,
    • Capsulated yeast:Cryptococcus neoformans
  • In Gram positive bacteria, it lies outside the murein layer whereas in Gram negative bacteria, it lies outside the outer membrane.
  • The capsule is composed of high molecular weight complex polysaccharides like levans, dextrans, and celluloses.
  • Capsule is not present in all the organisms and its production may depend on the environment and growth conditions surrounding the bacterial cell.
  • Capsule doesn’t function as an effective permeability barrier or add strength to the cell envelope but does protect bacteria from attack by phagocytic cells of the human body.
  • Capsule can make a bacterial cell more virulent and capable of causing disease.
  • It also facilitates and maintains bacterial colonization of biological (e.g. teeth) and inanimate (e.g. prosthetic heart valves) surfaces through the formation of bio films.


  • Capsule is non-ionic in nature and due to this non-ionic property of capsule, its staining is more difficult than other types of differential staining procedures.
  • In addition, the capsular materials are water-soluble and may be dislodged and removed with vigorous washing.
  • Smears of capsulated organisms should not be heated during capsule staining because the cells shrink and may create a clear halo zone around the organism which can be mistaken for the capsule.
  • Capsule staining uses two reagents.
  1. Primary Stain: Crystal Violet (1% aqueous)
  • A primary stain crystal violet is applied to a non-heat-fixed smear.
  • The cell and the capsular material both will take the violet color at this point.
  1. Decolorizing Agent: Copper Sulfate (20%)
  • The capsule is non-ionic but the bacterial cell wall is anionic. Hence, the basic dye (crystal violet) stains the cell but not the capsule. The stain gets adhered to the capsule but doesn’t bind to it.
  • Copper sulfate but not water (because capsule is water soluble) is used as a decolorizing agent. It washes the crystal violet out of the capsular material without removing the stain bound to the cell wall.
  • The decolorized capsule now absorbs the copper sulfate and appears blue whereas the cell still appears deep purple.
  • A negative staining technique can also be applied to visualize the capsule since they don’t absorb most basic dyes.
  • In fact, a capsule can be observed by the combination of positive and negative staining techniques.
  • The background is stained by acidic dye (India ink or Nigrosin) and the bacterial cell is stained by a basic dye. The capsule appears colorless between the dark background of the negative stain and the colored cell taking the color of the basic dye.


  1. Bacterial culture of Leuconostoc mesenteroides andKlebsiella aerogenes
  2. Reagents:1% crystal violet and 20% copper sulfate (CuSO4.5H2O)
  3. Micro-incinerator or Bunsen burner
  4. Inoculating loop or needle
  5. Staining tray
  6. Bibulous paper
  7. Grease free glass slides
  8. Microscope


  • Prepare thin smears of bacterial culture on a clean grease free glass slide.
  • Only air-dry the smear as heat-fixing can destroy the capsule or cause the capsule to shrink.
  • Apply 1% crystal violet on the smear and allow it to remain on the slide for 2 minutes.
  • Use 20% copper sulfate solution to gently wash off the crystal violet off the slide.
  • Blot the slide dry with bibulous paper.
  • Examine microscopically using 100X oil immersion objective.

Microscopic Examination:

  • The capsule appears as blue colored between deep purple background and cell wall.
  • In case of negative staining, the capsule appears colorless between the dark background of the negative stain and the colored cell.
  • Non-capsulalted: No clear halo zone around the bacterial cell.

Capsule staining: Principle, Requirements, Procedure and Microscopic Examination