Bacterial Capsule Demonstration Using Negative Staining Method: Introduction, Principle, Procedure, Result Interpretation and Keynotes

Bacterial Capsule Demonstration Using Negative Staining Method: Introduction, Principle, Procedure, Result Interpretation and Keynotes

Introduction of Bacterial Capsule Demonstration 

The capsule is the outermost layer of bacteria. It, usually is composed of polysaccharides, known as capsular polysaccharides (CPS) but it may be constructed of other materials. e.g. Bacillus anthracis capsule is made of poly-D-glutamic acid. Capsule Staining is a special type of staining. Bacterial capsule observation is sometimes cumbersome since the capsule is non-ionic, so neither acidic nor basic stain and that will adhere to its surface. Another fact is most capsule materials are water-soluble, simple stains will not adhere to them. Bacterial Capsule Demonstration is important because the capsule is a major virulence factor in the major disease-causing bacteria and thus it is essential to identify the strain. The most common encapsulated bacteria are Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis, and Klebsiella pneumonia.   The capsule is mainly of two types, micro, and macrocaspsule. Microcapsule size is less than 0.2 µ while macrocapsule is more than 0.2 µ. Applying a negative stain (Nigrosin), bacterial capsules can be easily demonstrated.

Principle of Bacterial Capsule Demonstration

A negative staining method differences a translucent, darker colored, background with stained cells but an unstained capsule. The background color is formed on the basis of negative stain (India ink/nigrosin/Congo red) used. Heat fixing is omitted since the capsule is fragile and can be diminished, desiccated, distorted, or destroyed. An application of a drop of serum is beneficial during smearing to enhance the size of the capsule that makes it more clear observation under the microscope.

Test Requirements for Bacterial Capsule Demonstration

Equipment and reagents required for bacterial capsule demonstration are-

  • Sprit lamp/Bunsen burner
  • Inoculating loop
  • Staining organism/ A given Culture
  • Control strains ( Negative control-Non capsulated strain of E.coli while as Positive control -Capsulated strains of Klebsiella pneumoniae)
  • Clean and grease-free slides
  • Serum (optional)
  • Nigrosin
  • Microscope
  • Cedarwood oil

Test Procedure of Bacterial Capsule Demonstration

  1. Transfer an aseptically a loopful of culture on a clean and dry slide
  2.  Mix it with a loopful of stain
  3. Make a smear by using a glass slide
  4.  Allow in to dry slowly at room temperature.
  5. Examine the smear under the microscope for the detection of the capsule (clear zones surrounding the cells) focussing at 10x objective and finally move on 100X (oil immersion field) using cedarwood oil.


Result and Interpretation of Bacterial Capsule Demonstration

  • Presence of Capsule: A clear zone surrounding the cell/Colorless ( hollow bodies)
  • Absence of Capsule: Lacking clear surrounding the cell
  • Background: Bluish black
  • Negative control (NC): Lacking capsule
  • Positive control (PC):  Presence of Capsules
  • Test organism: Either encapsulated or lacking the capsule

Keynotes on Bacterial Capsule Demonstration

  • Cryptococcus neoformans, a fungus is also encapsulated strain.
  •  Staining is the process of adding a dye to the smear. It is of simple staining, differential staining, and special staining.
  • Simple Staining is used for the demonstration of shapes and arrangements. e.g. Methylene blue.
  • Differential Staining helps to differentiate the organism on the basis of staining whether positive or negative, cocci or bacilli e. g. Gram staining.
  • Special Staining is applied for special structure observation e.g. Capsule (Hiss staining), flagella, spores staining. Dye is a colored substance that has an affinity to the substrate to which it is being applied.
  • Basic dyes are methylene blue, basic fuchsin, crystal violet, safranin, and malachite green that possess a positive charge.
  • Acidic dyes are eosin, rose bengal, and acid fuchsin which possess a negative charge.
  • Composition of Nigrosin stain: Following are the ingredients of nigrosin stain.
    • Nigrosin:  10.0 gm
    • Formalin:  0.5 ml
    • Distilled water:  100.0 ml

Further Readings on Bacterial Capsule Demonstration

  1. functions.html
  4. 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.
  5. Microbiology; 5th Edition; Lansing M. Prescott; October 2002.
  6. Rautemaa R, Meri S. Complement-resistance mechanisms of bacteria. Microbes and infection. 1999 Aug 31;1(10):785-94.


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