Capsule Staining: Introduction, Principle, Requirements, Procedure, Result

Capsule Staining: Introduction, Principle, Requirements, Procedure, Result Interpretation, Significance and Keynotes

Introduction of Capsule Staining

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. Capsule detection 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.

The Hiss Staining Technique is a type of positive staining method that stains the capsule and the bacterial cell with a brighter background. For the reason that a capsule appears as a light violet color between a dark violet-colored bacterial cell and colorless background.

Principle of Capsule Staining

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. Another counterstaining with dyes like crystal violet or methylene blue, bacterial cell wall takes up the dye while capsule appears colorless with stained cells against a dark background. 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.

Requirements for Capsule Staining

Equipment and reagents required are-

Sprit lamp/Bunsen burner
Inoculating loop
Staining organism
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
1% (w/v) crystal violet
Microscope
Cedarwood oil

Procedure of Capsule Staining

Put a small drop of a negative stain on the slide.
Aseptically add a loopful of bacterial culture to drop and mix it properly.
With another slide, drag the ink-cell mixture into a thin film along with the first slide and leave for 5-7 minutes for air drying (avoid heat fixing).
Flood the smear with crystal violet (basic dye) stain (this will stain the cells but not the capsules) for about 1 minute.
Drain the crystal violet by tilting the slide at a 45° angle and let the stain runoff until its air dries.
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 Interpretation of Capsule Staining

Presence of Capsule: A clear zone surrounding the cell
Absence of Capsule: Lacking clear surrounding the cell
Background: Dark
Negative control (NC): Lacking capsule
Positive control (PC): Presence of Capsules
Test organism: Either encapsulated or lacking the capsule

Significance of Capsule Staining

It helps to differentiate the bacteria whether capsulated or non-capsulated.
A capsule of bacteria acts as a virulence factor and gives pathogenicity to the bacterial cells.
The capsule helps the organism in various ways-

Capsule assists bacteria to resist phagocytosis.
It saves the organism from desiccation.
It acts as a food reserve when certain organic compounds are in excess.
A virulence determinant of pathogenic microbes. e.g. S. pneumoniae.
The capsule also helps the organism by preventing complement-mediated bacterial cell lysis.
It protects anaerobic bacteria from oxygen toxicity.
It also excludes bacterial viruses and most hydrophobic toxic materials such as detergents.
It aids bacterial attachment to surfaces of solid objects in aquatic environments or to tissue surfaces in hosts

Keynotes on Capsule Staining

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
Preparation 1% Crystal violet stain: Solution A for crystal violet staining reagent
Crystal violet (certified 90% dye content): 1 gm
Ethanol, 95% (vol/vol): 20 ml
Solution B for crystal violet staining reagent
Ammonium oxalate: 0.8 gm
Distilled water (D/W): 80 ml
Mix A and B to obtain crystal violet staining reagent. Store for 24 hours and filter through paper prior to use.
Capsule staining can be performed in two ways-A. Positive staining method –A capsule is stained. e.g. Hiss Staining Method
B. Negative staining method – The capsule is unstained but it is made visible by the colorless area of the capsule in between colored cells and colored background. e.g. Maneval’s method,

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