Capsule Staining

introduction image

Capsule staining is a type of differential staining method which creates contrast in the microscopic image either by staining bacterial cell and background or by staining the capsule only. Therefore, capsule staining can be done by two methods, namely positive and negative staining methods. Positive staining method either stain a capsule only or can stain both the capsule and bacterial cell. Negative staining method stains both the bacterial cell and its background.

The capsule staining is employed to know the presence or absence of capsule, based on which we could classify the type of bacteria, i.e. whether a bacteria is capsulated or non-capsulated. It is necessary to perform capsule staining because the presence of capsule indicates a virulent strain of bacteria that can cause disease. Thus, by knowing the presence of the capsule, we can determine the degree of pathogenicity.

Content: Capsule Staining

  1. Definition of Capsule Staining
  2. Bacterial Capsule
  3. Principle
  4. Methods of Capsule Staining
  5. Significance

Definition of Capsule Staining

A capsule staining can define as the special staining method that makes the use of differential capsule stain which either highlights or stains the capsule or stains both the bacterial cell and its background.

It is an important staining method, because some bacteria like Bacillus anthracis, Streptococcus pneumoniae etc. have a capsule, which can cause pathogenicity in many humans and animals. Thus, it becomes necessary to identify the presence of an extracellular capsule. Capsule staining can be performed by India ink, Anthony’s, Maneval’s and Hiss method.

Bacterial Capsule

A bacterial capsule can define as the mucilaginous coating that surrounds the cell wall of bacteria. The capsule also refers as glycocalyx as it is composed of glycoproteins. A cytoplasm of bacteria partially forms a capsule which then goes to the cell wall and surrounds it as a mucous or slime covering.

The capsule protects a cell from desiccation because of the mucous content. A mucus layer also protects a cell from phagocytosis. The capsule also acts as a virulence factor and responsible for the pathogenicity of many microorganisms like Streptococcus pneumoniae, Escherichia coli, Neisseria meningitis etc.

On the majority, a capsule consists of polysaccharides but besides this, also contains polypeptides or glycoproteins. The formation of a capsule is a process which is controlled genetically. Capsules can be easily visible under the light microscope, by the use of differential capsule stain.

Bacteria having capsule will refer as “Capsulated bacteria”, and those who lack a capsule will refer as “Non-capsulated bacteria”. Capsule are of two types, namely micro and macro capsule. Microcapsule has a size less than 0.2µ whereas Macrocapsule has a size more than 0.2µ.

The capsule is non-ionic in nature, i.e. it will neither stain by acidic nor by basic stains. The basic dye will stain the negative bacterial cell, and an acidic stain will stain the positive background. Thus, to stain a capsule, we need a special capsule stain that will focus on the capsule.

A capsule can be easily destroyed by a heat treatment that’s why a step of heat fixing is skipped while performing capsule staining. In addition to this, a step of washing or rinsing is also avoided because it can dislodge the capsule from the bacterial cell.

To enhance the size of the capsule or to increase its visibility, we can also add a drop of serum. The addition of serum provides a more unobstructed view of a capsule under a light microscope.

Principle

The principle of capsule staining is based on staining of background with an acidic stain and staining of bacterial cell with a basic stain. As a capsule is non-ionic, it will not stain by either of the two dyes. Thus a capsule staining creates a contrast by staining a bacterial cell and its background in between which, capsule appears as a colourless halo.

Methods of Capsule Staining

There are different methods for capsule staining, among which the most common methods are:

  1. India ink method
  2. Anthony’s method
  3. Maneval’s method
  4. Hiss method

methods of capsule staining

India ink Method

India ink method uses two types of stain, i.e. a basic stain (Crystal violet) and an acidic stain (India ink). Crystal violet being positive stain will stain the negatively charged bacterial cell. India ink being negative stain will stain the positively charged background. After staining:

  • The Background appears darker or black.
  • A Bacterial cell appears violet.
  • The capsule appears as a clear halo.

Procedure

India ink method involves the following steps:

  1. Take a clean, sterilized or grease free slide.
  2. Add a drop of crystal violet to the centre of the glass slide.
  3. Prepare a smear, by taking an inoculum from the bacterial culture and mix it with a drop of crystal violet.
  4. Then, allow the smear to air dry(Do not heat fix, as it can cause cell shrinkage and distortion of the bacterial capsule).
  5. Flood a smear with the India ink for 30 seconds and remove the extra stain by tilting a glass slide.
  6. Add oil immersion to the stained area and observe it under the microscope having a 100X objective.

india ink method

India ink method is a type of negative staining method, which stains both the bacterial cell and its background but not a capsule. As a result, a capsule appears as a bright halo between the violet bacterial cell and a darker background.

Anthony’s Method

This method makes the use of two reagents, namely crystal violet as primary stain and 20% of CuSO4 solution as a decolouring agent and counterstain.

Crystal violet will stain the bacterial cell and background. The CuSO4 solution will stain the non-ionic capsule. After staining:

  • A Bacterial cell appears violet.
  • The background appears light violet.
  • The capsule appears as a faint blue halo

Procedure

Anthony’s method involves the following steps:

  1. Take a clean, sterilized or grease free slide.
  2. Add a drop of crystal violet to the centre of the glass slide.
  3. Prepare a smear, by taking an inoculum from the bacterial culture and mix it with a drop of crystal violet.
  4. Then, allow the smear to air dry (Do not heat fix, as it can cause cell shrinkage and distortion of the bacteria).
  5. Flood a smear with 20% of CuSO4 solution for at least 30 seconds and remove the extra stain by tilting a glass slide.
  6. Add oil immersion to the stained area and observe it under the microscope having a 100X objective.

anthony's method

Anthony’s method is a type of positive staining method that stains the capsule along with the bacterial cell but not stains the background. As a result, a capsule appears as faint blue halo between the violet bacterial cell and purple background.

Maneval’s Method

This method makes the use of two stains, i.e. acidic stain (Congo red) and a special stain (Maneval’s stain). The composition of Maneval’s dye includes:

  • 10% Ferric chloride, which acts as a “Mordant”.
  • 5% phenol, which increases the penetration of the stain between the smear.
  • Acid fuschin, stains the bacterial cell being a basic dye.
  • Acetic acid, which decreases the pH of the smear to the acidic side.

Congo red appears red (at neutral pH) and appears blue in colour (at acidic pH). After staining:

  • A Bacterial cell appears bright red-pink.
  • The background appears dark blue colour.
  • The capsule appears as a clear halo.

Procedure

Maneval’s method involves the following steps:

  1. Take a clean, sterilized or grease free slide.
  2. Add a drop of 1% Congo red to the centre of the glass slide.
  3. Prepare a smear, by taking an inoculum from the bacterial culture and mix it with a drop of Congo red.
  4. Then, allow the smear to air dry (Do not heat fix, as it can cause cell shrinkage and distortion of the bacteria).
  5. Flood a smear with Maneval’s stain for at least 1 minute and remove the excess stain by tilting a glass slide.
  6. Add oil immersion to the stained area and observe it under the microscope having a 100X objective.

maneval's method

Maneval’s method is also a type of negative staining method, which stains the bacterial cell and its background but not a capsule. As a result, a capsule appears as a clear halo between the pink bacterial cell and blue background.

Hiss Method

This method makes the use of two reagents, namely crystal violet and copper sulphate solution. After staining:

  • A Bacterial cell appears dark violet.
  • The background appears brighter in colour.
  • The capsule appears as a light violet colour.

Procedure

Hiss method involves the following steps:

  1. Take a clean, sterilized or grease free slide.
  2. Add a drop of crystal violet to the centre of the glass slide.
  3. Prepare a smear, by taking an inoculum from the bacterial culture and mix it with a drop of crystal violet.
  4. Then, allow the smear to air dry (Do not heat fix, as it can cause cell shrinkage and distortion of the bacteria).
  5. Flood a smear with copper sulphate solution and remove the excess stain by tilting a glass slide.
  6. Add oil immersion to the stained area and observe it under the microscope having a 100X objective.

hiss method
Hiss method is also a type of positive staining method that stains the capsule and bacterial cell with a brighter background. As a result, a capsule appears as a light violet colour between a dark violet coloured bacterial cell and colourless background.

Significance

Capsule staining allows us to determine the presence of a capsule, which acts as a virulence factor to cause disease. Thus, it helps us to identify the pathogenicity of the bacteria by knowing its presence or absence, which in turn allows physicians or healthcare professionals to recommend the right treatment.

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