Staining Techniques

Bacteria have nearly the same refractive index as water, therefore, when they are observed under a microscope they are opaque or nearly invisible to the naked eye. Different types of staining methods are used to make the cells and their internal structures more visible under the light microscope. Microorganisms must be fixed & stained to increase visibility, accentuate specific morphological features.

Staining is technique used in microscopy to enhance contrast in the microscopic image.

A stain is a substance that adheres to a cell, giving the cell colour. The presence of color gives the cells significant contrast so they are much more visible.

Stain: An organic compound composed of

a benzene ring + a Chromophore + an Auxochrome group.

Benzene: organic colourless solvent molecule

Chromophore: Coloured molecules

Auxochrome: An auxochrome is a functional group of atoms with one or more lone pairs of electrons, when attached to a chromophore, intensifies the colour of the chromogen.

GRAM STAINING

Gram Staining is the common, important, and most used differential staining techniques in microbiology, which was introduced by Danish Bacteriologist Hans Christian Gram in 1884.

This test differentiates the bacteria into Gram Positive and Gram Negative Bacteria, which helps in the classification and differentiations of microorganisms.

Reagents Used:

• Primary Stain - Crystal Violet
• Mordant - Gram’s Iodine
• Decolourizer - Ethyl Alcohol
• Secondary Stain - Saffranin

Procedure of Gram Staining:

• Place slide with heat fixed smear on staining tray.
• Gently flood smear with crystal violet and let stand for 1 minute.
• Gently rinse with tap or distilled water using a wash bottle..
• The smear will appear as a purple circle on the slide.
• Decolorize using 95% ethyl alcohol or acetone.
• Rinse with water
• Gently flood with safranin to counter-stain and let stand for 1 min.
• View the dried smear using a light-microscope.
• Gram Positive: Blue/Purple Color.
• Gram Negative: Red Color

Mechanism & Principle of Gram Staining

When the bacteria is stained with primary stain Crystal Violet and the stain gets fixed by the mordant (Gram’s Iodine), crystal violet and iodine combine together inside the stained cells and form a CV-Iodine complex which gives blue/purple colour to all cells, But after washing of these cells with alcohol, gram negative cells become colourless and gram positive cells retains the stain and appeared blue/purple.

In case of Gram negative bacteria, When they are exposed to alcohol (here termed as decolourizer), cell wall (made of thin layer of peptidoglycan and thick outer layer of lipids) CV-Iodine complex gets washed off because decolourizer dissolves the lipids in the cell walls, which allows the crystal violet-iodine complex to leach out of the cells and cell become colourless. Then when again stained with saffranin, they take the stain and appear red in color.

But in case of Gram positive microbial cells, these cells resist the action of alcohol, because the cell wall of gram positive cells is made of thick layer of peptidoglycan and lipid content is low. this. Decolorizing the cell causes this thick cell wall to dehydrate and shrink which closes the pores in the cell wall and prevents the stain from exiting the cell. So the ethanol cannot remove the Crystal Violet-Iodine complex that is bound to the thick layer of peptidoglycan of gram positive bacteria and appears blue or purple in colour.

ACID FAST STAINING

The main aim of this staining is to differentiate bacteria into acid fast group and non-acid fast groups.

This method is used for those microorganisms which are not staining by simple or Gram staining method, particularly the member of genus Mycobacterium, are resistant and can only be visualized by acid-fast staining.

The Ziehl – Neelsen stain was first described by Franz Ziehl and Friedrich Neelsen, thus this stain is also termed as the acid-fast stain because in this method, the decolourization is achieved by using both acid and alcohol.

Acid-fast organisms are characterized by wax-like, nearly impermeable cell walls; they contain mycolic acid and large amounts of fatty acids, waxes, and complex lipids, because of the high lipid content of the cell wall. The phenolic compound carbol fuchsin is used as the primary stain because it is lipid soluble and penetrates the waxy cell wall.

Procedure of Acid-Fast Staining:

• Heat fixed smear, Flooded with Carbol Fuchsin stain
• Place a small piece of bibulous paper over the smear and steam for 10 minutes.
• Cool and Rinse with Distilled water
• Flood slide with acid alcohol (leave 15 seconds).
• The acid alcohol contains 3% HCl and 95% ethanol,
• Rinse with Distilled water
• Add Methylene Blue stain (counter stain).
• This stain adds blue color to non-acid fast cells and Leave the smear for 1 minute
• Rinse slide, make it dry and observe under oil immersion microscope.
• Acid fast Positive Cells: Red Color
• Acid fast Negative Cells : Blue Color

When the smear is stained with carbol fuchsin, it solubilizes the lipoidal material present in the Mycobacterial cell wall but by the application of heat, carbol fuchsin further penetrates through lipoidal wall and enters into cytoplasm. Then after all cell appears red.

Then the smear is decolorized with decolorizing agent (3% HCL in 95% alcohol) but the acid fast cells are resistant due to the presence of large amount of lipoidal material in their cell wall which prevents the penetration of decolorizing solution.

The non-acid fast organism lack the lipoidal material in their cell wall due to which they are easily decolorized, leaving the cells colorless. Then the smear is stained with counterstain, methylene blue. Only decolorized cells absorb the counter stain and take its color and appears blue while acid-fast cells retain the red color.

CAPSULE STAINING

The main purpose of capsule stain is to distinguish capsular material from the bacterial cell. A capsule is a gelatinous outer layer secreted by bacterial cell and that surrounds and adheres to the cell wall.

Chemically, the capsular material is a polysaccharide, a glycoprotein or a polypeptide. Capsule staining is more difficult than other types of differential staining procedures because the capsular materials are water soluble and may be dislodged and removed with vigorous washing.

The specimen does not need to be heat-fixed prior to negative staining. Bacterial capsules are non-ionic, so neither acidic nor basic stains will adhere to their surfaces.

Few methods are discovered to observe capsules through microscope and these methods are based on the staining of background and the cells, but not the capsule.

So in this way the capsular material / capsules getting differentiate from the cells.

Types of Capsule Staining

• Negative staining with Indian Ink
• Anthony’s Method (Positive capsule staining)

Negative Staining with India Ink:

In this staining method, the specimen appeared in contrast, darker colored background with stained cells (if present in the sample) but an unstained capsule. The darkbackground is formed with india ink or nigrosin or congo red.

When viewed under the microscope,

• The background will be dark as a result of India ink,
• Bacteria cells (if present in the sample) will be purple having taken the crystal violet dye
• The capsule appeared clear area against a dark background as it takes no stain.

Anthony’s Method:

Crystal violet stains both cells and capsules. On treatment with copper sulfate, capsules appear light blue while cells remain dark blue.

In this method, two dyes, crystal violet, and India ink are used. The capsule is seen as a clear halo around the microorganism against the dark background. This method is used for demonstrating Cryptococcus.

When viewed under the microscope, the background will be dark as a result of India ink, bacteria cells will be purple having taken the crystal violet dye while the capsule will be clear against a dark background given that it takes no stain.