Staining: Purpose, Mechanism and Dyes/Stains used in light microscopy

Purpose of staining:

  • In bright field or light microscopy, background appears bright and the microorganisms appear dark as they absorb some of the light.
  • However, most of the microorganisms are transparent and can’t be observed on the transparent bright background easily.
  • So, microorganisms at first are treated with some colored dyes or stains which give them characteristic color.
  • Colored microorganisms are easily seen on the transparent background.
  • This process of coloring of microorganisms with certain dyes is called staining and the major purpose of staining is to increase the contrast between the background and the microorganisms or their parts, so that they become distinctly visible.

Types of dyes or stains used in light microscopy:

  • Most of the dyes used in staining techniques are synthetic which are derived from aniline or nitrobenzene.
  • Each dye or stain consists of 3 components:
    • Benzene ring
    • Chromophore
    • Auxochrome
  • Benzene ring is the fundamental structural component of the dye and is colorless.
  • Chromophore is the functional group that gives color to the dye.
  • Auxochrome is the functional group that imparts ionic property to the dye.
  • Colored portion of the dye is called choromogen which is formed by the combination of benzene ring and a chromophore.
  • Based on the nature of chromogen, there are 3 types of dyes:
    • Acidic dye or anionic dye
    • Basic dye of cationic dye
    • Neutral dye

  1. Acidic dye or anionic dye:
    • In acidic dye, colored part is negatively charged and hence also called anionic dye.
    • It is used to stain basic (positively charged) components such as histone protein or background.
    • Since most of the bacterial cells are negatively charged on the surface, acidic dyes can’t stain them.
    • This is because the repulsion among the similar negative charges cannot fix the dye on the cell walls of bacteria.
    • e.g. Eosin, Nigrosine, India ink etc.
  2. Basic dye or cationic dye:
    • In basic dye, colored part is positively charged, hence called cationic dye.
    • It is used to stain acidic (negatively charged) components such as bacterial cell wall.
    • e.g. Methylene blue, Crystal violet, Safranin, Basic fuchsin and Malachite green.
  3. Neutral dye:
    • In neutral dyes, both anionic and cationic parts are colored.
    • These are in fact the salts of acidic and basic dyes.
    • e.g. Giemsa stain

Mechanism of staining:

  • The process of staining involves ion exchange reaction between the stain (dye) and the active site of the cell to be stained. E.g. bacterial cell contains large number of negatively charged proteins due to (COO) groups.
  • These negatively charged groups are balanced by positively charged ions (such as Na+) present outside the cell wall to form salt.
  • Therefore, a bacterial cell can be represented as (Bacterial Cell)Na+.
  • These negatively charged bacteria are stained by cationic or basic dyes like Methylene blue having positively charged chromogen that replaces the Na+ from the bacterial cell wall.
  • Methylene blue can be represented as (Mb)+Cl.
  • When bacterial cell is flooded with methylene blue, due to ion exchange mechanism acidic component of bacterial cell i.e. bacterial cell wall, becomes stained by the following reaction:
  • (Bacterial Cell)Na+ + (Mb)+Cl————–> (Bacterial Cell)(Mb)+ + NaCl

Staining: Purpose, Mechanism and Dyes/Stains used in light microscopy