Types of stains and Preparation of staining solution


  • It is difficult to visualize microorganisms in living state, because they are minute, transparent and colorless when suspended in aqueous medium.
  • Staining is done to visualize microbes and also helps to differentiate them into specific groups as well as aids in diagnosis of disease.
  • To stain = to color

Dyes (Stains)

  • Any colored compound that reacts with or is absorbed by or dissolves in another phase and renders that phase colored.
  • Stains are organic unsaturated cyclic compound with chromophore and auxochrome group.
  • Chromophore= responsible for color
  • Auxochrome= dyeing property of salt formation
  • Without auxochrome group the chromogen (colored compound) is not able to bind to cells or tissues or fibers.
  • The binding of stain (chromogen) with the cell is due to presence of electric charges on them.

Classification of Dyes

  • Based on the nature of chromogen, there are three types of stain.
  1. Acidic Dyes (Anionic stain)
  2. Basic Dyes (Cationic stain)
  3. Neutral Dyes (Compound dyes)
  4. Leuco Dyes
  5. Metachromatic Dyes
  6. Synthetic Dyes
  7. Natural Dyes

Acidic stain (Anionic stain)

  • Chromogen of acidic stain is negatively charged. so, it is also known as Anionic stain
  • Acidic stain are used to stain the positively charged components such as background staining.
  • Acidic stain can not stain bacterial cell due to repulsion of same charge.
  • Examples: Eosin, Nigrosine, India ink, Congo red, Acid Fuchsin, Rose bengal

Basic stain (Cationic stain)

  • Chromogen or colored part of basic stain is positively charged. so, it is also known as cationic stain.
  • Basic stain are used to stain negatively charged components such as bacterial cell.
  • Examples: methylene blue, safranin, malachite green, basic fuschin, crystal violet

Neutral stain:

  • In neutral stain, both cation and anion are colored, such that net charge is neutral.
  • Neutral stain are actually is a salt of acidic and basic stain.
  • Examples: Romanowsky’s stain (eg: Giemsa Stain).

Metachromatic Dyes

  • Dyes appear to be of different color in contrast to its original color.
  • Example: Methylene blue

Synthetic Dyes

  • Artificial dyes were first made by Willium Perkins from aniline.
  • Also known a Aniline Dyes/Coal tar dyes.

Natural Dyes

  • Derived from natural sources like plants.
  • Example: Saffron, Indigo, Litmus, Hematoxylin


  • Mordant are compounds which form insoluble complexes with the stain and the resultant complex intensifies the reaction of staining between the dye and the cell structure. And staining procedure with mordant is known as Indirect Staining.
  • Classification:
  1. Basic Mordant: react with acidic dyes (Ferrous sulfate, Cetyl pyridinium chloride)
  2. Acidic Mordant: react with basic dye (Picric acid)


  • Pre-mordanting (Dyar’s method of cell wall staining)
  • Post-mordanting (Gram’s Staining)
  • In conjugation with stain ( Leifson’s method of flagella staining)


  • They are also known as Intensifiers.
  • Agents which increase the intensity of staining.
  • Physical intensifiers: Heat
  • Chemical intensifiers: acidic or basic (Acetic acid, phenol, potassium hydroxide, formalin).


  • Used to remove excessive stain from overstained cells.

Different Purposes:

1.Use of excess mordant: When excess mordant is present outside, dye-mordant complex break-up, due to less mordant inside the cell, excessive mordant come out of the cell.

  • Example: Giemsa’s or Leishman’s staining method for staining of blood films.
  1. Use of Acid and Alcohol: highly effective decolorizers for many dyes as it has greater solubility of dye.
  2. Use of Oxidizing agent: compounds like Picric acid can oxidize the dye to a colorless compound.
  3. Use of acidic dyes: the dye of acidic charge removes the basic dye from the cell or its structure. (Use of Congo red in Maneval’s methods).

Use of Stains

  • Basic Staining Technique:
  • Preparation of glass slide
  • Preparation of smear
  • Heat fixation
  • Staining
  • Observation under microscope

Preparation of Staining Solution



Gram Iodine

iodine 1

Crystal violet


Methylene blue

methylene blue