Bacterial Cell Wall Staining by Chance's Method

  Bacterial cell is consisting of various structural components. Cell wall is one of the most important component. Cell wall present outside of the bacterial cell membrane. It gives rigidity, protection and shape to the bacterial cell.

  Based on the structure of cell wall, all bacteria are divided into two groups as

  • Gram positive and
  • Gram negative.
Cell wall of Gram positive cell is monolayered while cell wall of gram negative bacteria is bilayered.
Bacterial cell wall can be demonstrated by various special staining methods like Chance's method, Ringer's method & Dyers method. The most common cell wall staining method is Chance's method.

Requirements :

  • Clean grease free slide
  • Nichrome wireloop
  • 24 hrs old culture of bacteria
  • 0.5% New fuchsin/Basic fuchsin solution (Basic Stain)
  • 0.5 % Condo red solution (Acidic Stain)

Procedure & Steps

  • Prepare the smear on the slide under aseptic conditions with the help of wire loop.
  • Air dry the smear, but do not heat fix (Because the heat fixation changes the structure of capsule. therefore heat fixation is avoided here).
  • Apply 0.5% New Fuchsin for 3 minutes.
  • Remove the excess stain (but do not water wash)
  • Apply 0.5 % Congo Red for 4 minutes.
  • Gentle wash with water
  • Air dry and observe under oil immersion objective lens.

Machanism and Principle of chance's cell wall staining method

  New Fuchsin stain is a basic stain. Therefore it is stained cell wall as well as cytoplasm. Cell wall and cytoplasm both are acidic in nature and they are having negative charge on their surface. However here the strong staining of cell wall take place. Because cell wall is more acidic than cytoplasm due to the presence of free carboxylic groups on its surface.

  Congo red is a acidic stain and this acidic stain bind with basic stain which is already present on the cell and removes that basic stain. That means here Congo red acts as a decolorizer. but the  removal of basic stain(New Fuchsin) takes place only from the cytoplasm and not from the cell wall, because it is strongly bound to the cell wall.

  Therefore after water wash cytoplasm becomes colourless while cell wall becomes pink coloured. that means here the role of Congo red is to carry out the decolorization, But the decolorization of only cytoplasm takes place and decolorization of cell wall does not take place.

Microscopic Observation

Spherical and rod shaped bacterial call wall Pink in colour.
 Under the microscope you will observed pink colour cell wall and colourless cytoplasm.