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Safranin is used as a counterstain in some staining protocols, colouring cell nuclei red. This is the classic counterstain in both Gram stains and endospore staining. It can also be used for the detection of cartilage, [2] mucin and mast cell granules. Safranin typically has the chemical structure shown at right (sometimes described as dimethyl ...
Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50–90% of cell envelope), and as a result are stained purple by crystal violet, whereas gram-negative bacteria have a thinner layer (10% of cell envelope), so do not retain the purple stain and are counter-stained pink by safranin. There are four basic steps of the ...
Crystal violet stains both Gram positive and Gram negative organisms. Treatment with alcohol removes the crystal violet colour from gram negative organisms only. Safranin as counterstain is used to colour the gram negative organisms that got decolorised by alcohol. While ex vivo, many cells continue to live and metabolize until they are "fixed".
A number of other bacteria—that are bound by a single membrane, but stain gram-negative due to either lack of the peptidoglycan layer, as in the mycoplasmas, or their inability to retain the Gram stain because of their cell wall composition—also show close relationship to the gram-positive bacteria.
Schematic of typical Gram-positive cell wall showing arrangement of N-Acetylglucosamine and N-Acetylmuramic acid; Teichoic acids not shown.. The Gram-positive cell wall is characterized by the presence of a very thick peptidoglycan layer, which is responsible for the retention of the crystal violet dyes during the Gram staining procedure.
One commonly recognizable use of differential staining is the Gram stain. Gram staining uses two dyes: Crystal violet and Fuchsin or Safranin (the counterstain) to differentiate between Gram-positive bacteria (large Peptidoglycan layer on outer surface of cell) and Gram-negative bacteria. Acid-fast stains are also differential stains.
In Gram staining, crystal violet stains only Gram-positive bacteria, and safranin counterstain is applied which stains all cells, allowing the identification of Gram-negative bacteria as well. An alternative method uses dilute carbofluozide.
Malachite green is water-soluble so vegetative cells and spore mother cells can be decolorized with distilled water and counterstained with 0.5% Safranin. [7] In the end, a proper smear would show the endospore as a green dot within either a red or pink-colored cell. [2]