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Safranin O (C.I. 50240) is a CERTISTAIN dry dye that is used for the preparation of a staining solution for e.g. Gram stain procedures in smears and sections of human origin in bacteriology. The Gram staining allows a fast differentiation of bacteria in Gram-positive and Gram-negative.
It is mostly utilized for the identification of cartilage, mucin, and mast cell granules. The safranin stain works by binding to acidic proteoglycans in cartilage tissues with a high affinity forming a reddish-orange complex. The binding made cartilage tissues appear red when observed under the microscope.
SAFRANINE SOLUTION is a fundamental biological stain, a red-pink dye used in histology, cytology, and microbiology to color acidic cell components like nuclei, cell walls (in plants), cartilage, and mucin, often as a counterstain in techniques like Gram staining to highlight Gram-negative bacteria or in plant histology (with Fast Green) to differentiate lignified tissues. It binds to negatively charged molecules (like nucleic acids and proteoglycans) to make structures visible under a microscope, providing crucial contrast for identifying different cell types and tissues.
How does safranin affect Gram-positive cells? Safranin penetrates the cell wall, but not enough of it is retained to cause a color change...... In the Gram-positive cell walls, most of the spaces between the molecules that make up peptidoglycan are already occupied by crystal violet/iodine complexes.
What is the use of Safranine Solution in Gram Staining?
Following steps are followed for gram staining in which there is use of Safranine Solution also.
1. Prepare a thin smear on clear, dry glass slide.
2. Allow it to air dry and fix by gentle heat.
3. Flood with Gram's Crystal Violet (S012) for 1 minute. (If over staining results in improper decolourization of known gram-negative organisms, use less crystal violet).
4. Wash with tap water.
5. Flood the smear with Gram’s Iodine (S013). Allow it to remain for 1 minute.
6. Decolourize with Gram's Decolourizer (S032) until the blue dye no longer flows from the smear. (Acetone may be used as a decolourizing agent with caution, since this solvent very rapidly decolourized the smear).
7. Wash with tap water.
8. Counter stain with 0.5% w/v Safranin (S027) for 20 seconds and rinses off with water.
9. Wash with tap water.
10. Allow the slide to air dry or blot dry between sheets of clean bibulous paper and examine under oil immersion objective
Gram Safranin solution is used for contrast staining of bacterial species that did not retain their primary dye, i. e. Gram-negative bacteria. That enables differentiating the blue and purple-colored Gram-positive bacteria from the red-colored Gram-negative bacteria.