Gram′s safranin solution

Gram’s safranin solution, also known as Safranin T or Basic Red 2, is a ready-to-use staining solution that comprises 10% safranin in ethanol. Safranin is a basic, cationic, azine dye containing a planar, diaminophenazine chromophore, with a phenyl substituent attached to a ring nitrogen. The trimethyl cationic form of safranine is lipophilic, whereas the dimethyl forms are weakly hydrophilic. It is most notably used in bacteriology as a counterstain in Gram staining to differentiate between Gram-positive and Gram-negative bacteria.

Gram’s safranin solution has been used in the following applications:

• as a non-toxic replacement for crystal violet for the quantification of biofilm formation
• to develop a hyaluronic acid visualization method
• to study improved β-lactam susceptibility against biofilm-embedded staphylococcus aureus by 2-aminothiazole

Gram staining enables the differentiation of bacteria into Gram-positive and Gram-negative. The mureine structure of the bacterial cell wall is the basis of the color affinity. Bacteria are first stained with crystal violet, and then with iodine solution, which forms a dye-iodine complex. This complex is retained in the multi-layer mureine structure of the cell wall of Gram-positive bacteria after decolorization, imparting a blue-violet color. The cell wall of Gram-negative bacteria comprises a single-layered murein structure that does not retain the dye-iodine complex, allowing the uptake of safranine counterstain, and imparting a pink-red color.