VMC 211: General Veterinary Microbiology (1+1)

PRINCIPLE OF GRAM'S STAINING

• Gram’s staining is a differential staining method. This method was developed by Christian Gram.
• Based on this staining bacteria are grouped into two categories, gram positive and gram negative.
• In this staining method four different reagents namely
• primary stain,
• mordant,
• decolorizer and
• counterstain.
• The primary stain normally used in Gram’s staining is crystal violet.
• The mordant used is Gram’s iodine. The iodine in Gram’s iodine combine with violet and forms a complex. The crystal violet iodine complex binds to magnesium RNA components in the cell. The resultant magnesium - RNA – crystal violet – iodine complex is bigger and difficult to remove from the cell.
• The decolourising agent used is ethyl alcohol (95%) which functions as a lipid solvent and dehydrating agent.
• Safranin or dilute carbol fuchsin is used as the counter stain.