1. Gram staining is a method of differentiating bacterial species into two large groups(Gram-positive and Gram-negative).The Gram staining is almost always the first step in the identification of bacteria.It is a valuable diagnostic tool in both clinical and research settings, not all bacteria can be definitively classified by this technique. This gives rise to Gram-variable and Gramindeterminate groups as well.

2. The method is named after its inventor, the Danish scientist Hans Christian Gram (18531938), who developed the technique while working with Carl Friedlnder in the morgue of the city hospital in Berlin in 1884.In 1884, while examining lung tissue from patients who had died of pneumonia, Gram had discovered that certain stains were preferentially taken up and retained by bacterial cells. 3. Gram did not use a counterstain in his procedure. It was a few years later, that the German Pathologist Carl Weigert(1845-1904) from Frankfurt, added a final step of staining with Safranin.Gram himself never used the red counterstaining in order to visualize the gram negative bacteria. 4. BacteriaCells will be decolourized while some cells will retain the stainStained with Crystal VioletAll Bacteria will be stained PurpleGrams Iodine solution Add Decolourizer (Alcohol or Acetone)Stain will be fixed due to formation of complex of Crystal Violet & IodineStaining with Safranin(Counter Stain)Cells that retains the colour of Primary Stain are Gram positive. Cells that do not retains the colour of Primary Stain and takes up the colour of Counter Stain are Gram Negative. 5. Applying a primary stain (Crystal Violet) to a heat-fixed smear of a bacterial culture.The addition of Grams Iodine, which binds to crystal violet and traps it in the cell.Decolourization with Alcohol or Acetone, andCounter staining with Safranin 6. Prepare a heat fixed smear of the bacterial culture.Cover the smear with the Crystal Violet for 1 min.Add Grams Iodine, which washes the crystal violet stain.Rinse the slide in running water and add decolourizer(Alcohol).Again rinse the slide and cover the smear with the Safranin for 1 min.Wash off the safranin with water, air dry the slide and Observe under oil immersion lens. 7. GRAM-POSITIVE BACTERIAGRAM-NEGATIVE BACTERIA