1.  Prepare a separate smears of E. coli and S. aureus on two different slides. Fix these preparation by allowing them to dry by passing over the Bunsen Burner. When dry pass quickly through the flame one or two time to fix the smear.  Stain the smear with crystal violet stain (primary stain) for about one minute.  Rinse with water for a few seconds  Add few drop iodine solution (mordant) to both the smears Allow the solution to react for 30 sec.

2.  Rinse with water.  Dip in 95 % ethyl alcohol for 10 to 15 sec.  Counter stain both the smears with safaranin (counter stain) for about 20 to 30 sec.  Wash with water and blot dry using absorbant paper  Air dry the slides  Examine the smear first under HP. When you locate the bacterial aggregates, add a drop of immersion oil and observe under the oil immersion objective of the microscope.

3.  Crystal violet is the preferrd basic dye.  It reacts with the acidic components of the cell.  It takes about 10 seconds to react

4.  A mordant increase the affinity of the dye to the cell by chemically combining with it. Staining is deeper and can not be washed out easily if a mordant is used. Iodine solution is the mordant used in gram’s staining.

5.  As the name indicates it is an agent that removes the dye from a stained cell. Some stained cells are decolourized more easily than others (depending on their chemical composition)  E. g 90% alcohol or acetone-alcohol mixutre

6.  This is also a basic dye different in colour than the initial (primary one). The purpose of the counter stain is to give the decolourized cells a colour different from the first one. The microbes which are not decolourized by alcohol by alcohol retain the first stain while the decolourized ones takes up the counter stain (second stain)