The procure of gram staining is as follows:
1. Prepare the smear on a microscope slide and heat-fix it
2. Flood the smear with crystal violet on a staining tray
3. Gently and lightly rinse the smear with sterile water from a wash bottle
4. Flood the smear with iodine for a minute
5. Rinse the smear like in step 3
6. Pour 95% ethyl alcohol or acetone to decolorize for 10 seconds
7. Rinse like in step 3
8. Flood the smear with safranin (a counter-stain) for about 1 minute
9. Rinse the smear like in step 3
10. Leave the smear to air dry and observe under a microscope
Heat-fixing adheres the smear to the slide and hence makes it less amenable to being washed away during gram-staining procedure. Gram staining using crystal violet allows the peptidoglycan layer of the Gram + bacteria to pick up the dye and retain it even after decolorizing. Gram- bacteria lose the crystal violet color after decolorizing with alcohol since they lack the peptidoglycan layer. Safranin is a counter stain since it is picked by the Gram – bacteria that have lost the crystal violet dye. Therefore under a microscope and after the staining procedure, Gram+ bacteria appear violet/purple in colr while Gram – appear pink.
