Gram-Staining Procedure

Fixation

As you have done previously, fix your bacterial cells on a microscope slide. Again, if your source of cells is a colony from a petri dish, you will need to thin out the cells to get good staining and subsequent visualization.

Primary Staining

Stain your slide with crystal violet (the primary stain). Leave on for 1-3 minutes; expose for a longer time (i.e. 3 min.) the first time and modify your procedure as needed thereafter.

Rinse with water, being careful not to "flood" the slide. Rinse until the water runs clear.

Mordant (Increasing Dye Retention)

Add Gram's iodine for 3-4 minutes.

Destaining (Decolorization)

Decolorize with alcohol (95% to 100% EtOH, typically) again being sure not to "flood" your cells, washing away the unbound crystal violet and iodine.

Counter-Staining (Secondary staining)

Counter-stain with safranin for 3-4 minutes.

Rinse with water; again, ne flood pas.

Addendum

Carefully, blot dry

Observe under the microscope using, ultimately, oil immersion. Record your results.