3)Add iodine (1 min)
4)acid/alcohol wash (1 min)
5)Flood with safranine (1min)
6)Air dry and examine.
These times are for clinical microbiology and experimental methods employ optimal and more precise times (but overall its pretty close).
Down side of this method is that you must smear bacteria onto the slide and fix it by heating the underside of the slide with a bunsen burner. if they are pink then you have gram negative (Gram's stain didnt stick) if its purple then its gram positive(Gram's stain did stick) This is due to the peptidoglycan layers. Gram negative bacteria have only a thin layer of peptidoglycan as part of the cell membrane/wall where Gram positive have a very think peptidoglycan layer.Source(s):Medical Microbiology
