1. Materials - Crystal violet (primary stain) - Gram's iodine (mordant, to fix crystal violet in the cell wall) - Ethanol or Acetone (decolorizer) - Safranin (secondary stain or counterstain) - DI Water - Microscope slide 2. Procedure - Place a small drop of bacterial sample on a slide. Heat fix the bacteria to the slide by passing it through the flame of a Bunsen burner three times. Applying too much heat or for too long can melt the bacteria cell walls, distorting their shape and leading to an inaccurate result. If too little heat is applied, the bacteria will wash off the slide during staining. - Use a dropper to apply the primary stain (crystal violet) to the slide and allow it to sit for 1 minute. Gently rinse the slide with water no longer than 5 seconds to remove excess stain. Rinsing too long can remove too much color, while not rinsing long enough may allow too much stain to remain on gram-negative cells. ...
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