A magnification of 500x to 1000x may be needed to distinguish cell shape and arrangement. View the slide using a compound microscope.The gram-negative cells should be stained red or pink, while the gram-positive cells will still appear purple or blue. Gently rinse with water no longer than 5 seconds. Apply the secondary stain, safranin, and allow it to sit for 1 minute.However, if the decolorizer is left on too long, all cells will lose color! Study reveals widespread use of fentanyl among people who inject drugs in New York City, despite overwhelming preference for heroin. The gram-negative cells will lose color, while the gram-positive cells will remain violet or blue. Rinse the slide with alcohol or acetone about 3 seconds, followed immediately with a gentle rinse using water.Use a dropper to apply Gram's iodine to the slide to fix the crystal violet to the cell wall.Rinsing too long can remove too much color, while not rinsing long enough may allow too much stain to remain on gram-negative cells. Gently rinse the slide with water no longer than 5 seconds to remove excess stain. Use a dropper to apply the primary stain (crystal violet) to the slide and allow it to sit for 1 minute.If too little heat is applied, the bacteria will wash off the slide during staining. Applying too much heat or for too long can melt the bacteria cell walls, distorting their shape and leading to an inaccurate result. Heat fix the bacteria to the slide by passing it through the flame of a Bunsen burner three times. Place a small drop of bacterial sample on a slide.If the staining procedure is performed correctly, gram-positive bacteria will be purple, while gram-negative bacteria will be pink. Both gram-positive and gram-negative bacteria pick up the pink stain, but it is not visible over the darker purple of the gram-positive bacteria.
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