Gram Stain Micropod

Transcript for Gram Stain Micropod podcast by Marla Garrison, MCC Biology Instructor

The Gram Stain is a differential staining technique that allows us to differentiate gram negative cells from gram positive cells based upon the chemistry and structure of their cell walls.  After completing the staining protocol, gram positive cells appear purple while gram negative cells appear pink.  The gram stain utilizes crystal violet as the primary stain. This basic dye is positively charged and, therefore, adheres to the cell membranes of both gram negative and positive cells. After applying crystal violet and waiting 60 seconds the excess stain is rinsed off with water. Next, a mordant is used. The mordant is Gram’s Iodine. This binds to the crystal violet making a large complex that adheres to the cell membrane. Gram’s Iodine is allowed to sit for 30 seconds then the decolorizor, 95% ethanol is added.  Ethanol acts as a solvent which causes the crystal violet-iodine complex to dissolve away and rinse out of the cell wall if the cells are gram negative. Recall that gram negative cells have a very thin layer, about 20% of their cell wall, made up of peptidoglycan. They are, therefore, unable to retain the iodine-crystal violet complex. Gram positive cells, on the other hand, contain a very thick layer of peptidoglycan, making up about 80% of their cell walls. And, this contains the dye complex even though the ethanol attempts to remove it. After 15 seconds or so of decolorization the slide is rinsed to remove the ethanol and the counterstain, safranin, is applied. Safranin, another positively charged basic dye, adheres to the cell membrane. Gram negative cells, having no dye present at this stage of the staining process will bind the safranin and appear pink under the microscope. The gram positive cells have retained the crystal violet and, although they also bind the safranin, they still appear a dark purple.