Gram Staining Essay

2147 Words9 Pages
Introduction PART 1 In 1883 Hans Christian Gram discovered an important differential staining method that is used extensively today. The stain is called the Gram Stain. This staining procedure differentiates microbes into two basic groups: Gram positive microbes and Gram negative microbes. Gram staining consists of four components: * Primary stain (Crystal violet, methyl violet or Gentian violet) * Mordant (Gram's Iodine) * Decolourizer (ethyl alcohol, acetone or 1:1 ethanol-acetone mixture) * Counterstain (Dilute carbol fuchsin, safranin or neutral red) Procedure : 1. Prepare the smear and heat fix it. 2. Add crystal violet (primary stain) - 1minute. 3. Wash and add Gram’s iodine (mordant) - 1 min. 4. Drain off iodine, add alcohol (decolorizer) -15 seconds 5. Wash and add Basic fuchsin or Safranin (secondary stain)- 1 min. 6. Wash, dry and observe under oil immersion lens. Differential stains render one type of microbe one colour and other types of microbes another colour. In the Gram stain, Gram positive organisms retain the primary dye complex (crystal violet-iodine) whereas Gram negative cells loose the primary dye complex during the challenge rinse. Most differential stains have a challenge step that follows staining with a primary dye. In the Gram stain the challenge step is a rinse with either ethanol or acetone (either may be used). This step dehydrates and tightens the cell wall of Gram positives (mainly peptidoglycan) such that the rinse does not enter the cell. Gram negatives have mainly a lipid cell wall (even though they do contain peptidoglycan) that allows the challenge rinse to penetrate the cell and rinse out the crystal violet-iodine complex rendering the Gram negative cell colourless. Thus, the Gram negative cells must be stained to be seen, and this is done with the counter stain. The counter stain used in the
Open Document