1. The Gram stain is named for the Danish bacteriologist who first devised it in 1844 (History, 2004). It is usually the first test used to identify bacteria. The Gram stain is based on the ability of bacterial cell walls to retain crystal violet dye during solvent treatment. Bacterial cells with walls containing small amounts of peptidoglycan and lipopolysaccharide are Gram-negative; those containing relatively high amounts of peptidoglycan and lipopolysaccharide are Gram-positive (The Gram, 2004). Microorganisms which retain the crystal violet-iodine complex appear purple-brown under the microscope, and are considered Gram-positive. Those which do not retain the stain appear red and are considered Gram-negative. An example of a Gram-positive bacterium is Staphylococcus aureus. An example of a Gram-negative organism is Salmonella typhii.
The Gram stain is carried out on a smear of the sample containing the bacteria (History, 2004; The Gram, 2004). The steps are:
1. Prepare a smear by placing one drop of the bacterial culture on a glass slide and smear it onto the slide using a wooden stick or by using an inoculation loop, making sure the smear is thin enough to air dry in a few seconds. Thick smears will not stain properly.
2. Air dry the culture smear and fix it over a gentle flame.
3. Add crystal violet stain over the culture, and let stand for 10 to 60 seconds. If the smears are very thin, the stain can be poured off immediately. Pour off the stain and rinse the excess stain with a stream of water, being careful not to wash the culture off the slide.
4. Add the iodine solution to cover the smear and let stand for 10 to 60 seconds. This reacts with the crystal violet to form a dark shade of blue. Under the microscope, cells which are Gram-positive will appear blue-black. Pour off the iodine solution and rinse the smear with running water. Shake off the excess water.
5. Add a few drops of deco...