Gram staining is a common laboratory technique used to differentiate bacterial species into two broad categories: gram-positive and gram-negative. The procedure is named after Danish bacteriologist Hans Christian Gram, who developed the technique in 1884. Gram staining is widely used in clinical settings to quickly and accurately identify bacteria based on their cell wall composition.
To perform a Gram stain, the following steps are typically followed:
- Obtain a bacterial sample and prepare a heat-fixed smear on a microscope slide. This can be done by taking a bacterial culture and spreading a small amount of the bacteria onto the slide, then heating the slide to kill the bacteria and fix them in place.
- Flood the slide with crystal violet stain for one minute. This step is called primary staining, and it involves covering the bacterial cells with a violet-colored dye.
- Rinse the slide with water to remove excess stain.
- Flood the slide with iodine solution for one minute. This step is called mordanting, and it helps the crystal violet to adhere to the bacterial cells.
- Rinse the slide with water to remove excess iodine.
- Flood the slide with 95% ethanol for 10-15 seconds. This step is called decolorization, and it removes the crystal violet stain from the cells of gram-negative bacteria, leaving only the iodine behind.
- Rinse the slide with water to remove excess ethanol.
- Flood the slide with safranin counterstain for 30 seconds. This step is called secondary staining, and it adds a red-colored dye to the cells that were not decolorized in step 6. As a result, gram-positive bacteria will appear purple or blue, while gram-negative bacteria will appear red or pink.
- Rinse the slide with water to remove excess counterstain.
- Observe the slide under a microscope to examine the bacterial cells and determine their gram-staining characteristics.
It is important to note that the exact steps and specific reagents used in Gram staining may vary slightly depending on the laboratory and the specific bacteria being tested. Additionally, some bacteria may not retain the stain well and may be difficult to accurately classify using this technique. In these cases, additional tests may be necessary to determine the bacterial species.