Gram Staining Procedure
Introduction to Gram Staining:
Gram staining is a laboratory technique that is widely used to differentiate bacteria into two broad categories: gram-positive and gram-negative. Developed by Danish bacteriologist Hans Christian Gram in 1884, this staining procedure remains a crucial diagnostic tool in microbiology even today. This article will discuss the gram staining procedure in detail, including the reagents used, the steps involved, and the interpretation of results.
Principle of Gram Staining:
Gram staining is based on the principle that different bacterial cell walls have different chemical and structural properties. The cell wall of gram-positive bacteria contains a thick layer of peptidoglycan, which retains crystal violet-iodine complex during staining. The cell wall of gram-negative bacteria, on the other hand, has a thinner peptidoglycan layer, which is surrounded by an outer membrane containing lipopolysaccharides (LPS). The LPS layer of gram-negative bacteria is impermeable to the crystal violet-iodine complex, which is why gram-negative bacteria appear pink or red after counterstaining with safranin.
Reagents Used in Gram Staining Procedure:
The reagents used in gram staining include crystal violet, iodine solution, alcohol (usually ethanol or acetone), and safranin. Crystal violet is the primary stain, which imparts a purple color to all bacterial cells. Iodine solution is used as a mordant, which enhances the binding of crystal violet to the bacterial cells. Alcohol is used as a decolorizing agent, which removes crystal violet from the gram-negative bacteria but not from the gram-positive bacteria. Finally, safranin is used as a counterstain, which imparts a red color to the gram-negative bacteria that have lost their purple color.
The gram staining procedure consists of four basic steps: preparation of bacterial smear, staining with crystal violet and iodine, decolorization with alcohol, and counterstaining with safranin.
1. Preparation of Bacterial Smear:
A small amount of bacterial culture is transferred to a clean microscope slide using a sterile loop or swab. The bacterial cells are then spread uniformly on the slide to form a thin film. The smear is then allowed to air dry completely.
2. Staining with Crystal Violet and Iodine:
The slide with the bacterial smear is fixed by passing it through the flame of a Bunsen burner or by using a fixing agent such as methanol. The slide is then flooded with crystal violet solution and allowed to stand for 1 minute. The excess stain is then washed off with water, and the slide is flooded with iodine solution and allowed to stand for 1 minute. Iodine solution acts as a mordant, which fixes the crystal violet to the bacterial cell wall.
3. Decolorization with Alcohol:
The slide is then decolorized with alcohol by flooding it with 95% ethanol or acetone for about 10-15 seconds. This step is critical as over-decolorization can result in false-negative results, while under-decolorization can result in false-positive results.
4. Counterstaining with Safranin:
The slide is then flooded with safranin solution and allowed to stand for 1-2 minutes. The excess stain is washed off with water, and the slide is allowed to air dry. The slide is then examined under a microscope using an oil immersion lens.
Interpretation of Gram Staining Results:
The gram staining results are interpreted based on the color of bacterial cells under the microscope. Gram-positive bacteria appear purple or blue-violet, while gram-negative bacteria appear pink or red. Mixed cultures may show both gram-positive and gram-negative bacteria. In some cases, bacteria may also appear gram-variable, which means they stain differently at different points in the cell wall. Inclusion bodies also help to identify organisms in some specific bacterial infection.