|Gram stain ingredients are: Crystal Violet, Iodine, Alcohol and Safranin|
|Once your smear is dry and heat fixed, add Crystal Violet|
|Cover the smear with the Crystal Violet and let stand for 20 seconds|
|Rinse briefly with water|
|Cover the smear with iodine (the mordant).
Let stand for one minute
|Decolorize with alcohol until liquid runs clear|
|Rinse slide with water to stop the decolorization|
|Add Safranin (the counterstain)|
|Cover the smear with the Safranin and let stand for 20 seconds|
|Rinse slide with water until clear|
|Put slide on blotting paper|
|Blot the slide dry|
The Gram stain is the most commonly used differential stain for determining cell morphology. Differential stains allow for distinguishing certain characteristics of cells, and the stains commonly use two or more stains. The Gram stain, which divides most clinically significant bacteria into two main groups, is the first step in bacterial identification.
The Gram staining method, named after the Danish bacteriologist who originally devised it in 1844, Hans Christian Gram, is one of the most important staining techniques in microbiology. It is almost always the first test performed for the identification of bacteria. The primary stain of the Gram's method is crystal violet. Crystal violet is sometimes substituted with methylene blue, which is equally effective.
The microorganisms that retain the crystal violet-iodine complex appear purple brown under microscopic examination. These microorganisms that are stained by the Gram's method are commonly classified as Gram-positive or Gram non-negative. Others that are not stained by crystal violet are referred to as Gram negative, and appear red. Gram staining is based on the ability of bacteria cell wall to retaining the crystal violet dye during solvent treatment. The cell walls for Gram-positive microorganisms have a higher peptidoglycan and lower lipid content than gram-negative bacteria. Bacteria cell walls are stained by the crystal violet. Iodine is subsequently added as a mordant to form the crystal violet-iodine complex so that the dye cannot be removed easily. This step is commonly referred to as fixing the dye. However, subsequent treatment with a decolorizer, which is a mixed solvent of ethanol and acetone, dissolves the lipid layer from the gram-negative cells.
The removal of the lipid layer enhances the leaching of the primary stain from the cells into the surrounding solvent. In contrast, the solvent dehydrates the thicker Gram-positive cell walls, closing the pores as the cell wall shrinks during dehydration. As a result, the diffusion of the violet-iodine complex is blocked, and the bacteria remain stained. The length of the decolorization is critical in differentiating the gram-positive bacteria from the gram-negative bacteria. A prolonged exposure to the decolorizing agent will remove all the stain from both types of bacteria. Some Gram-positive bacteria may lose the stain easily and therefore appear as a mixture of Gram-positive and Gram-negative bacteria (Gram-variable). Finally, a counterstain of basic fuchsin is applied to the smear to give decolorized gram-negative bacteria a pink color. Some laboratories use safranin as a counterstain instead. Basic fuchsin stains many Gram-negative bacteria more intensely than does safranin, making them easier to see. Some bacteria which are poorly stained by safranin, such as Haemophilus spp., Legionella spp., and some anaerobic bacteria, are readily stained by basic fuchsin, but not safranin. The polychromatic nature of the gram stain enables determination of the size and shape of both Gram-negative and Gram-positive bacteria. If desired, the slides can be permanently mounted and preserved for record keeping. .