Gram Stain Set, 4 x 4oz bottles For Sale
This Gram Stain Set includes four chemicals, each housed in a four-ounce HDPE flip top dropper container.
Solution #1 consists of 0.5% Crystal Violet, 10% Ethanol, and 0.8% Ammonium Oxalate.
Gram’s iodine, PVP-Iodine combination (solution #2). (PVP-Iodine is a stable chemical compound of PVP and elemental iodine that is entirely soluble in water, ethanol, and other solvents.)
Acetone/Alcohol Decolorizer, a 1:1 mixture of acetone and ethanol.
Solution #4 contains 0.5% Safranin.
Because of the flammable alcohol, this staining kit can only be shipped to the 48 contiguous United States and Canada by Standard Ground. No air deliveries are permitted.
The Gram stain process was invented by the Danish physician Hans Christian Gram to distinguish pneumococci from Klebsiella pneumonia.
In brief, the process includes applying an iodine solution (potassium iodide) to cells that have already been stained with crystal violet or gentian violet. This technique results in the formation of “purple coloured iodine-dye complexes” in the cytoplasm of bacteria. Following the staining with crystal violet and iodine, the cells are treated with a decolonizing agent such as an ethanol/acetone mixture. The permeability of the cell wall to these “purple coloured iodine-dye complexes” when treated with the decolorizing solvent distinguishes Gram-positive and Gram-negative bacteria. Gram-positive bacteria retain purple iodine-dye complexes following treatment with the decolorizing chemical, whereas Gram-negative bacteria do not. A red counterstain, such as safranin, is applied following decolorization treatment to visualize decolorized Gram-negative bacteria.
Preparing the smear…
The first consideration is proper smear preparation. For viscous specimens, make a thin film of the substance on a clean glass slide with a sterile loop or swab. Allow to dry before running the slide through a flame several times (the slide should not get too hot to touch). Failure to follow these instructions may result in staining abnormalities and disruption of bacteria and cell shape.
Unfixed smears tend to be washed away during staining and washing, resulting in the absence of stained bacteria.
Procedure for staining…
For 10 seconds, flood the slide with crystal violet. (Wash with running tap water).
Flood for 10 seconds with Gram’s iodine. (Wash with water).
Decolorize the smear carefully with an ethanol/acetone mixture until the thinnest sections are colourless. (Wash with water). This is the most important stage, and it is also the one most impacted by technical changes in time and reagents.
10 seconds of safranin (pink colour) flooding. (Wash with water). Allow to dry naturally or blot with absorbent paper.
Results… Gram-positive organisms retain the violet-iodine complexes after washing in ethanol, but Gram-negative organisms lose this complex and stain red from the safranin counterstain.