Classic Gram staining: Gram-color
Crystal violet is the best dye for use with Gram staining. Gram-positive and Gram-negative cells both take up the dye in the same way by chemical binding between functional groups of the dye and the target structures in the cell. Iodine potassium iodide in aqueous solution permeates into both cell types then reacts with the bound dye and the unbound dye. Decolorization is the real differentiating step in the Gram stain method as Gram positive cells walls are impermeable to the purple iodine-dye complexes whilst Gram negative cell walls allow the dye to escape. A mixture of alcohol and acetone is one of the most efficient decolorizing agents, which yields excellent and reproducible results and enables a better standardization of the method.

Counterstaining is then required in order to be able to see the Gram negative cells that have become decolorized. The dye most frequently used for counterstaining is safranin.

In the preparation of the specimen material, heat fixation of the specimen smear is required as unfixed cells may be washed off the slide during the staining procedure. Merck offers a ready to use kit for classical Gram staining which is based on Gram’s 4 step method. A certified protocol is part of the kit and is the basis for clear coloration and reproducible results. The staining can be performed on a staining rack or in jars or a staining instrument. The kit contains 6 x 500 solutions and is sufficient for 250 slides, stained on a staining rack.

Modified Gram staining - Gram-color modified, phenol-free This kit operates on basis of Gram´s four steps but with a modification to the first step- a modified crystal violet solution which is free of phenol is mixed with sodium hydrogen carbonate, and used as a mordant or trapping agent, preventing the complex from leaving the cell. The mixture results in a pronounced color intensification in the stain of Gram-positive bacteria, which will appear as a deep blue color.

The iodine solution is an iodine/potassium iodide solution stabilized with PVP (Polyvinylpyrrolidone). The advantage offered by addition of PVP is that a complex is formed with iodine. The complex reduces the evaporation of iodine out of solution, thus keeping it more stable and decreasing the formation of iodine vapours at the workplace.

Decolorizing solution is a mixture of ethanol and acetone, which ensures a greater differentiation of the bacteria, even in the hands of relatively inexperienced user. Counterstaining is done by using a phenol-free fuchsin solution Fuchsin is chosen because the contrast to the intensively coloured gram-positive bacteria is made even more obvious - dark blue vs. magenta red. All solutions contained in the kit are packed in easy-to-handle 200 ml PE bottles and are sufficient for 65 -70 tests. The kit is designed for use on a staining rack.