Crystal Violet

Author: Kelsey Goldman, HTL (ASCP)

Last Update: August 26 2023

Cite this Page! -> Goldman, Kelsey. “Crystal Violet.” HistologyOutlines.Com, August 26 2023. Accessed (today)

Purpose:

Crystal Violet is an essential part of the Gram Staining protocal. Along with iodine, the Gram stain allows for differentiation of bacteria based on the bacterias physical (but not normally visable) propeties. The Gram stain allows for better idenification of bacteria which can inform the clinicial on what treatment options will be the most benifical (Suvarna et al.)

Control Tissues:

Staining Pattern:

Biochemistry Theory:

It is not compeletley understood what the mechanism of action of a postitive Gram Stained bacteria is. (Suvarna et al.)
The Gram Stain has three parts; Immerision into Crystal Violet solution, Iodine, and decolorization (as well as an optinal counter stain). (Suvarna et al.)
Crystal Violet is what leads to the blue to blue/black staining in positive cells. When the slide is first immersed into the crystal violet solution, the solution stains everything. The crystal violet staining is more tightly bound to the cell and thus resists decolorization more- leading to a positive signal. (Costa)
The iodine funtions as a mordant. Iodine forms a complex with the bound crystal violet. This complex makes it more resistant to decolization. (Costa)
There are two common destaining agents for the gram stain. The destaining agents are alcohol, acetone, and a mixture of the two. The destaining agent removes all not bound and mordanted Crystal Violet. (Jones)
staining is thought to depend on many factors. These factors include cell wall thickness, chemical composition, and funtional integrty of the cell wall of the gram positive bacteria. (Suvarna et al.)

Troubleshooting

Many errors occur at the point of decolorization. This will take practice to accomplish correctly. We recommend that you stop the decolorsation process in water and look quickly under the scope to verify that you have not removed too much color. The sample should retain some CV, but not be a purple mess. be careful not to dry out the section in the sight of the bulb (Jones)
Do not allow sampels to dry at any point of the staining will be uneven. (Suvarna et al.)
The Crystal Violet dye can form clumps over time that could effect staining quality. Filter the crystal violet solution if precipate occurs. Ensure that the Crystal violet solution is stored properly in a cool dark spot. (Costa)

Additional References:

Costa, Pedro Moura. The Handbook of Histopathological Practices in Aquatic Environments: Guide to Histology for Environmental Toxicology. Elsevier, Academic Press, an imprint of Elsevier, 2018.
Jones, Gilda. The Gram Stain : A New Look at an Old Tool. Rev March 1986, 1986, https://babel.hathitrust.org/cgi/pt?id=mdp.39015009124465&seq=5.
PubChem. Gentian Violet. https://pubchem.ncbi.nlm.nih.gov/compound/11057. Accessed 24 Aug. 2023.
Suvarna, S. Kim, et al., editors. Bancroft’s Theory and Practice of Histological Techniques. Eighth edition, Elsevier, 2019.

Sample Protocol