For Gram Stains

Documentation

Introduction

Crystal violet is an organic chloride salt that belongs to the triphenylmethane type alkaline dyes. This monochloride salt whose scientific name is hexamethyl chloride rose aniline has a wide bio and industrial applications including developing fingerprints, dying ink,  as an antifungal, antibacterial & anthelmintic agent, and as a histological stain. Other names used to describe crystal violet include Basic Violet 3, Gentian Violet, Hexamethyipararosaline chloride and Methyl Violet 10B.

Its powder form appears as a dark green compound with metallic luster but appears purple when dissolved. When in aqueous or alcohol solution, crystal violet has a (purple) blue-violet color, with the color largely depending on the acidity of the solution. The different colors of the dye are because of the different charged states of the dye molecule.

Brief history

Charles Lauth was the first scientist to synthesize methyl violet in 1866. Crystal violet is one of the components of methyl violet. Crystal violet was first independently synthesized by Alfred Kern while working in Basel. For the easier synthesis of this product, he entered into a collaboration with a German chemist Heinrich Caro. Kern also discovered that diethylaniline could be used to synthesize violet dye, which is commonly known as Basic violet 4.

Jakob Stilling, a German ophthalmologist, discovered the antiseptic properties of crystal violet in 1890. He collaborated with E. Merck & Co. to market the dye, which was probably a mixture of aniline dyes, as an antiseptic. Later in 1902, Conradi and Drigalski discovered that the effect of the dye was strong on most gram-positive bacteria but little effect on gram-negative bacteria such as Bacillus typhi (Salmonella typhi) and Bacillus coli (Escherichia coli). John Churchman published a more detailed study of the dye on the different strains of bacteria in 1912.

Chemical properties

Some of the chemical properties of crystal violet include:

• Light sensitivity
• Insoluble in ether but soluble in alcohol, chloroform, and water.
• Exothermic with reducing agents, releasing gaseous hydrogen.
• Reacts vigorously with oxidizing agents.

Crystal violet preparation

Crystal violet is prepared by using N, N-dimethylaniline. This product is condensed, chlorinated and added taken through other reactions.

It can also be synthesized via the reaction of N, N-dimethylaniline and Michler ketone in the presence of phosphorus oxychloride. It is then taken through azeotropic reaction with hydrochloric acid. After this, the compound is then recrystallized in hot water to generate the compound.

0.5% Crystal Violet Solution preparation procedure (100ml)

Reagents

• 20mL methanol
• 80mL distilled water
• 5 crystal violet (Sigma-Aldrich).

No sterilization procedures are required in this process. The crystal violet powder is dissolved into the distilled water; methanol is then added to this solution.

The crystal violet solution made should them be stored in the dark at room temperature. It should be used within 2 months.

Crystal violet staining of adherent cells

Crystal violet can be used to examine the effect of chemotherapeutics and other agents on growth and survival cells in a culture. Since during cell death, adherent cells normally detach from culture plates, this can be used to show differences in proliferation and quantify cell death on stimulation with death-inducing agents. Crystal violet binds to DNA and proteins in cells and as such can be used to detect the maintained adherence of cells. In this procedure, the dye works as an intercalating dye that enables the quantification of DNA which is always held proportional to the number of cells in the culture. The staining protocol takes about 6 minutes to perform. Crystal violet aids in the visualization of cells by staining the cell nuclei a deep purple color.

The staining procedure:

• Place the cells to be examined on the ice and wash them with cold PBS.
• Fix the cells with 100% ice-cold methanol for about 10 minutes.
• Then aspirate methanol from plates. After this, the cells can be stained with crystal violet or stored for future use. For storage, the cells should be covered with 50% glycerol in PBS, wrapped in a plastic wrap or parafilm and refrigerated.
• To stain the cells, move them to room temperature and add 0.5% crystal violet solution in 25% methanol. Then incubate the cells for about 10 minutes and remove the crystal violet stain by washing in water until the dye comes off. After washing, allow the cells to dry at room temperatures.

Applications of crystal violet

As an antibacterial and antifungal agent

Crystal violet can be used to kill or slow the growth of bacteria. It can also be used to destroy fungi by inhibiting their reproduction or ability to grow.

As an antiseptic drug

Crystal violet can be used as a disinfectant to inhibit the activity of microbes or to destroy them.

As an anthelminthic drug

Crystal violet can also be used to kill parasitic worms.

As a histological dye

Crystal violet is also used in the microscopic examination of cells to highlight and give contrast to specific features of the cell, e.g. nuclei and cytoplasm.

Other applications

• As an acid-base indicator in biological stains and non-aqueous titration. Color ranging from green (PH 0.5) to blue (PH 2.0).
• Crystal violet, in a hydrobromic acid medium, forms a colored chelate with thallium. As such it can be used as a thallium sensitive reagent. Crystal violet can also be used to detect metal ions such as antimony, zinc, mercury, tungsten, cadmium, and gold.
• Crystal violet can be used to check the viability of cells in culture.
• As an active component of Gram staining (primary stain).
• In DNA visualization in agarose gels. In this case, high concentrations of DNA are required.
• Crystal violet can also be used in staining of plant chromosomes (Flemming triple stain), chromatin (Lieb’s amyloid stain) and nucleoli (Newton’s crystal violet-iodine technique).
• Can also be used to stain cells for invasion and migration studies.
• Used as an external disinfectant to destroy cells.
• Dying of paper, wood, acrylic, inks, and silk.

Strengths and limitations

Strengths

Crystal violet is useful in assessing or detecting bacterial contamination in tissue samples.

It is also essential in assessing the cytotoxic/cytostatic effects on tumor cell lines as well as testing the different species of archaea.

Limitations

Crystal violet can lead to permanent pigmentation of the skin if it comes into contact with granulation tissue. On account of this, the dye should not be applied to ulcerative lesions of the face.

Crystal violet is not a strong sensitizer when used as an antiseptic agent.

Crystal violet may produce allergic contact dermatitis or necrosis in the intertriginous areas.

Its use for bacterial and pathogenic fungi treatment in animals such as Staphylococcus & Candida species has declined on account of reports on animal carcinogenicity.

Precautions

Crystal violet is toxic and should be handled with a lot of care.

When using crystal violet to stain cells, the examiner ought to pipette on the sides of the petri dish so as to reduce the chances of the cells detaching.

Several toxicology tests carried out in mice has shown dose-related carcinogenic properties on different organs. The reduced leuco moiety form induces hepatic, renal and lung tumor in mice. The FDA has classified crystal violet as unsafe for use in animal feed. The Federal Food, Drug, and Cosmetic Act prohibit its use in animal feed, and such feed is regarded as adulterated. Crystal violet is absorbed from water into the fish tissue and is then reduced into the leucocrystal violet (leuco) moiety.

A common side effect of crystal violet is the staining of skin and cloth; this can, however, be easily washed off by bleach and water.

Conclusion

• Crystal violet is a blue monochloride salt of crystal violet cation. This aniline-derived dye has various antimitotic and antifungal properties. It dissociates into positive and negative ions that can penetrate bacterial cells. Its photodynamic action is elicited via a free-radical mechanism. The positive ions of the dye interact with the negatively charged elements of the cell wall such as DNA, peptidoglycan, and lipopolysaccharide, thus, retaining the purple color of the dye.
• Crystal violet is also a mitotic agent and mutagen. It has been used for topical treatment in creams against fungal and bacterial infections. Its been used against some pathogenic fungi such as Candida species and some gram-positive bacteria, e.g. Staphylococcus species.
• Its use for tropical treatment has declined on account of reported animal carcinogenicity.
• Crystal violet also induces permeability of eukaryotic or prokaryotic membranes by dissipating the action potential of such membranes. This leads to inhibition of respiration in such cells and subsequent cell death.