METHYLENE BLUE: CHARACTERISTICS, PREPARATION, USES - CHEMISTRY - 2025

The methylene blue is a dye of organic nature with multiple functions. It is also known by the name of methylthionine chloride. Its molecular formula is C ₁₆ H ₁₈ ClN ₃ S. It was synthesized in 1876 to dye textile garments, however it did not take long for scientists of the time to discover its great usefulness in the field of medicine, especially for dyeing microscopic preparations.

This use is still preserved, as it is currently used in simple staining techniques for the diagnosis of certain infectious diseases, such as pityriasis versicolor, erythrasma or meningitis caused by Haemophilus influenzae.

Methylene blue in solution, Aspergillus sp and onion epidermis stained with methylene blue. Source: Public Domain Picture / Flickr / Viascos /

Its use as a contrast dye is also frequent, as for example in the Ziehl Neelsen staining technique, specific for the diagnosis of acid-alcohol resistant microorganisms. However, this has not been its only use, as methylene blue was later exploited for its antiseptic and healing power.

In addition, various investigations led to the use of methylene blue intravenously, as it was discovered that it could be used in this way, without toxic effects. In this sense, it was used to treat certain tropical diseases, such as malaria, as well as other diseases such as methemoglobinemia.

On the other hand, in addition to the therapeutic uses already mentioned, other very interesting properties have been discovered. Such is the case of its use as a vasopressor in patients with refractory vasoplegia or any other clinical condition that causes arteriolar vasodilation, hypotension and cardiac depression.

It has also been highlighted as a marker to locate the origin of digestive hemorrhages in the small intestine.

characteristics

This compound is popularly called methylene blue, but its chemical name is 3,7-bis (dimethylamino) -phenazationium chloride. Some bibliographic sources also call it tetramethylthionine chloride or methylthionine chloride. Its chemical formula is C ₁₆ H ₁₈ ClN ₃ S.

It has a molecular weight of 319.85 g / mol and a melting point of 100 ° C. In solution, it has a density of 1,757 g / cm³ and is completely odorless.

In its solid state, it appears as very fine dark green crystals with a certain bronze-colored luster, but in solution it takes on a strong blue hue. As solvent you can use water, chloroform or alcohol.

Preparation

It can be prepared in whatever concentration is needed. (See video 1 below)

For Loeffler's methylene blue technique (single staining)

- 0.3 g of methylene blue.

- 30 ml of 95% ethyl alcohol.

- 100 ml of distilled water.

- Dissolve and store in amber bottle.

It should be noted that previously, KOH was added to this technique in order to alkalize the colorant, before being used. Today this step is unnecessary, since the impurities that acidified the colorant were eliminated.

For the Ziehl Neelsen technique

- 0.5 g of methylene blue.

- 0.5 ml of glacial acetic acid.

- 100 ml of distilled water.

- Dissolve and store in amber bottle.

Video 1

Applications

- Diagnostic use

Microbiology

Methylene blue is widely used in the microbiological area, with different uses such as those mentioned below:

It is the contrast dye in the Ziehl Neelsen staining technique (specific stain for staining acid-fast bacteria). Ex: Mycobacterium tuberculosis and Mycobacterium leprae. Anything that is not acid-fast is discolored by acid alcohol and counterstains by methylene blue.

It is also used as the sole stain (simple staining) for the observation of bacteria and fungi.

- Bacteria

Methylene blue is useful used in the diagnosis of erythrasma. This disease is a superficial dermatitis of bacterial origin, caused by the bacteria Corynebacterium minutissimun. Bacteria stained with methylene blue appear as fine branching filaments or short isolated blue bacilli.

On the other hand, the metachromatic granules of the species Corynebacterium diphtheriae stain strongly blue with this dye.

Because Haemophilus influenzae stains slightly with the Gram stain, methylene blue is sometimes used to stain the cerebrospinal fluid sediment for this bacteria. With methylene blue they can be seen as blue-black bacilli.

- Mushrooms

In mycology it is used for staining samples taken with adhesive tape, where the presence of the causative agent of pityriasis versicolor (Malassezia furfur) is suspected.

Specimens stained with methylene blue reveal characteristic structures of the fungus, such as typically curved, clustered or isolated hyphae with abundant spherical cells in clusters.

Other mycological preparations can be stained with methylene blue.

- Therapeutic uses

Antiseptic

For years methylene blue has been used to clean wounds, due to its antiseptic and healing properties. In addition, onychomycosis can be treated with methylene blue, managing to eliminate the fungus.

On the other hand, its use extends to other species. For example, it is used in aquaculture, as this dye can treat freshwater ornamental fish, infected with the ciliated protozoan Ichthyophthirius multifiliis.

Methemoglobinemia

Methemoglobinemia is characterized by increased methemoglobin in the blood. This is unable to transport oxygen, therefore, the patient may present cyanosis.

Paccor et al. In 2018, reported a pediatric case of methemoglobinemia (35%), due to accidental use of dapsone. The child presented cyanosis in both extremities and on the face.

He was treated with 1% intravenous methylene blue, obtaining a favorable recovery. This treatment is recommended only when methemoglobinemia is greater than 30%.

It is important to determine that the cyanosis is caused by the high concentration of methemoglobinemia, since methylene blue is not effective if the cyanosis has another etiology.

Vasopressor

One of the most interesting properties of methylene blue is that it has been found to have a vasopressor effect, as it paralyzes the synthesis of nitric oxide by inhibiting the action of guanylate cyclase.

Nitric oxide is known to increase in patients with septic shock, as well as in other clinical entities, such as: anaphylactic shock, cardiopulmonary surgery, sepsis, among others. Endogenous nitric oxide causes arteriolar vasodilation, hypotension, and cardiac depression. Sometimes the use of classic vasopressors is not enough to stabilize the patient.

In this sense, Mora-Ordoñez et al. In 2006, described a case of a patient who presented vasorefractory vasoplegia after cardiac surgery, without response to the usual treatment. For this reason, the medical group decided to treat him with a single, low dose (2 mg / kg) of methylene blue, obtaining satisfactory results.

Methylene blue was administered parenterally, diluted in 5% glucose solution, in a time of 60 minutes.

Likewise, Carrillo-Esper et al. In 2010, treated a patient with refractory septic shock at the same dose, with an excellent response. Both studies widely recommend methylene blue infusion as a therapeutic option in these cases.

Detection of gastrointestinal bleeding

Methylene blue has been used as a marker to locate vascular lesions in the small intestine.

- Redox indicator

Methylene blue has an oxide reducing property. That is, in the presence of NaOH (alkaline pH) plus glucose, methylene blue is reduced to leucometylene blue (colorless). If oxygen is added to the reaction, the leucomethylene blue is oxidized, turning the solution blue. The reaction is completely reversible. (See video 2).

Due to these properties, its use is not only limited to the health sector, being used in the food and cosmetic industries, among others.

Video 2

Removal of methylene blue in wastewater

As already mentioned, methylene blue is one of the most widely used colorants in various fields. Among them is the textile, paper, pharmaceutical, food industry or in diagnostic, teaching and research laboratories, among others.

Therefore, the waste from these industries and laboratories contains a high amount of colorants, among which is methylene blue. These colorants are harmful to the environment.

That is why, many researchers in order to decontaminate wastewater, have proposed a variety of waste agricultural materials and therefore low cost, with great absorbent capacity. The proposed materials are coconut, rice or cassava shells, among others.

References

1. Mora-Ordóñez J, Sánchez-Llorente F, Galeas-López J, Hernández Sierra B, Prieto-Palomino M, Vera-Almazán A. Use of methylene blue in the treatment of vasoplegic syndrome in the postoperative period of cardiac surgery. Med. Intensive, 2006; 30 (6): 293-296. Available in: scielo.
2. Albis A, López A, Romero M. Removal of methylene blue from aqueous solutions using cassava shell (Manihot esculenta) modified with phosphoric acid. Prospect. 2017, 15 (2): 60-73
3. Paccor A, Matsuda M, Capurso C, Rizzo E, Lacorra M. Methemoglobinemia due to Dapsone: a pediatric case report. Arch Argent Pediatr, 2018; 116 (4): e612-e615.
4. "Methylene Blue" Wikipedia, The Free Encyclopedia. 10 Apr 2019, 21:17 UTC. 17 Aug 2019, 23:23. Wikipedia.org
5. Manual of primary care in poisonings. Volume II, Special Part. Ministry of Health of the Nation. Argentina republic. 2002. Available at: msal.gob.ar