Trypan Blue | Properties, Uses and Side effects

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What is Trypan Blue

Trypan blue is a type of acid azo dye. It is an organosulfonate salt that is used as a crucial stain in biosciences to selectively colour dead tissues or cells blue. Tissues with their cell membranes and cells that are alive are not affected by Trypan blue. Because cells are exceedingly discriminating in the substances that pass through the membrane, TB is not absorbed in a healthy cell; yet, it passes past the membrane in a dead cell. As a result, dead cells have a characteristic blue color under a microscope. Because living cells are not stained, this staining method is also known as a dye exclusion method.

It is also used as a direct dye for cotton textiles and as a fluorochrome.

Note for the reader – In a few instances, this article refers to Trypan Blue as TB.


  • Trypan blue is a bluish-grey to dark blue powder while its aqueous solution is deep blue with a violet tinge.
  • The molecular formula for Trypan blue is C34H28N6O14S4 and its molar mass is 872.88 g/mol.
  • Melting Point: > 300 °C (572 °F; 573 K)
  • Solubility: <0.1 mg/mL in water, 20 mg/mL in methyl Cellosolve (2-Methoxyethanol), and 0.6 mg/mL in ethanol.
  • It is made from toluidine, which is one of many isomeric bases that is taken from toluene, C14H16N2.
  • Trypan blue gets its name from its ability to destroy trypanosomes, the parasites that cause sleeping sickness.
  • Azidine blue 3B, Benzamine blue 3B, Benzo Blue bB, Chlorazol blue 3B, Diamine blue 3B, Dianil blue H3G, Direct Blue 14, Niagara blue 3B are some of the other names for TB.
  • The extinction coefficient of Trypan blue in methanol is 6⋅104 M−1 cm−1 at 607 nm.
  • In the year 1904, a German scientist by the name of Paul Ehrlich made the TB and Trypan red for the very first time in history.
  • When heated to decomposition, it emits very toxic fumes of nitric oxides, sodium oxide, and sulfur oxides.

Chemical Structure of Trypan Blue

Uses of Trypan Blue

Uses of Trypan Blue

Cell Counting

Trypan blue is used as one of many exclusion stains to measure the number of viable cells in a suspension, exploiting the principle that living cell membranes will resist permeation as they possess intact cell membranes. It stains dead cells by permeating their compromised membranes and binding to intracellular proteins which results in a dark blue appearance. Cell suspensions assayed with trypan blue will ideally display both cells with clear cytoplasm (live) and blue cytoplasm (dead). This facilitates simple cell counting using optical microscopy.

Thus, Trypan blue exclusion assay is extensively used in microscopy (for cell counting) and tissue viability testing in laboratory animals. However, the approach is unable to discriminate necrotic from apoptotic cells.

Used in Ophthalmology

Trypan blue is a dye used as a visualizing aid to stain the epiretinal membranes during ophthalmic surgical vitrectomy procedures, thereby facilitating the removal of the tissue. It is used in ophthalmic cataract surgery to stain the anterior capsule in the presence of a mature cataract. This is done to help visualize the continuous curvilinear capsulorhexis before it is created.

Antiparasitic agent

TB is the antiprotozoal medicine that was originally in use in 1909 to do the treatment of Babesia infection and is most widely used to do the treatment of Babesia canis even today. Other commonly used medications, such as imidocarb, have resulted from the complex chemical structure being gradually simplified. Trypan blue is given intravenously at a dose of 10 mg/kg as a 1% solution. Babesia is removed from the blood within 24–48 hours in dogs with uncomplicated cases, correlating to visible indications of recovery.

In Vitreoretinal surgery

Trypan blue is an important adjunct in vitreoretinal surgery that increases the effectiveness and safety of membrane identification and removal by colouring the internal limiting membrane, epiretinal membranes, and posterior hyaloid.

Quantitative analysis of morphological changes in yeast cells

The structure of the yeast cell wall is dynamic, and it can adapt to different physiological states or environmental situations. For inspecting morphological changes, selective staining with fluorescent dyes is a helpful technique. Staining of Saccharomyces cerevisiae cell wall with trypan blue was done. It facilitated cell wall analysis i.e., quantification of cell size and cell wall volume, by confocal and super-resolution microscopy. This allowed for the measurement of morphological changes during anaerobic growth and in the presence of chemicals.

Rapid detection of Cerebrospinal fluid sterility

According to current research, trypan blue staining can distinguish between live and dead Cryptococcus yeasts. We expected that using TB staining in conjunction with haemocytometer counting would allow us to quickly estimate the quantitative culture count and detect CSF sterility. 194 CSF samples were examined from 96 HIV-infected volunteers with cryptococcal meningitis in Kampala, Uganda. CSF cryptococcal antigen was used to diagnose cryptococcal meningitis (CRAG).

Side effects

Trypan blue is the staining selective tissue solution that has been used to help in ophthalmic surgery by coloring the anterior capsule of the lens. It commonly causes the following adverse effects:

  • Blue discoloration of the eye for a short period of time
  • Staining of implanted lenses
  • Carcinogenesis, mutagenesis, and fertility impairment

In rats, TB causes cancer. Wister/Lewis rats developed lymphomas after receiving 52 weeks of weekly subcutaneous injections of 1% TB at a dosage of 50 mg/kg (dose total estimated 1,250,000-fold the maximum suggested human dose of 0.06 mg per every injection in a given 60 kg adult, keeping this in mind the total absorption).


During the first few weeks after receiving this medication, your doctor will want to check on your progress at regular visits.

Some of this medication may linger in your eye for several days. It may cause your eyes or tears to seem blue. If you have any concerns, consult your doctor.

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Q. How does trypan blue work?

Trypan blue is a diazo dye that is extensively used as a vital stain to color dead tissues or cells blue preferentially. It has been barred from the intact cell membrane of live cells because of its negatively charged strong composition. It also can cross the membrane of the dead cell with membrane integrity reduced. Once within the cell, TB binds to intracellular proteins, turning the cells blue. In fluorescence microscopy and flow cytometry, TB is frequently used to test vitality.

Q. Is trypan blue the same as methylene blue?

Because trypan blue is blocked by live membranes, it colors dead cells blue. Methylene blue is a universal cell stain that should stain almost everything. To know more, you may refer here.

Q. Is trypan blue toxic to humans?

Trypan is found to be toxic to humans due to its carcinogenic and mutagenic properties.

Q. What does Trypan blue stain?

Trypan Blue is basically cell membrane impermeable and therefore only enters cells with compromised membranes. In a dead cell, trypan blue passes through the porous cell membrane and enters the cytoplasm and binds to intracellular proteins thereby rendering the cells a bluish colour. Thus, it is used to quantify live cells (cell viability measurement). The trypan blue exclusion assay allows for direct identification and enumeration of live (unstained) and dead (blue) cells in a given population.


The information provided here is based on general knowledge, articles, research publications etc. and we do not claim the authenticity of any of the information provided above. We do not claim or suggest/advise any medical, therapeutic, health or nutritional benefits of Trypan blue. We do not supply or promote our Trypan blue product for the applications which are covered by valid patents and which are not approved by the FDA.

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Macsen Labs is a manufacturer and supplier of high-quality Trypan blue.