Trypan blue

  Trypan blue is a vital stain used to selectively colour dead tissues or cells blue. It is a diazo dye.

Live cells or tissues with intact cell membranes are not coloured. Since cells are very selective in the compounds that pass through the membrane, in a viable cell Trypan blue is not absorbed; however, it traverses the membrane in a dead cell. Hence, dead cells are shown as a distinctive blue colour under a microscope. Since live cells are excluded from staining, this staining method is also described as a Dye Exclusion Method.

Additional recommended knowledge

Recognize and detect the effects of electrostatic charges on your balance

Essential Laboratory Skills Guide

What is the Sensitivity of my Balance?

Background & chemistry

Trypan blue is derived from toluidine, that is, any of several isomeric bases, C₁₄H₁₆N₂, derived from toluene.

Its CAS number is [72-57-1] and its SMILES structure is CC1=CC(C3=CC(C) =C(N=NC5=C(S(=O) (O)=O)C=C(C=C (S(=O)(O)=O)C=C6N) C6=C5O)C=C3)=C C=C1N=NC2=C(O) C(C(N)=CC(S(=O) (O)=O)=C4)=C4 C=C2S(=O)(O)=O.

Trypan blue is so-called because it can kill trypanosomes, the parasites that cause sleeping sickness. The dye is also known as diamine blue and Niagara blue.

Trypan red and Trypan blue was first synthesized by the German scientist Paul Ehrlich in 1904.

Uses of trypan blue

  Trypan blue is commonly used in microscopy (for cell counting) and in laboratory mice for assessment of tissue viability. The method cannot distinguish between necrotic and apoptotic cells.

It may also be used to observe fungal hyphae and Stramenopiles.

Protocol for vital staining

Place a suitable volume of a cell suspension (20-200 μL) in appropriate tube add an equal volume of 0.4% Trypan blue and gently mix, let stand for 5 minutes at room temperature. Place 10 μl of stained cells in a hemocytometer and count the number of viable (unstained) and dead (stained) cells. Calculate the average number of unstained cells in each quadrant, and multiply by 2*10⁴ to find cells/ml. The percentage of viable cells is the number of viable cells divided by the number of dead and viable cells.

References

• Chapter "Detection of Caspase Activation Combined with Other Probes of Apoptosis", Eurekah Bioscience Collection, NCBI bookshelf
• Protocol for use of the dye from Northwestern University (PDF)