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. (from:Wikipedia) 

Viable Cell Counts Using Trypan Blue (Invitrogen) 

Trypan Blue is a vital dye. The reactivity of trypan blue is based on the fact that the chromopore is negatively charged and does not interact with the cell unless the membrane is damaged. Therefore, all the cells which exclude the dye are viable. 

PROCEDURE: Trypan Blue Staining of Cells

1. Place 0.5 ml of a suitable cell suspension (dilute cells in complete medium without serum to an approximate concentration of 1 x 10^5 to 2 x 10^5 cells per ml) in a screw cap test tube. 

2. Add 0.1 ml of 0.4% Trypan Blue Stain. Mix thoroughly. 

3. Allow to stand 5 min at 15 to 30°C (room temperature). 

4. Fill a hemocytometer as for cell counting. 

5. Under a microscope, observe if non-viable are stained and viable cells excluded the stain. 

 Reference: 1. Freshney, R. (1987) Culture of Animal Cells: A Manual of Basic Technique, p. 117, Alan R. Liss, Inc., New York.