Materials

• Suspension culture of cells
  
• Sterile transfer pipettes
  
• Stock 0.2% (w/v) Trypan blue
  
• Hemacytometer and microscope

Procedure

1. Gently swirl a suspension culture to distribute the cells evenly. Aseptically remove a small sample (0.1 ml) of cells from the cultures. Place the sample in a separate test tube (it need not be sterile).

2. Dilute 4 parts of stock Trypan Blue with 1 part of 5X saline and add 0.1 ml of the diluted dye to your sample. Mix gently.

3. Set up a hemocytometer and cover slip. Immediately place a drop of the stain/culture combination on the hemocytometer (remember to use both sides of the hemocytometer) and wait one minute.

4. Observe the cells with low power microscopy. Count the total number of cells, and the number of stained cells.

5. Compute the concentration of viable cells per ml. of culture.

Notes

Trypan Blue is a stain that is actively extruded from viable cells, but which readily enters and stains dead cells. Therefore, the cells which are blue are dead. The difference between the total number of cells and the number of dead cells would be the number of viable cells in a given aliquot of your culture. Trypan Blue actually significantly overestimates the number of viable cells, but is sufficient for purposes of this lab.

Approximately 30% of the cells measured as viable with Trypan Blue will not be able to continue growth beyond a 24 hour period.