Starving Tetrahymena

In order to conjugate, Tetrahymena cells must satisfy the following requirements: a) they must be starved for at least one required nutrient; b) they must be of different mating type; and c) they must have reached a sufficient level of sexual maturity. 

Day 2 Materials Edit
 * Tetrahymena cultures (early log-phase)
 * Starvation Buffer (10 mM Tris-Cl pH7.5)

Measuring Cell Culture Density

 * 1) Measure the optical density (OD540) of the cultures. Cells are ready to be harvested when OD540 = 0.3 (corresponds to 2.5×10^5 cells/mL).

Starvation

 * 1) Determine the appropriate volume of cell culture necessary to yield OD = 0.3 and place this volume in 50 mL conical tube(s).
 * 2) Centrifuge at 1100g (2250 rpm in Beckman-Coulter centrifuge in the hall) for 3 minutes, and decant the supernatant.
 * 3) Resuspend cells in 100 mL of Starvation Buffer. Wash 2X.
 * 4) Resuspend cells in appropriate volume of Starvation Buffer and place cells for 12-24 hours in a 30 C platform shaker. Shaking is not essential for starving cells.