Starving and Mating Tetrahymena

''Mating (conjugation) in Tetrahymena is done by starving cells of different mating types and then mixing them together. This protocol outlines the procedure for mating large cultures of Tetrahymena and can be applied to smaller cultures just as well.''

Day 1 Materials

 * Tetrahymena stock cultures


 * SPP or NEFF medium

Method

 * 1) Inoculate NEFF or SPP medium with stock cultures of different mating types.
 * 2) Incubate overnight at 30 ° C in a platform shaking incubator at 110 rpm (generally for 24 hours). Allow cells to grow to early log-phase.

Day 2 Materials

 * 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 1100× g (2,250 rpm in Beckman-Coulter centrifuge in the hall) for 3 minutes, and decant the supernatant.
 * 3) Resuspend cells in 100 mL of sterile Starvation Buffer (10 mM Tris, pH 7.5). 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.

Day 3 Materials

 * Tetrahymena cultures (starved)

Conjugation

 * 1) Mix equal number of cells in a petridish or flask (depending on the volume of the mating) and place at 30 ° C in a moisture chamber. Do not shake cells as this inhibits mating.
 * 2) Check for mating efficiency (using a hemacytometer) at 2-3 hours post-mixing. If mating efficiency is >80%, then proceed with experiment.