Fluorescence Microscopy of Tetrahymena

This technique works for vegetative as well as mated Tetrahymena.

Day 1 Materials

 * 1×SPP medium or NEFF medium
 * Tetrahymena cultures

Method

 * 1) Start 1 mL cultures of cells by adding appropriate antibiotics to growth media in a 24-well plate.
 * 2) Add 10-50 μL (inoculum depends on density of cells in culture) of Tetrahymena cultures to the medium and place at 30 °C for 1-2 days to allow cells to reach mid to log-phase. If you are planning to mate cells, then starve cells for 12-16 hours in 10 mM Tris-Cl and then mix together to induce conjugation.

Day 2 Materials

 * Mid to log-phase cultures or mated Tetrahymena
 * Formaldehyde 37.5% or PFA (Paraformaldehyde 4%)
 * 10×PBS buffer
 * DAPI (10 mg/mL)

Method

 * 1) Pipette out 200 µL of cells into microfuge tubes. Fix cells with 5 µL of FA or 200 µL of 4% PFA and allow fixation to proceed for 30 minutes at room temperature.  Using PFA is recommended as it is a better fixative agent than formaldehyde as cells retain their rounded shape in PFA.  PFA is added to 1 volume of cells. So if 400 µL of cells are fixed, add an equal amount of PFA (400 µL) to the cell culture. 
 * 2) Spin cells in a bench-top microcentrifuge for 1 minute. Carefully aspirate or pipette out supernatant.
 * 3) Wash with 500 µL of 10×PBS buffer and allow to sit at room temperature for 10 minutes. Spin cells for 1 minute. Pipette out the supernatant.
 * 4) Add 1 µL of DAPI to the cell pellet and keep at RT for 10 minutes. During this time, gently tap the bottom of the tube to allow the DAPI to evenly penetrate the cells.
 * 5) Pipette out 5-10 µL of cell pellet onto a slide.
 * 6) Affix coverslip and observe under the microscope.   If the slides will be viewed later and need to be stored for later use, add 5 µL of Vectashield mounting medium to the cells on the slide. Then place the coverslip on top and seal the edges of the coverslip by gently streaking nail polish or any other sealant.