Isolating Tetrahymena in Drop Medium

This method can be used to isolate single cells or mated pairs.

Materials
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 * Vegetative or starving cultures of cells
 * SPP or NEFF medium
 * Penicillin G & Streptomycin Sulfate
 * Fungizome
 * Selective antibiotics (as required)
 * Petridishes
 * Drop-maker
 * Acetone
 * Needle-tube apparatus

Making drops of medium

 * 1) Place drop-maker into dish containing acetone and sterilize by placing over a flame. Ensure there is no residual acetone on the drop-maker and then wipe first onto a paper towel to remove residual medium that may have dried and settled between the pillars.  Gently place the drop-maker in acetone; do NOT soak it with acetone as it is likely to flare when heated over the flame. Acetone is very volatile so exercise safety pr Needle_Tube_Apparatus.pngotocol and wear personal protective equipment (PPE).
 * 2) Sterilize the drop-maker again using acetone and once it has cooled (wait 5 seconds), place the drop-maker in petridish containing medium (and required antibiotics).
 * 3) Gently place drop-maker in a new petridish. Tilt the head of the drop-maker sideways (from one side to another) a few times and lift the drop-maker at an angle.   Tilting ensures that there is no  vacuum action pulling the bottom of the Petri-dish when lifting the drop-maker out of the petridish. 

Placing cells in drops

 * 1) While looking under the microscope, place the needle end of the tube into the cell cultu    re to draw in cells.
 * 2) Then move the needle to the drop where the cell(s) need to be deposited and gently blow into the other end of the tube, the mouthpiece, to push out the cell(s) into a drop. Ensure that only pai  r or cell is deposited per drop.    If any extra pair(s) or single cell(s) escape into a drop, draw them in    again to the needle  as quickly as possible. If you wait too long, then cells will swim in outward regions in the drop making it difficult to draw them out.Isolating_Cells_in_Drop_Medium.png
 * 3) Incubate petridish containing drops at 30 °C.