Mating and Tetrad Dissection in Chlamydomonas

Materials and Reagents

1. Culture tubes and inoculating loops
2. Single edge razor blades
3. Sterile (autoclaved) toothpicks and bacterial spreader
4. Micropore tape (3 M, catalog number: 1535-0)
5. Tin foil
6. Microscope slides and coverslips, 18 x 18 mm, No. 1.5 thickness (VRW, catalog number: 48366-205)
7. Chlamydomonas reinhardtii strains of opposite mating type
8. TAP-agar plates (TAP medium supplemented with 1.5% agar, stored at 4 °C)
9. Iodine solution (1% iodine, 2% potassium iodide, catalog number: 869053)
10. Tris acetate phosphate (TAP) medium (see Recipes)
    1. Tris base
    2. Glacial acetic acid
    3. Ammonium chloride (NH₄Cl)
    4. Magnesium sulfate heptahydrate (MgSO₄·7H₂O)
    5. Calcium chloride dihydrate (CaCl₂·2H₂O)
    6. Potassium phosphate dibasic trihydrate (K₂HPO₄∙3H₂O)
    7. Potassium phosphate monobasic (KH₂PO₄)
    8. Hutner’s trace elements
    9. Sodium hydroxide (NaOH, 1 M) and hydrochloric acid (HCl, 1 M) to titrate pH
11. Mating medium (TP-N, see Recipes)
    

Equipment

1. Compound microscope (5x or 10x objective lens) equipped with a microdissection needle for tetrad dissection (Note 2)
2. Compound microscope equipped with a 40x objective for observing the mating process (Phase contrast optics is preferable for observing flagella)
3. Stereo microscope for picking colonies
4. Incubator holding 21-25 °C with ~50 μmol m^-2 s^-1 white light with a 12 h/12 h light/dark diurnal cycle (A diurnal light cycle is preferable, although not necessary, for routine culturing of the algal cells)
5. Sterile hood 
6. Shaker
7. Autoclave
   

Procedure