Culture Establishment – Spore Sowing and Density Calculations

Spore Sowing and Density Calculations

Through painstaking empirical analyses, we determined that there are about 1250 wild type C-Fern spores per mg, i.e. each spore weighs about 0.8 µg. One mg of C-Fern spores will inoculate 3.5 small Petri dishes with about 0.24 mg or 300+ spores per dish. For a typical 60 x 15 mm (about 22.5 cm²) Petri dish, this inoculation rate is about 15 spores per cm² of the agar surface. At substantially below this density, sex expression stabilizes and gametophyte growth is relatively unaffected. If the density is important, the actual area of Petri dishes should be empirically determined since actual dimensions can be substantially different for nominal dimensions.

Since known quantities of water are used to suspend spores for sowing and this suspension is delivered by drops from a Pasteur pipet, it is important to determine the drop volume from your given brand of glass or plastic Pasteur pipet. We use a plastic transfer pipet which, for a 1 ml inoculation volume will sow about 8 dishes at 3 drops per dish. If using other pipets, drop volume must be measured and factored in to achieve controlled sowings.

For liquid culture, spores can be sown into media without agar at a rate of 3 to 10 mg per 100 mL. Cultures should be placed on a rotary shaker at 50-100 rpm, depending on the volume of the solution. If not shaken, cultures will rapidly fertilize when gametophytes become sexually mature.