Box 1. Culture conditions

If there is a choice, use nonfermentable carbon sources such as glycerol or ethanol, rather than glucose or other sugars, to increase the yield of mitochondria and mtDNA. In the case of photosynthetic organisms, an appropriate light source is required. In addition, optimal aeration (enriched with CO₂ for algae) is often essential for high yield. In standing cultures (fragile species), the relative volume of liquid in the culture vessel should be small (<1/10) and the cover of the opening should allow sufficient air exchange (e.g., loosely attached alumina foil). When agitation of the culture is possible, volumes of up to 600 ml per 3-liter Erlenmeyer flasks can be used (e.g., on a rotary shaker at up to 300 r.p.m., depending on the robustness of cells). Alternatively, aeration with a pressurized sterile air supply that is connected to an aquarium stone is highly effective. Obviously, a dedicated fermenter may be used, although such equipment requires substantial care for sterilization and operation, and might be inefficient for fragile species that do not tolerate agitation of the culture (most flagellated protists and amoeba).