Axenic cultivation of Naegleria gruberi : Requirement for methionine

A simplified axenic medium for Naegleria gruberi strain NEG-M contains -methionine, dextrose, yeast extract, a macromolecular fraction of fetal calf serum, and phosphate buffer. Amoebae cultured in suspension in this medium grow with doubling times of 8–10 h (at 32 °C) to yield 2–4 × 10⁶ cells/ml. Amoebae from growing or early stationary phase cultures, transferred to nonnutrient buffer, differentiate synchronously into flagellates. Differentiation occurs reproducibly 80 min after initiation (time for 50% flagellates at 25 °C) if amoebae are taken from a culture maintained at pH 6.6.