Abstract
The effects of various physical factors on the growth of protoplasts of Entomophthora egressa were studied. In order to obtain growth in cultures shaken at 50 and 100 rpm using Grace's liquid medium, a fetal-calfserum (FCS) supplement of 28 ml per liter (2.7%) was required. Concentrations of FCS up to 50 ml per liter failed to support growth at 150 rpm. Analysis of Grace's medium indicated that it had an osmolality of 350 mOsM. This osmolality, except where designated, was maintained at the start of experiments in all media. The optimum sucrose concentration for protoplast stability (retention of spindle shape) was 350 mM (403 mOsM); for centrifugation, a 330-mM-sucrose-10-mM-MES buffer (pH 6.2) was optimum as based upon protoplast retention of shape and subsequent viability. The optimum temperature for growth (protoplast yield) was between 17 and 21 C. Of the three buffers tested (MES, MOPS and TES), MES at 10 mM was found to provide the best balance between buffering capacity and toxicity. The protoplasts grew over the pH range 5.2-8.2. The optimum pH for growth was 6.2.
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