A simplified, defined medium for growth of Entomophaga aulicae protoplasts

Abstract

A defined medium, which allowed growth in both stationary and shaken cultures in the absence of fetal calf serum, was developed for protoplasts of the fungus Entomophaga aulicae. The protein "requirement" was obviated and growth was enhanced by the addition of hematin (0.5 μg/mL) and oleic acid (1.0 μg/mL). The utilization and production of ninhydrin-positive compounds was monitored at various stages of medium simplification. The protoplasts were autotrophic for all vitamins and organic acids. Fructose was not required for protoplast growth; however, it could function as a carbon source. The optimum nitrogen level was 635.08 mg N/L, and the optimum carbon level was 280 mg C/L under the conditions utilized. The protoplasts did not utilize either inorganic nitrogen (ammonium ion) or sulfur (sulfate ion) at the levels tested. No amino acid could function as a sole nitrogen source. Analysis of the amino acid composition of a general (> 14 000 molecular weight) protein extract gave no evidence of a skewed amino acid content. The final simplified medium included 8 amino acids (aspartic acid, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, and threonine) of the 21 in the original medium. This medium is useful in isolating vitamin auxotrophs for use as markers in virulence and in protoplast fusion studies, and in studies on the chemical nature of the limiting membrane and its outer surface.