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Culture of Corn Stunt Spiroplasma in a Simple Medium. C. H. Liao, Graduate Assistant, Department of Plant Biology, Cook College, Rutgers University - The State University of New Jersey, New Brunswick, NJ 08903, Present address of senior author: Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403; T. A. Chen, Professor, Department of Plant Biology, Cook College, Rutgers University - The State University of New Jersey, New Brunswick, NJ 08903. Phytopathology 67:802-807. Accepted for publication 9 December 1976. Copyright © 1977 The American Phytopathological Society, 3340 Pilot Knob Road, St. Paul, MN 55121. All rights reserved.. DOI: 10.1094/Phyto-67-802.

A simple medium (C-3G) containing 20% agamma horse serum, 1.5% PPLO (pleuropneumonialike organism) broth, 12% sucrose, in distilled water met all the nutritional and physical requirements for the in vitro growth of corn stunt spiroplasma. The growth characteristics of one isolate established in C-3G medium was studied after triple cloning. A sigmoid type of growth pattern was observed by directly counting the cell helix under the dark-field microscope. The doubling time of the helices in the exponential phase was calculated to be 20 hr at 29 C in nonshaken cultures. Since the organism lost motility and helicity immediately when the pH in the culture medium dropped to 5.4 or lower, this suggested that the relatively short stationary phase (1-2 days) and the rapid decrease rate in the last phase (7 × 10⁶ helices/hr) most likely resulted from low pH rather than from exhaustion of nutrients in senescing cultures. This suggestion was further supported by the observation that the growth yield could be enhanced by the addition of 0.06 M of HEPES (N’-2- hydroxyethyl piperazine - N-2-ethanesulfonic acid) buffer to strengthen the buffer activity in C-3G medium. The granulated and occasionally fried-egg shaped colonies that appeared in the interior of medium solidified with 0.8% Oxoid Ionagar were characterized by a relatively smaller size, and somewhat diffused edge.