Angeles, Calif.), and Mike Y. Kubota. Continuous in vitro cultivation of spherules of Coccidioides immitis. J. Bacteriol. 87:468-472. 1964.-Spherules of Coccidioides immitis were grown in vitro in continuous culture for more than 4 years. Cultures were grown in modified Converse medium in a modified Lubarsky and Plunkett culture tube incubated at 40 C under increased CO2 tension. Spherules that had transformed from hyphae were freed from mycelial fragments, and were allowed to settle to the bottom of the tube. The mycelium which normally degenerated at 40 C was removed repeatedly with a syringe and needle until the culture was almost pure spherules. For continuous culture, 10 to 12 ml of old medium were remnoved, and were replaced by fresh medium previously bubbled with a mixture of 20% carbon dioxide and 80% air.
Study of potential pathogenicity of microbial L forms was done by the localized Shwartzman reaction. Stable L forms of Proteus mirabilis served as skin preparation in rabbits for induction of Shwartzman reaction by subsequent intravenous injection of either P. mirabilis L forms or Escherichia coli endotoxin. The intensity of the reaction was positively correlated to numbers of L forms in the skin. L forms also served as the intravenous challenge. In vivo multiplication of L forms was not a prerequisite for the reaction, as it could be produced with nonviable, osmotically lysed L forms. The reaction produced with L forms in the skin was more intense than that produced with the parent bacterial form. These latter observations, coupled with the demonstration that L forms disappeared from the skin (lysed?) after 4 hr, in contrast to bacteria which were recoverable for 72 hr (duration of study), suggest release of endotoxin by L forms as a pathogenic mechanism. ,WAlthough the role of microbial L forms in the pathogenesis of disease is uncertain, evidence has accumulated which suggests that L forms may play such a role. (Various terms are used to describe bacteria with all or part of the cell wall missing. These terms include L forms, L colonies, L-phase growth, bacterial variants, protoplasts, and spheroplasts. For purposes of this report, the terms are considered interchangeable. In literature citations the authors' terminology is used.
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