NEW SOLID MEDIUM FOR ENHANCED GROWTH OF PASTEURELLA TULARENSIS

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Abstract
The purpose of this work was to develop a solid medium for more rapid growth of Pasteurella tularensis, especially from small inocula comparable to those generally encountered in clinical materials. After titration of various ingredients, separately and in combination, the optimal base was found to be: 2.6% tryptose broth (Difco) with thiamine, 0.5% cysteine-HCl, 0.2% sodium thioglycolate, 1.0% glucose, dissolved by mixing without heat, and adjusted to pH 7.2 + 0.03. To this base 1.0% agar (Difco) is added and dissolved by heating in flowing steam for about 5 min. prior to autoclaving at 121 C for 20 min. After sterilization and cooling, 5.0% defibrinated rabbit blood is added aseptically. Plates of the completed medium are incubated at 37 C for 24 hr. prior to use. Colonies of P. tularensis approximately 1.0 mm in diameter are obtained on this medium after 26 to 29 hr. incubation if conditions of high relative humidity (90 to 100% saturation) are maintained. The mechanisms involved in the growth enhancement obtained on this medium are under study. The role of thioglycolate appears to be that of keeping in solution the relatively high concentration of cysteine-HCl required. The specific methods of preparation described, the incubation of plates prior to use, and final incubation under conditions of high humidity all are important for optimal results. This developmental work has employed only pure cultures. Attempts are being made to develop a selective medium, using this new preparation as base, for the direct isolation of P. tularensis from a variety of clinical materials.