Enhanced Growth of T-Strain Mycoplasmas with N -2-Hydroxyethylpiperazine- N′ -2-Ethanesulfonic Acid Buffer

The small size of T-strain mycoplasma colonies obtained on agar medium has been a drawback to the study of these organisms. The incorporation of 0.05 m N -2-hydroxyethylpiperazine- N′ -2-ethanesulfonic acid (HEPES), a new hydrogen ion buffer, into agar medium at p H 6.8 led to the production of colonies at least 60% greater in diameter than those obtained on media without HEPES at p H 6.5 or 6.0. In addition, the colonies often had the “fried-egg” appearance typical of “classical” large-colony-forming mycoplasmas. The addition of HEPES to liquid medium in the presence of 0.1% urea resulted in a slightly higher number of viable organisms and a corresponding increase in ammonia production. Rapid death of the mycoplasmas occurred on continued incubation of the liquid medium cultures even in the presence of HEPES. The larger colony size facilitated the study of hemadsorption and tissue-culture cell adsorption. Preliminary results of such tests in which these phenomena have been demonstrated are presented. The advantages and disadvantages of having larger colonies are discussed, and the terminology of T-strain mycoplasmas is considered in the light of the present findings.