Effect of HEPES buffer systems upon the pH, growth and survival of Mycoplasma mycoides subsp. mycoides small colony (MmmSC) vaccine cultures

Abstract The use of a buffer system based on N -[2-hydroxyethyl]piperazine- N ′-[2-ethanesulfonic acid] (HEPES), in conjunction with standard Gourlay’s culture medium was investigated for the growth and maintenance of Mycoplasma mycoides subsp. mycoides SC vaccine strain T 1 44. When the initial pH of the culture medium was adjusted to 8.0, 0.075 M HEPES–NaOH was found to be sufficient to prevent the pH falling below 7.1 at any stage during the growth cycle, even in the presence of 0.5% glucose. Compared to growth in standard unbuffered Gourlay’s medium, the final culture titre was found to be one log 10 higher, at 10 11 colour changing units (CCU) per ml, and considerably extended culture survival was observed at 37°C. The titre remained above 10 10 CCU ml −1 for 4 days, and above 10 8 CCU ml −1 in excess of 1 month. After 4 month’s storage at 37°C the titre had fallen to 5×10 4 CCU ml −1 . In contrast, no viable bacteria could be detected in standard unbuffered medium 3 days after the onset of stationary phase, at which point the pH had dropped to 5.4. No significant difference in growth rate between the two media was observed. Adoption of a HEPES–NaOH buffer system by African vaccine manufacturers should require minimal changes to current formulations and procedures, and should enhance both the final titre and thermostability of freeze-dried and liquid broth vaccines against contagious bovine pleuropneumonia (CBPP).