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

The use of a buffer system based on N-[2-hydroxyethyl]piperazine-N'-[2-etha nesulfonic acid] (HEPES), in conjunction with standard Gourlay's culture me dium was investigated for the growth and maintenance of Mycoplasma mycoides subsp. mycoides SC vaccine strain T(1)44. When the initial pH of the cultu re medium was adjusted to 8.0, 0.075 M HEPES-NaOH was found to be sufficien t 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 unbuf fered 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 ext ended culture survival was observed at 37 degreesC. The titre remained abov e 10(10) CCU ml(-1) for 4 days, and above 10(8) CCU ml(-1) in excess of I m onth. After 4 month's storage at 37 degreesC the titre had fallen to 5 x 10 (4) CCU ml(-1). In contrast, no viable bacteria could be detected in standa rd unbuffered medium 3 days after the onset of stationary phase, at which p oint the pH had dropped to 5.4. No significant difference in growth rate be tween the two media was observed. Adoption of a HEPES NaOH buffer system by African vaccine manufacturers should require minimal changes to current fo rmulations and procedures, and should enhance both the final titre and ther mostability of freeze-dried and liquid broth vaccines against contagious bo vine pleuropneumonia (CBPP). (C) 2001 Federation of European Microbiologica l Societies. Published by Elsevier Science B.V. All rights reserved.