THE EFFECT OF ROTATION AERATION ON THE GROWTH OF MYCOBACTERIUM TUBERCULOSIS

Abstract

It has been confirmed by the reported experiment that 7H9 Tween- or Oleic Acid-Albumin medium permits early growth from small inocula of tubercle bacilli.It is shown that this type medium is the most preferable medium as evidenced by a 13-13.5 hour generation time (Table 8). When actively growing cultures of tubercle bacilli were transferred in dispersed fashion in this medium and incubated in a stationary manner the culture was characterized by arithmetic linear growth, independent of the age of the seed culture, or amount of inoculum. It was ob-served that if 7H9 cultures of mammalian tubercle bacilli were aerated by con-tinuous rotation aeration the growth is much greater than is observed in stationary incubated cultures. Yields of bacilli were increased many fold over those obtained in stationary cultures (two-fold greater at 4 days, three to four-fold at 7 days). It was found that with rotation aerated cultures, the average values of generation time calculated from experiments using four different inocula (Table 3) was con-siderably shorter than that of stationary cultures.
In the reported studies, the generation time of mammalian tubercle bacilli cal-culated by the formula of Monod(6), using logarithmic plotting with small inocula, is approximately 20.0 to 24.2 hours during exponential growth of early stage (Tables 4 and 5). Under these conditions of lag phase growth, at least, the stationary culture and rotation aerated culture are approximately identical. When the Values of growth over a long incubation period were taken (Tables 4 and 5), the genera-tion time calculated from rotation aerated cultures was quite short as compared with that obtained from stationary incubation over the same period.
It was shown that the period of logarithmic growth varies and is dependent upon the available supply of oxygen: a good oxygen supply permits continuousrapid growth of M. tuberculosis strains. A 7H-9 Tween Albumin culture of tubercle bacilli (H37Rv-strain) inoculated in a shallow layer of medium such as 30 ml volume medium contained in a 250ml Erlen-Meyer flask and agitated gently by hand once each day, gave a generation time of approximately 17.2 hours during early stage of growth (24-64 hours). This was somewhat shorter than that observed in station-ary tube cultures or in rotation aerated cultures: with stationary cultures it was 22.2 hours and with rotation aerated cultures it was 20.0 hours (Table 6). In the present experiments described the effect on the growth rate, when cultures of tubercle bacilli were rotation aerated, indicated that significant differences in growth rate enhancement was greatest with M. tuberculosis var. bovis (Ravenel-strain), decreasing in the order of M. tuberculosis virulent H37Rv-strain and then H37Ra-avirulent strain (Table 10). The difference in degree of stimulation by aeration on the growth rate of these strains appears to be explainable by a difference in the rate of meta-bolic utilization of oxygen in the Tween Albumin medium employed. In this study, investigations have been devoted to the determination of the optical concentrations of glucose and glycerol employed alone, and in various combinations in aerated cultures. The addition of glycerol, as low as 0.2 per cent final concentration, showed a marked initial stimulatory effect on the growth of the M. tuberculosis strains tested. Higher concentrations of glucose and glycerol (0.5 per cent) are inhibitory in stationary cultures, stimulatory for aerated cultures. It is apparent that considerable increases in oxygen uptake depend on the concentration of carbon sources.
Better growth in the initial stage of growth was obtained in cultures with glucose alone than with the same concentration glycerol alone. It was clearly shown that the oxidative breakdown of glucose in the initial stages of growth was a far more efficient means of obtaining energy than when glycerol was used.