A Method for the Determination of the Rate of Growth of Tubercle Bacilli by the Use of Small Inocula.

Older observations on the nutritive requirements and metabolism of virulent tubercle bacilli do not have quantitative significance because of the lack of accurate methods for the estimation of the rate of growth. The recognition in recent years that these organism grow just as well when submerged in liquid media as on the surface of liquid or solid media has eliminated some of the difficulties inherent in measuring the amount of growth of tubercle bacilli. This type of culture eliminates many technical problems and permits the use of uniform homogeneous inocula. Employing subsurface growth, two methods have been described for the quantitative estimation of the amount and rate of growth of virulent tubercle bacilli. The first involves the use of micro-Kjeldahl nitrogen deterninations (Youmans, 1946; Sattler and Youmans, 1948). Though accurate, this method suffers from being somewhat laborious and requiring relatively large nunmbers of organisms. The second employs photometric turbidity readings on cultures grown in Dubos' medium (Wolinsky and Steenken, 1947; Smith, 1947). The disadvantage of the second method resides in the composition of the medium that must be employed. Dubos' medium contains polyoxyethylene sorbitan monooleate, a surface-active agent that promotes a more diffuse type of growth and permits turbidimetric measurements (Dubos and Davis, 1946). Unfortunately, this substance has been shown to have a retarding effect on the growth of virulent tubercle bacilli and, furthermore, may markedly affect the action of substances present in the medium on the multiplication of tubercle bacilli (Fisher, 1948; Wong, Hambly, and Anderson, 1947; Forrest, Hart, and Walker, 1947; Kirby and Dubos, 1947; Youmans and Youmans, 1948). Because of the importance, for purposes of diagnosis, of the isolation of small numbers of tubercle bacilli from pathologic material, very small numbers of tubercle bacilli have been used as inocula to determine the suitability of various media for the initiation of growth. This method has also been used to investigate the various nutritive requirements of the organism. However, because of the manner in which it has been employed, only approximate indications of the rate of growth have been obtained. Use of very small inocula for such purposes has suffered also from the lack of appreciation on the part of many workers of the sampling error involved (Drea, 1942). In using this method to determine the suitability of various media for the growth of tubercle bacilli, the usual procedure has been to inoculate tubes of media with one or more quantities of suspensions of tubercle bacilli which varied from 10-1 to 10-10 mg. Following incubation at 37 C for some arbitrary period of time, the tubes have been examined to deter-