Growth characteristics in HeLa cells of the rapidly growing acid fast bacteria, Mycobacterium fortuitum, Mycobacterium phlei, and Mycobacterium smegmatis.

With the techniques found suitable for the growth of tubercle bacilli in HeLa cells (Shepard, 1955, 1957a) little extracellular bacterial growth occurs when virulent strains are used. When rapidly growing mycobacteria (Gordonand Smith, 1953, 1955) are introduced into the tissue cultures in the same concentration, bacterial growth in the extracellular tissue culture fluid is rapid and soon results in death of the HeLa cells. To be able to observe whether the well known saprophytes Mycobacterium phlei and Mycobacterium smegmatis and the potentially pathogenic Mycobacterium fortuitum could grow in HeLa cells, it was necessary to modify the procedures in two respects. The first was the addition of 500 ,g streptomycin/ml of tissue culture fluid after the bacilli had been phagocytosed by the HeLa cells, so that the growth of bacilli remaining outside the cells would be suppressed. It had previously been shown that concentrations of streptomycin up to 2,000 ,ug/ml were without effect on intracellular growth of tubercle bacilli (Shepard, 1957b). The second modification was introduced because observations of stained smears revealed that many large clumps of organisms were present in the bacterial inocula prepared from the rapidly growing mycobacteria when the method found suitable for tubercle bacilli was followed (Shepard, 1955, 1957a). This change consisted of filtering the bacterial suspension through filter paper (Fenner, 1951). The classification of the rapidly growing acid fast bacteria, M. phlei, M. smegmatis, and M. fortuitum, has been placed on a firm basis by the studies of Gordon and Smith (1953, 1955). Wells et al. (1955) studied the pathogenicity in animals of a culture of M. fortuitum which they had isolated from a patient with cellulitis and lymphadenitis. A distinctive disease picture was produced in mice following intraperitoneal injection of this culture, as well as of 8 of 9 cultures of M. fortuitum which these workers received from Dr. Gordon.