L-forms of group A streptococci Size of viable particles and growth requirements
Filtrability is one of the characteristics of the L-phase of bacteria. The results of the filtration experiments reported in the literature indicate a diameter of the minimal reproductive unit of approximately 0.3t~. However, by measurements on photographs of slide cultures of the L-phase of several bacteria the diameter of the reproductive elements was found to be > 0.6/~ to 0.7t~ (Weibull and Lundin, 1962; Weibull, 1963). With the L-phase of two strains of group A streptococci the same discrepancy between the results of both techniques was obtained. Dependent on the expelimental conditions the filtration endpoint varied between 0.25/~ and 0.45u.. Slide cultures, observed by phasecontrast microscopy and photographed at regular intervals during incubation, showed that only elements having a diameter > 0.8~ developed into colonies. The possibility that the L-phase, due to its plasticity, is capable of passing through filters of smaller pore size than their diameter was investigated. Filtrates of 0.45~. filters were conceotrated by ultracentrifugation. Slide cultures inoculated with the sediment showed that the elements responsible for growth had a diameter ranging from 0.7tL-1.7tz. The results obtained indicate that the reproductive elements of the L-phase of the group A streptococci have a diameter > 0.8/~. Another property in which the L-phase differs from the parent strains is the need for special growth requirements (Sharp, 1954). However, results were obtained which indicate that these special conditions are not required during subculture. It proved to be possible to get luxuriant growth in a chemically defined medium of both the L-phase and the parent strains.
ISOLATION AND PROPAGATION OF MYCOPLASMA
We have used mainly media formulated according to the prescription of D. Herderschec ( 1963) with minor modifications. Their primary distinction from other media is a high saccharose content (8 % ). Besides, the blood for the serum is drawn into a 10% solution of trypsin (see further thesis J. Waldman, to be published). For the isolation of T strains, we have adopted the advice of M. C. Shepard (1965) and adjusted the pH of the medium to 6. The plates are incubated anaerobically in hydrogen with 20% COz. For other mycoplasma, the pH of the media is 8 and the plates or broth cultures are incubated anaerobically with 5 % COz, except for M . pneumoniae which is also incubated aerobically. Colistinum (80mgr/600 ml medium) is added in part of the media to suppress the growth of contaminating bacteria (colistinum methane sulfonate) .
Effect of pH on Human Mycoplasma Strains
Shepard, Maurice C. (U.S. Naval Medical Field Research Laboratory, Camp Lejeune, N.C.), and Carl D. Lunceford. Effect of pH on human Mycoplasma strains. J. Bacteriol. 89:265–270. 1965.—The optimal reaction of culture media for the cultivation of T-strain Mycoplasma of human origin was investigated. By use of a recently modified tryptic digest medium, the optimal reaction in either agar or fluid medium was found to be pH 6.0. In contrast, human classic (large-colony) Mycoplasma could be cultivated in agar or fluid medium over a rather broad pH range, and the influence of the reaction of the medium appeared to be primarily species-dependent. M. salivarium, for example, grew best in agar from pH 5.5 through 6.5. M. pneumoniae (Easton's agent) yielded largest colony numbers in agar and highest titers in broth at pH 8.0. In the case of T-strain Mycoplasma, both maximal colony numbers in agar and highest titers in fluid media were achieved at a reaction of pH 6.0. In addition, largest colony size of T-strain Mycoplasma was also achieved in agar at pH 6.0, and averaged 50 to 100% larger than that obtained by cultivation at pH 8.0 with the same medium. Although T-strains will develop in agar media over a pH range of from 5.0 through 10.0, the extremely small colony size and poor staining properties resulting from growth in an alkaline medium make their recognition in agar cultures difficult. Aerobic cultivation of T-strains was first achieved in agar adjusted to pH 5.5 to 6.0. In fluid medium, multiplication of T-strains occurred only within the limits of pH 5.0 through 8.0, with highest titers being reached at pH 6.0. Greater attention to the reaction of complete Mycoplasma media is stressed.