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Two bacteriological methods, one to demonstrate phosphatase production and the other to demonstrate aesculin hydrolysis, were tested on sterol-requiring My coplasma and sterol-nonrequiring Acholeplasma strains to determine their utility in differentiating species. The phosphatase test, which gave weak and variable reactions with most of the sterolnonrequiring strains, was judged ineffectual for differentiating these strains. The aesculin test distinguished all Mycoplasma strains and Acholeplasma granularum, both of which gave negative reactions, from most Acholeplasma laidlawii strains and from Acholeplasma axanthum, which gave positive reactions.T h e bacteriological characterization of Mycoplasma species by standardized methods was recently described by Aluotto et al. (1). Of the 22 species studied by these authors, half displayed distinctive patterns of biochemical activities. The remaining strains fell into groups containing several species with biochemically similar patterns. One such group included Mycoplasma gallisepticum, Mycoplasma histotropicum [now regarded as a strain of Mycoplasma pulmonis (3)] , Mycoplasma granularum, and Mycoplasma laidlawii. Application of the test of Razin and Tully ( 6 ) for determining cholesterol requirement readily permits differentiation of the sterol-requiring species in this group from the two sterol-nonrequiring species, M. granularum and M. laidlawii. [For these and other sterol-nonrequiring species, a new generic name, Acholeplasrna, was recently proposed (4) and will be employed hereafter in this report.] Acholeplasma granularum and Acholeplasma laidlawii can be differentiated from each other by serological methods and gel electrophoretic patterns (7, 8), but a simple biochemical test for distinguishing them has been lacking. These strains react somewhat differently from each other when tested for phosphatase production and hemolysis; however, neither test alone is capable of distinguishing the species. The phosphatase reaction was reported t o be weakly positive for A. laidlawii type A but negative for A . laidlawii type B and A. granularum (1). The type of hemolytic reaction for various A . laidlawii strains was examined (unpublished data) and found t o vary from strain t o strain. Now that a new and distinct species of Acholeplasma has been recognized (9), further biochemical tests for characterizing and differentiating them are all the more desirable.A survey of biochemical reactions applicable t o the problems of Mycoplasma and Acholeplasma characterization and differentiation pointed to aesculin hydrolysis as a possibility. A group of more than 22 types or representative strains of Mycoplasma and 32 Acholeplasma strains were, therefore, examined for ability to hydrolyze aesculin. In addition, the test for phosphatase activity was reexamined on all Acholeplasma strains t o clarify and expand the findings of Aluotto et al. (1). MATERIALS AND METHODS Organisms

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SUMMARYA bacteriological study was made of blood specimens taken repeatedly during a 2-year period from a child with subacute bacterial endocarditis who received intensive treatment with antibiotics. The conventional bacillary form of Corynebacterium sp. was present in the blood and bone marrow of the patient before the beginning of antibiotic therapy and on occasions when the administration of antibiotics was suspended. These were the periods when the patient showed overt symptoms of clinical illness. When antibiotic therapy was adequate to produce clinical remission of symptoms, the infecting organism was not eradicated, but persisted in the blood in a small granule-like form that could be demonstrated and cultured only by highly specialized techniques. The cultural procedures required to bring about reversion of the granule-like form to the conventional bacillary form and the morphology of the various transitional forms that the organism assumed during the reversion process are described and illustrated.

SUMMARY : A strain of a pleuropneumonia-like organism (PPLO) isolated from urethral exudate from a case of non-specific urethritis was studied in HeLa cell tissue cultures. Although the organisms entered the cell cytoplasm, they did not produce marked damage or proliferate luxuriantly until filtrate from a broth culture of Staphylococcus pyogenes or yeast extract was added to the infected tissue cultures. The organisms subsequently isolated from tissue cultures initially inoculated with PPLO and yeast extract showed conversion from PPLO form to L form of growth. Further culture of the L form, especially with the aid of mucin, resulted in conversion of the L form to a corynebacterium. This corynebacterium was indistinguishable culturally, biochemically and serologically from a corynebacterium isolated on rabbit blood agar plates which had been inoculated with a portion of the original urethral exudate from the same case of non-specific urethritis. The view is expressed that other human genital strains regarded a t present as PPLO may be found to be L forms of Corynebacterium spp. The criteria for identification of PPLO and L forms are discussed.During recent years pleuropneumonia-like organisms (PPLO) have been isolated with increasing frequency from the human urethra in the presence and in the absence of various inflammatory processes (Harkness, 1950; M e l h 8z Linnros, 1952;Nicol & Edward, 1953). The pathogenic significance of the PPLO in genital infections, however, is controversial, and most investigators emphasize the need for more information about the organisms themselves before an aetiologic role in genital diseases can be attributed to them. Keller & Morton (1954) reported that several human genital strains of PPLO produced no pathological manifestations in the developing chick embryo. No reports have come to our attention on the action of human PPLO on human cells in tissue culture, although Edward (1952) mentioned that tissue cultures might be useful tools for investigating the pathogenicity of PPLO. This investigation was undertaken, therefore, to obtain basic morphological data on the behaviour of a human urethral strain of PPLO in cultures of human cells. The manifestation of any pathogenic effects by the organisms upon the cells was sought. For comparison, the behaviour in tissue culture of a presumably non-pathogenic corynebacterium from the same source as the PPLO was observed. The role of secondary factors which might possibly alter the behaviour of the PPLO in the tissue cell environment was also investigated. The secondary factors chosen for 49-3

acid homologies of selected bacteria, L forms, and Mycoplasma species. J. Bacteriol. 90:1200-1204. 1965.-The molar per cent of guanine plus cytosine (G + C) in the deoxyribonucleic acids (DNA) of Proteus mirabilis, strain 9, and its stable L form was determined by thermal denaturation and found to be approximately 39.5%O G + C. The DNA homologies of this bacterium and its L form were estimated by the agar-column technique and were equivalent in their abilities to anneal and form specific duplexes. The next series of comparisons were performed between two Mycoplasma species and their often suggested bacterial parent. The G + C ratios of M. gallisepticun (32.7%o), 31. gallinarum (28.1%c), and Haemophilus gallinarum (41.9%) varied to a high degree. In the homologous system, the denatured DNA of H. gallinarum trapped in agar bound approximately 40%O of its sheared, denatured, and H'-labeled DNA. In comparison, the nucleic acids of M. gallinarum and M. gallisepticum were incapable of binding the labeled DNA of H. gallinarum. These findings provided evidence that the two strains of M1ycoplasma were not derived from H. gallinarum.