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To determine whether viruses or mycoplasma have either an inductive or provocative effect on the pathogenesis of the human leukemia, it is first necessary t o isolate candidate agents. Next, the agents isolated must be characterized to determine whether they are known human viruses or mycoplasma. Finally, evidence must be accumulated to indicate whether the isolated agents can be implicated in the cause or pathogenesis of human leukemia. Although our long term studies' have provided a number of interesting findings concerning the isolation of viruses from bone marrow specimens obtained from leukemic and nonleukemic patients, this report will summarize results on the isolation and characterization of mycoplasma.Isolation of Mycoplasma that it is possible to isolate mycoplasma from bone marrow specimens from leukemic patients by cell culture technique when direct cultural methods yielded negative results. Even when cell cultures were used for the isolation of PPLO, it became evident (TABLE 1) that agents were not isolated frequently enough to evaluate whether they could be implicated etiologically in human leukemia. However, these experiments made it possible t o classify cell lines according to their relative susceptibility to infection (TABLE 2) and, consequently, to use the sensitive cell lines to improve the isolation technique. Recent improvements in technique have increased the sensitivity of the assays so that it now appears possible to evaluate, on the basis of isolation studies, whether PPLO can play a significant role in the clinical course of leukemia. The methods used to isolate mycoplasma are outlined in TABLE 3. T h e key points of technique include the use of specimens that are less than three hours old, to make passages only from the positive tubes in a passage sequence, to keep the p H of cell culture fluid less than 7.6, and to avoid freezing either the clinical specimen or passage material. Results are positive most often with specimens obtained from leukemic patients in relapse or who have just had the disease diagnosed.Representative results of the P P L O isolation studies on bone marrow specimens obtained from leukemic children are presented in TABLE 4. Only children with leukemia in relapse or just diagnosed were studied. Isolations were done principally by the use of the Michigan sublines of Chang-liver and human amnion cells since they are most sensitive to infection.' Representative results of isolation studies done on bone marrow specimens obtained from adults with leukemia are tabulated in TABLE 5. Often specimens from a single patient were positive simultaneously in more than one cell line (TABLE 6 ) .Long-term studies have been carried out in an attempt to define a medium and procedure suitable for the direct cultivation of PPLO from bone marrow specimens.' During the period 1961-1965, more than 1000 marrow specimens were cultured for PPLO. A wide variety of media and procedures were used.' Beginning in August, 1965, it has been possible to repeatedly isolate mycoplasma from