PLEUROPNEUMONIA-LIKE (MYCOPLASMA) INFECTIONS OF TISSUE CULTURE

Abstract

The medium of Barile, Yaguchi, and Eveland was found to be a satisfactory medium for the culture of pleuropneumonia-like organisms (PPLO) in cell lines. Somewhat simpler to prepare and at least as efficient was 15% human blood added to blood agar base. A transparent medium giving even better growth contained human serum, yeast and red blood cell extracts. As a liquid medium it supported growth of PPLO with a generation time of 1.3 hours. Tissue culture medium in the absence of cells did not support the growth of PPLO. Most of the cell lines at this laboratory were infected with PPLO. Most of the virus strains propagated on cell lines and some of those grown in primary tissue cultures contained PPLO. Growth curves of PPLO in tissue culture were studied. In 48 hours the number of PPLO rose to a concentration of 107 ml in the fluid, and probably even more of these organisms were present in association with the cells. Fluorescent antibody stxins showed much PPLO antigen in clumps in a distribution that accorded with a location on the cell membrane. The antibiotic spectrum of a number of isolates of PPLO was studied with antibiotic sensitivity discs and no differences were found among them. The minimal inhibitory concentration for PPLO and the minimal toxic level for HeLa cells for tetracycline were determined. To rid a cell line of its infection, 2.5 [mu]g tetracycline per ml were incorporated in the medium for one passage. One cell line has now remained free of PPLO for 17 months (75 transfers) without further treatment with tetracycline. It was not possible to institute PPLO infections of cells lines by inoculating them with bacteria of a number of species, all susceptible to penicillin or streptomycin. The addition of saliva of laboratory personnel to the tissue cultures was likewise ineffective in starting PPLO infections. Serum seems not to be a frequent source, since the PPLO-free cell line has now been exposed to 750 ml of human serum from four different lots, each lot representing 20 or more people.