Growth of Mycoplasma hyorhinis cultivar alpha on semisynthetic medium.

Serial passage of Mycoplasma hyorhinis cultivar alpha (formerly noncultivable strains) has been accomplished in modified CMRL-1066 medium with fetal bovine serum. In modified CMRL-1066 liquid medium, cultivar alpha strains grow at a similar rate and to equivalent titers when compared with BTS-7, the type strain of the species. Further experiments with BTS-7 demonstrate that the extent of growth obtained in the semidefined medium was comparable to growth in conventional mycoplasma medium. M. hyorhinis strains, including cultivar alpha strains, grow in serial passage when fetal bovine serum is replaced with bovine serum albumin, palmitic acid, and cholesterol. The results of these studies show that M. hyorhinis cultivar alpha strains are not nutritionally more fastidious than other mycoplasmas but that they are noncultivable on standard mycoplasma media because they are sensitive to high levels of inhibition activity by medium components.

Antibiotic sensitivities and elimination of mycoplasmas from infected cell cultures.

Invaluable or irreplaceable cell lines infected with mycoplasmas can be cured. We have had success with a protocol that utilizes a combination of anti-mycoplasma immune serum and pretested antibiotics. A panel of antibiotics was evaluated by examining inhibition of agar growth of a variety of cell culture mycoplasmas. Resistant strains were found with certain drugs and intraspecies heterogeneity was seen in drug response. For these reasons drug regimens must be tailored to individual strains. When possible, two effective antibiotics were chosen to minimize chances of outgrowth of drug-resistant subpopulations. Cell lines were diluted and cultured in treatment cocktail consisting of growth medium plus antibiotics and antiserum. After a short exposure, cells were subcultured into fresh growth medium. Routine culturing practices were resumed and mycoplasma monitoring was begun. This procedure has been effective in curing eight cell lines, of which five were monolayers and three were suspension cultures. Mycoplasmas from four species comprised the contaminants. Two cures were of mixed infection caused by strains of two mycoplasma species. No failures have been encountered with this procedure thus far. Cultures have remained free of mycoplasmas and have retained properties of interest.

Uracil phosphoribosyl transferase activity of mycoplasma and infected cell cultures.

Human H.Ep-2 and mouse 3T6 cells infected by Mycoplasma hyorhinis showed an increase in [3H]uracil uptake and a more than 20-fold increase in the activity of uracil phosphoribosyltransferase (UraPRT). Uninfected cell cultures gave background levels of this enzyme activity. A survey of 16 strains of mycoplasma showed 13 to possess UraPRT activity. Rabbit kidney cells (RK13) were infected with eight different strains of four mycoplasma species known to be common cell culture contaminants. Seven of the eight cell cultures showed elevated UraPRT activities four days after infection. This enzyme activity may be of value in monitoring cell cultures for mycoplasma and aid in classification.