Comparative studies between microbiological culture and uptake of uridine/uracil to detect mycoplasmal infection of cell cultures

Abstract

Controlled, prospective studies were performed to compare detection of cell culture mycoplasmas by ratio of uptake of tritiated uridine (UdR) to tritiated uracil (U) and by microbiological culture. Culture was by standard agar and broth inoculation with aerobic and anaerobic incubation; immunofluorescent staining of indicator cell cultures was used to detect M. hyorhinis. The ratio of uptake of UdR to U (UdRU) and interpretation of test results were by standard published methods and performed in triplicate. 115 cell cultures were simultaneously assayed by the two techniques. 84 cultures (73.1%) yielded agreement between the 2 methods; 2 cultures (1.7%) yielded conflicting results, and 29 cultures (25.2%) yielded UdRU results in the questionable range. Conflicting results consisted of two negative UdRU tests in mouse cell cultures infected with M. orale. In separate studies, 3T-6 cultures freshly infected with M. orale yielded negative UdRU results 3 days after infection, questionable results after 10 days and a positive UdRU 17 days after infection. UdRU detected infection in fibroblast, epithelial, and lymphocyte cell cultures. Highest UdRU ratios were detected in human skin fibroblasts at early population doubling levels (PDLs), 4064 in one culture at PDL4. UdRU was determined for IMR-90, a human diploid fibroblast at 12 different PDLs using the same lot of media. UdRU gradually decreased throughout the life of the culture, from 2 125 at PDL6 to 340 at PDL36. Cultures in phase III and others exhibiting poor growth frequently yielded questionable or false-positive UdRU results and were not included in tabulations of these results. UdRU determined in endothelial cell cultures decreased as population density increased. In a representative experiment performed over a 4-day period, the UdRU values were 1 808, 955 and 356 when the number of endothelial cells in culture were 5.3 × 105, 6.6 × 105 and 1.1 × 106, respectively.