Detection Of Mycoplasmas In Cell Cultures Research Articles

Quantitative detection of cell culture Mycoplasmas by a one step polymerase chain reaction method

A rapid, sensitive assay was developed that can detect the six species of Mycoplasmas that account for the vast majority of cell culture infections. This assay, a modification of the method published by Wong-Lee & Lovett [1], allows direct evaluation of culture medium by a single-step PCR method that utilizes primers complementary to conserved 16S rRNA sequences. Extensive testing of medium from uninfected cultures spiked with purified Mycoplasma DNAs showed that the method described in this report can detect the equivalent of one Mycoplasma in 15 μl culture medium; thus, evaluation of a single culture sample allows detection of Mycoplasmas in cultures infected with the equivalent of 10 or more Mycoplasmas per 15 μl (or ≥6.7×102 Mycoplasma equivalents/ml) with greater than 99.99% confidence. Comparison of results obtained with this PCR-based assay and a standard biological colony-forming assay revealed that the PCR assay is capable of detecting 0.0015-0.015 colony forming units, suggesting that the PCR assay may also be detecting nonviable Mycoplasmas. The high level of amplification achieved with this method allows direct detection of amplification products by ethidium bromide staining of agarose gels, and thus allows rapid screening of cell cultures.

Fast and simple procedure for the detection of cell culture mycoplasmas using a single monoclonal antibody

The detection of mycoplasmas in cell cultures is still a problem, especially in those laboratories in which the detection and identification of microorganisms is not established as a routine procedure. In our laboratory, monoclonal antibodies (mAbs) have been prepared to Acholeplasma laidlawii, Mycoplasma hyorhinis, Mycoplasma orale, Mycoplasma arginini and Mycoplasma salivarium. 30 mAbs were obtained and one of these, designated CCM-2, was shown to bind to all five mycoplasma species. It also bound to Mycoplasma fermentans, Mycoplasma hominis and to all wind types ( n = 54), isolated from cell cultures submitted to our laboratory. The mAb was used in a immunofluorescent assay (IF) and the method correlated with the microbiological assay. Using this mAb immunofluorescent staining of cells is a fast and simple procedure for mycoplasma detection in cell cultures.

Detection of cell culture mycoplasmas by a genetic probe

The utility of a genetic probe prepared against mycoplasmal ribosomal RNA was evaluated. The probe consisted of a tritiated DNA probe prepared with reverse transcriptase against Mycoplasma and Acholeplasma. Using a batch process without Southern blots and autoradiography, the probe assay detected mycoplasma infection in 27 of 52 cell cultures, an incidence of 51.9%. No false-positives were detected. Two false-negatives occurred; these were unusual mycoplasma infections of cell cultures grown in serum-free media. The sensitivity of the probe varied with mycoplasma species, from 9.3 × 10 3 for M. arginini to 2.6 × 10 6 for M. orale. The assay is rapid, taking only 2–3 h.

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

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.