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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.

Uridine phosphorylase activity has been used to detect mycoplasmas in cell cultures by measuring formation of14C-uracil from14C-uridine. In this report we show that all species ofMycoplasma, Acholeplasma, andUreaplasma tested exhibited uridine phorphorylase activity. Among the genusSpiroplasma, serogroups I-1, I-3, I-5, I-7, I-8, IV, XIII, and XIV lacked uridine phosphorylase activity.

A helical mycoplasma, Spiroplasma mirum strain SMCA, produced malignant transformation in mouse NIH 3T3 cells and monkey kidney CV-1 cells. The transformed cells exhibited morphological changes consistent with the transformed phenotype, grew in soft agar and produced tumors in athymic and BALB/c mice. Transmission electron microscopy revealed structures morphologically similar to mycoplasmas present in the cytoplasm of transformed but not untransformed 3T3 cells. The time of inoculation of S. mirum SMCA to 3T3 cells and the passage level of 3T3 cells affected transformation.

A radiochemical method was developed for the assay of aspartate aminotransferase and alanine aminotransferase activities in Mollicutes. Using [1-C14]α-ketoglutarate as the amino group acceptor in transamination, we found that the fermentative speciesAcholeplasma laidlawii MG of the family of Acholeplasmataceae, the fermentativeMycoplasma pneumonia FH of the family of Mycoplasmataceae, and the nonfermentativeMycoplasma salivarium VV, also of the family of Mycoplasmataceae, all had aspartate aminotransferase and alanine aminotransferase activities. The radioactive product was identified as [1-C14]l-glutamic acid.Mycoplasma pneumoniae andM. salivarium had very low activity of alanine aminotransferase. Both aminotransferases had a partial requirement for pyridoxal 5′-phosphate and were strongly inhibited by 0.1 mM aminooxyacetate.

Studies were performed to compare three methods to detect mycoplasmal infection of cell cultures. The methods included microbiological assay by inoculation into broth and onto agar with anaerobic incubation, fluorescent DNA staining by Hoechst 33258, and mycoplasmal mediated cytotoxicity by 6 methylpurine deoxyriboside (6MPDR). Fluorescent DNA staining and 6MPDR assays were performed in an indicator cell culture system. A total of 2589 cell cultures were assayed. Mycoplasmas were detected in 174, an incidence of 6.7%. Species isolated were: Acholeplasma laidlawii, Mycoplasma orale, M. arginini, M. hyorhinis, M. fermentans, M. pirum, and M. pneumoniae. In separate studies, 6MPDR also detected infection with Spiroplasma mirum when this organism was deliberately inoculated into cell cultures. The efficiencies of microbiological testing, fluorescent DNA assays, and 6MPDR were 43.1, 98.8, and 97.1%, respectively.

The most frequent causes of cell culture contamination are poor techniques and housekeeping and inadequate sterility testing of supplies and culture media. The most common contaminants of cell cultures areMycoplasma, Torula sp., andPseudomonas sp. Routine procedures used in this laboratory to control or prevent accidental contamination include the use of filtered laminar air flow in transfer rooms, elimination of antibiotics wherever possible, careful execution of aseptic procedures, periodic review and discussion of techniques, sterility testing of all media components, use of approved clothing, and effective cleaning and disinfection procedures.