To Submit Cell Cultures for Mycoplasma Testing
The TCF can screen your cell cultures for the presence of pleuro pneumonia-like organisms (PPLO) ie. mycoplasma and acholeplasma contamination. The routine test that we offer is a staining type assay that is a modification of the Hoechst stain assay (DNA fluorochrome 33258) described by T. Chen (Experimental Cell Research 104:255-262, 1977). This procedure is based on the observation of a fluorescent staining pattern of DNA in the cytoplasm of indicator cells (VERO) that are known to be mycoplasma negative. These indicator cells are inoculated with medium and a few cells from the sample submitted and incubated for approximately five days. The proper staining pattern and color indicates the presence of mycoplasma contaminating the surface of the cells that would have occurred from the sample. Because of our use of an indicator cell line and experience in performing the test, the system is relatively sensitive (approximately 100 organisms per ml). However, there can be artifacts associated with the assay and the results should be considered a presumptive indication of PPLO contamination and not an infallible diagnostic test. This test is subjective and will err on the side of false positives. That is, very "clean" preparations that stain negative are almost always accurate, whereas preparations with cell debris or lysed cells can contribute to artifact background staining and could lead to a positive reading. We can also use the Gen-Probe Rapid Detection System as a confirming test. This test is a nucleic acid hybridization system using a DNA probe homologous to twenty (20) species of mycoplasma and acholeplasma ribosomal RNA. Six (6) of these have been suggested to be the organisms present in 98% of laboratory PPLO contaminations. The sensitivity of this test is about 10,000 infectious organisms per ml. The advantages of the test are objectivity of interpretation and speed of results. A positive result will almost always be accurate whereas a negative result should be considered presumptive. Very low levels of PPLO may not be detected. By using both tests, we are able to achieve a high degree of certainty that can together be confirming of the presences or absence of mycoplasma contamination.
Cultures should be grown for at least seven days without antibiotics, longer if antibiotics such as gentamicin, kanamycin, tylosin, and certain others are used and if increased sensitivity is desired. Media should not be changed the last four days prior to testing. Cultures should be submitted in 25 cm2 flasks on Wednesday the week of testing. Testing is completed on Friday and results are available on the following Tuesday.