Mycoplasma contaminations are more prevalent in cell culture than many workers realise. The reason for this is that mycoplasma contaminations are not evident under light microscopy, nor do they result in turbid growth in culture. Instead, the changes induced are more subtle and manifest themselves mainly as a slowdown in growth and in changes in cellular metabolism and functions. However, cells generally return to their native morphology and normal proliferation rates relatively rapidly after eradication of mycoplasma.

The presence of mycoplasma contamination in cultures has in the past been difficult to determine and samples had to be analysed by specialist laboratories. This is no longer the case and techniques such as enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction ( PCR) and DNA staining using the fluorescent dye Hoechst 33258 are now available for detection of mycoplasma in cell culture laboratories. The gold standard, however, is the microbiological culture technique in which cells in sus pension are inoculated into liquid broth and then incubated under aerobic conditions at 37 °C for 14 days. A non-inoculated flask of broth is used as a negative control. Aliquots of broth are taken every 3 days and inoculated onto an agar plate, which is incubated anaerobically as above. All plates are then examined under an inverted microscope at a magnification of 300× after 14 days of incubation. Positive cultures will show typical mycoplasma colony formation, which has an opaque granular central zone surrounded by a translucent border, giving a ‘fried egg’ appearance ( Figure 1 ). It may be necessary to set up positive controls in parallel, in which case plates and broth should be inoculated with a known strain of mycoplasma such as Mycoplasma orale or Mycoplasma pneumoniae.

Fig1. Photograph of mycoplasma, showing the characteristic opaque granular central zone surrounded by a translucent border, giving a ‘fried egg’ appearance.

The DNA binding method offers a rapid alternative for detecting mycoplasma and works on the principle that Hoechst 33258 fluoresces under ultraviolet light once bound to DNA. Thus, in contaminated cells, the fluorescence will be fairly dispersed in the cytoplasm of the cells owing to the presence of mycoplasma. In contrast, uncontaminated cells will show localised fluorescence in their nucleus only. The Hoechst 33258 assay, although rapid, is of relatively low sensitivity when compared with the culture technique described above. For this assay, an aliquot of the culture to be tested is placed on a sterile coverslip in a 35-mm culture dish and incubated at 37 °C in a cell culture incubator to allow cells to adhere. The coverslip is then fixed by adding a fixative consisting of one part glacial acetic acid and three parts methanol, prepared fresh on the day. A freshly prepared solution of Hoechst 33258 stain is added to the fixed coverslip, incubated in the dark at room temperature to allow the dye to bind to the DNA and then viewed under ultraviolet fluorescence at 1000×. All positive cultures will show fluorescence of mycoplasma DNA, which will appear as small cocci or fi laments in the cytoplasm of the contaminated cells ( Figure 2b ). Negative cultures will show only fluorescing nuclei of uncontaminated cells against a dark cytoplasmic background ( Figure 2a ). However, this technique is prone to errors, including false-negative results. To avoid the latter, cells should be cultured in antibiotic-free medium for two to three passages before being used. A positive control using a strain of mycoplasma seeded onto a coverslip is essential. Such controls should be handled away from the cell culture laboratory to avoid contaminating clean cultures of cells. It is also important to ensure that the fluorescence detected is not due to the presence of bacterial contamination or debris embedded into the plastics during manufacture. The former normally appear larger than the fluorescing cocci or fi laments of mycoplasma. Debris, on the other hand, would show a non-uniform fluorescence owing to the variation in size of the particles usually found in plastics.

Fig2. Hoechst 33258 staining of mycoplasma in cells. (a) A Hoechst-negative stain, with the dye staining cellular DNA in the nucleus and thus showing nuclear fluorescence. (b) A Hoechst-positive stain, showing staining of mycoplasma DNA in the cytoplasm of the cells.

ELISA detection  of mycoplasma is also commonly used and can be carried out using specifically designed kits following the manufacturer’s protocol and reagents supplied. In this assay, 96-well plates are coated with the antibodies against different mycoplasma species. Each plate is then incubated at 37 °C for 2 h with the required antibody or antibodies before blocking with the appropriate blocking solution and incubating with the test sample(s). A negative control, which is sim ply media with sample buffer, and a positive control normally provided with the kits, should also be included in each assay. A detection antibody is subsequently added to the samples, incubated for a further 2 h at 37 °C before washing and incubating with a streptavidin solution for 1 h at 37 °C. Each plate is then tested for mycoplasma by adding the substrate solution and reading on a plate reader at 405 nm after a further 30 min incubation at room temperature. This method is apparently suitable for detecting high levels of mycoplasma and could also be used to identify several species in one assay.

As with the ELISAs, commercial PCR kits are also available that contain the required primers, internal control template, positive control template and all the relevant buffers. Samples are generated and set up in a reaction mix as instructed in the manufacturer’s protocol. The PCR is performed according to manufacturer’s protocols and the products generated analysed by electrophoresis on a high-grade 2% agarose gel. Typically, cell lines are checked every 1–3 months by ELISA and PCR.

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مواضيع ذات صلة

Laboratory Diagnosis of Infections of the Eyes

Laboratory Diagnosis of Central Nervous System Infections

Diagnosis and Differential Diagnosis of Paroxysmal Nocturnal Hemoglobinuria

Differential Diagnosis of Pancytopenia

Diagnosis of Aplastic anemia

Diagnosis of Upper Respiratory Tract Infections

tuberculosis testing (TB testing, Interferon gamma release assay [IGRA, T-Spot.TB], QuantiFERON-TB Gold [QFT, QFT-G, TB Gold test, TB blood test], Nucleic acid amplification for TB [NAAT], TB antibody)

thyrotropin-releasing hormone stimulation test (TRH stimulation test, Thyrotropin-releasing factor stimulation test [TRF stimulation test])

Laboratory Diagnosis of Lower Respiratory Tract Infections: Specimen Processing

Laboratory Diagnosis of Lower Respiratory Tract Infections: Specimen Collection and Transport

thyroid scanning (Thyroid scintiscan)

thyroid fine needle aspiration biopsy (FNAB, Skinny needle thyroid biopsy, Fine-needle aspiration [FNA])