Mycoplasma are simple bacteria that lack a cell wall, and they are considered the smallest self-replicating organism. Because of their extremely small size (typically less than one micrometer), mycoplasma are very difficult to detect until they achieve extremely high densities and cause the cell culture to deteriorate; until then, there are often no visible signs of infection.

Detecting Mycoplasma Contamination

Some slow growing mycoplasma may persists in culture without causing cell death, but they can alter the behavior and metabolism of the host cells in the culture.

Chronic mycoplasma infections might manifest themselves with decreased rate of cell proliferation, reduced saturation density, and agglutination in suspension cultures; however, the only assured way of detecting mycoplasma contamination is by testing the cultures periodically using fluorescent staining (e.g., Hoechst 33258), ELISA, PCR, immunostaining, autoradiography, or microbiological assays.

Figure 2.4:  Photomicrographs of mycoplasma-free cultured cells (panel A) and cells infected with mycoplasma (panels B and C).  The cultures were tested using the MycoFluor™ Mycoplasma Detection Kit, following the kit protocols.  In fixed cells, the MycoFluor™ reagent has access to the cell nuclei, which are intensely stained with the reagent, but the absence of fluorescent extranuclear objects indicates that the culture is free from mycoplasma contamination (panel A).  In fixed cells infected with mycoplasma, the MycoFluor™ reagent stains both the nuclei and the mycoplasma, but the intense relative fluorescence of the nuclei obscure the mycoplasma on or near the nuclei.  However, the mycoplasma separated from the bright nuclei are readily visible (panel B).  In live cells, the MycoFluor™ reagent does not have access to the nuclei, but readily stains the mycoplasma associated with the outside of cells (panel C).  The images were obtained using 365 nm excitation and a 100/1.3 Plan Neoflaur® (Zeiss) objective lens coupled with a 450 ± 30 nm bandpass filter.