Hello Bintang Tedjoabagaskara

Unfortunately, it is challenging to quickly and reliably detect mycoplasma contamination in cell cultures. In early stages of contamination, mycoplasma does not typically cause pH changes in the medium nor do they have significant toxic effects on mammalian cells.

However, the visible signs of mycoplasma contamination that you will notice is a reduced rate of cell proliferation. This is because mycoplasma will compete with your cells for nutrients in the culture medium. Other indications will include cell aggregation and morphological changes.

But your iPSC culture seems to have great morphology!

Though it is difficult to detect visible signs of mycoplasma contamination, there is one way which you may try. You may show the presence of mycoplasma by light microscopy using oblique illumination, which renders the mycoplasma cells visible as white foci due to light scattering. Try it out!

Oblique illumination is achieved by means of a sliding diaphragm that shields the center of the light beam to produce a partially coherent light source, which is projected obliquely onto the specimen, producing a high contrast image. The diaphragm position is controlled by means of a rotating knob that can be employed to adjust the obliquity of illumination. More information on oblique illumination may be found in the link below.

https://www.microscopyu.com/techniques/stereomicroscopy/oblique-illumination

If you are not successful, then there are several different techniques to identify if your cell culture is contaminated with mycoplasma. These include histochemical staining, ELISA, DNA fluorochrome staining, microbiological culture, biochemical / bioluminescence methods, and PCR.

Best.