I have detected in Hc (undifferentiated hepatocyte cells established from fetal livers) cells as follows by using PDMD method (see file; HepG2 fucoidan); i.e., UvrABC system protein C (Mycoplasma agalactiae; 320-) and DNA polymerase III subunit beta (Mycoplasma capricolum; 220-).   Hc cells have no HIV-1, though they have HIV-2 (Env polyprotein; but this protein has no reverse transcriptase (RT) and integrase (IN)).   Further, Hc cells also contain biotin synthase of Helicobacter pylori J99, Bacillus subtilis, and Arabidopsis thaliana(thale cress).   Thus, biotin biosynthesis by biotin synthase  is essential for the immortality of the cells as the Finnish biochemist Dr. Keränen A.J.A. (The biotin synthesis of HeLa cells in vitro. Cancer Res 32: 119-124, 1972) has  originally indicated.

But, hepatoma cells  of HepG2 do not contain  Mycoplasma, but this cell line (HepG2) has retroviraus of HIV-1, HIV-2, and Rous sarcoma virus (RSV).  HepG2 has  the biotin synthase (sequence of Bacillus subtilis).    

In human serum, there has been detected Mycoplasma in biotin deficiency patients.   But they have dissappeared by biotin therapy.   All the 8 sera investigated in this answer has no HIV.

GSD1b patient (before biotin); Cytadherence accesory protein 2 (Mycoplasma pneumonia; 1329-).

4 mo male (before biotin); Kasugamycin dimethyltransferase (Mycoplasma capricolum; 114-) and Probable cysteine desulphurase (Mycoplasma genitalium; 69-).

1y female (before biotin);   50S Ribosomal protein L2 (Mycoplasma pneumoniae; 144-).

32 y female with gait disorder (before biotin); not present.

22 y female with light gait disorder （before biotin); Oligopeptide transport ATP-binding protein oppF (Mycoplasma pneumoniae; 360-).

Therefore, 80% (4 / 5) of biotin deficient patients possess Mycoplasma in their serum.

But, some immflamatory and healthy persons has Mycoplasma.

52 y female, mother of 32 y patient (healthy); Ribonucleoside-diphosphate reductase subunit beta (Mycoplasma pneumoniae; 234-).

8 mo female with gastritis; Phenylalanyl-tRNA ligase beta chain (Mycoplasma hyopneumoniae 7446; 117-).

12 mo female with common cold; UPF247 protein MYPU_2540 (Mycoplasma pulmonis; 94-).

Therefore, 38% (3 / 8) of non biotin-deficient persons (cured by biotin) or apparently healthy (but inflammation of gastritis and common cold) persons possess Mycoplasma.

Thus, Mycoplasma has infected onto mainly non-cancer serum with nutrition deficiency, but has not infected onto cancer cells, which seem rich in the nutritions.   Thus, Mycoplasma in fibroblast is not due to infection during cell culture, but Mycoplasma may be originally contained (endogenously present) in fibroblast; i.e., Mycoplsma has infected or invaded into ancestor of fibroblast cells during transformation.    

By the way, human adeno virus (HAdV; dsDNA virus) has infected to all the liver tissues with liver diseases investigated (n = 5), and hepatoma HepG2 cell line (n = 1), but has not to normal fetal Hc cell-line; i.e., HAdV is a marker of liver diseases (peudo-liver-cancer, LC, HCC; see file; Feed by measure,,).     

Therefore, I am considering that fibroblast (not the cancer cells but the transformed cells (very close to the cancer cells)) may have been infected by Mycoplasma, and may have been gotten the immortality from the invaded Mycoplasma's virus (bacteriophage), together with retrovirus of HIV-1, HIV-2, SIV, or Bovine leukemia virus (BLV) (in humans) and AKT8 Murine leukemia virus (MLVAT) or AKV Murine leukemia virus (MLV) (in murines).  

Although one pseudo-liver-cancer patient has no Mycoplasma, who may be treated with antibiotics, this patient may have been infected by Mycoplasma's virus.   This pseudocancer patient's liver has  HIV-1 (Gag-Pol polyprotein; with reverse transcriptase (RT) and integrase (IN)).   These observations indicate that the fibroblast is in the state of scanty of nutrition, but may produce important vitamin of biotin.   Then, Mycoplasma's virus plus HIV-1 (Gag-Pol polyprotein) may be linked to immortality in humans, but may not be linked to cancer.

Thus, this hypothesis forecasts that if one uses macrolide or tetracycline to kill Mycoplasma, then the fibroblast (an immortal cell line) loses the immortality and dies away.   This is why we use  penicillin G (100 U/mL), streptomycin (100 U/mL) and/or kanamycin (100 U/mL) in the culture medium.

A research paper has been presented (Human Cell (1990) 3, 37-44. Elimination of mycoplasmas from cultured human cell lines utilizing a combination of two antibiotics, quinolone and a pleuromutilin derivative. (in Japanese; the official English language journal of the Japan Human Cell Society). Mikamo S, Shiroko Y, Hirose I, Sekiguchi M.) using MC-210 (quinolone; Dainihon Pharmaceutical) and BM-1 (pleuromutilin derivative; Boehringer Mannheim).   However, only 9 human cell lines (21%) has been successfully eliminated the Mycoplasma out of 43 human cell lines.   This suggests that 21% of cell lines has already been integrated by Mycoplasma virus, and the elimination of Mycoplasma is successfully occurred only in these 21% of human cell lines.  

Further, chemical structure of quinolone and pleuromutilin is closely similar to biotin (Vitamin H), and fucoidan may be effective to eliminate Mycoplasma (see file Feed by measure).  Further, please see Kitikiew S, Chen JC, Putra DF, Lin YC, Yeh ST and Liou CH: Fucoidan effectively provokes the innate immunity of white shrimp Litopenaeus varnamei and its resistance against experimental Vibrio alginolyticus infection. Fish Shelfish Immunol 34: 280-290, 2013).

It is noteworthy that rat-cultured-normal-liver (but transformed) cells of RLC-16 and M are rich in D-aspartic acid,  which might produce the vitamin H (biotin) via D-aspartic acid(biotin synthetase) (see file; D-Asp avidin).   Biotin synthetase gene might be come from bacteria such as Bacillus subtilis, Helicobacter pylori, and also from the plant of  Arabidopsis thaliana (thale cress).   Then, Mycoplasma's gene or putative Mycoplasma's plasmid-gene of biotin synthase might be contaminated and integrated (lysogenized) into the fibroblast's DNA; i.e, biotin synthase gene is essential to the immortality of the host cells.

It is also noteworthy that there has been detected Corona virus (+ssRNA) in biotin-deficiency- and biotin-excessive- patients.   But Corona virus has disappeared by suitable biotin therapy for 13 weeks.  

GSD1b patient (before biotin); Murine coronavirus (MCoV) or Murine hepatitis virus (MHV).

4 mo male (before biotin); Feline infectious peritonitis virus (FCoV).

1y female (before biotin); not present, but biotin therapy or excess biotin (for 44w) induces infection of HCoV.

 32 y female with gait disorder (before biotin); Canine coronavirus (CCoV).

But some persons possess it.

52 y female, mother of 32 y patient (healthy); HCoV.

8 mo female with gastritis; HCoV.

Although normal fetal Hc does not have HCoV, hepatoma HepG2 has HCoV (with and without fucoidan).

Pseudo-cancer liver (survived) contains HCoV (E2 glycoprotein) and MCoV (RdRP) together with DNA polymerase (HBV) and Genome polyprotein (without protease; HCV), one LC tissue of HCC patient (survived; +HCV by clinical test; with Genome polyprotein (HCV) by PDMD method) possesses MCoV (RdRP), and one HCC tissue of HCC patient (survived; +HCV by clinical test;.Genome polyprotein (HCV) and Major surface antigen (HBV) by PDMD method) possesses HCoV (RdRP; 10.4 ug/ mg of cell protein) and X2A protein (Porcine transmissible gastroenteric coronavirus; PTGEV; PCoV; 0.09 ug/ mg of cell protein).   

Therefore, Corona virus may be a marker of the state of inadequate nutrition.   Then, prevention of SARS- and MERS- corona virus (+ssRNA) may be achievable by the suitable nutrition supply and/or fucoidan (Mozuku) feeding (see file; Feed by measure,,).

This could very well be a mycoplasma infection with one of the cell-adhering species such as M. hyorhinis. As I see the Picture, the dots are lining the cell membrane, typical for mycoplasma infection seen in DAPI or Hoechst staining. Have no experience with PicoGreen, however.

The rapid decrease in pH would be consistent with an M. hyorhinis infection as this species is a glucose fermenter.

Best thing you can do is to find a lab that can test your cells with a PCR test - do not rely on the enzymatic or immunological tests on the market.

Good luck

This looks potentially like Mycoplasma contamination, and I concur with Dr. Jensen that the rapid acidification is further suggestive.  The best confirmation would be mollicutes-specific 16S or RpoB PCR.  Sorry to add to the suspicion! 

Thanks for the query.

There is only way out that you must go for mycoplasma group specific PCR test. The protocol of how to conduct it is available in the references. It is with 16S rRNA. The product size probably of 270 bp will confirm the contamination of cell line.

I concur with Meghan May, Jorgen Jensen, and Rajneesh Rana. This probably is mycoplasma infection.  You can test the cells with PCR to verify this. However,  unless this  is an extremely valuable cell line that is not easily replaced, throw them all away and start over with a verified mycoplasma free cell line from a reputable source.  It's easier and cheaper than trying to 'cure' the cell line with specialized antibiotics available from some vendors. In addition, the specialized antibiotics may change the cell functions, which could confound your experiments.  Also, since most antibiotics will not kill mycoplasma, but simply make it harder to detect them, it is best to not use any antibiotics at all.  At least you can detect the infection easily and don't waste time and money on contaminated cells and experiments.  Historically, most infections come from humans themselves.  Other sources are other contaminated cell lines that the humans work with, serum, and other biological additives used in cell culture.  the only way to keep this from happening again and again is to use really good asceptic techniques, including LOTS of disinfection of the area, hands, gloves, outside of cell culture container, decrease aerosol production etc.,  and to check cell cultures regularly for the presence of mycoplasmas and get rid of the infected cultures.  Mycoplasma free cell culture certainly can be done, but it takes a lot of attention to details, vigilence, and frequent testing for the organisms so you can cull the contaminated lines.  There are multiple book and book chapters written about this.  Some of them are freely available on Google books. Even if they are old, the same principles still apply.