Your picture is way over exposed. Mycoplasma, when present, are visible even with a "normal" exposition. To be able to compare you could include a non infected cell line as reference. What strikes me, is the very homogeneous distribution (soup) of the cytoplasmic signal; usually micoplasma form rather coarse distinct dots. Moreover, on the picture taken with a shorter exposition time no cytoplasmic signal is seen. What you see could well be an artifact due to over exposition.

When cells get old (lots of passaging), they will also tend naturally to grow slower and get more difficult to transfect. Your problem may not necessarily be micoplasma...although it could.

Thiago,

I use to work with fluorescent dyes in living cells and I can tell you that healthy mitochondria should look like a fibers around nucleus instead of dots. On the other hand, stressed mitochondria could render a punctuated pattern but still you have these dots at the cellular border, so for me these correspond to mycoplasm. It makes sense with slow proliferation and morphologic change you describe as well.

Regards, OP.

treat it 2 weeks with plasmocin and try again ;)

Your picture is way over exposed. Mycoplasma, when present, are visible even with a "normal" exposition. To be able to compare you could include a non infected cell line as reference. What strikes me, is the very homogeneous distribution (soup) of the cytoplasmic signal; usually micoplasma form rather coarse distinct dots. Moreover, on the picture taken with a shorter exposition time no cytoplasmic signal is seen. What you see could well be an artifact due to over exposition.

When cells get old (lots of passaging), they will also tend naturally to grow slower and get more difficult to transfect. Your problem may not necessarily be micoplasma...although it could.

Cyrille is probably right. Hoever PCR testing is recommended.

Usually mycoplasma is quite well visible with a normal exposure, with nucleus well visible with clear borders and the additional staining from mycoplasma more concentrated at the cell borders, as mycoplasma likes to be connected with cell membrane. However, in this case two photographs are overexposed and two underexposed, so I cannot say. The best it is to look directly under the microscope and when you see nucleus clearly, then look for mycoplasma.

As say Cyrille your picture is and mycoplasma, when present, are visible with a normal exposition. I recommended you PCR testing to be sure that it is mycoplama. I think is better do not treat the cells with plasmocin if you can use a new aliquot of the cells.

Looks like mycoplasma to me. In my experience it has to be quite bad already to see it with hoechst/DAPI, so even when not visible this way, they could be there. This is why it's important to test by PCR just as the others have recommended.

I have previously found that fetal hepatocyte Hc has already been invaded by bacteria and microbes (please see file; HepG2 fucoidan).

Either Arabidopsis thaliana (thale cress), Caulobacter vibrioides, Lesionella pneumophila, Mycobacterium avium, Treponeme pallidum, Yersinia enterocolitica, Rickettsia canadensis, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Mycoplasma agalactiae, Streptococcus pyogenes, Mycoplasma capricolum, or Streptococcus mutans may be possible.

Further, old established cell-line such as HepG2 (established in 1975) may have no bacterium due to antibiotics, but Bacillus subtilis, Helicobacter pylori, or Enterococcus faecalis may be present.

Furthermore, fresh bloods of Japanese without cancer have such microbes as follows; i.e.,

Bacteria; Streptomyces avermitilis, Streptomyces griseus, Mycobacterium leprae, Mycobacterium vanbaalenii, Mycobacterium bovis, Mycobacterium avium, Nocardia farcinica, Corynebacterium efficiens, Lactobacillus brevis, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus helveticus, Lactobacillus delbrueckii, Mycoplasma pneumonia, Mycoplasma hyopneumoniae, Mycoplasma pulmonis, Mycoplasma genitalium, Bacillus anthracis, Bacillus cereus, Haemophilus influenza, Haemophilus somnus, Escherichia coli, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio cholerae, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, Listeria welshimeri, Listeria innocua, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Enterococcus faecalis, Streptococcus pyogenes, Rickettsia prowazekii, Rickettsia typhi, Richettsia felis, Wolbachia sp., Burkhoderia sp., Flavobacterium sp., Pseudomonas syringae, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas mendocica, Burkholderia cenocepacia, Cronobacter sakazakii, Buchnera aphidicola, Candidatus Baumannia cicadellinicola, Candidatus Blochmannia floridanus, Candidatus Sulcia muelleri, Campylobacter concisus, Arcobacter butzleri, Chlamydophila felis, Chlamydophila caviae, Chlamydia muridarum, Helicobacter pylori, Wolinella succinogenes, Bradyrhizobium japonicum, Azorhizobium caulinodans, Clostridium beijerinckii, Gluconobacter oxydans, Yersinia pseudotuberculosis, Pseudoalteromanas haloplanktis, Shewanella baltica, Shewanella sediminis, Shewanella halifaxensis, Moritella abyssi, Thermoplasma volcanium, Thermoanaerobacter tengcongensis, Enterobacter cloacae, Brucella ovis, Trichodesmium erythraeum, Coxiella burnetii, and Sodalis glossinidius.

Fungi; Encephalitozoon cuniculi, Yarrowia lipolytica, Candida albicans, Neurospora crassa, Pichia angusta, Ajellomyces capsulatus, Pneumocystis carinii, Filobasidiella neoformans, Aspergillus terreus, Aspergillus fumigatus, Aspergillus niger, Ustilago maydis, Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Kluyveromyces lactis.

Protozoa; Eimeria tenella, Ascaris suum, Entamoeba histolytica, Plasmodium lophurae, and Plasmodium falciparum.

Nematode; Caenorhabditis elegans.

Brine shrimp (sea monkey); Artemia salina.

Plant; Pisum sativum (pea), Cajanus cajan (pigeon pea), and Solanum lycopersicum (tomato).

These results of contamination or infection are uniquely obtained by the quantitative PDMD (Protein-Direct-Microsequencing-Deciphering) method. Please, do not use non-quantitative PCR, Western Blotting, and ELISA.