I was trying to expand a cell line provided from a colleague but i noticed more than normal cell death in the culture just after the cells where thawed and attached to a t25 flask. Since then, the cells where able to reach confluence , although it took more time than it normally would, but in the mean time i noticed that everytime before changing media and observing the cells, a lot of cells and cell debris where floating in it.
More recently i noticed that the cells where showing a different morpholgy and at the same time i was testing my asceptic techniques using antibiotic free media (in different falsks).
To my surprise i found that the cell line that was showing changes in morphology and cell death was completely contaminated with what appears to be a bacteria when grown in antibiotic free media, suggesting that the cell line was carrying some antibiotic tolerant bacteria (media alone was used as negative control and was fine).
Are this cell lines still usefull for growth inihibition assays using drungs and propper controls? If they do, how can i get rid of this tolerant bacteria?
I check the cells grown in antibiotic-free media searching for this bacteria and found some of them, although they are few in number.
RG_PX: HCC1954 Passage 3
RG_PX+3: HCC1954: Passage 6
DMEMF12 10%FBS in both cases. P/S 1% in the earlier passage and 2% in the later. I just noticed this difference in antibiotic concentration and not sure if this could be generating this changes.
I would suggest to do bacterial culture and to choose the best antibiotic with the recommended concentrations if you still need this cell line.
I would suggest to do bacterial culture and to choose the best antibiotic with the recommended concentrations if you still need this cell line.
You can use contaminated cell line to make growth inhibition assays only when you get rid of the bacteria. Otherwise you will get false results - bacteria affect cell proliferation and viability.
The symptoms sound like mycoplasma, but those bacteria are too small to see at this magnification. I would not use this cell line.
Use less serum 0.5% fbs. Or serum starvation for 12-18 hours
also change media dail. And split cells more frequently rather waiting for more conflunce.
I vaguely remember that Tetracyclines were used in cell cultures to get rid of Mycoplasma, but some products of this class do have an anti-cancer activity. Penicillin, Beta-lactams, have no toxic action on animal cells, but it may contain added things such as potassium, preservatives, with a definite action on cells.
Perhaps this infectious diseases aphorism is applicable to cell cultures:
‘Bugs are much too clever, and if you get rid of all the bacteria, you’re going to get funghi. If you get rid of all the funghi, you’re going to get algae. If you get rid of all the algae, you’re going to get something worse’.
Doc. Sidney M. Finegold (U.C.L.A.),
or this other:
‘Der Eber ist stets missgestimmt, weil seine Kinder Ferkel sind. Nicht nur die Frau -die Sau- alleine, auch die Verwandten, alles Schweine!’.
'Kein Schwein ruft mich an, keine Sau interessiert sich fur mich!'
Gesund +
I am not involved in tissue culture for last two decades.
But from a common sense, I am advising you. You may try Lincomycin, as it has effects on Mycoplasma.
But before that, you have to collect detail information on use of that drug in tissue culture, proper concentration required, availability in usable form etc.
Penicillin - streptomycin, Beta lactams can be for your problem. But before that, as Saeb Aliwaini sir said better to do antibacterial test.
All the Best
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