Hello every:
My stable cell line selected by G418 (selection concentration500ug/ml) have been contaminated during culture (G418 150ug/ml). I haven’t preserved them. Is G418 can kill the bacteria? What is the concentration need to kill the bacteria?
Any suggestion is welcome!
Thank you

Hey,

G418 interferes with protein synthesis in eukaryotes as well as in prokaryotes, so in principle you can kill the bacteria with G418.

The question is if your cells will survive an increase of G418-concentration in the medium or if they just can stand a quite low concentration (150 µg/ml).

Is it possible to increase Pen/Strep in your medium?
Maybe you could split your cells and test different concentrations and see which one is the upper limit for your cells and can kill the bacteria.

Would it be a great effort to repeat the experiment? If it´s an important experiment I wouldn´t want to rely on transfected cells which were contaminated, though you could kill your bacteria.

Anyway I think a possible way is to examine which concentration will work of G418 or Pen/Strep or both.

Good luck
Cheers

huh...
i would better use penicilin /streptomycin in order to kill bacterias... moreover, assuming that G418 acts on both bacterias and cells at protein synthesis level, i think that in order to kill bacterias and avoid resistant apparition, you may be forced to use too high concentrations for cell survival...
but if your cells are not ireplacable, maybe can you just send them to garbage and start with others...

Hi,
theoretically it's possible to use G418, but I would start anew for 2 reasons
1) it's very hard to get rid of bacterial contamination in culture, I have tried on a few occasions, it just tends to come back after a while
2) you'll have to use a high concentration of G418 to avoid having resistant bacteria, but that'll probably kill your cells too.

Also I agree with Bomber in that cells contaminated by bacteria will have activated some genes to fight it, which wouldn't be active in a "normal" state, so your results might be questionable.
Good luck

Thanks all of you for your kindly help!
It will take me about 3 weeks to repeat the experiment :-(. But I will repeat it just as you said 'results from these contaminated cell may be questionable'.

At present,I must use this contaminated cell to do some experiment.So, I still want to retrieval the cell to do some pre-experiment.

Hi, Bomber, you suggested me increase Pen/Strep in the medium, what concentration should be used uaually to kill the bacteria? My cell could survive 600ug/ml G418 and I now use 500ug/ml. The bacteria number didn't change evidently after 24h culture. But I still could find some active bacteria by microscop.

Hi Bluemoon,

hhhmmm...concentration of Pen/Strep... ?
Difficult to estimate, I would use twice the concentration you usually take and see the effect.
The problem is Penicillin is just working for activley growing cells and so it´s not for sure that you will get rid of all bacteria (see point 1 from "kant").

If you know that your cells can stand 600 µg/ml G418 than I would use it. This should ensure that you select your cells quite stringent and maybe helps to free the culture from contamination.

One important point: I´m sure you already did that but anyway:
Do you know the source for your contamination? Get rid of everything that was used for your cells as everything could be the source. This may prevent another contamination in the next experiment.

It sounds that you need the cells quite urgent, however even for pre-experiments I wouldn´t want to use them due to 2 reasons:

1) you have no idea what is changing in your cell culture due to the contamination

2) maybe more important point: you provide a source of contamination in the hood for others who work with cell culture and this should not be: better get rid of the cells .


Whatever you decide good luck with it
Cheers

Thanks Bomer and others,
You are right. The contaminated cell is still questionable even for pre-expriments. Don't let one mistake lead to a series mistakes. Begin another expriment, no other way to go.
Thanks again!