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Contamination? - (Sep/21/2008 )

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HI all,

I'm culturing GI cancers stem cells from patients, however there are somethings which are either thin layer or mass floating on the medium. Their amounts are increasing time by time. Anyone can tell me what is it? is it contamination? if contamination, are they fungus? Thanks!

-Leonard-

If you culture your medium alone (without any cell), do you get the same thing? Any antibiotics added?

-pcrman-

QUOTE (pcrman @ Sep 22 2008, 12:43 PM)
If you culture your medium alone (without any cell), do you get the same thing? Any antibiotics added?


I'm trying to culture medium alone. I have used 2x P/S usually. In some wells, I have used 5x Kanamycin.

-Leonard-

For primary culture I always use P/S, Kana and Amphotericin B for at least 3 days with daily exchange of medium. Do you get your cells from someone else or are they also primary culture?
If it is a fungus, P/S and Kanamycin alone won't help. You might want to try Amphothericin B. But from my expericence it is almost impossible to remove fungi from a culture.


-vista-

QUOTE (vista @ Sep 22 2008, 06:54 PM)
For primary culture I always use P/S, Kana and Amphotericin B for at least 3 days with daily exchange of medium. Do you get your cells from someone else or are they also primary culture?
If it is a fungus, P/S and Kanamycin alone won't help. You might want to try Amphothericin B. But from my expericence it is almost impossible to remove fungi from a culture.



I get the cells from digestion of patients' tissues. I don't change medium frequently because it will resist the sphere formation of the cancer stem cells. Therefore I just add medium every two days.

-Leonard-

For primary samples including Amphotericin B (Fungizone) is very much necessary. We also have observed this which we thought earlier to be because of stressed cells but later turned out to be fungal contamination!

-Calvin*-

QUOTE (Calvin* @ Sep 23 2008, 12:13 PM)
For primary samples including Amphotericin B (Fungizone) is very much necessary. We also have observed this which we thought earlier to be because of stressed cells but later turned out to be fungal contamination!


But I have used Fungizone in the tissue digestion medium.

-Leonard-

I have now added fungizone with concentration of 1ug/ml in the growth medium 2 days ago, however they are still here.

-Leonard-

QUOTE (Leonard @ Sep 26 2008, 05:33 AM)
I have now added fungizone with concentration of 1ug/ml in the growth medium 2 days ago, however they are still here.


Just take some of your medium and put it in a new well and add fresh medium without antibiotics, culture over night and tomorrow you will see if this is a contamination.
Adding antibiotics will help preventing a contamination, but once there is a contimination, you can keep it down for a little while with antibiotics, but eventually, the contamination will overgrow.
When I isolate primary cells from tissues, I also put pen/strep and fungizone in the dissection media.

-aspergillie-

QUOTE (aspergillie @ Sep 26 2008, 04:39 PM)
QUOTE (Leonard @ Sep 26 2008, 05:33 AM)
I have now added fungizone with concentration of 1ug/ml in the growth medium 2 days ago, however they are still here.


Just take some of your medium and put it in a new well and add fresh medium without antibiotics, culture over night and tomorrow you will see if this is a contamination.
Adding antibiotics will help preventing a contamination, but once there is a contimination, you can keep it down for a little while with antibiotics, but eventually, the contamination will overgrow.
When I isolate primary cells from tissues, I also put pen/strep and fungizone in the dissection media.



yup...I just added fresh medium without fungizone in a well few days ago... the medium is still clear today. However I also added P/S and fungizone in the dissection medium.

-Leonard-

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