11 replies to this topic

[Neurite]

Hello everybody,

I need your experienced comment here...

I found yeast in my medium and unfortunately, I had already place that medium in my cultures.... I cannot simply throw them away as they just arrived from ATCC and I dont have more vials... So, I washed the plates 3 times with medium with 1% antibiotics and then, left the plates 10 minutes with antibiotics directly from the stock... Then put fresh medium...

Today, after 15 hours, I checked my cultures and they were so clean... I couldnt see any yeast...

Today I washed them twice with medium and then put fresh medium again and left them...

So, my question is, how long should I wait to really see if the cultures are clean ?? I mean, is 15 hours enough for yeast to become visible ???

Can I assume my cultures are yeast free now or, how many hours should I leave the same medium to allow yeast to grow in case they are still there ??

Thanks a lot !!!!

[klinmed]

Neurite, on Jun 28 2009, 02:16 AM, said:

Hello everybody,

I need your experienced comment here...

I found yeast in my medium and unfortunately, I had already place that medium in my cultures.... I cannot simply throw them away as they just arrived from ATCC and I dont have more vials... So, I washed the plates 3 times with medium with 1% antibiotics and then, left the plates 10 minutes with antibiotics directly from the stock... Then put fresh medium...

Today, after 15 hours, I checked my cultures and they were so clean... I couldnt see any yeast...

Today I washed them twice with medium and then put fresh medium again and left them...

So, my question is, how long should I wait to really see if the cultures are clean ?? I mean, is 15 hours enough for yeast to become visible ???

Can I assume my cultures are yeast free now or, how many hours should I leave the same medium to allow yeast to grow in case they are still there ??

Thanks a lot !!!!

Unlike bacteria, yeast can grow quite slowly. You may not be safe yet!

I would grow the cells in the presence of x1 antibiotic (nystatin or amphotericin) for at least 8-10 passages. At each passage you should plate some cells in an additional flask WITHOUT antibiotic.  Incubate this flask for at least one week to check for yeast growth (then discard).  If the antibiotic-free cultures remains sterile at 3 consecutive passages you should be fine. You can also try plating the culture medium onto some agar medium (like YPD) at each passage to check for yeast.
Good luck, but be warned that this could be tricky.

Hope this helps.

[Neurite]

Oh !! Thanks for the info klinmed !!!

I will follow your advices !!!
Ah ! Silly yeasts !! Hate them !!! Why in my new cells, why !!!

One more question, do yeasts kill the cells or, I have time to try to eliminate them ?

Oh, Im having a hard time...

[Neurite]

2 more questions:

Has anybody here used the recipe that comes at the Freshney book ? With dissection BSS ? Did it work ? Did the cells "liked" that buffer ?

And, do yeasts centrifugate at the same speed and time like cells ?? Could centrifugation help me eliminating more yeasts ?? Im thinking cells are heavier ??

Thanks !!
Please help me !!

[bob1]

yeast and mammalian cells will sediment at almost the same rate in a centrifuge, as density rather than mass is the critical thing there.  You are better off changing the medium with antibiotic frequently and doing as Klinmed suggested.

Actually, you are best to get fresh cells from the ATCC!!!!  Antibiotics that kill yeast are also quite damaging to other eukaryotic cells.

[Neurite]

Yes you know, if the lab and the money were mine, I would buy more cells but we are a poor lab and I think my boss would first kill me and then buy me more cells

They had just arrived last monday !!!

Will try with antibiotics. I have to

bob1, on Jun 28 2009, 06:30 PM, said:

yeast and mammalian cells will sediment at almost the same rate in a centrifuge, as density rather than mass is the critical thing there.  You are better off changing the medium with antibiotic frequently and doing as Klinmed suggested.

Actually, you are best to get fresh cells from the ATCC!!!!  Antibiotics that kill yeast are also quite damaging to other eukaryotic cells.

[GeorgeWolff]

Appreciate your cocnern but what would be (will be?) your boss's reaction if he understood you had contaminated the cells and perhaps compromised the integrity of the culture and subequent work to clean it up?  Perhaps it's better to be thought of as a (momentarily) sloppy scientist with high expectations than the same willing to compromise to save face.

[rhombus]

Neurite, on Jun 28 2009, 01:16 AM, said:

Hello everybody,

I need your experienced comment here...

I found yeast in my medium and unfortunately, I had already place that medium in my cultures.... I cannot simply throw them away as they just arrived from ATCC and I dont have more vials... So, I washed the plates 3 times with medium with 1% antibiotics and then, left the plates 10 minutes with antibiotics directly from the stock... Then put fresh medium...

Today, after 15 hours, I checked my cultures and they were so clean... I couldnt see any yeast...

Today I washed them twice with medium and then put fresh medium again and left them...

So, my question is, how long should I wait to really see if the cultures are clean ?? I mean, is 15 hours enough for yeast to become visible ???

Can I assume my cultures are yeast free now or, how many hours should I leave the same medium to allow yeast to grow in case they are still there ??

Thanks a lot !!!!

Been doing cell culture 31 years and have never been sucessful in washing away a yeasy or bacterial contamination.

Using antibiotics/antimycotics doe not work either AND YOU ARE PUTTING ON A SELECTION PRESSURE ON YOUR CULTURE.....selecting cells that are resistant to bugs AND resistant to the chemical agents you are adding.


GET CLEAN CELLS FROM THE ATCC

Kindest regards

Rhombus

[Neurite]

2 weeks later...

My cells are growing now in antibiotic free medium and still, no yeast....

If anybody once, has this same problem, I suggest you try to save your cells...

Good luck everybody.

[GeorgeWolff]

I suggest you start over - think scientific rigor.

[eldon]

Neurite, on Jul 12 2009, 07:15 PM, said:

2 weeks later...

My cells are growing now in antibiotic free medium and still, no yeast....

If anybody once, has this same problem, I suggest you try to save your cells...

Good luck everybody.

the next time you pass your cells spin down the remaining culture, aspirate, resuspend in 200 uL water and plate on YPD at 30C for 10 days. use sterile DMEM for a neg. ctl.

you'll know if there's yeast.

better yet, follow the best advice about starting again with fresh cells.

[leelee]

I agree with bob, George, eldon and Rhombus- start over, you can't trust any results you get with your cells now.

Edited by leelee, 13 July 2009 - 10:34 PM.