8 replies to this topic

[gummybeary]

Dear all,
Could you please tell me whether the small round dot among the hepatocytes are yeasts?
my cells are cryopreserved hepatocyte. After 3 days plating, I've seen this round dot among them
and it proliferate overnight in the incubator. I suspect it's yeast infection.
If so, how to clean up the incubation and hood!!

Thanks alot!!!

[gummybeary]

Dear all,
Could you please tell me whether the small round dot among the hepatocytes are yeasts?
my cells are cryopreserved hepatocyte. After 3 days plating, I've seen this round dot among them
and it proliferate overnight in the incubator. I suspect it's yeast infection.
If so, how to clean up the incubation and hood!!

Thanks alot!!!

[bob1]

It may or may not be yeast - yeast typically bud - so if they are yeast you should see a larger cell with smaller ones coming off the sides, sometimes forming chains of 3-4 cells.

It could be cell debris , which should increase as time passes with culture (if you are not splitting or otherwise diluting the cells) as cells die and leave bits behind.

If it is yeast - it is best to throw out your cells!
Give the incubators a good clean down, especially the water tray with detergent, autoclave the parts that can be and ethanol spray the bits as you put them back into the incubator.  Hoods can be wiped down with IMS, Virkon or Trigene, so long as they are wiped down further to remove detergents from the surfaces... Virkon will corrode the surface if left on for too long.

Make sure your sterile technique is good - have an experienced person watch you to point out any parts where you may have got slack (as happens to the best of us occasionally).  If you brew your own beer or make your own bread, you could be bringing yeasts into the lab with you.

[gummybeary]

Hi Bob,

Thank so much for your reply.
I think I posted twice, the post you replied doesn't come with the attachment which is a picture of the cell culture.
Could you look at my second post with the attachment and let me know what you think?

thanks

[gummybeary]

Please help!

[protolder]

gummybeary, on 04 May 2011 - 03:14 PM, said:

Please help!

Hola, Sorry as Bob1 says you have to trow away your culture, it´s clearly contaminated. Have you add to the medium any fungicide?if you have more frozen aliquots you could cultivate shortly with fungizone, but knowing that you coudn´t cure the cells and have any  hided infection.Buena suerte

Edited by protolder, 04 May 2011 - 10:06 PM.

[rhombus]

protolder, on 04 May 2011 - 10:05 PM, said:

gummybeary, on 04 May 2011 - 03:14 PM, said:

Please help!

Hola, Sorry as Bob1 says you have to trow away your culture, it´s clearly contaminated. Have you add to the medium any fungicide?if you have more frozen aliquots you could cultivate shortly with fungizone, but knowing that you coudn´t cure the cells and have any  hided infection.Buena suerte

Bob1 is again "spot on the money". The photo shows a classic very heavy yeast contamination. Once contaminated with yeast the cells WILL change in some way....Morphology/phenotype etc.

Amphotericin B/fungizone and Nystatin are antifungals which HAVE THERE OWN TOXIC EFFECTS ON CELLS and will therefore further affect the cells as well.

The old adage is " if in doubt chuck them out"


Kindest regards.

Uncle Rhombus

[gummybeary]

Thank you very much for your reply. I would think it's yeast contamination too. So I guess I need a major cleanup of incubator
and hood. Should i just follow BOB's suggestion, using trigene or virkon?

Thanks, and your advise is always appreciated
!!

[bob1]

Yep, definitely yeast... throw the cells out, treatment is almost never fully effective and alters how the cells behave quite a lot as yeasts are eukaryotic and so are mammalian cells - similar pathways activate.