2 replies to this topic

[Suhas]

Ok this is weird and driving me into an asylum

I grow CHO cells that express myoglobin (Wild Type) and those that are knocked out for myoglobin (Rec)

The media I use is Ham's F-12 supplemented with 10% FBS, 1% PS, 1% buferall, 0.02N NaOH and Geneticin

I thaw out frozen vials of CHO cells from liquid nitrogen >> culture them in T-75 flasks (20ml working volume)

Next day I see the entire media has turned green and kinda smelling bad. The cells are all floating around.

Maybe it turned green coz of the pH of media (its got an indicator or something right) ... i dunno what the smell is and i dont suspect contamination although i could be wrong

I have checked the pH of my media - its around 7.4

The incubator is set at 5% CO2 and it works fine... others using different cell lines have no issues

Is there a problem with my media? Am i missing something? I have tried different frozen vials, some which I froze, some which a post doc froze, tried all techniques in the book to thaw them... i don't think its any issue with dmso coz my post doc doesn't do any mistakes

Weird thing is... not all flasks turn green at once. Half of them turn green overnight... remaining turn green in course of time. And this does not happen all the time... i have a set of cells growing in petri plates and they are growing fine. I feel like quitting PhD just coz i can't get this simple damned thing right!!!!! HELP

[bob1]

This indicates contamination I suspect.  Try plating a small amount into bacterial medium and see what grows.

[sbyrne27]

Put it down as contamination and set up a new bank. i wouldnt bother trying to work out what it is as the time could be used better

cheers

Suhas, on 16 December 2010 - 01:12 PM, said:

Ok this is weird and driving me into an asylum

I grow CHO cells that express myoglobin (Wild Type) and those that are knocked out for myoglobin (Rec)

The media I use is Ham's F-12 supplemented with 10% FBS, 1% PS, 1% buferall, 0.02N NaOH and Geneticin

I thaw out frozen vials of CHO cells from liquid nitrogen >> culture them in T-75 flasks (20ml working volume)

Next day I see the entire media has turned green and kinda smelling bad. The cells are all floating around.

Maybe it turned green coz of the pH of media (its got an indicator or something right) ... i dunno what the smell is and i dont suspect contamination although i could be wrong

I have checked the pH of my media - its around 7.4

The incubator is set at 5% CO2 and it works fine... others using different cell lines have no issues

Is there a problem with my media? Am i missing something? I have tried different frozen vials, some which I froze, some which a post doc froze, tried all techniques in the book to thaw them... i don't think its any issue with dmso coz my post doc doesn't do any mistakes

Weird thing is... not all flasks turn green at once. Half of them turn green overnight... remaining turn green in course of time. And this does not happen all the time... i have a set of cells growing in petri plates and they are growing fine. I feel like quitting PhD just coz i can't get this simple damned thing right!!!!! HELP

Edited by sbyrne27, 09 April 2011 - 03:56 PM.