Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Cell Biology and Cell Culture
Register Search Today's Posts Mark Forums Read

Cell Biology and Cell Culture Cell Biology Forum. Cell Culture Forum. Post and ask questions about cell culturing, cell lysis, cell transfection, cell growth, and cell biology.


cell culture help

cell culture help - Cell Biology and Cell Culture

cell culture help - Cell Biology Forum. Cell Culture Forum. Post and ask questions about cell culturing, cell lysis, cell transfection, cell growth, and cell biology.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 11-03-2009, 03:12 AM
jtl jtl is offline
Pipette Filler
Points: 9, Level: 1 Points: 9, Level: 1 Points: 9, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2009
Posts: 2
Thanks: 1
Thanked 0 Times in 0 Posts
Default cell culture help



Thanks in advance for any advice offered!

Our lab has just suffered the departure of our long time cell culture technician and now we are experiencing problems with our cell cultures. Most apparent is the pH change in the cultures that appear to be converting to acidity to quickly. We generally change media every three to four days depending on cell line. Have tested for bacterial contamination-negative. I am concerned the quality of the media has been compromised. The new technician began turning very bright flourescent lighting on in the storage area that was not lit previously. Does anyone think this could cause our dramatic lack of thriving cells? I have read that deterioration of components could create toxic by products. This has been going on over the course of a month. Some cell lines ok, some totally dead, generally not thriving.
Reply With Quote
  #2  
Old 11-03-2009, 02:32 PM
Warthaug's Avatar
Post-Doc
Points: 2,383, Level: 31 Points: 2,383, Level: 31 Points: 2,383, Level: 31
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2009
Location: London, Canada
Posts: 203
Thanks: 1
Thanked 68 Times in 61 Posts
Default Re: cell culture help

A fluorescent light in a storage area is not an effective way of controlling bacterial growth - the intensity of the light drops off with the square of the distance, meaning unless something is close to the bulb it'll not get sanitised.

The UV could be breaking down your media, although for the above reason's I suspect that's not the issue.

Did you do microplasma testing? Your problem sounds like micro, and they wouldn't appear on normal bacterial tests. If you haven't done that, I'd recommend MP testing.

Also, have you tried replacing any of the cell lines with frozen stocks? If so, are the frozen stocks also not growing well?

Finally, are you sure you're using the right media for the right cell lines? Some cells are quite sensitive to the type of media and serum concentration.

Oh, and one last thing, are you heat-inactivating your serum first?

Bryan
__________________
[Only registered users see links. ], here at Molecular Station.
Reply With Quote
  #3  
Old 11-04-2009, 02:21 AM
jtl jtl is offline
Pipette Filler
Points: 9, Level: 1 Points: 9, Level: 1 Points: 9, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2009
Posts: 2
Thanks: 1
Thanked 0 Times in 0 Posts
Default Re: cell culture help

Thanks for the ideas!

The lighting was not intended for sanitizing purposes-the tech just liked the additional lighting and left them on nine hours a day-quite intense.

We have gone back to our frozen lines, with similar problems.

I suspect a problem in the production of our MEM. We add Sodium Bicarb and RO water to a powdered MEM and go through a filtering process. We store this and make up a 10% or 2% FBS media as needed.

I am also concerned about the mycoplasma contamination potential.

Thanks again
Reply With Quote
  #4  
Old 11-04-2009, 05:20 PM
Warthaug's Avatar
Post-Doc
Points: 2,383, Level: 31 Points: 2,383, Level: 31 Points: 2,383, Level: 31
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2009
Location: London, Canada
Posts: 203
Thanks: 1
Thanked 68 Times in 61 Posts
Default Re: cell culture help

Have you considered trying prepared MEM? You'd still have to add serum, but it would eliminate any preparation issues in terms of the pH, filtering, etc.

Personally, I make up my media with the serum added to the desired concentration, which eliminates the possible measuring error when adding serum to smaller amounts of media. But I doubt that kind of error would cause the problems you describe.

I'd check for MP before anything else. That, and maybe try prepared media.

Oh, and one last thought - are you certain your plastics are tissue-culture grade? Bacterial and chemical grade plastics often contain components toxic to mammalian cells, and may also be poor substrates for cell adhesion. You may want to double-check that they are tissue-culture quality.

Oh, and one other last thought - in the past we've had issues with some batches of serum not supporting good cell growth. If you haven't done it already, it may be a good idea to try serum from another company, or at least a different lot from the same company. I think the quantities of growth factors can vary largely batch-to-batch...

Bryan
Reply With Quote
The Following User Says Thank You to Warthaug For This Useful Post:
jtl (11-05-2009)
  #5  
Old 02-22-2012, 07:54 PM
Banned
Points: 157, Level: 3 Points: 157, Level: 3 Points: 157, Level: 3
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2012
Posts: 46
Thanks: 0
Thanked 1 Time in 1 Post
Default Re: cell culture help

thanks
Reply With Quote
Reply

Tags
cell , culture


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Human Cytome Project - Update 24 Jan. 2005 Peter Van Osta Cell Biology and Cell Culture 1 08-01-2010 02:18 PM
Human Cytome Project - an idea - Update 19 April 2005 Peter Van Osta Cell Biology and Cell Culture 1 06-01-2009 02:17 PM
A Human Cytome Project - an idea - Update 14 March 2005 Peter Van Osta Cell Biology and Cell Culture 0 03-14-2005 02:27 PM
Human Cytome Project - Update 6 Jan. 2005 Peter Van Osta Cell Biology and Cell Culture 0 01-06-2005 11:18 AM
Tissue Engineering & Stem Cell Research 2005 Andrew Oliver Protocols and Methods Forum 0 12-02-2004 11:38 PM


All times are GMT. The time now is 07:59 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.19200 seconds with 16 queries