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A solution to a weird problem - pH


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#1 Suhas

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Posted 20 December 2010 - 07:13 AM

Few days back I had posted a problem on this forum that my CHO cells are dying coz of some mysterious reason and the media appears lemon green in color and stinks

Although i suspected contamination, it was actually a pH issue - it was around 6.5 making the media very acidic and hence the cells failed to adhere and died

We buy commercial Hyclone Ham's F-12 media and supplement it with 10% FBS, 1% PS, 1X Bufferall, Geneticin, 100mM NaOH and 10mg per ml Hemin - this leads to a pH of 6.5. This is weird coz i never remember adjusting pH for commercially bought media anywhere - normally the pH is always mainted at 7.4.

Moreover, I'm scared to take freshly prepared media out from the hood coz our pH meter is a lab across and I fear contamination. I normally filter sterilize the media after pH adjustment but will this suffice? My post-doc who made this protocol for media preparation refuses to believe that pH needs to be adjusted but I have checked the pH and its always acidic. I HAVE to adjust the pH unless i'm doing something stupidly wrong here. Coz once i make the media, its golden in color. The moment i add a few drops of NaOH - it becomes pink.

Give a look at my media constituents? Is something wrong? And will taking my media bottle out to the adjacent lab, adjusting pH and re-sterilizing it by filtration still risk contamination?

SuHas

#2 bob1

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Posted 20 December 2010 - 01:25 PM

pH 6.5 is quite acidic for cell growth. What is the Hemin dissolved in? Cell culture medium should not smell any worse than bacterial medium, though the hemin may change this (I've never worked with it) - I still suspect that you have contamination!

#3 lab rat

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Posted 20 December 2010 - 09:25 PM

If the solution turns immediately after adding the components, I would start troubleshooting by evaluating your prep method.

I see you add 10 mg/ml hemin. Bob is on track with the prep question. Is this prepared in a basic solution, then added to the medium? Or is it added directly to the medium? What happens when you add the all the other supplements but not the hemin? After adding FBS, the medium should be orange-red. Clear PS and (yellow?) Geneticin shouldn't effect a significant color change. I'm not familiar with Bufferall.

Good luck.

Edited by lab rat, 20 December 2010 - 09:26 PM.

42..."An immutable fixed-precision number of unlimited magnitude." <a href="http://en.wikipedia....amming_language)" target="_blank">http://en.wikipedia....amming_language)</a>, accessed 25June2009.

#4 Suhas

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Posted 20 December 2010 - 11:45 PM

I dissolve the hemin in 100mM NaOH and then add into media

I just realized something!

My media preparation protocol asks me to add 2ml of 5M NaOH into 500ml media, but i have been adding 2ml of 100mM NaOH by mistake!!!!!

Do you think this is the problem of why my media is ending up being acidic?

Suhas

#5 phage434

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Posted 21 December 2010 - 05:04 AM

Yes.

#6 bob1

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Posted 21 December 2010 - 01:45 PM

What about the smell - does hemin smell?

#7 Suhas

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Posted 22 December 2010 - 09:07 AM

Actually no.

I'm still not sure about the smell.

I guess it happens because earlier, the media would get very acidic and all my cells would die - and cause the smell? Maybe they secrete some byproducts. I dunno.

Now after i adjusted pH to 7.4 in my freshly prepared media, my cells are happy and i'm getting good results

so it may not be a contamination issue. Btw i did not use hemin in my new media (coz hemin doesn't dissolve easily and its black in color and discolors my media). my cells still grow fine




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