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Quick question about making culture media.


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5 replies to this topic

#1 Wek

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Posted 26 December 2012 - 06:16 PM

I was wondering if a slight difference is serum concentration (say 9.7% vs 10%) and supplements matters when making culture media. The media that I use the most is MEMa plus 10% ESFBS and 1% supplements. The way I make it is by removing 50mls from a 500ml bottle and add 50ml of ESFBS, 5ml of glutamine, 5ml of PenStrep, 500ul of 2-Mercaptoethanol and 5ml of NEAA. I was taught to make it this way and haven't had any problems growing cells but does the 15ml make any difference? Do you guys make your media by removing and adding the exact amount?

#2 Myworkismyplay

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Posted 26 December 2012 - 07:10 PM

I know plenty of people who only add supplements (like FBS, A/A, etc.) without removing any of the base media (DMEM, a-MEM, RPMI, etc). The correct way to do it would be to proceed like you are making a buffer, starting with 60-80% volume of base media (like with water for buffers), add any supplements like 5-20% FBS, antibiotics, antimycotics, etc., then q.s. to final volume with your base media.

The difference in the concentration of serum you are using is very minor, I would not worry about it. If you want to be absolutely sure you would have to repeat your experiments with the correct concentration, of course. The important thing right now for your experiments, if you are in the middle of one, is to be consistent -- make it the exact same way each time throughout the entire experiment so if it does have an effect, it won't be a variable for analyzing your experimental results.
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#3 Wek

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Posted 26 December 2012 - 09:31 PM

How do you exactly make complete media the same way you would make a buffer (start with 60-80% base)? Wont you have to pour out the base media and measure it, increasing the chances of contamination? The media that we buy comes in 500ml bottles so we make 500ml of complete media every time.

#4 madelingirly

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Posted 08 January 2013 - 06:54 PM

if u did not remove medium before adding FBS, cocn of FBS would be 9 % not 10 %.
as it would be 50 FBS in 550 ml medium volume
for accuracy, it should be the same to repeat ur experiment even after years
I seen people just added volume of FBS/supplement without removing any part of medium.
I guess this is not correct.
as if u would write in ur published work I used 10 % FBS medium it must be 10% not 9%.
I guess what
Myworkismyplay


means
That u remove equivalent volume to ur added constituents in clean sterile container, in the clean bench, then added ur supplements (FBS/Antibiotic), the final volume should be 500 ml, or u must complete it to be 500 ml
this is how I understand her words

#5 leelee

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Posted 08 January 2013 - 10:15 PM

I always just add my additives to my media without removing any of the media first. Most people I know (in my lab and others) do the same. I don't think it is a big deal for components such as serum, particularly if you are consistent.

But obviously you need to be accurate when adding a compound that you are wishing to test.

By the way, best practice is to culture without any antibiotics or antifungals in your media. These will mask low level (or "cryptic") contaminations and have an impact on the way your cells behave. If you use correct aseptic technique they are unnecessary.

#6 rhombus

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Posted 09 January 2013 - 08:43 AM

How do you exactly make complete media the same way you would make a buffer (start with 60-80% base)? Wont you have to pour out the base media and measure it, increasing the chances of contamination? The media that we buy comes in 500ml bottles so we make 500ml of complete media every time.


Dear All,

What I do is add 55 mls to the 500ml bottle of DMEM or RPMI. I only add x 100 L- Glutamine (i.e. 5.5mls) and do not use any antibiotics. Therefore the serum concentration is as near to 10% without having to remove 50 mls of precious media that I have payed for. Also as others have said, each addition or manipulation increases the chances of introducing contamination.

Kindest regards and happy new year to everyone

Uncle Rhombus




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