Is it necessary to pre-warm medium? - (Apr/02/2006 )

In my lab, I was told to pre-warm medium in 37oC water bath first, and then add the warm medium to cell culture. But I'm a bit concern about the fact that medium change color too quick when it is warmed so I just leave the medium on bench for 30min then add it to my cell culture. I've done it for a month and my cells look normal. Just wondering: Is the cold medium gonna hurt cells in a way that can't be visualized such as desensitize surface receptor, messy up metabolism pathways, etc?

And: My medium is F12+DMEM+FCS+penicilin. It is red when I newly made it. Is it still good if it turns to pink?

I've split cells using pre-warmed medium and also using cold medium and didn't really notice any big difference. Maybe for sensitive cells it matters, but as long as your cells behave normally (morphology, growth characteristics, behaviour in assays or transfections etc.) I wouldn't really worry about it.

If your medium turns pink, you have some pH problems and I wouldn't use it anymore. (Your medium is red due to the presence of phenolred, a pH indicator that'll change color when medium becomes too acid or too alkalic).

depending on the cell type, your cells might unstick from your plate when you add cold medium.

If you see no difference between room temperature and 37°C medium, then it's fine.

You should not warm the complete bottle of medium, I would rather make a liitle aliquote of 50 or 200 mL depending on the time you will need to use the complete aliquote.

another thing to note...some medium is light-sensitive and warming it on the counter will cause breakdown of some constituents, leading to change in pH, leading to pink color, leading to unhappy cells...

a smaller aliquot is really best and it only takes a few minutes if you aliquot the whole bottle at once.

we take a 500ml bottle of media and aliquot it into 25-50 ml volumes right after adding the supplements, all at one. then we store the little aliquots in the fridge away from light. we warm them in the incubator (it's clean, dark, and the right temp) and it only takes a moment with the smaller amount

I would also be concerned about the benchtop, not just because of the light exposure, but also the more your media sits around in regular lab air the easier it is to contaminate it...I would feel that both of these issues would supercede the temperature thing

That's been very helpful. Thanks everyone!!!

HI,
in my opinion, i avoid changing medium temparatures from cold to warm or to 37C. we never know what kind of minute changes will happen inside cell, may be after several repetitions the effects may come into action. i feel prevention is better than cure :-)
come to second point, i support aimikins medium aliquoting method. in my experience i found that if the medium is exposed to air then it start turning into pink color(may be because of CO2 in the air).
i observed that 500ml medium bottle with 400ml of medium does not change to pink color, it does when i have 200ml around. this happend with me several times, based on this observation i assumed, may be air contents are making some reaction with phenol red and changes its color.
members can correct me if my assumptions are wrong.

thanks
gud luck

I am not sure if anyone has replied in regards to this point (I apologise otherwise). If u add cold media, it can stimulate various signalling pathways such as the stress pathways of MAPK cascades (p38 etc). Therefore, it may be experimentally important to use prewarmed media if u are going to study signal transduction and effects of signal transduction.

In referrence to other points. I think light exposure does alter the constituents of the media, however not to any bad effect. For example, I get the impression that the indicator changes colour (from red to pinky/purple). However, this change in colour has had no negative effects on my cells (I generally use DMEM +10%FCS +gentamycin).

I have been lazy and used cool media for routine cell culture, however not for cell culture tasks leading up to experiments (ensuring cells are exposed to minimal stress).

I think aimikins point concerning smaller aliquots is valid if you use just little medium at a time. This way, your medium would be warmed and cooled very often, leading to changes. If you only warm and cool your medium 5 times or so (because having loads of cells in culture) the changes will be minor so I wouldn't bother aliquoting.

you are right Vairus, if you use 500 mL in 3 days, no use to aliquote it !

about the pink color: it's due to pH change, because CO2, which acidify the pH is disappearing in the atmosphere.

if you put this medium back in the CO2 incubator you will see that it become red again.
However, I would not put it on cells if I need them directly for an experiment.