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Use-by-date of antibiotic & serum in media


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6 replies to this topic

#1 science noob

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Posted 19 September 2012 - 06:55 PM

How long can we store media containing 10% serum and antibiotics (e.g. pen/strep) for? if media bottles were stored at 4C?
Or is it advisable to add fresh stocks to aliquoted media when you need it.

#2 leelee

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Posted 19 September 2012 - 09:45 PM

I think it is advisable to only add media supplements to media as you need it. Because I go through quite a bit of media, I add serum and L-glu to 500ml media at a time. I would top up the L-glu after a few weeks, but in reality my media is gone by then.
I'd use media with supplements added up to a month ago (topping up the labile L-glu), but probably no longer than that. And even then it would depend on what I was using my cells for. I'm happy to use older media for flasks I'm infecting to make viral stocks, as I don't much mind if the cells are less than happy. They'll be infected and frozen down for virus extraction in a few days anyway.

As for media antibiotics, it is advisable to not use them at all.
They can have an effect on your cell behaviour, and may cover up cryptic contamination. If your aseptic technique is good, you won't need them anyway- and if your aseptic technique is bad enough to require antibiotics, then you'd probably want to know that so covering it up with antibiotics is no good.

(But of course, I'll defer to whatever Uncle Rhombus says on the matter- he is the gold standard for tissue culture advice Posted Image )

Edited by leelee, 19 September 2012 - 09:46 PM.


#3 science noob

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Posted 19 September 2012 - 11:11 PM

As for media antibiotics, it is advisable to not use them at all.
They can have an effect on your cell behaviour, and may cover up cryptic contamination. If your aseptic technique is good, you won't need them anyway- and if your aseptic technique is bad enough to require antibiotics, then you'd probably want to know that so covering it up with antibiotics is no good.

(But of course, I'll defer to whatever Uncle Rhombus says on the matter- he is the gold standard for tissue culture advice Posted Image )


I've heard two fields of thoughts on media antibiotics. One is the 'better safe than sorry' philosophy - add antibiotics to all medium and keep the bugs away regardless of how aseptic you are. Second philosophy is that antibiotics makes you 'aseptically lazy/slack' and could possibly change the phenotype of the cells you're working with. I belong to the former and has kept consistency since.

Anyone else have any thoughts on this?

Edited by science noob, 19 September 2012 - 11:12 PM.


#4 leelee

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Posted 19 September 2012 - 11:26 PM

Yeh I don't know about the "better safe than sorry" approach, IMHO a person's aseptic technique has to be pretty damned bad to be getting consistent contaminations anyway.

If I'm honest, my main reason for not using antibiotics is because I'm lazy Posted Image

Its one less thing I have to source, order and add to my media. Everything else is a bonus Posted Image

This does seem to be a recurring topic though, and one for which people are firmly on one side of the fence or other. I don't know what the majority of peeps do, so I'd be interested to see what everyone says.

Edited by leelee, 19 September 2012 - 11:28 PM.


#5 bob1

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Posted 20 September 2012 - 01:10 AM

I don't use antibiotics, for the reasons Leelee said. Used to use them when I started cell culture, but finally stopped it about 4 years ago, and don't have problems with contamination.

#6 rhombus

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Posted 20 September 2012 - 05:25 AM

How long can we store media containing 10% serum and antibiotics (e.g. pen/strep) for? if media bottles were stored at 4C?
Or is it advisable to add fresh stocks to aliquoted media when you need it.


Dear Science noob,

When I train staff in the "dark arts" of cell culture, media preparation, usage and storage are an important part of having cells that are happy AND to get reproducable experimental results. The following points may help:-

Always purchase liquid media. In the old days (and some poeple still do this) researchers made up media from powder and then filter sterilised. Purchasing liquid media that has been QC is essential i.e. to reduce adding variables into your experiments.

Aliquot ALL media additives and store at -20oC. Glutamine, non essential amino acids (NEAA) and serum should be stable for upto 3 years. I know from experience that Foetal calf serum/Foetal bovine serum that is not sold by the manufacturers and is past it's expiry date IS RETESTED and given a further 3 year expiry window. Therefore if you have serum that has past it expiry AS LONG AS YOU HAVE STORED IT CORRECTLY (-80oC) you can still use it

ALWAYS BATCH TEST YOUR SERUM

NEVER USE ANTIBIOTICS IN BASIC CELL CULTURE.

#7 rhombus

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Posted 20 September 2012 - 05:47 AM

Sorry pressed the wrong button.......to continue:-

Antibiotics.......are very dirty compounds and will have an effect on your cells. They will put a "selection pressure" on your cells and mask background "Cryptic" contamination.

The basic media's (RPMI and DMEM) are formulated to be buffered with CO2 in the incubator at 5 and 10% respectively. When using the media in the Class II cabinet AND having the bottle open, the pH of the media will be affected. Atmospheric CO2 levels are approximately 0.038%. Thus every time you open your bottle AND keep it open, the pH of the media will become more and more alkaline. When your bottle is full (550ml) the affect is small. However as the volume decreases this effect will be more dramatic. I have seen researchers with 50-100mls of media left in the bottle.....the phenol red now no longer red but purple .....indicating a massive change in pH.
pH again will put a selection pressure on the cells and potentially add variability into your experimentation.

I always purchase media that requires the addition of L-Glutamine (2mM final concentration). Glutamine is one of the most unstable media components. I personally never use the so called stable Glutamax. It's advantage, say the manufacturer, is that you do not get a build up of ammonia in the media. If you use your bottle within a couple of weeks ammonia build up is not seen.

Do not store media in light conditions as it has been reported that fluorescent light increases the free radicals in media. Some of us use silver foil to protect our media from the light.

When warming up media in the waterbath only warm for 5 minutes. Many times I have seen media warming up for hours. THIS IS EXTREME BAD PRACTICE.

Learn good asetic technique from one of the old technicians that have been doing it for many years.

Hope some of this is useful

Kindest regards

Uncle Rhombus




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