Prepare the media? mammalian cells

[sotellme]

Where should I prepare the media? In the laminar flow hood or on an opened table (risks for contamination?) ?

 

[Sho'Nuff]

Unless you add a lot of antibiotics and antifungal stuff, I'd say the flow

 

[sotellme]

Should I always prewarm my media before adding to cells? And at what temperature?

Thanks.

 

[Sho'Nuff]

Sometimes I am lazy and add cold medium, cells can cope with that allthough they'll need a night or so to recover (restart transcription of genes and the sort) you just put them on hold for a bit so if its for passaging,... no problem,... but if your doing an experiment then I would not do that since you'll be studying effects in cells that have all sorts of stress responses activated.

If your not too lazy prewarm at 37 degrees centigrade...

 

[Monique]

You can take out the medium out of the refrigerator a few hours before you start the experiment, or heat it in a warm bath that has disinfectant in it. You should always work in a laminar flow hood, media for mammallian cells are often rich in nutrients so it is very easy to get contaminations. Adding antibiotics is not always desirable. I've never used antibiotics and never had a problem. I know other people who DO use antibiotics and get infections, you working method has to be clean.

Remember that you sterilize your hood and the outside of the bottle containing the medium and other things with alcohol, also your hands even if you wear gloves, before you start your experiments.

 

[Monique]

Work with E. coli I always do on an open table, as sterile as possible, they grow on minimum medium so there's a low risk of infection.

 

[DocToxyn]

Sometimes it may not be practical in terms of space, time or money to assemble your culture medium in the hood. All of oyur components must be purchaseed already sterilized and then aliquoted into usable quantities and stored under the proper conditions. You may not be able to do this for all media. You can regularly mix all the required ingredients for media on the bench, pH it, and then move to the hood and filter it through a 0.2 micron filter into a clean, sterile container. Once this is done standard sterile practices regarding your handling of the medium apply.