8 replies to this topic

[alvaro]

Dear all, I have a question...may be a stupid question, but I hope somebody could help me. I have penicillin G sodium podwer and I dont know exactly how to prepare it for culture medium. Yes!!! I know that if I work with care in aseptic conditions, I will not need antibiotics...but my room for cell cultures is not the best room to do that, but is the only solution my institution offer to me. So I prefer using antibiotics (pen and strep) in my cultures.

I need to know how to prepare penicillin and Strep. Is recomended to use 100 units/ml of penicillin and 100ug/ml of Strep. I have each other in powder so... I have to take the exact quantity and put directly to the medium and then filter all...or is better to have aliquots of penicillin and use this aliquots for adding to the medium and then filtter??

I dont know how to do this. I hope somebody could help me!! thanks!!

[protolder]

Hola, although some people prefers don´t use antibiotics because they can hide latent contaminations, it is safe and extend life of cells. In my experience is better gentamycin that pen/strep but if you have them, you could prepare the mix 100 times concentrated, sterilize by o,22um filter making aliquots of 10 ml to add to 1 l. of medium. In the case of penicillin you have to remind the potency that varies from 1400 to 1800 units/mg. Good luck

[Ameya P]

Dear all, I have a question...may be a stupid question, but I hope somebody could help me. I have penicillin G sodium podwer and I dont know exactly how to prepare it for culture medium. Yes!!! I know that if I work with care in aseptic conditions, I will not need antibiotics...but my room for cell cultures is not the best room to do that, but is the only solution my institution offer to me. So I prefer using antibiotics (pen and strep) in my cultures.

I need to know how to prepare penicillin and Strep. Is recomended to use 100 units/ml of penicillin and 100ug/ml of Strep. I have each other in powder so... I have to take the exact quantity and put directly to the medium and then filter all...or is better to have aliquots of penicillin and use this aliquots for adding to the medium and then filtter??

I dont know how to do this. I hope somebody could help me!! thanks!!

Alvaro,

If you are asking how to make solutions of penicillin and streptomycin, then you should dissolve the antibiotic in ddH2O/ ethanol (I dont know what you use for Pen and strep.) and obtain desired concentrations of the antibiotic. You should filter it with 0.22 uM filter and store it in the fridge (in aliquots)and add it to the medium when required.

[Piersgb]

You could just buy Pen-Strep 100x solutions from somewhere like Invitrogen. They're not very expensive at all! As these products are the standard used in cell culture, you wouldn't need to worry about validating how you made up your own Pen-Strep solutions. I think it could save you some time and effort..!

http://products.invi...=search-product

[alvaro]

Ok, I think I will buy it already prepared from any company...anyway, the question now is:

the pen/strep solution should be made with H2Odd or with culture medium?? I supose it should be diluted with culture medium (DMEM in my case) because at the end I will add it to 500 ml of complete DMEM, so I will need 440ml of DMEM + 50ml of FBS (10%)+ Pen/Strep diluted in 10ml of DMEM ...isn't it?

another question is if I have to filter the p/s solution (10ml) before adding it to the DMEM or I can add to the DMEM (500ml) and then filter it all together???

thaks so much!

Edited by alvaro, 30 September 2010 - 06:35 AM.

[protolder]

Hola, Yes you have prepare P/S with water, sterile filter and freeze aliquots at -20ºC untill their use at 5ml/tube for 500 of medium or 10ml for 1l of medium. If you buy the sterile medium in 500 ml bottles, you add the sterile P/S to the sterile medium, but if you prepare your own medium from powered components, you can add solid P/S and sterilize by 0.22um filter all together.As the commercial sterile medium comes in 0.5 l bottles, everybody add the 50ml of serum and the 5ml of antibiotics sol, having a final volume of 555ml. Good luck

[alvaro]

Hola, Yes you have prepare P/S with water, sterile filter and freeze aliquots at -20ºC untill their use at 5ml/tube for 500 of medium or 10ml for 1l of medium. If you buy the sterile medium in 500 ml bottles, you add the sterile P/S to the sterile medium, but if you prepare your own medium from powered components, you can add solid P/S and sterilize by 0.22um filter all together.As the commercial sterile medium comes in 0.5 l bottles, everybody add the 50ml of serum and the 5ml of antibiotics sol, having a final volume of 555ml. Good luck

Ok, I have DMEM comercial medium in 500ml bottles. What I had thought is filter 440ml of DMEM and store the remaining 60ml and then add 50ml of FBS (10%). By other side prepare aliquots of P/S solution (x50) with 10ml of DMEM (taken from the remaining 60ml of DMEM), filter it, and then add one aliquot to 490ml of DMEM+FBS.......is good????

P.D: why I have to use H2Odd to prepare aliquots of P/S instead of culture medium??

thanks for all!

Edited by alvaro, 01 October 2010 - 12:48 AM.

[sushik]

P.D: why I have to use H2Odd to prepare aliquots of P/S instead of culture medium??

This is just a guess, but I think that you might run the risk of the pen/strep not fully dissolving in culture medium, as culture medium already has a multitude of salts and nutrients already dissolved in it.

Furthermore, having a pen/strep solution in ddH2O means that you can use it for other types of media, not just the medium you're intending to dissolve it in.

Edited by sushik, 05 October 2010 - 02:32 PM.

[alvaro]

P.D: why I have to use H2Odd to prepare aliquots of P/S instead of culture medium??

This is just a guess, but I think that you might run the risk of the pen/strep not fully dissolving in culture medium, as culture medium already has a multitude of salts and nutrients already dissolved in it.

Furthermore, having a pen/strep solution in ddH2O means that you can use it for other types of media, not just the medium you're intending to dissolve it in.

OK, thanks for your answer!