Antibiotics in TC media

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made_in_the_80s
made_in_the_80s's picture
Antibiotics in TC media

Hi,

I have recently been having some contamination problems in my tissue culture. I have not been using anitbiotics in the media as I know it's not good practice in the long-term (damage to cells and increasing chances of antibiotic resistant bacterial strains etc.).

However, it has been suggested that I could hit the contamination with a treatment of pen/strep for a short while until the contamination is sorted and my cells are happy again.

Having searched the net though, I can't find the answer to a question I ask out of curiosity really: Why is a pen/strep combo used as standard in tissue culture media? why not gentamicin or kanamycin? I've come across a couple of (very old!) papers which say that gentamicin is better and a guidance sheet on invitrogen states that gentamicin and kanamycin have slightly broader antibiotic spectrums.

Thanks!

Ravin
Ravin's picture
Hey I have been using

Hey I have been using Gentamicin @ the concentration of 2mg/L in my cultures since long. The antibiotic is added in my initial revival medium also. The results are good however if you have issues pertaining to fungal contamination pen/strep combo is a good option. About usage of combination I am unaware!!

Shubhangi
Shubhangi's picture
Penicillin-kills gram

Penicillin-kills gram positive while Streptomycin Gram negative bacteria. Its standard antibiotic mixture that controls most types of bacteria.
It has no activity against fungi or mycoplasma.
This product is useful both as a preventive agent and as a treatment for bacterially contaminated cultures.though later i will not recommend.
However, organisms resistant to one or both antibiotics are becoming more common in cell culture laboratories.
One can also use gentamycin or kanamycin or any other. pen/strep is being used "traditionally"
I have been using it since past 5yrs.

Arefeh
Arefeh's picture
In our lab, we use pen/strep

In our lab, we use pen/strep for routine culture, because as you mentioned gentamicin or kanamycin have a wider spectrum and are stronger, so we prefer not to have resistancy against them! In the case of contamination we use gentamicin instead of pen/strep.

PaulaB
PaulaB's picture
Do you keep frozen stocks of

Do you keep frozen stocks of cells? I don't use antibiotics in the media at all and have multiple flasks going on different days that are split on different days. That way if there is contamination the dodgy flask and/or media can be chucked and in a last resort if the other flask becomes contaminated I return to frozen stocks.

heehawmcduff
heehawmcduff's picture
I would certainly avoid

I would certainly avoid antibiotics if possible as I have seen detrimental changes in morphology and behaviour when using pen/strep

Jason King
Jason King's picture
Our lab is also considering

Our lab is also considering switching to using media that is free of pen/strep. The reason for this is that we find that low level contaminations can exist without us realising and they result in altered cell function. In particular the presence of very small levels of LPS in macrophage cultures is a problem.
 
I guess we all keep our TC media in a fridge when not in use. If the media is being used over weeks and months, this makes sense as it protects the more sensitive constituents. However, if you use media as fast as I do there might be a case for leaving the media at RT or even at 37%. Doing this means that there is no danger of using contaminated media without realising it!

g a
g a's picture
made_in_the_80s wrote:Hi, I

made_in_the_80s wrote:

Hi, I have recently been having some contamination problems in my tissue culture. I have not been using anitbiotics in the media as I know it's not good practice in the long-term (damage to cells and increasing chances of antibiotic resistant bacterial strains etc.). However, it has been suggested that I could hit the contamination with a treatment of pen/strep for a short while until the contamination is sorted and my cells are happy again. Having searched the net though, I can't find the answer to a question I ask out of curiosity really: Why is a pen/strep combo used as standard in tissue culture media? why not gentamicin or kanamycin? I've come across a couple of (very old!) papers which say that gentamicin is better and a guidance sheet on invitrogen states that gentamicin and kanamycin have slightly broader antibiotic spectrums. Thanks!

pen/strep are broad spectrum antibiotics and are used for gram + and -ive contaminants. anti fungal supplements are amphotericin B. Logic of using pen/strep is that they are common antibiotics , are comparatively cheaper than gentamicin / kanamycin etc. and secondly if a resistant strain develops for the pen/strep one can always switch over to the more stronger antibiotics in form of kanamycin sulphate and gentamycin etc that have more broader spectrum of effectiveness and are not coomon and hence are unlikely to have resistant form rather than switching it over vice versa. from pen/strep combination.