Has any one successfully decontaminated a contaminated cell culture - (Aug/15/2006 )

Hi everyone,

I've noticed motile microbes in my cell culture lately. It's giving me lots of headaches trying to solve this problem. Can't throw away the cells and so tried decontamination methods.

I was wondering if anyone had ever succeeded in decontaminating a motile microbes or any type of contamination before. Can't really tell if they're bacterial or protozoan. But from the size, I'd guess it's bacteria.

Do appreciate if you could share your experience regarding the following important question...

How long during the decontamination process will you see a significant reduction in contaminating microbes from the cell culture?

Grateful for all your feedbacks.
Thanks.

Some questions to ask yourself :

i) What effect are the bacteria and the toxins released having on the cells ?

ii) What effect are Antibiotics/Antifungals/Mycoplasma removal agents having on the cells?

iii) Why have you got contamination in the first place ?


Everybody gets contamination at some point. However if you are transfecting cells you should at that stage take all precautions when handling them until you have a frozen master stock. If you are handling primary cells, chuck them and start again.
I have never tried to "Clean up" contaminated cells and have never understood the science behind wanting to.

Hi MSG
Rhombus is right. It's preferable to work with uninfected cultures wherever possible and if you continue you need to be confident that the antibiotics/antifungals will not adversely influence your experimental results. There are plenty of comments throughout Bioforum where an adverse outcome has been experienced following the use of antimicrobials.

However, I also understand that sometimes you need to proceed with a contaminated culture. For example, in my own field, the results are not influenced by the presence of antibiotics and it is usually not possible to simply obtain another culture. Sometimes the samples are contaminated before we receive them for testing (eg. miscarried products of conception, some stillbirths etc).

It is possible to keep bacterial and some fungal (eg. yeasts) contamination under control. Purchase a combined broad spectrum antimicrobial solution (eg. pen/strep/fungizone soln) and use at the recommended concentration when required. If you see contamination and you must keep the culture, remove all medium, wash the vessel twice with PBS, replace with fresh media and add antibiotics. Check the culture the next day for evidence of reinfection (you may see some residual infection initially, if so, repeat the wash steps).

All things being equal you should not see evidence of contamination again but you will need to keep adding antibiotics with each new media change. Assuming you subculture into new vessels you might try withdrawing the antibiotics after a couple of weeks but if there is even one viable bug somewhere in your vessel the contamination will reoccur. If your culture appears resistent to the antibiotics I would dispose of it immediately.

lots of thanks.
Guess I'll just have to figure out what can be done to re-transform the primary cells.
Oh ya... can't really pin-point the source of infection but most probably waterbath. I've made a point to myself to have a parafilm sealing the cap of bottles, tubes and whatever immersed into waterbath. Also I've gotten a sound advice from a friend to place a large beaker with distilled water into waterbath and let it equilibrate to 37C before placing bottles into it. That'll ensure that the content in waterbath will not mix with content in beaker.

Cheers,