I am doing some In Vitro callogenesis from explants (internods) on Petri dishes (MS/2 medium), but I got several fungal contaminations during and after incubation period (30 days) despite washing my plants in calcium hypochlorite (Ca(OCl)2), containing few drops of Tween 20.
What can I do to avoid these fungal contaminations? Any help I would be appreciated.
Dear Karim,
I think that is not a problem of the plant treatment.
Sometimes is to difficult avoid contamination problems. I supposed that you working under stricted sterile conditions. When you work with in vitro explants is recommendable that use a laminarflow cabine in which only is working with plants, in order to avoid bacterial or fungal contamination, and also under the fire flame. You can close the petri dishes with paraFilm generating thus a protective athmosphere inside the petri dishes. Its also important the storage conditions for your petri dishes, avoid places wets, which are favorable for fungi germination.
Good luck!
Thank you Elias Hurtado-Gaitán for you reply.
Effectively, I will be follow your recomandations.
But during my experimental no antifungal had added.
Furthermore, all contaminations will be appear with droplet of water in petri dishes (culture chamber) after incubation time.
Attached below a picture that show an exemple of contamination.
Wich type of antifungal can I will be used to avoid these contaminations?
Dear Karim,
You can use mercury chloride. Other good option is disecting the parts of the explants which have been in directly in contact with the calcium hypoclorite, or combine the washed with 96% ethanol previosly to the calcium or sodium hypoclorite treatment.
Good luck!
Handling and surface sterilization is very important, as well as, closing the petri dishes tightly by parafilm under aseptic conditions. So, I agree with Elias, you have to pay more attention for washing internode explants under current water, then dip them in alcohol or MC (mercuric chloride) for a few seconds, finally, with sodium hypochlorite and rinse them in sterilized D.water for at least three times......good luck
Dear Karim,
Do you cool properly your Petri dishes after pouring the medium inside? Sometimes, condensation makes microbial contamination easy. I usually place all the plates containing medium separately inside a laminar flow cabinet until no mist remains and then I put them upside-down inside their own bag (properly sealed) in the fridge until use. Anyway, you can also try PPM as disinfectant, as it it easy to use and remove.
Hope it works!
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