tissue culture and its problem??? - (Oct/27/2009 )

Hi my friends
I want to ask u question


I try to do plant tissue culture and every time there is specific type of fungs apper to me even I consider the aseptic condition so the culture failed

How can I solve this problem??

To complete mywork??

hi noor albayan,

if you're getting contamination it basically is because your conditions are not sterile enough. the problem you're facing might be due to many different reasons, so try to discard each at a time. first, make sure the tissue you're using has been properly treated (e.g. sodium hypochlorite), also you should do all your tissue culture in a laminar flow cabinet, wear gloves, have a bunsen burner near you and once you start try not to take your hands out and seal everything before you move it outside the cabinet.

hope this helps.

maybe due to the environment? Why not discuss with your labmate. if they also experienced the same problem, ask your PI to fumigate the room...

I agree that perhaps you are not using sterile enough conditions but we once had an instance of a yeast contamination on one person's shelf in the growth room that spread to everyone so we had to clean the whole room out.

But be sure you flame all your forceps/blades properly, not just to get rid of bacteria/fungi but also to remove any traces of plant tissue (sometimes callus can get stuck in the grooves of forceps).


Another thing I do is pour all my media at least four days in advance of using it. That way, if it's contaminated I should start to see something growing before I put my plants in there.

Does anyone else have the same problem?

I would suggest to optimize merchuric chloridle that is used for surface sterilization.
Cheer!

Surface sterilize with 99% ethyl alcohol before using mercuric chloride. May be you need to increase the treatment time with mercuric chloride. Please make sure laminar flow is working nicely and you are wearing aseptic dress during inoculation. Hope this help!

Can you try to identify the fungus? From here, you can choose to find out the proper way of disinfection.

Just my 2 cents.
Adrian