sryknr

Hi everyone,

I have such a big problem unfortunately. I would like to share it and hope that someone will help me.

Whatever i did about sterilization about the suckers, it did not work properly. I mean there are so many contaminated suckers at growth chamber. Last time I tried two stages of sterilization, first the suckers were washed under the sink for 30 mins than i dipped the suckers in %96 ethanol solution for 30 seconds, than %25 NaOCl solution for 15 mins, than rinsed 3 times 1-2-5 mins distiled water. At the end of first sterilization stage the outer layer of suckers are removed. Than at the second stage of sterilization starts and the suckers are waited in %10 NaOCl solution, than tinsed 1-2-5-10 mins distiled water. Than we remove one more layer of banana suckers, than we placed them on the culture media.
But almost all suckers are darkening and bacterial contamination never ends
We used Gentamicin and Na-ampicilin with AgNO3 but it was not enough.

The other problem is multiplication. I mean we use BAP and 2IP but there is no shoot forming, rarely we observe green leaves and elongation but this is also not what we want.

If you have any idea about solution, that would be great.

dries666

Hello, I used to do an internship in a tissueculture lab for bananas last year and I was wondering about your working area. It doesn't matter how much u sterilize if u can't properly work in a sterile environment... how do u handle the sterilised meristems when putting them into the culture recipients? One other thing I find is that there might be an issue of oversterilising? In a way, if u completely sterilise something, u do this by killing off every living cell... including banana cells, so maybe the sterilising method is too effective, not only killing off the germs but also the banana meristems? Another thing is the antibiotics u add to your medium. I never recalled the lab using antibiotics in the culture medium, I might be wrong but I think they only autoclaved the medium after it was prepared and just made sure they were working in a horizontal flow cabinet to Ensure nothing contaminating would enter the culture tube.

So I'm not really sure what's going on at your place from your description, I would also think pictures of your cultures would help a lot in figuring out the problem. Anyway, my pointers are just some of the things that might be going wrong in the culture... I think it's not hard to master but it takes some trial and error, together with the right knowledge.

Friendly greetings and good luck with your cultures,
Dries

merce3

to create a sterile working environment you can take a large see-through tupperware container, cut holes and tape some gloves so that you can put your hands inside. then clean the inside thoroughly with alcohol, and work with your material inside the box.

__________________

sryknr

I did all works with sterilized tools in a laminar flow cabin, but it was not enough

obdiah

new to tc my self just beginning to experiment but I noticed you rinsed in distilled water distilled water dosent necessarily mean sterile water depending on how it s handled after distillation I usually place a a quart or two in the pressure
canner i use to sterilize my media and keep it sealed until use that way I know its sterile even the best tissue prep dosent work if the rinse isn't sterile keep us posted on your progress

sryknr

all of the water that i used was sterilized.....the problem is endogenic bacteria

obdiah

very interesting next questions to ask and bear in mind I am an uneducated hillbilly high school drop out so if I say something stupid ignore it . is the lack of shoot multiplication and the bacterial problem related in any way? and do you have any way of identifying the bacteria and could the bacterial contamination be internal to your x plants? as in cellular?and not destroyed by an external dip ? think out side the box it gets results
good luck and hang in there