Tried some flasking recently - Orchid Board

Tindomul · #1 11-04-2006, 12:46 AM

Tried using homemade medium today.
I used a blender to chop the potatoes cuase I have no grater. Worked well. I cooked the potatoes till they got a consistancy of goo, the smell of cooking potato and its skin is interesting. I ended up burning alot of the stuff at the bottom of the pot.
I added 15 grams of agar, a whole banana, chopped up as well, and 5 grams of honey.

In anycase, here is some of what I did:
I cooked the potato and banana and honey water till it was boiling, then added the agar, then boiled it again. Finally I filled jars and sterilzed the stuff in an autoclave.

and here it all goes into the autoclave.
While I waited for that to get sterilized, I prepared the seeds.
I used Aarons method:
First I got some bottles with metal lids, poked a hole in each lid. Filled it with Silicone so that when I punture it with the syringe (that will be filled with seeds) the hole will close itself.
I sterilized the seeds using a drop of non anti-bacti soap, water and some sugar, left it over night. Sucked up any extra water, gave the seeds a bath in 3% Hydrogen Peroxide for 10 Mins, washed them with sterile water.

Tindomul · #2 11-04-2006, 12:51 AM

Okay, fast foward a few days:
Bad news! Three of my 6 cultures have mold! Thats 50% mortality rate!!
WHy ME!! Why ME!!! Oh the HUMANITY!!!
Just kidding.
LEts cross those fingers for those last three who show no sign of mold. WHich leeds me to ask, how long do I have to wait before I can breathe a sigh of relief that I have no mold?
I think I went wrong when I was using the syringe to shoot the seeds into the cultures. I remember accidentaly sucking up air with the syringe and pushing the air with seeds and all into the cultures. The air around me must be really contaminated. No wonder I'm always sneezing.

Tindomul · #3 11-04-2006, 12:52 AM

Okay, fast forward another day or two and:
Well, Here is an update. Two of the flasks are mold free for now. Thanks for the prayers, they seem to be working.
Two of the ones that got mold got it all over. Two others have one little spot of mold, and I sprayed it (using the syringe) with hydrogen peroxide. Do you think its a lost cause???
Pictures will be posted tomorrow.

ScottMcC · #4 11-04-2006, 07:20 AM

were you working under a sterile hood? if not...then you're probably hosed. if so...then you may have a chance.

fungal contamination of tissue cultures is bad, and hard to get rid of.

sorry to be the big downer.

Marco · #5 11-04-2006, 08:26 AM

Hey Tindo. If I ever get seeds I'm coming over to the lab! I hope alls well with the flasking attempts

Tindomul · #6 11-04-2006, 10:56 PM

Quote:

Originally Posted by ScottMcC

were you working under a sterile hood? if not...then you're probably hosed. if so...then you may have a chance.

fungal contamination of tissue cultures is bad, and hard to get rid of.

sorry to be the big downer.

Can you refrase that whole post. Whats a hosed? There were no tissue cultures as far as I understand it. Just seeds.
I did work under a hood. How sterile it was is another story.

Marco · #7 11-05-2006, 07:25 PM

hosed = screwed in not so pleasant way

Tindomul · #8 11-05-2006, 11:30 PM

lol! Ha, I didn't know that.

Marco · #9 11-06-2006, 09:49 AM

Lol @ tindo. Question...where did you get the agar recipe from? did you actually read that book I sent you

I tried opening it the other day and well....i got past a page then

and that was pretty much it

littlefrog · #10 11-06-2006, 12:49 PM

I'd say that the problem with mold is not necessarily your technique, assuming you operated your autoclave properly. Make a new batch of medium and let it sit for a couple weeks, to check the initial sterility. If you cool your flasks the wrong way you will suck in contaminants from the atmosphere (physics!). Lots of things to think about.

It is very difficult to get dry seed surface sterilized. Especially your first time... You might want to try Calcium hypochlorite or even bleach as a sterilant (protocols all over the web). I had really good luck with bleach (I can try to find my notebooks for the protocol, but it was standard).

If your seed was stored for a long time, or stored damp, you will have a very hard time getting it clean. Try again starting with fresh seed. Actually to really narrow down your problems, you will want to try a few different batches of seed.