Homemade medium?

[Tindomul1of9]

I ended up using 15 grams of agar which gave me the desired consistancy of jello. I'm crossing my fingers that there is no contamination. But if there is, its not a big deal becuase this is just my practice culture using NOID Phal seeds. A learning experience. All I know is that the Phal has white flowers, of the typical moth shape.

[Tindomul1of9]

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.

[Korxi]

thats just to bad.. I really hope the rest will survive!

That bit of air is probably the problem! I dont know how you did the whole sugarwater, peroxide thing, but I read a guide that I really think is a good guard against contamination, here is how it goes:

1. Put the seeds in 5% sugarwater in a small cup or glass, cover and leave for 12 hours or so.
2. Tak out your syringe and suck up as many seeds as you would like to sow (without the syringe).
3. Hold the syringe with the top pointing upwards (the seeds should be on the bottom - if not reverse) and push out as much sugarwater as possible.
4. Now suck up aprox 3 ml. peroxide. Now you need to get all bubbles out, you do this first of all by tapping the syringe, then you put your finger on the tip of the syringe (still without needle!!) and compress the peroxide and then make a vacuum (watch out it could spray out or suck in alot of air if you are not carefull!) do this a couple pf times till all bubbles are completely gone.
5. Leave the seeds in the peroxide for about ½ hour, and then puch out most of the peroxide, you can leave a bit behind (it will just turn into water and oxygen in the jar/flask)
6. Now you put on the needle and push until a bit of peroxide sprays out.
7. Disinfect the silicone plug in the lid of your jar/flask with alcohol and push the needle through, push out as many seeds as you like and tilt the jar/flask a bit to get the seeds dispersed.
8. Pull out the syringe and seal the silicone plug with a bit of silicone or tape.

In this way I think you are prety sure the seeds won't get contaminated when sowing. - At least I hope cause that's what I'm going to do :P

Christian

[Diane]

Oh the HUMANITY!!!

Shouldn't that be Orchanity???

[Tindomul1of9]

Hmmm, I didnt do all that. Well there is always next time. This is just insane how sterile everything has to be.

[Tindomul1of9]

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???

[Korxi]

I have no idea, but I'm keeping my fingers crossed Any signs of protocorms yet? can we get a picture btw?

[ATester]

thats just to bad.. I really hope the rest will survive!

That bit of air is probably the problem! I dont know how you did the whole sugarwater, peroxide thing, but I read a guide that I really think is a good guard against contamination, here is how it goes:

1. Put the seeds in 5% sugarwater in a small cup or glass, cover and leave for 12 hours or so.
2. Tak out your syringe and suck up as many seeds as you would like to sow (without the syringe).
3. Hold the syringe with the top pointing upwards (the seeds should be on the bottom - if not reverse) and push out as much sugarwater as possible.
4. Now suck up aprox 3 ml. peroxide. Now you need to get all bubbles out, you do this first of all by tapping the syringe, then you put your finger on the tip of the syringe (still without needle!!) and compress the peroxide and then make a vacuum (watch out it could spray out or suck in alot of air if you are not carefull!) do this a couple pf times till all bubbles are completely gone.
5. Leave the seeds in the peroxide for about ½ hour, and then puch out most of the peroxide, you can leave a bit behind (it will just turn into water and oxygen in the jar/flask)
6. Now you put on the needle and push until a bit of peroxide sprays out.
7. Disinfect the silicone plug in the lid of your jar/flask with alcohol and push the needle through, push out as many seeds as you like and tilt the jar/flask a bit to get the seeds dispersed.
8. Pull out the syringe and seal the silicone plug with a bit of silicone or tape.

In this way I think you are prety sure the seeds won't get contaminated when sowing. - At least I hope cause that's what I'm going to do :P

Christian

Hmmm, sound like you use a similar process that I do! As for the contamination, it sounds like you either didn't get up to temp for long enough when autoclaving, or you don't have a tight seal on the flask lid and that allowed the nasties to get in. As far as the length of time you need to let things sit before breathing a sigh of relief...about a week should do. Lately, when I have been amking flasks, I make up more than I need to since I have found I end up flasking some seed for either for myself or for someone who drops an e-mail. Either way, the flasks don't sit around long enough for them to become stale (which I have no idea what the time frame is for that...and I hope I never do!

[Korxi]

I read aprox. 1 year in the fridge... So no worries

[Tindomul1of9]

Sounds good! Now I just hope I get to see some action. For now, its still all brown. Or moldy, depending on the flask.