Media preparation for home orchid flasking. Part 2... Replating Media

[newflasker]

Originally Posted by Phalguy

Hello all!
5 days later, all flasks are contaminated ! Sterilization has failed ! Back to square one !
Phalguy

I did many times almost the same as you do without any problem. The only differences are:

- I use pressure cooker instead of oven.
- I put the tape on before sterilize.
- I close the lid right away when I get jars out of the cooker when jars are still hot.

I could be wrong but I believe that when you let the jar cool down it sucks contaminated air in without the protection of a bandage tape.

[Phalguy]

What kind of media do you use? Tell me about your technique!

[newflasker]

I use knudson C modified medium for germination and replating. It's good. I replate my protocorms/seedlings in a carton box. I clean the box by Clorox and spray 70% isopropanol then transfer seedlings to a new jars/bottles.

[bogdan]

Originally Posted by newflasker

I use knudson C modified medium for germination and replating. It's good. I replate my protocorms/seedlings in a carton box. I clean the box by Clorox and spray 70% isopropanol then transfer seedlings to a new jars/bottles.

What do you use for seeds sterilization? I have H202, might it work? (I prepared the sowing media already and I'm afraid to sow the seeds, as I have no contamination and I don't want to ruin them ) )

[newflasker]

Originally Posted by bogdan

What do you use for seeds sterilization? I have H202, might it work? (I prepared the sowing media already and I'm afraid to sow the seeds, as I have no contamination and I don't want to ruin them ) )

Hi Bogdan,
I use Clorox (Sodium Hypochloride, bleach) for seed sterilization. 10 % of Clorox for 12 minutes. You live in Romania so your bleach may be different. In US Clorox has 5% sodium hypochloride with ~ 70 % chlorine. I never try H2O2 but I read in several articles people use H2O2 and they like it. The following links may help you:

Explant Sterilization

http://www.orchideenvermehrung.at/do...0peroxide1.txt

I remember I saw an article which provides more detail on using H2O2 for sterilize orchid seed. It said that you deep the seed in sugar solution for 12 hours before using H2O2 to improve germination. If I found it I will let you know. One suggestion, if you use dry (not green) seeds don't use all at a time; If one got contaminate you can try next. Hope you successfully germinate seeds. Cheers.

[newflasker]

Hi Phalguy,
A question for you. Do you see any good on your potato flakes on orchid seedlings? Some articles said that potato starch doesn't work but potato does. In my homemade medium, if I don't use potato then seedlings have light green color but if I add potato then seedlings look normal dark green and seem get better. I hate to prepare fresh potato; I have to peel, cut, weigh, cook then measure. If potato flakes work it will be more easy for me to make medium.

[Phalguy]

fo me, potato flakes works well.. I try it with an agar base media with success and I tried it with a cornstarch base media with the same success.
I`m having sterilization problem with my vented flasks.... I think I will try the pressure cooker instead of the oven !

Phalguy

[Phalguy]

Hello BogDan!

HEre`s a link:
http://www.orchideenvermehrung.at/cgi-local/framebreaker/reload.pl?english/nodes/in%20vitro/index.htm

[bogdan]

Thank you for that site, this is the website that put me in touch with in-vitro propagation of orchids.
I will prepare another media and will see what happens and then I will post picture of protocorms/mold

[aussieorchid]

Phalguy sorry I took so long to get back to you about those reuseable vent spots I have been looking for the link I am sure it was Home Page - PhytoTechnology where I saw them but I have not been able to find them again on their website although they are listed on their Australian distributor site that I buy things through.


As for sterilisation techniques I find chlorine works best the additive free stuff you use for the pool works well and is cheap. Most of these are around 10% sodium hypochlorite so I mix 100mL of this with 900mL clean water to give a 1% chlorine solution. Make a little packet out of filter paper or coffee filter bags and put the seed into this. Place the paper packet into the chlorine for approx 10mins and shake it around during this 10mins. A drop of liquid detergent will also help the chlorine sterilise the seed but only a drop or two otherwise the chlorine will come out of solution. After 10mins move to the sterile laminar flow or cabinet you are using remove the seed packet and place it into sterile water to rinse. Try to have enough water to give the packet 3 good rinses. After this you can carefully open the packet and dab the seed onto your media. Depending on the amount of seed you used you should get a few flasks out of each packet.

i think I coverd all the steps but feel free to give me a shout if I missed any steps.