Stigma Turning Brown?

[gixrj18]

Soak them in H2O2 (Hydrogen peroxide) afterwards. I use a small amount of H2O2, so you can pour it right in the flask....seed and all. It's okay to put a little in the flask, because it turns to water anyway.

[Royal]

Originally Posted by vmax3000

What's that? Seriously, I followed recommendations from another orchidist who had successfully plated. His instructions were, loosely, as follows: Sow the sterile seed petri dishes until the first hint of "green", then, transfer them to a "flask" (pace picante jar), then, when they had gotten a plant-like structure, transfer them to their final "flask"....I had gotten to the plant-like structure phase with the phals. The angrecoid seed molded in the petri dish. These were both green pod, so this time will be different in that the pod has begun to open. So, I will be sterilizing seed this time. Should the 5% bleach soln be rinsed off the seed? Should the seed be sowed wet? You guys have me thinking......stand back!!

Your petri dishes were essentially mother flasks. I'm sure they work great, but I just use baby food jars. They seal well, cheap or free, and are made to take autoclaving. They are also tiny, so you can cram a bunch into a small pressure cooker.

The process you describe sounds about right. Just remember that there are very few absolutes. Some won't require replating twice. Some might need it more to grow out longer. Some people sow seed on a multi use media and don't replate at all. Really, the reason we replate is because germination requires a different media composition than is optimal for growth of roots and leaves. They'll just turn green on a lean media, but replate them on a richer substrate and they go nuts.

A glove box is key to maintain sterility. You can just cut holes in a big clear rubbermaid storage bin and attach gloves to the inside. In a clean room with still air, you may not even need the gloves. In a dusty drafty room, you may want to tape the cracks shut. Only open flasks inside a disinfected glove box. Disinfect it with a strong bleach solution or 70% alcohol in a spray bottle. I've used both with good results. I recommend thermal disinfection (autoclave or p-cooker) for your tools and glassware.

I try to rinse the seed after sterilizing it. I think I really just dilute it a little by adding water. I try to decant as much water as I can, then dump it in or use a dropper.

[Becca]

So this may be a silly question....but once you get your materials inside the glove box, say I am flasking a mother flask for the first time and not replating, so I need to spray everything down with a 70% bleach solution, the inside of the container, the inside of the mother flasks with the medium in it, the seeds and all? I need a little video to watch...lol...I am a visual learner. How expensive are pressure cooker...and is autoclaving the process of putting everything in the pressure cooker and sterilizing it? I am so confused...lol...sorry!

[vmax3000]

Becca,
The materials have to be sterilized....flasks, tools and media in flasks can be sterilized in either a pressure cooker (standard off of the shelf number) or an autoclave, if you have access to one. The seed pod or seed, themselves if the pod is open, have to be disinfected using a bleach solution. If the seed is autoclaved....it's cooked!! Mmm, what's for dinner?!? They won't germinate! Once the flasks are sterilized by autoclave they don't need to be opened until you are ready to sow the seed. Royal is talking about wiping down the inside the container where you will be opening the flask and sowing the seed, which is, hopefully, isolated from the rest of your environment.
Royal,
When I sowed the second time, I just used the same media (in a new flask, of course), but no concentration difference... Should I make up a more concentrated form of the media or add banana to the second flask? Thanks guys!

[Royal]

Pressure cookers aren't too expensive, much cheaper than an autoclave! Both increase the pressure to achieve higher than boiling temperatures, sterilizing everything. Look in the canning section at Sprawl-Mart.

At least 70% Alcohol. NOT 70% BLEACH! If you use bleach, mix house hold bleach with water at about 1:4 or 1:3. Bleach fumes kill me, which is why I tried alcohol. Just don't use the weaker alcohol that they sell. The stronger the better. I've even used grain alcohol (98%), but that is a little pricey. What ever you choose, this will be to disinfect the surfaces of everything in the glove box, including the OUTSIDE of the flasks. Use a cloth to make sure the lids and necks are wiped down. Dust is your enemy.

Disinfecting seed is different, and you use a much weaker solution. Forget about the alcohol here, use chlorine or hydrogen peroxide. I've never used peroxide so I won't speak to that. You'll need to put the seed in a vial/tube/jar and add your sterilant. Decant or pipette, with or without a rinse, into the flask, making sure not to touch the inside of the jar at all - with anything but the seed.

The jar/flask/petri dish with the media has already been autoclaved or sterilized in the p-cooker, so it is already completely sterile. I make up my flasks 7 - 10 days in advance so if one is contaminated I won't waste seed on it.

[Royal]

Originally Posted by vmax3000

Royal,
When I sowed the second time, I just used the same media (in a new flask, of course), but no concentration difference... Should I make up a more concentrated form of the media or add banana to the second flask? Thanks guys!

I still have lots to learn about all this, especially the media. I'd look for a "replate" or "maintenance" medium for your replate flasks and "germination" media for your mother flasks. Phyto Tech has a lot, but it gets a little confusing. Look on the OSP site. They re-sell some of the same stuff and he ships for a lot less. Plus, you can order a few test tubes or some seed!