Flasking -101

[Ania]

This looks like a lot of scientific fun. You put together a nice tutorial.



I have a Rhyncholaelia digbyana seed pot to give away to ANYBODY who would like to try growing orchids from seed. It just crocked open and looks like its full of seeds. PM me if you want it!

Ania

Ps. You can search for my post if you want to see it in flower.

[Feed_me_Seymour]

What exactly is your agar mix? Is it simply activated charcoal and agar? Also if you add a little H2OH to the mix it will keep comtams down a bit. 14.9mL solution to 1-1.5mL of household hydrogen peroxide works. (For fungi at any rate) the idea is to keep spores from being able to germinate. I wonder if this trick could work for the task at hand... Did you experiment with agar mixes at all?

[mycologist]

So far so good:

These are tiny TINY baby orchid embryos (20x-30x magnification)

C. cristata


D. antennatum (you can kind of see the lines of cells)


I lost three of the flasks (of 9) to seed borne contamination the first round, but all of the species still have at least one clean flask and they have all germinated. For the rest I used a sugar pre-soak to encourage spore germination prior to sterilization. For those, only 1/12 was contaminated. I wish I had used clearer tubes, but hopefully they will get enough light until I replate.

ho boy
i just saw this thrid now!!!!!
cool lab man!!!!!
i use the same lab-welcome to the war against fungus!!!!-most with seeds that dosent come from green pods!!!!!
i cut the area with hot sterelized knife the fungus and put some oxigen\bleech\or any solusion that starilized for 12-20 hours and suck it back and hope that i did good
i dont have 100% luck but somtimes it works
u must cut abit from the good agar that didnt got infected too - to make shure for more luck-and i say luck,cause fungus spread the spores and somtimes the spores infect the rest of the petrry
good luck
kfir

Kfir,
Thanks for the info! I just discarded the contaminated tubes since I had made enough replicates. For plants that are further along, I have read that you can sterilize them (dip in 3% peroxide IIRC) and replate them onto clean agar. Pre-soaking in sugar solution made a huge difference! BTW, the fungi in the first set of photos are symbiotic and were inoculated on purpose.

This looks like a lot of scientific fun. You put together a nice tutorial.



I have a Rhyncholaelia digbyana seed pot to give away to ANYBODY who would like to try growing orchids from seed. It just crocked open and looks like its full of seeds. PM me if you want it!

Ania

Ps. You can search for my post if you want to see it in flower.

Ania,

It looks very nice! I would be interested but I haven't worked with any pods yet. If no one else takes them let me know.

What exactly is your agar mix? Is it simply activated charcoal and agar? Also if you add a little H2OH to the mix it will keep comtams down a bit. 14.9mL solution to 1-1.5mL of household hydrogen peroxide works. (For fungi at any rate) the idea is to keep spores from being able to germinate. I wonder if this trick could work for the task at hand... Did you experiment with agar mixes at all?

The media contains everything including the kitchen sink. I haven't experimented with media - the formula worked for all six of the species I tried. There is a lot of literature and work already done so I will rely on that until I meet something stubborn. They also have good Cyp. media I will try next. If you go to the website for the company you can get the info sheet from the product number and it will tell you everything that is in it.

I haven't ever used peroxide in media (but use it for seed sterilization regularly). I don't see how it would work because it breaks down to water when exposed to oxygen in the media. With the opposite approach using a sugar pre-soak to germinate the spores and make them vulnerable prior to sterilization contamination was minimal.

[mycologist]

Update - the majority of the seeds didn't germinate. After referring to a book that shows the development of the embryos I now realize the pic I posted above is an ungerminated seed. I have some green protocorms in only one flask.

I picked up a bunch more fresher seeds and will try again soon with the tropical ones.

I ended up with a several green pods of local Cyp. acaule and started some mother flasks last week - no contamination so far so wish me luck. Of course it had to be the most difficult one