Flasking -101

[Brutal_Dreamer]

This all looks very complex! Let us know how it works.

Cheers,
BD

[mycologist]

Think of it like instant cupcakes - just add water and warm to mix, then put it into the cups and bake. It took 2 hours total, and 90 min. of that was cooking time. Hagem's media would be on the souflee end - a bunch of stock solutions to prep and more than a dozen ingredients.

I was actually fairly dissapointed that I would not have to grow a fungus with the seeds.

[Brutal_Dreamer]

Think of it like instant cupcakes - just add water and warm to mix, then put it into the cups and bake. It took 2 hours total, and 90 min. of that was cooking time. Hagem's media would be on the souflee end - a bunch of stock solutions to prep and more than a dozen ingredients.

I was actually fairly dissapointed that I would not have to grow a fungus with the seeds.

Hahah... you are funny. Well, to an arts oriented guy, that all still looks pretty complex. I had a few flash-backs to bio lab when looking at the photos. I am happy it was easy for you and hope you have excellent results. I bet you will.

Cheers,
BD

[mycologist]

Hahah... you are funny. Well, to an arts oriented guy, that all still looks pretty complex. I had a few flash-backs to bio lab when looking at the photos. I am happy it was easy for you and hope you have excellent results. I bet you will.

Cheers,
BD

Thanks BD, glad you were quick to pick up on my dry humor.

The methods I am presenting are tried and true, although some adjustments may be necessary. So if it doesn't work I will have to take the blame!

The major items I am relying on are an autoclave (a big pressure cooker but you just press a button and come back later) and laminar flow hood (or biosaftey cabinet like mine). If you have any connection (or can make friends easily) with a biology department I would bet you could find someone with access who is interested (botanist, mycologist, or microbiologist). Otherwise, a flow hood just requires a hepa filter, a fan, and a working space. The filter goes in the back and the sterile air blows over the working area constantly. You can also use a "glove box".

You may want a drink before I post the next part.

[mycologist]

Sowing seeds:

Prep work - Sterilized filter apparatus, 5-10% bleach solution with a tiny tiny drop of soap in sterilized flask, autoclaved distilled water, sterile pipettes & handle, sterile disposable petri dishes, sterilized microspatula, tweezers, alcohol burner (with 95%), 95% alcohol in dipping well, 70% alcohol in sprayer & LABELED mother flasks with species and date. My filter apparatus has a magnetic seal that sandwiches in the filter disk. This keeps the filter from floating up (which would cause major seed loss). It's pretty cool, you'll see it in a bit after a preview of my bald spot. You want a filter that is not porous or felty to trap the seeds, and that will drain fast. You should not need to pull a vacuum on them.

And the best part - seeds.

Spray your hood with 70% alcohol and wipe, allow to dry. Organize your space, and don't forget the burner like I did. Flame the spatula.

By far the most difficult step - opening the seed package without flinging them.

Put some in a petri plate. A little goes a long way. These Den. antennatum were huge compared to the others.

I did not do a pre-soak in sugar water. It sounds like a good idea, but I didn't fit it in.

Add bleach solution (I used 6%) and stir for recommended time (I did 6 min.). They like to clump up so get them spread apart so all surfaces are exposed and keep them wet.


Pour & rinse petri plate (with sterile water) into filter apparatus, drain, rinse a few times with sterile water. Rinse them all down from the sides of the apparatus and onto the filter disk.


The top of the filter apparatus pops off, the magnetic ring is around the filter area (the whole thing goes in the autoclave with the filter installed before starting).

These are the Coelogynes IIRC - they look like pollen. Hopefully they are sterile now!


Flame tweezers. Open lids on the appropriate mother flasks & pour out any condensation (it will just keep you from spreading the seads around easily). Using just several drops of water, rinse a portion of the seeds into a flask. I made three flasks per species from the one petri plate.

Swirl the seeds around to spread across the surface, close the cap tightly & parafilm, and lay down (they are like weebles, they always end up rolling to where the media is down).

That's it, I just have to put them in a good spot, check for contamination, and wait.

Advice and input is always appreciated.

[mtequine]

WOW, this is a great thread! Makes me miss being a bio teacher! Although I never had access to such fun equipment and facilities! Keep us posted on your progress!

[muis01]

All these pictures are great and show perfectly how it works, especially for someone who doesn't have the time or infrastructure to do this.
I hope you will send some updates now and then, so we can follow the changes and growth!

[kfir from israel]

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

[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?