Flasking -101

[mycologist]

Well, I appreciate so much all that I have learned here, and thanks to searching and all, have received so much help here (even though you have never heard of me), I thought I would share this with you all:

I have no experience what so ever with flasking orchids. Nonetheless, I thought it might be entertaining for some to watch as I start cranking out some baby orchids (knock on wood). Basically my motivation here is largely conservation driven (I have some grant ideas on Cypripedium that I am interested in), plus I really like orchids and especially their interactions with fungi. Who knows, maybe a few will get around the forum here as well.

Since I have been doing this type of thing for some time (although not with orchids evar), I have a few items at my disposal:

Autoclave
Lab in a University with:
Class III biosaftey cabinet
Ph meter
Balance
Everything from agar to biotin
Incidentals: everything needed to sequence fungi from the roots of just about any plant.
Also:
Growth chambers (limited)
Greenhouse

Plus I ordered a bunch of orchid media on my own dime.

So, here is a teaser for now:



So why should you care?

One time I went to the arctic tundra and isolated a bunch of mycorrhizal fungi from ericoid plants, grew them out into sterile culture and sequenced and identified them and characterized how they provide organic N to the plants:

Fungus emerging from root


Fungus on its own:


Then I grew them back into the plants, again in completely sterile conditions. There were only a couple so it was easy (this is one of three walls):




Here is a picture a student of mine then took of the fungus growing back in the root:


Any help on this new project would be very much appreciated! I hope to have students do all the stages in a cummulative fashion so that they get to take a plant home at the end. I.e. they do each step but they depend on the previous year's class to keep it going (or what I have started for now).

[smartie2000]

sounds like you have all the equiptment and experience with in vitro techniques. Cypripedium can be trickier since the plantlets still need their winter rest.

[mycologist]

sounds like you have all the equiptment and experience with in vitro techniques. Cypripedium can be trickier since the plantlets still need their winter rest.

Thanks, I am editing the post right now because the point was to ask for help!

[Brutal_Dreamer]

Wow - looks good. I hope you get lots of help on this!

Cheers,
BD

[magooba]

Wow. I get to use a similar room soon, for my noids !! But i really am an amateur, so I'll have to come to this thread, and ones like it again and again.. Great work. Best of luck with your endeavour !! J

[Bluefirepegasus]

Wow...I am so jealous of all your stuff! If I had access to a lab like that.....oh my! I have a horticulture degree (agh! unfortunately have not been able to get a job in my field. Would have loved to be a lab person doing what you are doing with plants) and worked in a lab in college doing grunt work like autoclaving, mixing media, tissue culturing etc. I liked it.

Wow....the artic tundra! Well, good job! I can't offer help......but I do remember an experiment that a co-worker of mine did with orchids. He mixed up some regular agar media and put charcoal in it and then of course went through the sterilization process and then tissue cultured some orchids into the flasks. Then, he set the flasks gently on a shaker. I assume he did this to promote flow/circulation. You could try the same and see what it does for you. Now, he only put them on the shaker after they were well established. Who knows.....maybe it did not turn out for him. I don't remember. But I do know the charcoal agar worked well.

Good luck and don't sneeze in the hood!

Bluefirepegasus

[Gin]

I'm jealous too , just got done flasking my Phal. stobartiana X equestris pod , I smell like a bleach bottle .
I would offer help but know nothing about flasking in a Lab. Gin

[mycologist]

I probably should have waited to start this until I actually had the time I thought I had. I did get some good media and should have everything I need though. I've always felt kind of stupid about the thread title because I am just a beginner and wasn't intending that this would be an authoritative introduction to flasking - I was thinking more about trying to do this with students at school when I wrote it.

I'll get cracking and post up some pictures soon though. Getting students involved is the real trick, the methods I use will just be whatever makes sense and probably wouldn't help much outside of a lab environment.

[Brutal_Dreamer]

I probably should have waited to start this until I actually had the time I thought I had. I did get some good media and should have everything I need though. I've always felt kind of stupid about the thread title because I am just a beginner and wasn't intending that this would be an authoritative introduction to flasking - I was thinking more about trying to do this with students at school when I wrote it.

I'll get cracking and post up some pictures soon though. Getting students involved is the real trick, the methods I use will just be whatever makes sense and probably wouldn't help much outside of a lab environment.

Looking forward to it!!

Cheers,
BD

[mycologist]

Finally some progress. Hope my seeds haven't expired.

Step I: Make some Mother Flasks

Get or make media, and containers. Baby Food jars were recommended but I had some Nunc 50ml centrifuge flasks that are autoclavable. I used the following (cropped to remove supplier's info, but search and you will find):


It contains everything including the agar. I have agar and ingredients, but that is for later and this didn't come without agar.

I used half a bottle to make 500ml. Use a big beaker or container to eliminate loss in the autoclave etc.



You need to dissolve this stuff, so you can heat and stir, or I just tossed in the autoclave for a 3 min. cycle (stupid new fancy autoclave wouldn't open for another 35min. though.). After it is dissolved, dispense into your containers. I shake it thoroughly (with a lid) - if you use foil or something hold it low or the steam by the lid will burn you through terry cloth gloves when you shake it. Swirl between pours or you may get uneven agar distribution (some tubes will be too soft). I used ~25ml each, but it's a big flask and its hot so just do whatever. A drip ring (plastic deal around the top of the flask) helps a ton. If you slop, put on the lid loosely and wash the outside of the mother flask off. Media on the bottle and especially through the thread region can cause icky stuff to happen.



Keep the lids LOOSE and autoclave, I did 20 min. at 121 C on a liquid cycle. Take to HEPA hood and leave hood running for a bit until they cool mostly (they suck in a lot of air as they cool and contract). Close the sash to cool completely. When solid, close the lids tightly and parafilm so they don't dry out. Put in fridge until seeds get sterilized. I had a good fitting rack and wanted to make slants for increased area in the tubes, so I tipped them to cool.



VoilÃ**, 20 mother flasks ready for use.