Under Pressure - preparing flasks

I am continuing to collect equipment and supplies for my Orchid Flasking adventure and have taken another couple of steps closer to success.  A key component of that success will be sterility, and a key tool for that is an autoclave.

Mine was actually designed as a home pressure canner for preserving food (a giant pressure cooker), but that process has the same end in mind: a final product completely free of bacterial and fungal contamination.  As long as it can sustain at least 15 psi of pressure and 250° F it will accomplish that.  In my mad-scientist way, I rather enjoy the anthropomorphic thought of contaminating organisms landing in the freshly made nutrient media and "thinking" they have hit the jackpot, only to be utterly destroyed by this vintage piece of kitchenware.

The Beast - My Giant Pressure Cooker Canner
©2011 ColinFraser

Speaking of vintage, this thing is a classic.  It is a huge 20-Quart Presto from the 1970s and doesn't have any of the annoying safety interlocks of its modern counterparts.  The "Harvest Gold" enamel color attests to its age!  I bought it on eBay for $31 (plus some serious shipping - it's heavy), and I think I got a great deal because the "Harvest Gold" color has fallen out of style.  It reminds me of a Gibson Les Paul guitar from the 1950s whose mustardy color came to be known as "TV Yellow."  It was designed to show up well on black-and-white TV, but wasn't so popular after color caught on.

So, what goes in this monster?  Well, I ordered some pre-measured P668 Orchid Seed Germinating Media from the Orchid Seedbank Project.  Aaron sells it in packets that you just add to a liter of water and dissolve - simple.  I have also been collecting jars to put the media in.  My boss has been kind enough to set aside empty "palm hearts" jars for me, and I think those will make excellent replate flasks, but the most popular thing for Amateur Orchid Seed Germination ("mother flasks") is probably the 4oz baby food jar.  I don't, and never have had, kids; how else would I have time for all this Orchid Madness?!  So, I went back to eBay looking for empty jars.

Cleaning Jars in the Sink
©2011 ColinFraser

I ended up ordering some, and with shipping it worked out to a little over $0.50 per jar (the cheapest I could find them).  During the days while I waited for them to arrive in the mail, I actually looked at baby food in stores - the first time since I had to buy pureed fruit for my breeding Day Geckos years ago.  Imagine my surprise when I discovered that Walmart (I know, I know) has cheap baby food for $0.50 per jar; the food inside is worth less than the glass jar is!!  I could buy full jars, not wait for them to be shipped, feed the contents to my Scottish Terrier, and have them ready to go for the same price and less time than ordering them empty online!  What a crazy world . . .

Preparing to Fill the Clean Jars/Flasks
©2011 ColinFraser

I bought and emptied a few more jars, and my eBay purchases eventually arrived.  I removed the labels and any remaining glue, rinsed them well, soaked the jars and lids in hot bleach water in the sink for a while, and then rinsed them again.  Now my jars were ready to become flasks.

I added the media to a liter of distilled water, brought the mixture to a boil on the stove, and stirred while everything dissolved.  I let it cool slightly and then dispensed it into the jars with a "flavor injector" syringe.  There are almost 34 ounces in a liter, so it was no problem filling 30 jars with one ounce each.  I then put the lids on LOOSELY and arranged them all in the pressure cooker.

Following the manufacturers instructions (which I found online), I sealed it up and then vented the chamber for 10 minutes.  Once steam had displaced all of the air inside, I placed the pressure regulator over the vent, and the pressure began to build.

Sterilizing Heat and Pressure
©2011 ColinFraser

 When the gauge reached at least 15 psi and the regulator began to "jiggle,"  I let it run for at least 15 minutes at that level, before turning off the heat and letting it cool slowly.  My media was now autoclave sterilized and my "jars" had been transformed into "flasks."

The Agar in the Media Turns to Gel
©2011 ColinFraser

As the flasks cooled the media solidified, and I tightened the lids as I removed them from the pressure cooker.  I also intentionally opened one flask and exposed the media to the air for a few moments.  This will serve as my "contamination control" flask; it is virtually guaranteed to be contaminated.  When growth of contaminants is detectable in this flask, I can check it against the other ones, which will still hopefully be pristine.  At that point, I know that the remaining flasks are safe to use for Orchid Seed Sowing.  Neat huh?

Clean and Sealed
©2011 ColinFraser

Finally, I placed the cooled and sealed flasks into my still unfinished glove-box and misted bleach solution on and around them.  I let them sit for a bit; then I donned a pair of gloves, wiped each one, and wrapped some cling film around the lids.  Hopefully, this will prevent the entrance of contaminants if the flasks "breathe" a bit with changes in temperature and pressure.

Now all that's left to do is wait a couple of weeks and see how much, if any, contamination develops.  I have a couple of seed pods that are nearing readiness, so these flasks should be ready just in time!

Dendrobium Magnification

In my continuing quest for Orchid seed pods to practice culture techniques on, I am still pollinating anything that blooms in the Orchid Zone.  Today, that means Dendrobium Hamana Smile 'Autumn Splendor' is up.

Den. Hamana Smile 'Autumn Splendor'
© 2011 ColinFraser

This is a lovely pinkish/purplish Australian Dendrobium Hybrid (with kingianum and speciosum in its background).

It is a compact growing plant with the fragrant sprays of small flowers typical of this group.  Pollination of these is fairly straightforward, even for my first attempt with this genus, but the small blooms mean some difficulty in seeing what you're doing.

Bigger is Better!
© 2011 ColinFraser

I found that using a "helping hands" magnifying glass, which I picked up inexpensively at Harbor Freight Tools, was indeed helpful.  Since none of my other Dendrobiums are blooming, this one is a "selfing;" that is, I pollinated it with its own pollinia.  That means any offspring would still be considered Hamana Smile, but they would no longer be 'Autumn Splendor'.

Here is a helpful video from youtube that shows, in up close detail, how to pollinate this type of Orchid:

Glove Box Construction

I made up my mind that I want to try growing Orchids from seed at home.  I am on a tight budget, so fancy laminar flow hoods with expensive HEPA filters are out of my reach for now; that leaves the "glove box" or "still air chamber" as the best option for maintaining the sterile conditions necessary for starting seeds in flasks.  I have started to build my own DIY version, so here are some photos of the progress so far:

My Mad Scientist Glove Box!
© 2011 ColinFraser

PVC cleanout fitting
© 2011 ColinFraser

Cutting holes in a plastic storage bin.
© 2011 ColinFraser

PVC cleanouts installed
© 2011 ColinFraser

Long Dish-Gloves attached to fittings with hose clamps
© 2011 ColinFraser

Add forceps and scalpels for a sciency look!
© 2011 ColinFraser

I also cut a window in the lid to accommodate a clear sheet of acrylic for easy viewing from above, but I have yet to secure the window to the lid . . .

Pollination Madness

As my very first two Orchid seed pods were developing, I began to think about sowing the tiny seeds inside.  I knew it was more complex and difficult than with most flowers, but I like a challenge, and with my Biology background, setting up a small lab sounded fun (in a geeky sort of way).  It briefly crossed my mind that I could send out the seed pods to a laboratory that would start them for a fee, but then I would miss out on all of the fun . . .

So, I began to collect knowledge and equipment for Orchid Flasking at home - more on that in the next post.  For now, just know that I am getting excited about Asymbiotic Orchid Culture!

I knew that there would be some trial and error while I learned the techniques and procured the materials needed to grow Orchids from seed at home, and I had been thinking about how I could practice.  Where was I going to get a bunch of Orchid seeds to make mistakes with until I got it right?  Then I realized two things; first, I was unlikely to be making award quality plants at this stage no matter how carefully I selected parents from amongst my stock, and second, there was a high probability that some (if not many) of my early attempts could fail to grow or die anyway.

To the left of this Photo you can see the blooming LC Schilleriana I used below.
© 2011 ColinFraser

With this in mind, I decided that quantity was the priority for the time being and made up my mind to pollinate everything that bloomed, in order to start some more "practice pods" growing.  Here are the pollinations I attempted during the first two weeks of november (pod parent always listed first) :

LC Schilleriana (a pink one) x Self = LC Schilleriana  -  There were two flowers, and after sefling one I was going to put L anceps pollen on the other one.  I accidentally touched the pollen I was removing from the second flower to its stigma, so rather than worry about who the parent was, they both ended up as selfings.

Laelia anceps 'Irwins' flowers about to be pollinated.
© 2011 ColinFraser

Laelia anceps 'Irwins' x the same pink LC Schilleriana = LC Schloat Blue (1997)  -  Right now, if two remotely compatible Orchids are blooming at the same time, I am probably going to cross them for practice and see what happens.  I looked up this cross on the RHS website and was amused at the name.  My version reverses the original pod and pollen parents, and both are more or less pink . . . it seems unlikely any offspring would live up to the "blue" moniker.

LC Behrensiana - the other flower met a slug.
© 2011 ColinFraser

C (lc) Behrensiana x C (lc) Aylene - and the reverse  -  These "blue" Cattleyas were both blooming, so it seemed like an obvious pollen swap.

Laelia anceps 'Irwins' x an orange Epidendrum  - weird, but fun . . . 

Epidendrum
© 2011 ColinFraser

I also selfed an extra flower on the Behrensiana as well as the anceps, just to have more chances for more "practice pods."

S. Longifolia flowers
© 2011 ColinFraser

Finally, a small surprise.  I had recently brought home a cute Stenoglottis longifolia from an orchid society meeting raffle.  As I inspected its little flowers, I noted that some appeared to have swelling ovaries.  Sure enough there are little seed pods developing!  Either this one can self pollinate, or some of my local insects are doing the job.  Either way, I decided to try one myself since there are so many sequentially opening flowers on the spikes.  I could barely see the pollinia, even with a magnifying glass!  I'll leave that one to the bees!  I also have no idea how long these take to develop, so I will have to just guess about harvesting/sowing them.

S. Longifolia seed pods
© 2011 ColinFraser