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Breeding Catts: A Photo Journey

This is a discussion on Breeding Catts: A Photo Journey within the Breeding & Hybridization forums, part of the Orchid Propagation category; Aaron's taking himself way too seriously......

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  1. #31
    Piper's Avatar
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    Default Gowning up...

    Aaron's taking himself way too seriously...
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  2. #32
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    Default

    Now he's getting into it!

    I'm including the third picture for two reasons: so you can admire the red band running across Aaron's forehead, and to prove that while he does a great goof look (photo 2), he's really a cute guy!

    We apologize for the glove's rude gesture in the second photo. We weren't aware of it at the time.

    McJulie

    PS - Got some work to do. Stay tuned as our story continues later this evening...
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  3. #33
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    Default Additional comments

    I think Julie did a great job explaining some of the basics. Here is a run down of the fevered mayhem a week ahead of time...


    The fun part (well, its actually ALL fun), and by far the easiest part is the construction of the flasks. Some people like to drop the extra "bank" on fancy pyrex material lab-ware, and while it is perfectly suited to creating your mother and replate flasks, they are A) more expensive and B) you end up breaking them to remove your precious cargo (which is what was my mantra at all of the idiot drivers my 2 1/2 hour trek back home, "I have Precious Cargo! Don't get me in a wreak!"

    Mother Flasks are the first flasks you "plate" or "plant" your orchid seeds on the nutrient agar.

    The Replate flasks are the ones that you move your semi developed plantlets onto fresh agar and space them out a bit so that they have more room to grow. Often this is done more than once.

    the Agar is made up of mostly, you guessed it, Agar (made from kelp normally) that also contains a mixture of sugars, hormones, vitamins and other goodies to help your little ones to grow. It can also have charcoal (which makes the media look black) to absorbe some of the natural by products from the plant development and to help maintain a neutral pH

    I opt to use jelly jards since they are autoclavable, and will maintain a sterile environment. I also opt to use a presure canner instead of an autoclave.

    The process is fairly simple. First boil your jars, lids rings, or flasks and stoppers, in distilled water to sterilze them. The length of time varies depending on what altitude you live, but I boil for a minimum of 20 mins, sometimes longer depending on what stage I am with cooking the agar.

    Distilled water is simply that, just water. Normal tap water comes with its own peculiar mixture of natural salts and minerals (and in the case of treated water) and small amounts of chlorine that could have adverse effects on the developing seedlings.

    I purchase commercial agar, you can check on the web, there are many places to choose from, and people develope a preference as they go along. I found a modified agar w/ charcoal enhanced with banana powder (this is specifically for most epyphitic orchids...except Pleuros) all premixed, so all I have to do is add distilled water, cook and I am pretty much set to go. The commercial media is also pre-packaged to make 1000 mLs, but keep in mind, don't add 1000 ml of water to the media because it will never jell and instead make a sludge. When I cook my agar, I measure out about 800 mL, pour into a CLEAN! stainless steel pan (I don't suggest using Aluminium since it is reactive and could potentially cause problems down the road). I bring this to temp (it doesn't really boil until I put the agar in), and immediatly start stirring (I have the jars still in hot water/maybe a low boil at this point). For stirring the agar/distilled water I use one of those battery operated hand held cocktail mixers since it moves in almost an "orbital pattern" (for those of you with a KitchenAid stand mixer, you will know what I mean), and keeps everything in suspension in the water. After boiling for about 10 minutes, I decant the agar mixture into a 1000 mL volumetric flask where I already have a pre-made mark for 1000 ml. I just simply add warm (otherwise it causes some settling of the agar and you end up with "floaties") water to bring my final volume to 1000. I put in a generous amout of media (some may argue too much), I wipe off the jar lips, cap them, put the rings on loosley, (so I don't end up with exploded flasks) and place aluminium foil on stop and smooth it down , and place them in my pressure cooker set for 15 psi. The pressure cooker has about 3 inches of distilled water in the bottom. After I bring the canner up to temp, and hear steady but not loud hissing, I start my timer. Some people say 10 minutes at pressure is enough, I usually go to 20 since I have to adjust the temperature to maintain steady pressure and not have the pressure release valve sending out jests of steam everywhere.

    I also usally pour a test amount of agar into a saucer and stick it in the fridge for a minute or two to make sure that I have good results with the gelling of the agar (and it is also great fun to tease your cats and spouse with while you're playing with it).

    After the time is up, I just simply let the pressure in the canner drop to zero, crack the lid open for five minutes to start the jars cooling, and tighten down the bands to seal the jars (you end up hearing a little popping sound as the lid sucks down).

    That's it for the flask making part. The reason why I do this a week ahead of the actual flasking is to make sure that the jars sealed correctly and that the White Fluffy Forest doesn't grow and ruin the months and years worth of work that is in store.
    Last edited by ATester; September 28th, 2006 at 05:27 AM.

  4. #34
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    Default Mr Serious

    Quote Originally Posted by Piper View Post
    Aaron's taking himself way too seriously...
    Ehhh, it is the glasses that make me look so serious. I know, I know...Elvis Costello wants his glasses back

  5. #35
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    Default

    Amazing details! Thanks Julie and Aaron for taking the time to put down such details regarding your orchid seed sowing adventures. This thread is going to be one of the gems of this forum, I can tell already.

    Great pictures, stories and info as well. Julie just cracked me up again with her picture milking (ahem ... squeezing the nipple of) the udder made out of the plastic glove. It is so funny!!! It reminds me of my younger days when I had to milk my dad's goats and cows! Great pictures overall. It looks like you guys were having fun. That is the most important thing.

    Keep up with the postings and the pictures. You guys know that we read this thread with the utmost interest!
    Cheers. Doc Spock!

  6. #36
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    Wow, this is great! I love this thread. Thanks to you both for this information and the laughs too!

    Cheers!
    BD

  7. #37
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    I'm sure your efforts will yield productive, interesting results, but if nothing else, we all get pics of you two lookin' cool in your lab coats! I can see that you are both grave and serious scientists. Thanks for letting us in on the whole project!

    Reminds me of a previous life of mine as a medical technologist. Lab stuff, all. But what we had to use the hood for was much yuckier--sputum and stool samples and other nastiness. I think orchids are much more esthetic!

  8. #38
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    Default On to the flasking...

    The first order of business when we got to the hood was to put all of our crap inside and sterilize it. An ethanol spray bottle and the sterile gauze pads came in very handy. First you spray and wipe down Aaron's gloved hands, then the jars. The test tubes, petri dishes and scalpels were sterile in their packages.

    Then you take the seed pods and trim both ends so you just have the capsule itself. I called them the footballs. You trim the excess stem off because it gives you smoother surfaces less place for germs to hide and gives you less to sterilize.

    For green pod harvesting that is, when the pods are cut before they crack open on their own you can assume the seed inside the pod is sterile. If they split open on their own, you have to sterilize the seeds as well. The seed pod on my Catt gaskelliana got a puncture wound several months back some critter was involved, so we had to treat it as though the seeds weren't sterile. More on that in a bit.

    After wiping down the pods, Aaron sliced each one into sections. Because the pod hasn't dried out in prepreparation of the wind to scatter the seeds, the seeds are tightly packed in what we called the chaf. Essentially the pod or capsule's packing peanuts. The chaff is hard to distinguish from the seed in a green pod.

    First a pic of all our junk in the hood. Jar screw tops to the left, jars to the right. Test tubes and petri dish in the center. The little jar with the orange top has sterile water, and there's a bunsen burner in the back on the right which we used later to sterilze a tool.

    Second shot is of Aaron wiping down the footballs with ethanol.

    Third pic is him slicing open one of the footballs.

    The fourth is a closeup of the pod slice. Aaron scraped most of the seed out, and then handed it to me hence, no gloves. We had a dissecting microscope and I examined it under the scope. The seeds were tough to make out, but you could just. The light from the microscope caused the browning that you see on the edges of the slice. We did the Mildred Rive capsule first. Note how thick the walls are! Everything about that plant is huge!

    McJulie
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  9. #39
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    Default Seed, seed and more seed!

    After slicing the pod open, Aaron then scraped out the seed. The point here is to separate the seed from the chaf. Because the seed is heavier, it will sink to the bottom of a test tube, if suspended in water. So that's what you do.

    Aaron started to scrape the seed directly into the plastic test tubes in the first two pics. But you can see how awkward that was. He needed more hands. I would glove, spray and reach in to help, but that didn't work to well, and was tempting contamination. I noticed that my bead bracelet was lying on my glove and hadn't been sprayed...germy! So then Aaron scraped out the seeds into a petri dish, and then transferred them into the test tube. This worked much better. Second two pics.

    Once the seed/chaf is in the test tube, you fill it with sterile water, cap it, shake like hell and then wait for the seeds to settle to the bottom. That was for the sterile seed from the Mildred Rive capsule. Remember I mentioned the gaskelliana capsule had a puncture? We had to sterilize those seeds.

    Normally that would be done by suspending the seeds in hydrogen peroxide, rather than plain water. You may recall from my earlier posts that we forgot to bring the hydrogen peroxide. Ethanol will also sterilize the seed, but too much will kill them. We didn't really know what strength and length of time would be sufficient to sterilize and still safe for the seeds. So we winged it. We tried roughly 5-10% ethanol and left the seeds in it just until they settled to the bottom of the test tube (several minutes.)

    Our procedure was an adaptation of the home flasking/sterilize with bleach method. Because we couldn't use bleach in the hood the guy letting us use his hood couldn't risk the bleach fumes killing his cells, and we were using ethanol, we didn't know the appropriate concentration for sterilizing the seed. Hope we got it right!

    McJulie
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  10. #40
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    Default Final steps...

    After the seed had settled in both test tubes, Aaron poured off the water and chaf. He then used a non-reactive wire loop it was platinum, I believe to spread the seed slurry across the top of the agar in each jar. In the first pic he's lighting the bunsen burner to sterilize the loop.

    In the second photo he's smearing seed.

    Third photo shows the finished flasks. We had more seed from the Mildred Rives pod, so we have three jars of that which we remebered to label! and two from the gaskelliana pod.

    He splashed a little of each batch of seed into petri dishes so I could inspect the seeds. If they don't have an embryo, they're not viable. Even with an embryo they may not be viable, but you have a chance if you see embryos. Both pods' seeds had embryos!

    I couldn't get photos of the microscope view, but here's a picture fourth photo I've shamelessly lifted off the Web, showing what orchid seed with embryos looks like under a microscope.

    McJulie
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