The Mad Orchidist: contamination

Showing posts with label contamination. Show all posts

Wednesday, February 29, 2012

Deflasking Seedlings

While waiting for some of my own orchid seeds to germinate and grow, I have been busy shopping. Recently, I ordered flasks of several Schomburgkia (Laelia) hybrids from a grower on eBay:

Schomburgkia hybrid flasks from eBay
©2012 MadOrchidist.com

Flasks often get a little "scrambled" in shipping, so the seedlings usually need to come out fairly quickly. One of these four was exceptionally jumbled, so it's where I'll start.

Orchid Seedlings and Agar in a Scrambled Flask
©2012 MadOrchidist.com

I had some nice New Zealand sphagnum moss on hand, so I decided to try it for potting these new seedlings. I used some lava rocks in the bottom of the pots to bring the level up - no need to fill the whole pot with expensive moss!

Deflasking Materials
©2012 MadOrchidist.com

When the delicate little orchid seedlings first come out of the flask, conditions in the "real world" can be shocking to them; especially the lower humidity. Many growers use plastic tubs or bags to maintain high humidity, and then "harden off" the seedlings by gradually introducing them to regular growing conditions. I decided to use an old aquarium I had in the garage to this effect. It already had a nice compact fluorescent lamp in the hood too - bonus!

Seedling Humidity Chamber
©2012 MadOrchidist.com

OK, so it's time to get the little guys out of glass. Rather than breaking the bottle, I simply ran some lukewarm water into it and swirled it around a bit. Because this particular flask was already pretty jumbled up (and had a wide mouth), it was not difficult to flush all of the agar and plant material out into a large pyrex bowl. The others I will probably have to break.

So here is what I got:

Flask Contents Rinsed into Bowl
©2012 MadOrchidist.com

I then began the arduous task of teasing the seedlings apart and rinsing as much agar media as possible off of them. As expected, there were a handful of "heroes" (nice big growing seedlings) and also many smaller plants. Here are the largest of the group separated out:

After picking out everything that looked like it was worth trying to save, I was left with a bowl of media, broken roots and shoots, and some plants too tiny to bother with . . . the whole mess went right down the disposal in the kitchen sink.

Deflasking Leftovers
©2012 MadOrchidist.com

The remaining plants went into Community Pots, or "compots." I very carefully tried to get all of the roots covered in moss and then placed the pots into the humidity chamber. There is a layer of lava rock on the bottom of the aquarium so that I can pour water in it to maintain high humidity. I also tossed in a length of heat cable I had laying around; If I like how it works, I'll bury it in the lava.

Seedlings in Community Pots ("compots")
©2012 MadOrchidist.com

So there you have it. Buying flasks is not only an economical (although patience requiring) way to get a lot of plants, it is the best way to get in on the "ground floor" with new hybrids.

One flask done, three more to go . . . .maybe tomorrow . . .

Tuesday, January 17, 2012

Safety In Numbers

This post is mostly about contamination, but I figured I had better start out with a picture of a beautiful orchid, lest I scare away any readers:

LaelioCattleya Frank Shadle
©2012 MadOrchidist.com

This is LC Franks Shadle, happily blooming away in (what I consider) frigid conditions outside right now (I am pretty sure it hit freezing last night). This kind of temperature tolerance should definitely be selected for, and I have already pollinated the flower (as if there would be any question).

Anyway, on to the real issue at hand. A little while ago, I had a marathon session in the glovebox flasking up some seeds I ordered on eBay. I opted to make two flasks of each species as insurance against contamination.

Contaminated Flask
©2012 MadOrchidist.com

It is a cruel irony of orchid culture that if something goes wrong, you will probably find the unpleasant result very quickly, but if something goes right, it may take months (or years) on end to reward you. Such is the case here.

Ascocentrum and Gastrochilus flasks contaminated
©2012 MadOrchidist.com

Two of the 8 total flasks in this group have grown a white fluffy fungus - one Gastrochilus bellinus and one Ascocentrum ampullaceum. These are the two in which I was most interested, so I am glad to have a backup flask of each that appears uncontaminated. Really, the fact that some flasks have turned up contaminated means I can probably breathe easy about those remaining. After more than 2 weeks, none of this cohort has yet shown any signs of germination, but as with all things orchid, it could take a while . . .

So, having sown seeds in 18 mother flasks thus far in my laboratory career, two of them have grown something rather unpleasant. That is a contamination rate of about 0.11 -- improvable, but also acceptable to me at this stage.

Of the remaining seed flasks, three Epidendrums have begun growing, and everything else shows no sign of life - either desirable or undesirable. More waiting.

I have long been growing Orchids for their beautiful flowers, so I understand the waiting game, but there is a trick for the impatient like me. If you have enough plants, the likelihood that something will be blooming at any given moment increases dramatically. I remember becoming aware, several years ago, that I had reached this "critical mass" of Orchids. My off the cuff estimate is that once you reach a couple of hundred different plants (provided they are of a variety of varieties), you can pretty much count on having at least something blooming all the time.

The Mad Orchidist in his natural habitat

I think that the same phenomenon will eventually kick in with my laboratory operation. Right now, because I have a modest number of flasks and seed pods going, it sometimes seems as if nothing is happening. If this adventure picks up momentum, which I hope it will, then at some point in the no-so-distant future it will seem that there is always something interesting going on. Safety, and excitement, in numbers!

Thursday, January 5, 2012

Little Victories

Progress! The tiny green dots in the blurry photo below are actually what this blog madness is all about:

Epidendrum Orchid Protocorms In Vitro
©2012 MadOrchidist.com

These Epidendrum seeds have germinated; at this stage, the little green balls of undifferentiated and dividing cells are called protocorms. Most importantly, there are no obvious signs of contamination! All three of the flasks I made up with these seeds (dry and then sterilized) are looking good; here is another one:

Succesful Orchid Germination
©2012 MadOrchidist.com

In this case, the orchids themselves (reed-stem Epidendrums with no ID) aren't that exciting, but that fact that I was able to sterilize, flask, and germinate them successfully at home is. In fact, of the more than a dozen mother flasks I have sown with various seeds so far, none are showing any contamination (yet) - a good sign that my home-grown laboratory and procedures are viable.

Further progress toward my eventual goal of actually breeding my own new orchids is also being made. Today I noticed that the seed capsule of the first cross I made back in July 2011 has matured. It is turning yellow and has just begun to split at the end where the flower was. This eliminates any chance of sowing these with the "green pod" method, since I must presume the seeds are contaminated now that they have been exposed to the outside world. I will definitely get on to flasking them up soon.

LaleoCattleya Seed Capsule Maturing
©2012 MadOrchidist.com

These will hopefully be the fist new orchids that are completely my own, from pollination to germination. This was just the shot of encouragement I needed to stay excited about this orchid odyssey . . .

Monday, December 19, 2011

Flasking My First Orchid Seeds

This is what it's all about; sowing some Orchid seeds in flasks! A couple of my own crosses capsules' are nearing maturity, but I couldn't pass up the opportunity to practice on some that just came along.

Epidendrum seed capsules ready for flasking
©2011 MadOrchidist.com

The other night was the Orchid Society Holiday Party. Our host had some Reed-stem Epidendrums out in the yard that he says set seed all the time. As it happened there was a dry pod that had split open and a green one still sealed, both on the same plant. I have been considering whether I should go with the "green pod" method or wait for my pods to spit naturally and then sterilize the seed, and here was an opportunity to try both and compare! He let me take them both to sacrifice to the learning process in my lab (thanks Van!).

Pod Parent Tag
©2011 MadOrchidist.com

Given that the Epidendrum was probably pollinated by an insect, I have no way of knowing what the pollen parent was. All I know for sure is that the pod parent is pink; its tag is pictured above, plus I saw a few of the flowers on another spike. Since this is sort of an experiment, I am not really worried about the pedigree. If I end up growing anything from these they will be good for landscaping; Reed-stem Epis are practically bulletproof outdoors in this climate. Let's hope the seeds are hardy too:

Epidendrum Orchid seed capsule dried and split open (dehisced), fine seeds visible
©2011 MadOrchidist.com

I decided to deal with the open capsule first. Once an orchid seed pod dries, it splits open to release the thousands upon thousands (sometimes even millions) of tiny seeds inside. After this happens, the seeds must be considered contaminated and their surfaces sterilized before sowing.

Epidendrum Orchid Seeds being sterilized in a syringe
©2011 MadOrchidist.com

I opted to use household bleach in a ratio of one part bleach to nine parts distilled water as my sterilizing solution. Several online tutorials and discussions recommended using a syringe to clean the seeds. I bought dispensing syringes with caps on Amazon.com and made one cap into a filter by melting a couple of holes in it with a heated pin and then placing a small piece of coffee filter inside.

Basically, I drew up about 10mL of the sterilizing solution through the filter cap and then banged the syringe on my hand and the table for at least ten minutes to try and eliminate all of the tiny air pockets that cause the seeds to float and prevent the solution from doing its job. Below is a close up photo of the tiny orchid seeds in the syringe of sterilizing bleach solution:

Tiny Epidendrum Orchid seeds floating in sterile solution
©2011 MadOrchidist.com

I put the syringe, several jars of autoclave sterilized distilled water (for rinsing seeds), a few pre-made flasks with seed sowing media, and a jar of stronger bleach solution (to clean the outside of the syringe and any tools I used) into my Glove Box and sealed it up.

Tools and materials in the glove box
©2011 MadOrchidist.com

It is unfortunately impossible for me to have my hands in the gloves working and take pictures at the same time. At some point a small tripod and/or an intrepid volunteer will enable me to post more photos of what goes on inside the sterile glove box.

Briefly, I squirted out the bleach solution, drew up a rinse of distilled water a couple of times, and then squirted a small remaining amount of water with the seeds into a few flasks. The result is pictured below:

Tiny Orchid Seeds in Flask
©2011 MadOrchidist.com

I got some seeds on the sides of the flask, but there are plenty in there on the agar media too. I also ended up with more water than I would have liked in the flasks, so we'll see if that hurts things any.

I'll try to get to the green pod tonight or tomorrow so that I can make a fair comparison of the results. I also received some seeds today in the mail that I ordered from Thailand on eBay. I think I will give today's flasks a few days to see if contamination develops, so I can change my methods if necessary before trying again. For now, those seeds will go into my pollen storage unit in the refrigerator.

As for these flasks, now we wait . . . it's a theme with orchids . . .

Failure is Always an Option

Well the point of this blog is, at least partly, to keep track of my Orchid culture progress, so I would be remiss not to include the occasional failure. Here are a few things that have not gone according to plan lately.

These first few photos are of flowers that were pollinated, but then withered away without developing a seed pod. I am willing to give up enjoyment of the flower in return for a seed pod, but when you lose both it really stinks!

Cattleya flowers that were pollinated, but never went anywhere
©2011 MadOrchidist.com

Another Orchid Dud!
©2011 MadOrchidist.com

Laelia anceps that never developed pods - bummer, I had high hopes for these . . .
©2011 MadOrchidist.com

These next few pictures are of the Dendrobium nodes that I flasked up in this post. All three of them developed contamination.

contaminated Dendrobium node flask
©2011 MadOrchidist.com

The same flask as above, a few days earlier - that stuff grows fast!
©2011 MadOrchidist.com

The other two flasks developed fuzzy fungus
©2011 MadOrchidist.com

I had acknowledged previously that stems are tough to get completely sterile for this type of culture; now I know first-hand. I could try to clean and replate them, but they were really just for practice and I'll probably let them go.

So, with some failures under my belt, I feel like I am making progress. Next, on to some seed sowing . . .

Wednesday, December 7, 2011

Node/Keiki Culture - Dendrobium Lifeboat

I have mentioned previously that my Grandfather was an Orchidist, and even though he passed away years ago, my Grandmother still keeps a few orchids around the house. These days they are mostly store-bought Phalaenopsis in the windowsill and a few random things out on the patio.

When I went to visit Grandma the other day, I noticed some dry Dendrobium canes leaning in an empty pot outside. She is nearly 90, so she doesn't make it out there to water very often, and between that and the recent cold, this plant had wasted away. The roots were dry and cracked (not even potted in any mix), and there wasn't a leaf to be found. There were even a few well developed keikis that had made a start, withered, and died. Even so, I noticed a few nodes that were developing a last ditch effort at new plants.

Dendrobium lifeboat!
©2011 MadOrchidist.com

I was offered the plant if I thought I could save it. No way, but I realized these developing nodes would make a good experiment in my new mad laboratory. I knew Phalaenopsis could be cultured in vitro from nodes on flower spikes and figured the same would be true of these nascent keikis, especially since they had already started to develop. There's only one way to find out!

I brought it home and cut the canes up, leaving just a bit on either side of the three nodes with developing keikis I thought looked viable. I realize now that I should have made more effort at this point to get all of the dry papery covering off, as this may be a source of contamination later - we'll see.

I soaked them in a mild bleach solution for about 10 minutes, and prepared some sterile distilled water while I waited. I already had empty flasks made up with media, and I didn't want to run the pressure cooker autoclave for just one jar of water, so i microwaved some distilled water in a jar. I actually set the lid on too, which I know is not usually a good idea in the microwave, but its smooth surface did not allow any sparks to occur. While not ideal, I hope this water is sterile enough to rinse my cuttings without adding contamination.

Once the water cooled, I placed it, the cuttings (in sterilizing solution), and the necessary implements into my glove box, closed it up, and sprayed everything with bleach.

Below is my view through the clear acrylic window in the top of the Glove Box:

Basically, I just scrubbed the cuttings with a toothbrush, cut off a little more at each end, and then placed each one into its own baby-food-jar flask.

Here is the result:

There was actually one small "new lead" developing at the base of the withered mother plant too. It had new root tips developing, and I opted to pot it up in sphagnum moss and try to grow it alongside the node flasks for comparison.

I have read that other folks doing this kind of culture at home have had a hard time with contamination due to the difficulty of completely sterilizing the cuttings. Only time will tell if these proto-orchids will grow in the flasks, or something gross will grow instead!

Saturday, December 3, 2011

Anceps and Updates

December is definitely Laelia anceps season. This hardy orchid from Mexico blooms in the winter, despite the coldest temperatures of the year, and will routinely survive frost events.

In addition to all of the wonderful hybrids this species has parented, there are innumerable varieties of the pure species itself. I probably have six or seven different kinds and often feel the "collect the whole set" urge to get more. In fact, I am toying with the idea of mounting them all on one large cork tube to make a Laelia anceps tree; it would be quite a sight for the holidays! I am fortunate to live near one of the worlds premier anceps growers and hybridizers, The Santa Barbara Orchid Estate. Their Laelia collection is second-to-none, and most of my specimens of that genus have come from there, whether directly or indirectly.

So, here I am looking for blooming orchids to pollinate in December, and it isn't surprising that these beauties are on the list:

The one on the left is the variety 'Veitchiana' and is the pod parent in this cross. On the right is the 'Irwin's' that I used in a previous cross; it's flowers have already faded after being pollinated previously (pictured below), but I kept some of its pollinia in the refrigerator. I also placed the 'Veitchiana' pollen in folded up paper and added it to my pollen "cold storage" in the refrigerator.

A couple of my earliest attempts have yielded nice fat seed pods, and at least one is looking very ripe. I am still deciding if I want to use "green pod" seed sowing methods, or wait for dehiscence (the natural splitting open of the pod) and sterilize the seeds afterward. This pod may make that decision for me by bursting! Given that it has begun to turn yellow, it is probably ready for "green pod" sowing now, but I want to give my empty flasks at least a full week or more to be sure they are not contaminated.

Speaking of contamination, the "contamination control" flask from my last post still looks clean, let alone the ones that are supposed to be clean. However, I did make up some Petri Dishes with the excess agar media mixture, and those are growing some interesting (or perhaps unsettling, depending on your viewpoint) things. The first one I left exposed to the air, just like my "contamination control" flask, but for longer.

The second one was wiped with a small spatula that my Scottish Terrier, Dr. Watson, had licked. Not surprisingly, his mouth is not sterile (even though he has been sterilized)!

Both plates show representatives of the two major groups of contamination: bacteria and fungus. The fungus initially shows up as fine hyphae (threadlike structures) and eventually gets "fuzzy", while the bacterial colonies tend to look "slimy."

Hopefully none of this will show up in my batch of baby-food-jar flasks, and I'll be sowing seeds before the end of the month!

UPDATE:

Here is a photo of the same Laelia anceps 'Veitchiana', 24 hours after pollination. You can clearly see that the petals are folding forward over the column. To speak teleologically for a moment, the plant doesn't "want" pollinators visiting a flower that is already pollinated, so it immediately makes it less attractive. Flowers are "expensive" to maintain (especially in terms of water loss), so the sooner one gets pollinated and then discarded, the better from an evolutionary biology standpoint.

However, I can see why most orchid hobbyists are reluctant to pollinate a beautiful flower that they want to enjoy for a while . . .