Redneck Orchid Flasking

[vmax3000]

Originally Posted by John

Hi Vanessa, sorry to hear about your flasks. In the begining all I could grow was mold. Did the mold start on the seeds or elsewhere on the media? That may help narrow the route of contamination. If it started on or near the seeds then probably you didn't get the seeds sterile enough. If it started as spots on the media then it could be spores that were dropped into the flask from your tools, hands, or the edge of the flask. After you sterilized the pod how did you sterilize the seeds? I have not worked with pods, just dry seeds so if you like, I can tell you how I have handled the seeds and prepared the flasks. I am far from being an expert but I am learning more everytime I flask.
John

Hey John,
Sure enough, the fluffy mold is strictly around the seeds. Lesson learned. How long does a dry pod need to soak before it's sterile? Ballpark? I just soaked the pod (it had started to dehisce) in hydrogen peroxide. I let it soak until the major bubbling had slowed...think after you've poured a glass of soda or bubbly, when there are still bubbles, but few are seeking the surface - vapor pressure has been almost equalized. I didn't even think about sterilizing the seed . Goes to show that even with the fancy hood, I still have a bit to learn Please, indulge me on how to treat seeds. I will add it to my limited, but growing repository of 'chid knowledge Thanks for your input!!

[Royal]

I think we might be getting two techniques confused. Once a capsule opens, even just a crack, the seed is 'contaminated'. That's not to say it's ruined, just not sterile. So, with dry seed one must disinfect the seeds prior to sowing them on the nutrient medium.

There is another technique called 'green capsule' method. The idea here is that a green capsule that has not started to open still contains sterile seed. It is then possible to disinfect the outside of the capsule and, in a sterile environment, open it up with sterile tools to sow. If the cap was disinfected well and your tools are clean, the seed in a green capsule will not need any disinfection or treatment. I

If a capsule starts to crack open, it is too late for the green capsule method. Harvest and use the dry seed. Lots of folks prefer the green cap method, but I don't really know why. I know that some genera (Paphs and Cyps) get better results when using an immature seed capsule, but this doesn't speak to the technique, but rather, the seed morphology and maturity. Mature, dry seed is no harder to disinfect than a green capsule. Also, with dry seed, you can split it up and save some in the fridge for ?? years. This allows for multiple attempts.

Hope that clears things up.

[Gin]

Good take on the dry capsule and storing some of the seed . A side note all the information read here is very appreciated and interesting .Sorry about the flasks Vanessa know how it is Gin

[vmax3000]

Thanks Royal. Yeah, I used my green pod knowledge on the open, dry capsule...there's the rub. I really don't know how to treat seed...well, my current flasks confirm that! John asked about the location of the mold, and sure enough, it is located around the little grains of what used to be B. nodosa babies SO, the good news, my sterility techniques appeared to work on the media, tools, et cetera!
Here's something that bites....I THREW THE POD AWAY . I don't know why. I know that all plants preserve their progeny, hence their longevity through their seed. But somehow, the pod, with loads of seed still in it, was wet and I thought I was done... and I didn't really know what to do with it...save the pod in the fridge...how novel...

[korxi]

A while back I wrote a seed sowing guide on another forum, it also contains some media preparation and so on.

DIY seed sowing

I hope this could help, I've never gotten any infections with it yet

Christian

[John]

Hi Vanessa, I liked Christian's presentation. It is very well thought out and the pictures are great. I do it a little different but not better. I use media from Hills or Phytotech and after wisking it into boiling distilled or RO water and bringing it back to a boil, I then laddle it into waiting canning jars. I put the lids on snug because if the lid is vented, there will not be a vacuum in the jar and if the lid isn't vented, I will be opening it in the flow hood in a stream of sterile air so I feel it won't suck in spores. I put aluminum foil on the jars before they go into the canner. My thought here is that the foil will be sterilized along with the jars and media and it will protect the lid area while the jars are in storage.
For seed sterilization, I have also been using syringes. The ones I use are 3ml and they have a tapered tip on them not the one that a needle screws onto. I pull out the plunger and with my finger covering the tip, I shake the seeds into the syringe. The plunger goes back on and I push it in to a point where the seeds are all near the tip. I then asperate about 2ml of 10% bleach solution that has a drop or two of tween in it(dish detergent also works). I then do as Christian does and vigorously shake the syringe with my finger over the tip. I also do the negative/positive pressure thing and the flick with a finger thing. The syringe then sits upright for 10 minutes. during the ten minutes, I shake it several more times. From here the syringe goes to the flow hood and into a pan that hold 10% bleach solution. I do this to sterize the outside of the syringe. I then take a paper towel wet with 10% bleach solution and I put it over the tip of the syringe and push out most of the bleach solution. Then I asperate some sterile water, shake the syringe, push that out into the towel, aspirate about a ml of sterile water and that is pushed out into an open flask. If not all of the seeds are pushed out, I will aspirate a little more water and push it into the flask again. When I take the lid off of the flask, I place it into a pan of 10% bleach solution. After the seeds are in the flask the lid is put back on and the flask comes out of the flow hood. The foil is waiting(right side up) to go back on the jar and I write on it the date, media, and type of seeds. I generally will sow a third of the seeds first to see if the sterilization works and then another third holding back some seeds just in case of disaster. I think I will stop here and see if you have any questions. I know there are a lot of others out there doing things differently and that's the beauty of this. We can all try different methods and share what we find works.
John

[Royal]

Originally Posted by John

I know there are a lot of others out there doing things differently and that's the beauty of this. We can all try different methods and share what we find works.
John

Exactly!

Try this tip given to me by a friend: The reason for drawing the vacuum (and the surfactant) is to allow the disinfectant to make complete contact, or fully wet the seed. The positive/negative pressure will work, but so will ultrasonic waves. They totally break the surface tension and your seeds sink fast. Sinking means the disinfectant has fully infiltrated the seed testa.

I use small test tubes, add seed, disinfectant, and a few drops of wetting agent (two drops of Dawn in 100 ml). Cap, shake, and dip into an ultrasonic cleaner for about 30 seconds at a time shaking between "treatments". Once they sink, your good.

[dsim]

i have been following this thread... the flasking, sterlisation, etc (which i m interested)... but makes me wonder how do the seedlings of orchids in the wild survive all the "unwanted contamination"? is it not possible to just get the seeds to germinate without all these? ie. just put the seeds onto some place for it germinate? sorry, if that a truely blunt question.

and i found this one... DIY - Guide to orchid propagation - Orchid Article Library

looks interesting and simple to follow.

[vmax3000]

Originally Posted by dsim

i have been following this thread... the flasking, sterlisation, etc (which i m interested)... but makes me wonder how do the seedlings of orchids in the wild survive all the "unwanted contamination"? is it not possible to just get the seeds to germinate without all these? ie. just put the seeds onto some place for it germinate? sorry, if that a truely blunt question.

and i found this one... DIY - Guide to orchid propagation - Orchid Article Library

looks interesting and simple to follow.

Dsim,
I think the reason that orchids literally pack their pods with seed, without any nutrition (seed coat - think corn or rice, most of the body of a grain of rice or corn is starch needed for the germinating seed..or, our meals!). This speaks to the fact that only a very few will make it to fruition. But, the plants have succeeded, as we are enjoying them, presently! Also, and some of the wiser ones will have to back me or correct me, the mould, in the wild, serves the purpose of actually nourishing the seed, but is checked by something in the natural world which isn't found on these little sterile plates we make in the labs or our kitchens. The media we supply is supposed to substitute for the mould. I seem to recall it is a symbiotic relationship.

[Undergrounder]

orchid seeds need a very specific type of 'mould' to germinate in the wild, which won't be just floating around in your kitchen.

But in the wild they don't have a thick cake of agar, which is what the bad mould is attracted to, not the seeds themselves.