interestibg method. are other types of sterilization agent can be used? such as alcohol, hydrogen peroxide? or it just the one which has volatile properties. thank you.
regards,
yanto

only gaseous agents such as chlorine, can perform this type of sterilization

---------- Post added at 06:18 AM ---------- Previous post was at 06:13 AM ----------

sowing with very few seeds, I think there are too many big pieces

Just want to report that i had my first success using this method on the first try! ill post pictures along with details later on once the seedlings are a little more visible since i just was just able to see germination today 8/24/13.

Please help me :p
 

Hey guys, would anyone be able to tell me what NAA would mean. Thanks
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is an Auxin
1-Naphthaleneacetic acid - Wikipedia, the free encyclopedia

I am planning to flask some Cattleya seeds this week.

I have some jars ready that I plan to sew using more conventional methods, but I also want to try this method; I have some extra medium left over, and of course, plenty of seeds.

My medium is home-made, but a different recipe. Google "How to grow orchids from seeds - kitchen style", you will find it.

roby - thank you so much for posting this method!

Hi Roby,
I am new in the "In vitro" sowing and I find the classical method a bit complicated. I have some jars ready for 3 weeks and, so, would like to try the way of sterilization you talked about. I have a solution of sodium hypochl. at 3,6% (12° Chl.). Do you think that the concentration is enough? What level of concentration do you use? Thanks a lot,

[/COLOR]Adding my two-cents to the above, which is a GREAT guideline...

If dealing with fragrant orchids (such as Catt's) wait to do pollinating/harvesting of pollen until the flower in question is fragrant (I've had luck with warm days, mid-morning when I begin to notice strong fragrance... skip overcast/cool days)
I'm supposing the plant knows when it is most fertile and sends out the fragrance to attract pollinators.
Also (I would imagine) that nighttime fragrant orchids may be best pollinated at night. :)

-LVD

Use what talent you possess - the woods would be very silent if no birds sang except those that sang best.
Henry Van Dyke

Trying it out!
 

Just completed a series of replates using the gaseous sterilization method described above (with bleach soaked cotton balls for pads, without the use of a hood)... *fingers crossed*

A thought I had while replating:
It seems beneficial to mix a bit less water into the usual agar/nutrient solution so that it firms-up more than usual in the autoclave process.

This should cut down on the amount of plant material that sinks into the agar during replating, thus increasing the amount of plant material exposed to gas exchange for sterilization.

Another wards - a replate plantlet sitting on top of a firm agar surface won't sink.

Flask seedlings being replated:
C. Jose Marti 'Mother's Favorite' x self
This seemed like a perfect opportunity to experiment with this new technique since the flask had around 100 mini seedlings.

(I read about the Jose Marti and Mother's Favorite both being a registered cross of the same parents but registered by different people while the parents had different names, boy oh boy does the naming get confusing!)

-Laura

Use what talent you possess - the woods would be very silent if no birds sang except those that sang best.
Henry Van Dyke

The Cattleya alliance seeds (Cattlianthe Golden Wax) that I posted about earlier were sewn later, September 4, I think, but 4 of 6 flasks remain uncontaminated & germinated well (3 to 3.5 hours with the bleach fumes). The protocorms are now developing little leaf points.

I also tried 3 flasks of the same seeds using conventional sterilization and sewing methods, 2 failed quickly, the third germinated, but later developed contamination. Roby's method works much better , IMO.

I've done some more flasks with seeds that I can afford to lose with this method and I struggle to keep them sterile. :/ Argh!

But I struggle equally much trying produce sterile flasks in my home-made glove box as well.

How strong is the bleach solution you're using? I'm consciously using really small glasses nowadays to make sure that the seeds and the growth medium is exposed to the fumes, but within a week there has been contamination.

I'm just playing around with NOID phal seeds and other home-grown pods so it's not as if I'm losing precious plants here, and it is meant as trial and error tests.

During the weekend I opted for pre-sterilizing the seeds before I put them into the flasks, by rinsing them in bleach solution as described by Lincoln Orchid Society.

They still look clean, but it's only been a couple of days and way to early to tell yet.

Hmmm... I'm a bit at a loss as to what to change next if they also mold up...

Silje, the bleach I used is a generic type from one of our chain discount stores (Walmart). Their standard bleach is 6.0% sodium hypochlorite, the one I used is advertised as "33% more concentrated", so that would make it 8.0% sodium hypochlorite.

Although I have had good success with using this bleach for this type of disinfection (I leave exposed to the fumes for about 3 to 3.5 hours) for the first few months, I have noted that mold has suddenly developed in some of my flasks 3 to 4 months later. The seeds have developed into small seedlings with tiny leaves but no roots. I tried re-plating some onto fresh agar, followed with more bleach fume exposure, but this killed the tiny seedlings. Others I have experimentally transferred into jars with heat-sterilized moist sphagnum, and these remain alive, but still no roots yet. I have a few that are a bit older (bigger leaves, still no roots) that I may try experimentally growing on beds of live moss in glass jars, sort of like tiny terrariums.

I have one flask/jar that remains contamination-free. I will keep that jar going as long as I can, hopefully until I get some roots growing.

Something worth trying for your tiny seedlings living is sterilized sphag is perlite, also boiled and heat sterilized. Keeps moist, but not soggy and personally I find that it remains algae free and fresh longer, but that's just personal preferences.

Talking about bigger seedlings, I've been puzzling how on earth to do the re-plating and manage to produce clean flasks in that process. Like you say, the bleach will kill the seedlings... hmmm...this obviously still needs some fine-tuning.

I do like the idea, because working in a home-made glove box is frustrating, I think, but I'm leaning towards trying to find a friendly lab where I might be allowed to use a proper laminar flow cabinet for this kind of work.

If you have localized infection in a flask, get hold of a magnifying glass. Take the flask outside on a sunny day, and use the magnifying glass to concentrate the sunlight on the infection.

This burns away the infected area, but leaves the rest of the flask intact.

Thanks Silje, I will see if I can find some perlite locally. It might be easier to find once spring returns here. I have also considered using sterilized coarse sand or fine charcoal.

The original experiment, to try to grow seeds using this fume method, seems to be spawning numerous side-experiments. Necessity is truly the mother of invention!

---------- Post added at 10:53 AM ---------- Previous post was at 10:48 AM ----------

Fairorchids, have you done this successfully? I would have thought this would be difficult through the side of a flask, but I would like to hear how this worked out for you.

My dad had an orchid nursery (in Denmark, 1950-1977). He flasked in the kitchen, and used the magnifying glass successfully (on glass flasks). Not sure whether it will work on plastic flasks, but it should.

For the cellophane, we use to just cut a tear off sandwich bag and cut down one side. Then hold one side of the bag in your fist with about 1/8 to 1/4 inch sticking out, then just cut it off. When you open it will look like a hole in a square parachute. I use 10% bleach and distilled water solution with a drop of any liquid dishwashing soap and wet the sandwich bag to kill any bacteria, which might be on it.
One major thing though is using fresh house hold bleach and quality bleach. Not all bleaches are equal in their strength, and any liquid bleach can lose it strength, different countries have different standards for bleach so lab results can vary dramatically.

Sunlight through magnifying glass might actually burn and poke a hole in the plastic flask. We used to poke holes in plastic and burn small piece of paper during science experiments at home with help of magnifying glass.

can i use this method on replating
 

can I do this with replating as my seeds arnt growing and lots of condensation in jars, they are approx. 1mm and I did them in November.

Are you replating in a seald jar or flask?

If you focus the beam on the plastic wall, yes, that will burn a hole. If you focus the beam on mold that is one or two inches inside the wall and on top of agar, I don't think that there will be damage to the flask.

Anyway, if a flask is infected, it is worth trying this instead of tossing it in the trash.

a glass jar

You can try cotton wool plug in the lid or tape some cotton wool over the hole. But you need to leave a small part untapped over the hole, so the jar can breathe.

Not necessarily. It has been shown that it is not necessary

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Wow~ this is so cool!!!

How are your seedlings now???

I tried this method. My conclusions : very easy, a lot easier for me than the usual system. 80% success. It worked with laelia, phalaenopsis and ascocenda hybrids. I am very happy with it. :)
Maybe to adapt for disa seeds ; no infection but no germination, maybe the disa seeds are not strong enough for 4h sterilisation...
Thanks a lot to Roby !

I used this method & posted on this thread some time ago. Those tiny seedlings have now been moved to compots, though I never was able to successfully replate with this method. I went from mother flask to deflasking straight away.

As I replate in a glove box from a sterile flask to another sterile flask, I just transfer the small plants without any more sterilisation. It works usually. Only one infection in a flask after 1 month replate, so I deflasked and put the plants (Laelia Purpurata Mandayana) in sphagnum. They are slowly growing now. T find the sterilisation of the disa seeds difficult, so I try to sowe green as much as possible.

Deflasking
 

Well. From the above to this:

https://lh6.googleusercontent.com/-4...o/100_1009.JPG

The one in the middle towards the front and the one just to the right in the front are two of the Bulbophyllum orectopetalum seedlings pictured in my previous post. The non-bulbo seedlings pictured are a super-secret primary hybrid which I've grown on for the person doing the breeding.

All of the seed sterilisation and replating was done using Roby's method. It's been amazing and I can't wait to do more.

Just tried again with new seed. It's so simple! Lets see if I did work clean.

Eleven days now and no contamination. That is very good. But still to early to discearn with naked eye if anything moves.

Good luck.

sterilization of homemade media
 

hi i am new here. please anyone help me how to sterilize germinating media with a microwave oven. As far as i got first need to mix all ingredients of the media,then put needed amount of media in jar, keep the lid of jar little open, put the jar in microwave, set the temp in 150C and keep the oven run for 30 minutes. Am i right?

It sounds like you're mixing up oven sterilisation with microwave sterilisation. If you want to sterilise in an oven, then, then that would be about 150C for 30 minutes, and if you're using plastic containers you'll need to make sure they're oven safe.

thank you NatalieS. Would you please tell me that the process i described is proper order or not? what is the difference between ordinery oven and microwave oven sterilization process?

Well, basically the oven takes longer. In both cases you'd make up your medium over the stove first. Many people have written very good information about the different methods of sterilisation, and I doubt I could improve on the info they've provided. I recommend taking a look at this site as a first step:

Lotte & Thomas Orchids

Lots of info about different sterilisation methods, media preparation, etc.

Also, try searching on YouTube for 'microwave sterilisation of orchid medium'. There are some very good videos there.

Lot of thanks NatalieS

please help me with 100% home made media for cattleya / brassavola seed germination. 10 days ago i have sown some den. pulchellum seed in ready made media by Roby 's described method and i am happy still no sign of contamination.only three flask. please pray for success.

here is my first atempt flask. seeds are turning green now.