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 Post subject: Now It's My Time
PostPosted: Fri Jan 10, 2014 4:42 am 
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Finally I designed and built my own glove box! The hexagonal design happened because I wanted easy angles for how to put my arms inside the box.

Having finally my own glove box, I felt it was a good time to boldly go where I never went before: germinating orchids! I found this instruction for the orchid germination medium on the Internet.


90 ml Water
4 g Mashed Peeled Banana
1 g Agar Agar
2 g Sugar (I use icing sugar dust instead)
10 ml Fresh Peeled Crushed Tomatoes (I use 12,5 g tomato paste instead)
0.12 g Thiamine (Powderized Vitamin B Tablets)

But instead of fresh tomatoes, I used tomato paste. Where other mediums are almond-colored, my on e has the pleasant color of a brick red. But I hope, it does its job the same good as other germination mixtures. It is my very first attempt.

I boil my mixture in a steam pressure cooking pot for 30 minutes (I gave that longer time to make sure to kill any bacteria or fungus) to fully sterilize.
I kept in pot until next day, and then closed the lids.
I kept bottles in clean supermarket bags (the transparent ones from the vegetable department) to avoid surface contamination.

And now I am waiting 5 to 6 days with seed propagation, in order to check if no bad fungus or bacteria came in.

I want to try germinating vanilla seeds. But how to sterilize the vanilla? I don't want that the seeds bring any bad fungus or bacteria into the germination mixture. Who has an answer for me? Also: can a vanilla bean from the supermarket be used as germination candidate?

Any advices, suggestions or hints? Comments are welcome, too. Thanks.

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above: wheeled carrier construction and glove box
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above: the glove box from different angles
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above: the empty glove box is now filled with the germination flasks which contain the germination mixture, and my germination candidate - a vanilla bean from the supermarket

After the seeds have been placed on the surface of the germination mixture and the lids are closed, I will use candle wax to seal the lid area against any unwanted infestation of fungus or bacteria. But for now I will test them for one week for any unwanted invaders. The wetness on the glass is caused by the spraying of hydrogen peroxide to sterilize the box from the inside.

QUESTIONS

How to avoid that the seeds carry any unwanted bacteria or fungus inside the flasks? is there any way to sterilize the seeds, too?

Instead of sugar, I used icing sugar because it is much easier to mix. Should I use icing sugar?

Instead of fresh tomato, I used tomato paste. Could that cause any problems?

Can I use hydrogen peroxide to sterilize the box from the inside?

How long do you keep the filled flasks for contamination test before putting the seeds inside?

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 Post subject: Re: Now It's My Time
PostPosted: Fri Jan 10, 2014 10:03 am 
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What are the ingredients of the tomato paste? This all sounds very bizarre to me... :-/

I think supermarket vanilla beans are treated with steam abd dried - almost zero chance of germnating.

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 Post subject: Re: Now It's My Time
PostPosted: Fri Jan 10, 2014 1:04 pm 
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Sven, I admire your enthusiasm and can recommend trying to grow your own crosses but I would put in a few words of caution. All sorts of things can go wrong when trying to germinate orchid seeds and if you do not control your experimentation (and that is what you are doing by changing various things) then you will have no idea why it did not work. For instance the media you have made. Presumably someone has found that the recipe you quoted worked but then you have changed at least two ingredients in it so how will you know what went wrong if your seeds do not germinate?

I would suggest getting hold of some tried and tested media if at all possible so that if the seeds fail to germinate ( and there is no contamination) you can deduce that either the seeds were not viable, and this is quite possible, or you perhaps killed them in your sterilizing process.

I will try and answer some of your questions:-


How to avoid that the seeds carry any unwanted bacteria or fungus inside the flasks? is there any way to sterilize the seeds, too?

You can wash the seeds in a 3% solution of Hydrogen Peroxide (H2O2) for about 12-15 mins. Add a single drop of washing up liquid to the H2O2 and shake the seeds every few minutes. Do not be tempted to give them a longer wash as you could kill the seed as well as any spores etc on the outside of the seed.

Instead of sugar, I used icing sugar because it is much easier to mix. Should I use icing sugar?

I don't see why icing sugar is easier to mix particularly if you are boiling your mix for 30 mins. I refer you to my comment about changing things.

Instead of fresh tomato, I used tomato paste. Could that cause any problems?


I refer you to my comment about changing things.


Can I use hydrogen peroxide to sterilize the box from the inside?


Yes a 3% solution should do fine for that.

How long do you keep the filled flasks for contamination test before putting the seeds inside



I would leave them in a warm place for about 10 days. If no contamination by then they should be OK.

It will be great if you can manage to germinate some seeds so good luck.




























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 Post subject: Re: Now It's My Time
PostPosted: Fri Jan 10, 2014 3:36 pm 
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I've not done this myself, so not an expert. I think the icing sugar would be OK: I think it's just ordinary sugar ground finer? The tomato I'd be more worried about. I've not seen any recipe including it before. I'd assume paste would include the skins (and possibly a few bits of leaf and stem) which might change the composition a bit. It would also depend on the ingredients in the paste: if it's just paste you would probably be OK I think, though Cricketerry is right, but if it's got any other ingredients or preservatives then you could have problems. (Looking at the bottles in the picture, I'm really hoping you haven't used Heinz Tomato Sauce!)


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 Post subject: Re: Now It's My Time
PostPosted: Fri Jan 10, 2014 8:49 pm 
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Hello, Without sounding too negative I think you have got your work cut out. Like Terry, I think you are starting off with many variables so when the inevitable will happen you won't know where to start to rectify and progress.

You would have been best using a sugar solution instead of agar to test your protocol and box, this elimates waste agar etc.
I tried a home made glove box, failed miserably and didn't have the patients to mess about with it, couldn't be doing with the mess and lack of confidence in its ability to remain contaminant free. I will add that I didn't give it long before I gave up, two occassions of trying it.
Hence I purchased a flow hood from http://www.standardhepafilter.com

Link to my flow hood article http://www.koolorchids.co.uk/flow-hoods/

I haven't tried making my own media mix, but I may do in the future, it does take a really big variable away though if you buy the media to suit the task in hand.
Good luck with it.

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 Post subject: Re: Now It's My Time
PostPosted: Sat Jan 11, 2014 9:34 am 
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Hey you all, thanks a lot for your hints, advices and suggestions! Well appreciated!

I actually tried to be close to the recipe of the germination mix. I got it from a web page of a botanical orchid garden, here"s the link:
http://dokmaidogma.wordpress.com/2011/0 ... hen-style/

I had to switch to pure tomato paste and to icing sugar, because my supermarket just was out of tomatoes and regular sugar (white), and I don't wanted to use our cane sugar (I live in Jamaica). So I purchased what was next to what I tried to get. Well, another thought was that icing sugar should mix easier.

As a beginner, I am not sure how small or big the tolerance of orchid seeds is, if most of the ingredients are otherwise there. I used less tomato paste than the recipe called for plain tomato, because tomato paste is concentrated tomato (water removed). I even researched online (Google) the weight of 1 gram of tomato paste to make sure I am not too wrong with the amount.

Well, everything's done now - the seeds are spread. Let's see what happened. I gave a 7-day-contamination-test-period, hope that was still okay. I didn't see any contamination. But to be honest, the question is, if I failed with the seed treatment and spreading them. Here i have to improve as I encountered some problems.

One of the problems was, after the seeds were been for 10 minutes in that 3% hydrogen peroxide and water and detergent solution, how to get them out and into clean water. That was my obstacle. I might have failed here. I used a kitchen paper towel as filter and then tried to get the seeds from the paper into a waiting sterilized water bowl. But not sure if I succeeded. I also couldn't repeat the water cleaning, that was a next problem. I should have had more bowls with sterilized water inside the glove box, and also more paper towel. Or maybe no paper towel at all.

Fact is, now as it has been done, all I can do is to wait and check each day the flasks.

For the seeds, I used a purchased supermarket vanilla bean, and a fresh vanilla bean which I received from a vanilla orchid farm in the countryside here in Jamaica. So, let's see what happens. In any case, I won't give up but improve and try again. It was and is just too much fun and excitement... :-D

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 Post subject: Re: Now It's My Time
PostPosted: Sat Jan 11, 2014 10:23 am 
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SvenLittkowski wrote:

One of the problems was, after the seeds were been for 10 minutes in that 3% hydrogen peroxide and water and detergent solution, how to get them out and into clean water. That was my obstacle. I might have failed here. I used a kitchen paper towel as filter and then tried to get the seeds from the paper into a waiting sterilized water bowl. But not sure if I succeeded. I also couldn't repeat the water cleaning, that was a next problem. I should have had more bowls with sterilized water inside the glove box, and also more paper towel. Or maybe no paper towel at all.

................................It was and is just too much fun and excitement... :-D


Sven the beauty of using H2O2 instead of a chlorine based bleach is that you do not have to wash it off the seeds. This is because H2O2 breaks down quickly, in the presence of light, to hydrogen and water. When you try again (and I hope you will) then leave the seeds to settle at the bottom of the H2O2 and using a pipette or dropper of some sort suck up as many seeds as you can with as little liquid as possible. Then just squirt this mixture onto the surface of your medium, seal the jar and then tip it to spread the seed over the surface. Leave somewhere in the light, but not direct sun, and you should be OK. If you have access to a syringe then you can do the sterilizing in it, gradually get rid of the liquid as the seeds settle and then squirt some of the seed mixture onto the medium.

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 Post subject: Re: Now It's My Time
PostPosted: Sat Jan 11, 2014 11:52 am 
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You need the Carol Stiff manual, I will see if I can find the pdf file for you.
I do the peroxide/syringe method and it was easy but I did lack confidence my first time.
Put seed and peroxide in the syringe then shake, leave for sterilization(time this) Put a really small peice of paper towel between syringe and needle to act as the filter to remove the peroxide and leave about 1ml in the syringe, you do two flask with 1ml. So you could leave 2ml in the syringe and do 4 or 5 flasks maybe. If you do this remember to keep the seeds distributed in the liquid to help even ditrubution in the flasks.

Edit- Please go to my facebook group https://www.facebook.com/groups/633055240086601/files/ and ask to join and you can download the Micropropagation handout, it really is good, I cannot stress this enough. There are other files that you can download may also interest you.

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 Post subject: Re: Now It's My Time
PostPosted: Sun Jan 12, 2014 3:18 am 
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This is a good forum here! Thanks once more for the newest advises!

Yes, I used hydrogen peroxide. I just didn't knew that light would have been the solution. How bright, or what kind of light, should it be to have a rapid break-down into hydrogen and water?

My hydrogen peroxide solution recipe called for 1 part 3%HP to 9 parts of water. Since I could get only a 6% HP, I used 1 part of 6%HP to 18 parts of water. Hope that was still acceptable.

Also, the suggestion of a pipette or dropper is something so logical I wonder why it didn't occur to me in time. Thanks, too. And yes, I WILL do. :-)

I joined that Facebook group, but have to wait now to be accepted, before I can access those files.

I also heard about an e-book from Joseph Arditti with the title "Micropropagation of Orchids", but seem not to be able to find it anywhere online. And I need some opinions or feedback about the software "OrchidWiz Encyclopedia". Is anyone using it?

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 Post subject: Re: Now It's My Time
PostPosted: Sun Jan 12, 2014 10:45 am 
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Hello, You now have access to the group, read the manual and I think it will give you more understanding and confidence. Yes you can have your seed/3% peroxide in a glass phial and pipette it out. I may try this way next time as it may give more control over the syringe method.
You can use 6% peroxide and dilute with water( would use pure distilled water or RO)

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 Post subject: Re: Now It's My Time
PostPosted: Sun Jan 12, 2014 11:46 am 
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SvenLittkowski wrote:
.....................................Yes, I used hydrogen peroxide. I just didn't knew that light would have been the solution. How bright, or what kind of light, should it be to have a rapid break-down into hydrogen and water?

My hydrogen peroxide solution recipe called for 1 part 3%HP to 9 parts of water. Since I could get only a 6% HP, I used 1 part of 6%HP to 18 parts of water. Hope that was still acceptable.



Normal daylight, by a window without letting the sun hit the jar and heat it up would be fine. It only needs to be for a few hours.

I always use 3% H2O2 straight from the bottle for sterilizing my seeds and equipment. I would be surprised if diluting it with 9 parts water as your recipe suggests would be effective.

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 Post subject: Re: Now It's My Time
PostPosted: Sun Jan 12, 2014 12:58 pm 
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Regards sterilizing seeds, if are getting contamination even after bleaching them, try soaking them in a sugar solution with a drop of washing up liquid for 24 hours before you sterilize them, it allows the spores to germinate so that you can kill them. it also reduces the seeds clumping when they are transferred to the media.

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 Post subject: Re: Now It's My Time
PostPosted: Sun Jan 12, 2014 4:14 pm 
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cricketerry wrote:
SvenLittkowski wrote:
.....................................Yes, I used hydrogen peroxide. I just didn't knew that light would have been the solution. How bright, or what kind of light, should it be to have a rapid break-down into hydrogen and water?

My hydrogen peroxide solution recipe called for 1 part 3%HP to 9 parts of water. Since I could get only a 6% HP, I used 1 part of 6%HP to 18 parts of water. Hope that was still acceptable.



Normal daylight, by a window without letting the sun hit the jar and heat it up would be fine. It only needs to be for a few hours.

I always use 3% H2O2 straight from the bottle for sterilizing my seeds and equipment. I would be surprised if diluting it with 9 parts water as your recipe suggests would be effective.


Hmmm, good spot Terry, why would you dilute 3% peroxide for seed sterilizing and that recommendation is very weak.
You only needed to dilute your 6% by half, so this would have meant just adding the same amount of water to peroxide in one go so it is always ready to use, or dilute the 6% peroxide by half everytime needed.

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 Post subject: Re: Now It's My Time
PostPosted: Tue Jan 14, 2014 10:32 am 
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Thanks. I got most of the instructions from this document:
http://dokmaidogma.wordpress.com/2011/0 ... chen-style

That is, why I diluted the HP with water. Hopefully I succeeded to kill any bad contaminants. Let's see...

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 Post subject: Re: Now It's My Time
PostPosted: Tue Jan 14, 2014 12:41 pm 
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SvenLittkowski wrote:
Thanks. I got most of the instructions from this document:
http://dokmaidogma.wordpress.com/2011/0 ... chen-style

That is, why I diluted the HP with water. Hopefully I succeeded to kill any bad contaminants. Let's see...


Sven that 9 to 1 dilution is given for a chlorine based bleach not hydrogen peroxide (Mix 9 parts distilled water + 1 part bleach (household, 5-6% NaOCl depending on brand, I used Rimping ‘Haiter’ Bleach) + 1 drop detergent as many seeds repel water. We call this the sterilization liquid.)
this is also why they say to wash the seed after putting in the bleach.

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 Post subject: Re: Now It's My Time
PostPosted: Tue Jan 14, 2014 9:20 pm 
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cricketerry wrote:
SvenLittkowski wrote:
Thanks. I got most of the instructions from this document:
http://dokmaidogma.wordpress.com/2011/0 ... chen-style

That is, why I diluted the HP with water. Hopefully I succeeded to kill any bad contaminants. Let's see...


Sven that 9 to 1 dilution is given for a chlorine based bleach not hydrogen peroxide (Mix 9 parts distilled water + 1 part bleach (household, 5-6% NaOCl depending on brand, I used Rimping ‘Haiter’ Bleach) + 1 drop detergent as many seeds repel water. We call this the sterilization liquid.)
this is also why they say to wash the seed after putting in the bleach.


I haven't looked at the article but that makes perfect sense, hence why I chose hydrogen peroxide over bleach, less fuss!

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 Post subject: Re: Now It's My Time
PostPosted: Wed Jan 15, 2014 12:32 am 
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Thanks! I thought HP is more aggressive. But having read your two posts, I will do it again using HP with NO dillution. I am just so afraid to destroy these tiny seeds within a few seconds, that's why.

Even worse, all my own orchid seeds (vanilla) are now used up, I am out of seeds. But I need seeds in order to test new formulas and new approaches.

Does anyone have orchid seeds? Thanks.

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 Post subject: Re: Now It's My Time
PostPosted: Sun Feb 02, 2014 8:19 am 
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After some weeks, there are new developments which I want to bring to you all here now.

Of the eight flasks, three had to go into Nirvana because there were contaminated with fungus. But five of the flasks are clean up to now.

Inside one of these five flasks, I believe to see tiniest dots of ... green? And not (yet?) more than a fraction of a mm big.

Please have a look by yourself, and tell me what you think. Could that be embryonic orchids?

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 Post subject: Re: Now It's My Time
PostPosted: Sun Feb 02, 2014 10:49 am 
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Well first of all congratulations on having 5 flasks free of contamination. After several weeks you can be sure that they are sterilised properly so that part of your process worked ok.

As far as the green bits are concerned they certainly could be germinating seeds, time will tell on that one but anything green inside a sterile flask should be seen as a positive thing (assuming it is not mould or fungus).

I am not sure, looking at your pics, what angle you have taken them from. Are those green bits on your medium or on the sides of the jar? If on the sides I would suggest holding the jar by the lid and swinging it to get the seeds down onto your medium.

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 Post subject: Re: Now It's My Time
PostPosted: Wed Feb 05, 2014 4:58 pm 
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Thanks a lot, everyone! More photos will come as development (hopefully) continues.

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