Chlorine fumigation seed method for Paphs

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robx

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Hi everybody,

I am very happy to share this picture of green dots in a jar! This is my first batch of Paphs ever, P. bellatum and P. spicerianum, and half of them I did with a new method I wanted to try - chlorine fumigation.

Chlorine fumigation sterilization is described here:

http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0102-05362006000200019

Instead of the usual sterilization in a syringe of some bleach solution, you just sprinkle the seeds in prepared jars, cover the jar with a piece of plastic wrap with a thumb sized hole. Put a bleach soaked cotton ball (somewhat wrung out) on the plastic wrap over the hole, and put another piece of plastic wrap and then the lid. Let the chlorine fumes disinfect the jar for 3-4 hours, then use a forceps to quickly remove the cotton ball. You are done.

The idea is the bleach produces enough chlorine fumes to kill the spores, but not enough to kill the seeds. The paper says keep the cotton ball in for 3-4 hours, I kept the cotton ball in 3.5 hours.

The first batch of jars I tried half with the standard syringe method, half with this "Cl fumes" method. Both had success, and a contamination rate of about 1 in 8 (13%). Terrible I know but good enough for me. The syringe method was done in a big clear plastic tub and I am terrible at the syringe method, so uncoordinated, and as I do small batches I'm afraid I waste seed in the syringe. After I had success with the Cl fumes" I did the next batch with only the fumes method.

Let me know if you want any more details. I will say test that your cotton balls don't disintegrate in the bleach. (I used the concentrated bleach (8.25% sodium hyperchlorite) - I was careful to use fresh, unopened bleach). With the second batch I used a newer bag of cotton balls and they fell apart when i went to pull them out with the forceps. I ended up having to take the lid off and remove all the plastic. Surprisingly my contamination rate was the same the previous batches. Both bags of cotton balls said 100% cotton.

I hopes this encourages other to try paph seeds. I did all this in a regular kitchen. The next week I made ginger bread cookies and that seemed a lot harder! For other things like making media I read a lot of the great posts here and followed their instructions (although for Trithor's methods I substituted iced tea for wine :) ). I got the media from the internet and the coconut water from the overpriced health food supermarket in a little refrigerated bottle, 100% raw it said. Hurray for people who will eat and drink anything for health!

I still have to worry about getting a sterile environment for replating. The university said I could use laminar flow hood, but I do like to do things on my own and I'd like to say, "These are from my crappy glove box!"

Some jars seem to have few enough in them maybe they don't need to be replated. Has anyone taken plants from seed to compot in one jar? I'd probably have to make holes in the lid at some point. Six months? a year? maybe for the ones with plastic wrap still on take off the lid, secure the plastic wrap and poke holes in the wrap and cover with a plastic bandage. Any suggestions are appreciated.
 

Trithor

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Congratulations on your achievement. I think that the high contamination is the result of the iced tea substitution:p
(Your level on contamination is not bad at all for a glove box. Have you made a set of control flasks? If you do a couple of controls for each process involved (all exactly the same except no seed), you can often identify at which stage the contamination is occurring. I know it sounds like a tremendous waste of medium, but it helps identify a problem. I also make my flasks two weeks before I use them to ensure that the flasks are sterile to start with)
 

robx

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Congratulations on your achievement. I think that the high contamination is the result of the iced tea substitution:p

Ha! Glad you caught that!

(Your level on contamination is not bad at all for a glove box. Have you made a set of control flasks? If you do a couple of controls for each process involved (all exactly the same except no seed), you can often identify at which stage the contamination is occurring. I know it sounds like a tremendous waste of medium, but it helps identify a problem. I also make my flasks two weeks before I use them to ensure that the flasks are sterile to start with)

No, I didn't make control flasks, although I did let them sit for two weeks to make sure they were clean, and I had no visible contamination in those two weeks. Controls would be a good idea but I'm not unhappy with this level of contamination given how seat-of-the-pants everything was.

The first batch I didn't cook the agar long enough and it came out runny and I had to dump half the jars. I fixed that with the second batch and got perfect black jello. I made the second a small batch (200mL) as I didn't have a lot of seed and I wanted all the jars to fit in my pressure cooker so i could do them at once and leave them in the cooker until I was ready for them. Seemed to work out.

How often do you cook and how big a batch? That sounds like it came from an episode of "Breaking Bad"!
 

Trithor

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I cook about 2 liters of germination medium (not crystal) or 5 liters of replate medium at a time. That quantity of germination medium makes about 60 small germination flasks. I autoclave the small flasks with the medium in them (20 to a pressure cooker) I then pack 10 small bottles to a clear plastic container, so 6 plastic storage containers. I use clear storage containers to pack the germination flasks until I need to use them, so I can easily spot contamination problems and remove problem flasks. I use 3 different germination medium variations, and generally plate seed onto two or three of each variation and a control flask (so generally 10 small flasks at a time per pod) I find it very easy to use the syringe method as I can just squirt a small amount of suspended seed into each flask (except the control flask which is opened, but receives no seed and is sealed at the same time as the seeded flasks) these ten flasks then go back into the same clear storage container so they are isolated from the other flasks that I worked on at the same sitting. I inspect for contamination at two day intervals for the next month when they then are moved to the lighting grow racks (first thirty days dark for paphs) If I get high contamination and the control is clear, then obviously it was a problem with surface sterilization of the seed. Then I redo that seed using two approaches, a portion I soak in sugar solution over night prior to chlorine sterilizing, and another batch I chlorine treat for an extra 10 minutes over the first set.
The replate medium I sterilize in liter batches in the autoclave or pressure cooker and pour into pre gamma irradiated plastic tubs. One liter pours about ten tubs. These ten are treated exactly the same as the germination flasks (packed ten to a clear plastic storage container for two weeks prior to using)
And yes, in order to cook and handle the two liters of germination medium or the five liters of replate medium takes about the same time, generally a bottle of wine, so I try not to cook every day, but the temptation is there!
 

eteson

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Great! Congratulations, and thanks for sharing.
I switched from chlorine to H2O2 because I was killing too much seed during the stetrilization process and now i am very plased.... but Since I love to try new things i would test this method also.
Good luck during replating!
 

robx

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Thanks for the congratulations everyone and thanks for the details Trithor - it really helps a lot to hear how other people handle the details!
 

myxodex

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Interesting method. I'm encouraged by others doing their own sowing here on ST. Thanks for posting.
 
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