how to contain secondary contamination while replating?

[Joseph Stanly]

Can Any one enlighten me on"How to prevent contamination during replating of PoBs{Protocorm Like Bodies}?.

[jerrymeola]

No secrets. Good general sanitary practices. Disposable gloves are a cheap investment. Clean sterile instruments to handle the plants. Being careful about what you do.

[Lucifer]

Originally Posted by jerrymeola

No secrets. Good general sanitary practices. Disposable gloves are a cheap investment. Clean sterile instruments to handle the plants. Being careful about what you do.

Jerry is right. Good technique is your best bet. Once you learn them, they will become second nature. A laminar flow hood really helps. I'm a big fan of flaming tools, if you are to, make sure you cool the tool off by dipping it into the media before you handle the protocorms.

Kyle

[Joseph Stanly]

dear lucy,
I do have a laminar flow, and take al the precautions as mentioned in the text books. But I am worried about the % of contimination as high as 60%. I have some very rare species which sets pods rarely but the contamination spoils the lot. Thank you and Jerry for the nice advice!

[Lucifer]

Hmmm. How are you sowing the seeds? Dry or green. What genus are you dealing with?

Another tip is to use a product called PPM. Its a antifungal/bacterial type additive (sorry can't think of better words for it...). In my experience, it really cuts down on contamination when using highly contaminated seed or explants.

Another tip is to soak dry seed for 24 hours in a sugar solution. This 'awakens' dorment spores and allows the disinfecting solution a better chance of doing its job.

Keep trying, and keep a record of what you've done (disinfection time, concentration, protocols) so that you can duplicate it.

Good luck!

[Joseph Stanly]

Dear Lucy,
In fact PPM is plant protection media and I am using it. Your suggestion of soaking in sugar solution is try worthy. I have no contamination in green seeds but dry seeds gives me trouble.
I have succeed in germinating spathoglottis affinis, and minature dendrobium [phalenopsis type] only. Now I am going to try some other generas like vandas and intergenerics, dendrobium{phal types] cats, phalenopsis,cym etc But I need seedswhich I am unable to get.
Thanks, Keep in touch.
Stanly.J

[Lucifer]

Hi Joe,

From what you've told us, it looks like your sterilization procedures are the problem. It could also be that you're seed is so contaminated that is makes sterilizing nearly impossible.

What are you using to sterilize? Bleach? H2O2? I find bleach works good. Maybe up you concentration by 10% or increase the duration. Or do both. Do you rinse seed with sterile water?

Experiment! and keep records so you can duplicate the successful outcome.

Good luck and keep trying!

[awaxa]

Hi Joseph,
I have recently started dry seed germination myself and noticed that I get better results when I soak the dry seeds overnight in a concentrated sugar solution or exposed the seeds to a vacuum in a syringe. You can try the vacuum method. Sterilize the seeds in a syringe with the needle hole blocked at the top before it is connected to the syringe with a small piece of cotton. After you have pulled up your sterilizing agent and shook for a while, you will then be required to insert the needle into a spare rubber stopper and then pull the syringe plunger out as hard as you can. That produces a vacuum inside the syringe that allow to sterilizing agent to work on the seeds more efficiently. Do that at least 3-times. 100% success.
I wish you luck.

[Charles]

vacuum trick is note worthy too.
A wetting agent might be necessary. Have you added the small drop of dish soap? If your spores are not getting fully wet, you might be allowing some to "hide" from sterilization.

[Kevin Western]

Originally Posted by awaxa

Hi Joseph,
I have recently started dry seed germination myself and noticed that I get better results when I soak the dry seeds overnight in a concentrated sugar solution or exposed the seeds to a vacuum in a syringe. You can try the vacuum method. Sterilize the seeds in a syringe with the needle hole blocked at the top before it is connected to the syringe with a small piece of cotton. After you have pulled up your sterilizing agent and shook for a while, you will then be required to insert the needle into a spare rubber stopper and then pull the syringe plunger out as hard as you can. That produces a vacuum inside the syringe that allow to sterilizing agent to work on the seeds more efficiently. Do that at least 3-times. 100% success.
I wish you luck.

Hi Newbie,
Your comments re the use of a syringe to remove gas from the micropyle of the seed is truly brilliant.
I have a scientific background and I have been engaged as a Manufacturing Pharmacist in compounding specialised pharmaceuticals every day of my working life . I've also successsfully used vacuum technique to remove air bubbles from hairy tillandsia seeds before sowing by aseptic technique.
Despite all those years it has never occurred to me to use a syringe to pull a vacuum on the seeds and 'inject' sterilant back into the micropyle.
I think you may have made it possible for me to get a sterile result from some very valuable, hitherto impossible to sterilise seed. That is the Idea of the decade for me! Many thanks.
Kevin Western

[awaxa]

Hi Charles,
Yes, I use a wetting agent for all sterilizing. The trick also is the sugar, which causes the spores to become active and hence they can be destroyed easier especially under pressure.

[calypsogrower]

Hello,

I have tried sucking on the tubes with the seed inside in solution. It worked ok but lots of work! I am trying to find a way to use a hand pump vacuum. I posted elsewhere before I found this more appropriate thread...all help is appreciated! thanks, calypsogrower