Go Back   Orchid Forum Orchid Care > Orchid Care > Orchid Flasking and Seed Germination

Reply
 
LinkBack Thread Tools Display Modes
  #1 (permalink)  
Old 06-01-2006, 09:06 AM
Junior Member
 
Join Date: Feb 2006
Location: South India, kanyakumari dist.
Posts: 17
Thanks: 0
Thanked 2 Times in 2 Posts
Joseph Stanly is on a distinguished road
Arrow how to contain secondary contamination while replating?

Can Any one enlighten me on"How to prevent contamination during replating of PoBs{Protocorm Like Bodies}?.
Reply With Quote
  #2 (permalink)  
Old 06-10-2006, 05:28 AM
jerrymeola's Avatar
V.I.P Member
PhotobucketPhotobucketPhotobucketPhotobucket
 
Join Date: Nov 2005
Location: SW Florida - Fort Myers
Posts: 2,958
Images: 650
Thanks: 0
Thanked 1,239 Times in 532 Posts
jerrymeola has a reputation beyond reputejerrymeola has a reputation beyond reputejerrymeola has a reputation beyond reputejerrymeola has a reputation beyond reputejerrymeola has a reputation beyond reputejerrymeola has a reputation beyond reputejerrymeola has a reputation beyond reputejerrymeola has a reputation beyond reputejerrymeola has a reputation beyond reputejerrymeola has a reputation beyond reputejerrymeola has a reputation beyond repute
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
Reply With Quote
  #3 (permalink)  
Old 08-08-2006, 01:17 PM
Junior Member
 
Join Date: Jul 2006
Location: Hell
Posts: 11
Thanks: 0
Thanked 0 Times in 0 Posts
Lucifer is on a distinguished road
Quote:
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
Reply With Quote
  #4 (permalink)  
Old 08-10-2006, 01:30 AM
Junior Member
 
Join Date: Feb 2006
Location: South India, kanyakumari dist.
Posts: 17
Thanks: 0
Thanked 2 Times in 2 Posts
Joseph Stanly is on a distinguished road
secontary contamination

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!
Reply With Quote
  #5 (permalink)  
Old 08-11-2006, 04:02 PM
Junior Member
 
Join Date: Jul 2006
Location: Hell
Posts: 11
Thanks: 0
Thanked 0 Times in 0 Posts
Lucifer is on a distinguished road
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!
Reply With Quote
  #6 (permalink)  
Old 09-05-2006, 02:17 AM
Junior Member
 
Join Date: Feb 2006
Location: South India, kanyakumari dist.
Posts: 17
Thanks: 0
Thanked 2 Times in 2 Posts
Joseph Stanly is on a distinguished road
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
Reply With Quote
  #7 (permalink)  
Old 09-05-2006, 11:55 AM
Junior Member
 
Join Date: Jul 2006
Location: Hell
Posts: 11
Thanks: 0
Thanked 0 Times in 0 Posts
Lucifer is on a distinguished road
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!
Reply With Quote
  #8 (permalink)  
Old 10-20-2007, 05:04 AM
awaxa's Avatar
Junior Member
 
Join Date: Jun 2007
Location: Trinidad & Tobago
Posts: 22
Images: 16
Thanks: 4
Thanked 3 Times in 3 Posts
awaxa is on a distinguished road
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.
__________________
awaxa
Reply With Quote
  #9 (permalink)  
Old 10-28-2007, 07:43 AM
Charles's Avatar
Junior Member
 
Join Date: Sep 2007
Location: Florida, USA
Posts: 26
Thanks: 1
Thanked 9 Times in 2 Posts
Charles is on a distinguished road
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.
__________________
Nuthin Fancy.
Reply With Quote
  #10 (permalink)  
Old 10-29-2007, 09:51 PM
Junior Member
 
Join Date: Sep 2006
Location: South Australia, Australia
Posts: 34
Thanks: 0
Thanked 2 Times in 2 Posts
Kevin Western is on a distinguished road
Truly brilliant!

Quote:
Originally Posted by awaxa View Post
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
Reply With Quote
  #11 (permalink)  
Old 11-05-2007, 05:51 AM
awaxa's Avatar
Junior Member
 
Join Date: Jun 2007
Location: Trinidad & Tobago
Posts: 22
Images: 16
Thanks: 4
Thanked 3 Times in 3 Posts
awaxa is on a distinguished road
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.
__________________
awaxa
Reply With Quote
  #12 (permalink)  
Old 11-08-2007, 01:31 PM
calypsogrower's Avatar
Junior Member
 
Join Date: Nov 2006
Location: Pacific Northwest California
Posts: 24
Thanks: 0
Thanked 0 Times in 0 Posts
calypsogrower is on a distinguished road
vacuum for seeds

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
Reply With Quote
Reply



Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On







Free Vote Caster from Bravenet.com Free Vote Caster from Bravenet.com


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
SEO by vBSEO 3.6.0
Orchid Forum

vBskin developed by: CreationLab