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By Bud
Posts:  275
Joined:  Thu Mar 26, 2009 9:45 pm
#57517
Hi all,

Hope some of you remember me, I took of a sabbatical due to some home renovating, and trying to complete a Koi pond project. ( I have been popping by to see what’s been happinin)

Well my traps are just about a bloomin and about to spurt their flowers, so I want to put the stalks in culture. I never did stalks before so I want to this right as I have a few unique varieties. My understanding is the best part of the stalk is where it just below the flowers, and the buds should be trimmed off. My questions are; Should I trim off the tips of the buds BEFORE disinfecting, and after disinfecting further trim off a bit to account for damage from bleach, OR just disinfect the whole flower stalk and top, then trim off the buds after disinfecting. Any other hints or details will be helpful, as I want to have the best chance of success on some of these more unique cultivars. I will be making up media tomorrow, and also trying to decide on using small amount of kinetin or BAP, or just go PGR free. If there are any updated thoughts on that, please share.

Thanks much, and hope your plants are healthy and prospering

Bud
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By Matt
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Posts:  22523
Joined:  Mon Apr 21, 2008 11:28 pm
#57586
Hey Bud, it's been a while! Good to see you back posting again.

I prefer to sterilize the stalk with the buds on and then trim them off before flasking. That minimizes the damage to the tissue.

For media, virtually anything seems to work to initialize Dionaea. I'd go low on PGRs rather than high on them.
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By Bud
Posts:  275
Joined:  Thu Mar 26, 2009 9:45 pm
#57624
Thanks Matt,

I thought that may be best but wasn’t sure; I may add 0.5 ml of Kinetin or BAP. My only TC successes have been with various seeds so far, so hoping these will take.

Thanks again

Bud
By goldslinger
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Posts:  772
Joined:  Thu Jul 09, 2009 1:31 am
#57633
You'll do fine, and welcome Back !

Gary :D
By Bud
Posts:  275
Joined:  Thu Mar 26, 2009 9:45 pm
#57798
goldslinger wrote:You'll do fine, and welcome Back !

Gary :D

Hey Gold man,

Thought you would have a mega culture lab be now, called Agri-Gary

Actually I’m off to a bad start; didn’t heat my Agar well enough, and my media was too inconstant, so got to mix another batch today-not happy about it. Thinking about trying Gelcarin, heard it’s much more soluble at lower temps.

Hope things are going well

Bud
By goldslinger
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Posts:  772
Joined:  Thu Jul 09, 2009 1:31 am
#58208
Bud wrote:Thought you would have a mega culture lab be now, called Agri-Gary

I like the ring of that !

Actually, I do have more toys; more than necessary.

I'm actually starting to kick back and taking a bit more relaxed approach as I learn more. I don't see it as much as an intimidating challenge, but more of an enjoyable one now.

Still have a long way to go.

I really like My Gelcarin GP812; it is partially water soluable and gels when autoclaved. I'm finding that 7.5 - 8.0 Grams/Liter is a happy medium (no pun intended) for baby food jars and larger, while 7 Grams is sufficient for vials.

Do Not Warm the media and add the Gelcarin; it will take an eternity to dissolve; keep it room temperature and stir and the baby turds will dissolve soon enough. It will be a cloudy golden color and smell of sea water. After dispensing and autoclaving, it will be crystal clear with maybe a slight golden hue.

the GP812 will raise the ph of the media alot.

For 1/2 MS, full Vitamin, adjust the PH (with the Gelcarin added) to 4.7 and the end autoclaved product will be 5.5 - 5.6. I know this to be true.

Gary
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By Matt
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Posts:  22523
Joined:  Mon Apr 21, 2008 11:28 pm
#58212
goldslinger wrote:For 1/2 MS, full Vitamin, adjust the PH (with the Gelcarin added) to 4.7 and the end autoclaved product will be 5.5 - 5.6. I know this to be true.
I've been adding the GP812 and then adjusting to pH 5.5 to 5.6! I wonder what my final pH is? I suppose that I should check it in one of the vessels after autoclaving. I haven't ever done that. The plants seem to grow fine even if the pH is a little higher than it should be. Though I'd be interested in seeing how they grow when I adjust the pH to 4.7 before autoclaving.

Thanks for sharing this Gary!
By goldslinger
Location: 
Posts:  772
Joined:  Thu Jul 09, 2009 1:31 am
#58298
Hey, Matt.

Yea, I've tested several times now with a thawed frozen sample of autoclaved media (froze to liquify it without changing chemistry like microwaving it would do). I've been doing this to 'hone' in on the proper starting PH so I don't have to do that extra step and waste media. I used a $200.00 Hannah calibrated tester that I have now, and the results are consistant.

I only use Distillied water so results should be same for everybody. I do use citric acid to lower ph, so maybe a slight variable to the end result there, don't know.

It's a good idea to know and record the before PH on Your batches before autoclaving. that way, if the PH is off from the norm, then You KNOW You left something out or added wrong amount, doubled it, etc. and You need to throw it out. Good advice from Dr. Fossard.

There has only been One person (on the list serv)Whom swears that the PH doesn't change after autoclaving, but that information was given to Him by a sales rep as He never mentioned His own results. I've always been suspicious from remembering way back to Frank's first video in which he had to compensate ph for the Agar, so testing with GP812 was a given to Me. i would be very interested in hearing Your results.

Gary
By Bud
Posts:  275
Joined:  Thu Mar 26, 2009 9:45 pm
#58346
goldslinger wrote:[
I really like My Gelcarin GP812; it is partially water soluable and gels when autoclaved. I'm finding that 7.5 - 8.0 Grams/Liter is a happy medium (no pun intended) for baby food jars and larger, while 7 Grams is sufficient for vials.

Do Not Warm the media and add the Gelcarin; it will take an eternity to dissolve; keep it room temperature and stir and the baby turds will dissolve soon enough. It will be a cloudy golden color and smell of sea water. After dispensing and autoclaving, it will be crystal clear with maybe a slight golden hue.

the GP812 will raise the ph of the media alot.

For 1/2 MS, full Vitamin, adjust the PH (with the Gelcarin added) to 4.7 and the end autoclaved product will be 5.5 - 5.6. I know this to be true.

Gary
Thanks much Gary for the detailed info, it is very helpful, and It gives me a good idea where to start and what to expect. I have questions on the PH; as you indicate that the PH raises after autoclaving. First, did you check the PH after adding the GP812, but before autoclaving while the media was still as a solution, did it stay the same or raise?
Also not a chem major here, but are you saying the act of heating through autoclaving, changes the PH? And so I understand this, it sounds like you checked the PH by freezing it to return it to a liquid state?

Thanks again
By goldslinger
Location: 
Posts:  772
Joined:  Thu Jul 09, 2009 1:31 am
#58357
Thanks much Gary for the detailed info, it is very helpful, and It gives me a good idea where to start and what to expect. I have questions on the PH; as you indicate that the PH raises after autoclaving. First, did you check the PH after adding the GP812, but before autoclaving while the media was still as a solution, did it stay the same or raise?
Yes, tested after adding the Gelcarin; it will not clog Your meter. When adding the GP812, it raises significantly, from say 4.8 to 6.2 This is before autoclaving.
Also not a chem major here, but are you saying the act of heating through autoclaving, changes the PH? And so I understand this, it sounds like you checked the PH by freezing it to return it to a liquid state?
Since GP812 is only partially water soluable, You still have solids that aren't affecting the PH when first testing, only to have them fully in the picture when fully dissolved AFTER autoclaving, so it raises it more. This is My hyposthesis and I'm not a Chemist either.

Barry on the listserv turned Me onto the idea of freezing the media after autoclaving as the ice crystals physically break up the bond and doesn't chemically change it possibly affecting the results. It won't be fully liquid, but liquid enough that You don't have to macerate.

This is all 'for what it's worth' as I've used the first batches that tested 6.2 or whatever (because I didn't know to compensate) with seemingly no noticeable affects on established cultures, but I didn't pay that much attention; maybe the growth is slower, maybe more yellowing; I don't know for lack of history.
It certainly can't hurt to get it right, though and I'll continue to strive for that 5.5

Gary

Gary
By ahicks51
Location: 
Posts:  133
Joined:  Fri Jan 15, 2010 7:13 pm
#58365
Bud wrote: Also not a chem major here, but are you saying the act of heating through autoclaving, changes the PH?
pH will almost certainly change after autoclaving. At the very least, heating drives off carbon dioxide; pure water has a pH of 7.0; however, atmospheric air has a few hundred ppm of carbon dioxide, so once equilibrium is established, the pH drops to about 5.4 to 5.6, as the carbon dioxide in solution forms carbonic acid. Heat it up, or add salts, or whatever, and you change that equilibrium.

With only 2-3 components, it becomes fairly easy to calculate the pH of a solution. Start adding 10-20-30, and the equilibration can take centuries. Sure, the major changes take place in a few seconds, but it's a very complex solution- even moreso with sparingly soluble or insoluble fractions.

Heating that up changes the pH because it can change where individual components reach equilibrium. And when it settles back down, things may have changed even further.

Just look for consistency: make it the same way, every time, and when changes are made, make them in a repeatable fashion. It's the most we can hope for.
By Multiclone
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Posts:  186
Joined:  Sun Sep 27, 2009 2:37 pm
#58384
I have adjusted the ph between 5.5 and 5.8 before autoclaving for years with the Gelcarin added, for everything. Never had a growth problem yet.
Geoff
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By Matt
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Posts:  22523
Joined:  Mon Apr 21, 2008 11:28 pm
#58403
Multiclone wrote:I have adjusted the ph between 5.5 and 5.8 before autoclaving for years with the Gelcarin added, for everything. Never had a growth problem yet.
Thank you for posting this Geoff! It put my mind at ease so much. I've been adjusting the pH to 5.5 to 5.7 after adding Gelcarin but before autoclaving and my plants seem to grow fine on it as well. But I've been wondering what the final pH of the solution is since reading Gary's post.
By goldslinger
Location: 
Posts:  772
Joined:  Thu Jul 09, 2009 1:31 am
#58426
That makes Me feel better, too. I will still compensate on My batches, since I know where I want it to be.

It appears that PH isn't super critical to VFT'S. I guess with some plants it is.
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