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Post by flytrapcare.com on Nov 20, 2008 16:27:49 GMT -5
How long does it typically take for a culture to show contamination if it is indeed contaminated? (Assume that there is no contamination in the seed)
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Post by hackerberry on Nov 20, 2008 17:03:35 GMT -5
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Post by flytrapcare.com on Nov 20, 2008 17:13:54 GMT -5
Hey hackerberry, thanks for the reply. For some reason I thought contamination would show in 2 to 3 days for explants. Is this not true?
I actually have the "Plants from Test tubes" book, but I haven't read it yet. But if you point me to the correct page, I'll take a look.
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Post by hackerberry on Nov 20, 2008 17:42:14 GMT -5
There is a chapter talking about contamination and sterilization but it's really broad. There are some tips that are very useful but the rest is up to your experiment.
HB
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Post by lloyd on Nov 20, 2008 17:52:45 GMT -5
Surface contamination 2-3 days. Internal contamination (systemic fungus) 2-3 weeks or when seed coat opens (germination).
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Post by hackerberry on Nov 20, 2008 17:54:52 GMT -5
Hey Lloyd,
Can you say that "No Damp" systemic fungicide? I also use it to soak seeds overnight and it works. thanks!
HB
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Post by flytrapcare.com on Nov 20, 2008 19:58:05 GMT -5
Thanks for all the information guys. So far I don't see any contamination in any of the cultures I made. It's been 48 hours now since I finished them. I did about 50 cultures, and half of them were seed. I seriously doubt that none of them have any surface contamination. I did really good with sterilizing explants and seeds and keeping the sterilize area free of air movement, but I think it would take a great deal of luck to not get *any* surface contamination.
I do see some discoloration in the media around the explant pieces. However, I think this is from the residual PPM that was on the pieces when I put them in the media.
BTW - what is phenolic bleeding?
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Post by lloyd on Nov 20, 2008 21:04:07 GMT -5
I recently did try soaking some explants with No-Damp and 4% PPM in MS and there was no contamination. However the explant just sat there.
Phenolic oxidation is the discolouration of the agar around the explant. It is toxic. You can move the explant to a new area of the agar. I've tried activated charcoal and potassium citrate/citric acid and keeping the bottle in the dark. They did keep the phenolic oxidation down. However every one of my explants has failed so I'm hardly the one to give advice.
If there is a spreading sphere of colour around the explant it is phenolic exudation. I've seen it so bad that the caps turned yellowish-green. If the discolouration is a greyish white cloudiness it is mould.
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Post by flytrapcare.com on Nov 20, 2008 21:22:04 GMT -5
Ah, I see. Nearly all of my explants have a sphere of color spreading around them into the media, so it looks like nearly all of my explant cultures have phenolic oxidation. Does this mean that they are doomed? What causes it? Over sterilization?
Can placing the cultures too close to fluorescent lights cause it?
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Post by lloyd on Nov 21, 2008 14:03:01 GMT -5
Maybe too much light, trauma? Try moving it to a fresh patch of agar or putting it in the dark for a while.
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Post by flytrapcare.com on Nov 21, 2008 17:58:41 GMT -5
Every single piece of explant had at least a little bit of phenolic oxidation around it. I was reading the "Plants from Test Tubes" book and in the Media Problems section it said that phenolic oxidation can be a result of dull knives, old cultures or too liquid a medium. My medium didn't turn out well in the first batch because the agar didn't gel well, so today I made a fresh batch of media and transferred nearly all of the plant pieces to fresh media. I guess I'll see what happens.
The ones that I didn't put in fresh media, I tapped them around in their jars until the were on a fresh area of media. It'll be interesting to see how they do.
I do have the explants placed pretty close to the light source. I think I've read that 12 inches is a good distance, but is it that important? I guess it could result in a higher oxidation rate if they're too close?
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Post by lloyd on Nov 21, 2008 21:30:50 GMT -5
I've read that you can leave the explants in the dark for a few weeks to avoid phenolic oxidation.
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