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 Post subject: Sterilizing Stems
PostPosted: Mon May 28, 2007 7:00 pm 
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For those of you doing your own stem props, how are you sterilizing them. I did some recently and am not satisfied with the results.

Heres what I did:


Surfaced sterilized with 100% bleach with a Q-tip with the bract still on.

I would remove the bracts and place the stems in a 5% bleach solution for 15 minutes.

I would remove the plants and put them in a 3% H2O2 for 5 minutes to rinse them. Place into flask.


I suspect that I could go as high as 15% for the bleach solution and not do the rinse.

Any thoughts?


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PostPosted: Mon May 28, 2007 9:20 pm 
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Location: Kanab, Utah
After many years of doing this I finally settled on the following:
l. cut stem into individual node pieces with at least 1" of stem above and below the node.
2 apply an antibacterial "soap" solution to the stem with the bract intack and work this around with the fingers.
3 carefully remove bract with a blade dipped in 15% bleach (15% dilution of standard off the shelf "clorox"). I usually catch the corner of the bract with the scapel blade and lift it off. Don't do a lot of cutting to remove bract tissue.
4. carefully reapply antibacterial soap to bract area being careful not to damage the tender growing point below the bract.
5. Rinse in running tap water to remove traces of soap.
6. Place in tube with 15% clorox solution for approaximately 15 minutes. This is about right for most stems but tender fresh stems will not do well with this lenght of time.
7. Remove from bleach and rinse in a container of sterile R/O water or other suitable lab quality water.
8. pick up with sterile forceps and place in container with stem prop medium.
9. Works most of the time but some stems from some plants will have endogenous bacterial/fungal contaminants that will prevent success. I do not wet my plants when watering and I run my greenhouse a little drier than most do so I tend to have less of a problem with contamination. Never place stem in container of water while you are getting ready to process it as it will tend to pick up contaminants.
Dean

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 Post subject:
PostPosted: Sat Jun 02, 2007 6:54 am 
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Location: Canada
Thanks!

Do you think its Acceptable to substitute H2O2 for sterile water? Or do you think it is even neccessary to rinse off the bleach solution?

Quote:
Never place stem in container of water while you are getting ready to process it as it will tend to pick up contaminants.


You mean keep them in a glass or vase of water until your ready to use them? Thats good to know.

Do you have any experience re-sterilizing contaminated stems?

Kyle


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PostPosted: Sat Jun 02, 2007 8:47 am 
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My collection isn't as clean as Dean's, I use a slightly different protocol ... I usually leave that last clorox dilution on the stems when I stick them in the media; that is 3% clorox in sterile distilled water.

If the stem is systemically contaminated, it is probably a waste of time to try and re-sterilize it. If it is just surface contamination, I have had good luck resterilizing with 10% clorox, rinse with 3% ...

I used to place the stems into 50-50 H2O2 and water, while processing them. I was told the H2O2 would clean up some systemic pathogens. I have quit doing this though, because it was very hard on the softer tissue, and I am not convinced it really helped with systemic contamination. I have yet to find anything that really works for that.

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 Post subject:
PostPosted: Sat Jun 02, 2007 9:13 am 
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Robert,

I don't fully understand what you mean by systemic contamination? Do you mean that the fungis (or whatever) is actually in the tissue?

I like the idea of rinsing with the 3% bleach. I'm not always confident with the concentration of the H2O2 I use.

When you sow dry seeds, do you also use 3% bleach as your final rinse?

Kyle


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 Post subject:
PostPosted: Sat Jun 02, 2007 10:05 am 
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kyle wrote:
I don't fully understand what you mean by systemic contamination? Do you mean that the fungis (or whatever) is actually in the tissue?


Precisely. You can often see the fungus growing out of the cut end of the stem, when it is systemically infected like that.

kyle wrote:
When you sow dry seeds, do you also use 3% bleach as your final rinse?


No, with seed I use sterile distilled water as the final rinse.

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 Post subject:
PostPosted: Sat Jun 02, 2007 10:10 am 
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rbedard wrote:
You can often see the fungus growing out of the cut end of the stem, when it is systemically infected like that.

No, with seed I use sterile distilled water as the final rinse.


I have seen the fungus on the end of the stem. Usaully a white 'fluffy' one. Interesting.

In the past, i came the conclusion that my 'sterile' water, or the container it was in, was the source of contamination for both my seeds and my stems.

Do you think there would be harm done by using a 3% solution as that final rinse with seeds?


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 Post subject:
PostPosted: Sat Jun 02, 2007 7:21 pm 
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Location: Kanab, Utah
Several years ago I ran a large series of tests on decontamination of stems and concluded that the final rinse should be sterile water. I also had pretty convincing data that peroxide was not a useful way to sterilize stems because it tended to be difficult to remove from the tissue by a simple rinse. I found no significant positive effects of a rinse or soak with peroxide vs diluted bleach treatment. I did find negative effects.

Treatment of seed with peroxide was also not as successful as with bleach but the seed coat is much more resistant than stem tissue and the peroxide breaks down usually before the seed germinates. I see no good reason to use peroxide with either seed or stem protocols.

There are products that are an aid in combating endogenous bacterial and fungal contamination of stems and other tissues but I have not used them enough to be confident with them. I have use an antibiotic and fungicide mixtue that is expensive but quite effective when used properly. I belive that the best way is to use clean stems from clean plants and hope for the best.
Dean

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 Post subject:
PostPosted: Sun Jun 03, 2007 6:08 am 
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Location: Mt Jackson, Va
Here are some things I have found useful when doing stems:

1. A couple of days to a week before doing the lab work, give the plant and stem a good spraying with a systemic fungicide.

2. A the first signs of contamination, pull it out of the tube and repeat the whole sterilization process. Some dormant spores are very had to kill.

3. When all else fails try PhytoTechs PPM. Phals do not seem to like it very much, but at the lower concentrations the stems will grow. Sometimes it will clear up a problems in a few weeks, but in most cases it just slows the contamination growth enough that the node has a chance to produce plantlets.

Pat


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 Post subject:
PostPosted: Sun Jun 03, 2007 12:38 pm 
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Thanks for the tips Pat. I have also used PPM and it can be a lifesaver if you are struggling to save a very valuable stem from something difficult to stem prop.
Dean

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 Post subject:
PostPosted: Mon Jun 04, 2007 8:43 am 
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Dean, how big of a difference in development did you notice between a 3% final rinse and sterile water final rinse?

I use the 3% final rinse like Robert and I'm getting generally good results. I could see with smaller, younger and/or more delicate nodes that it might make the difference between frying or not frying the node.

I haven't tried any of the anti-fungal products yet. But I have also had reasonable good results re-sterilizing stems that contaminated the first time as long as you get to them before it gets out of control. The best chance is always to start with clean material in the first place. But sometimes you don't have that luxury.


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 Post subject:
PostPosted: Mon Jun 04, 2007 11:07 am 
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Rob, I never tested a 3% final rinse in my test series. Just thinking about it though, what good does it do? You have already treated the stem with more concentrated bleach, a final 3% rinse adds nothing and may retard the develpment of the node as it does with seed. I do have data that shows that it will retard seed germination and lower the germaination rate.

With lab protocols one must ask at each step what a particular procedure does for the protocol and is it needed. Many years ago, in a famous research lab, I took their protocol for chromosome treatment for "C-banding" and reduced it from a three hour procedure to a 15 minute procedure that was much more effective by just eleminating all of the multitude of steps and alcohol rinses that to me were meaningless.
Dean

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 Post subject:
PostPosted: Mon Jun 04, 2007 11:33 am 
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The primary reason I use it on my end is I don't normally keep sterilized water handy at all times. I have 3% premixed that I can use as needed.

Its not really a good reason to use a 3% rinse and probably something I should change in the future. But I'm getting the results I need right now, so there has not been motivation to change the process.


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 Post subject:
PostPosted: Mon Jun 04, 2007 1:15 pm 
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The reason I use the 3% final is because it was part of a protocol that was recommended to me, and I wanted to use it the way it was described, until such time as I had a reason to alter the procedure. The rationale behind it is, a final cut on the bottom of the stem is done immediately before flasking to have a freshly cut surface in contact with the media, and this cut is done in the 3% solution. Any time you cut tissue, there is a possibility of contamination from inside the tissue itself. I have a much higher success rate using the 3% bath for this final cut than using plain distilled water. It is possible that I could have an even better rate of success if I followed that with a sterile water rinse. But I have never had a reason to do so, as the vanishingly small amount of 3% clorox doesn't seem to have a negative effect. (I rarely sterilize the nodes to death.)

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 Post subject:
PostPosted: Mon Jun 04, 2007 2:14 pm 
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Robert, the 3% bleach treatment damages the stem tissue at the site of the new cut and causes more phenol production. It is not needed nor recommended by anyone with a background in tissue culture. The sterile water rinse is what is best here, but who am I to try to correct old habits (have a few of my own).
Dean

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