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 Post subject: Re: Showing of my cloning attempt...
PostPosted: Sun Feb 10, 2019 2:19 pm 
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raybark wrote:
Just a quick note on agar and gelling.

Once you have the right volume of liquid and have raised it to a proper temperature and given time for total solubility, you're left with a bunch of small molecular clusters. I heard a great description of "octopi with tentacles pointing in all directions". Getting them to gel properly involves growing those octopi tentacles linked together in bigger and bigger clusters, with liquid trapped within.

I speculate that if they cool too rapidly, you get smaller, loosely-bound clusters that makes the whole not appear to gel well, or it let's the liquid separate too easily from that network. That would suggest that a very slow cool is required.

As I type this, it has reminded me of generating color in glass - something I actually DO know something about: it's a similar procedure, involving melting to get every oxide in solution, then a slight cooling to initiate the crystallization of the color-forming nuclei, then a slight reheating to allow them to grow, enhancing the depth of color before allowing it to fully cool.



I think you might be on to something here. Normally I make batches 100ml minimum at a time, as it make the math much easier. When making the tubes, I add the water/agar, cook it for a couple minutes in the microwave, (careful not to overboil the container, as it makes a mess) then add the P793, stir, and distribute to the tubes, which are usually in the wooded block holders so I don't knock them over. The wood probably acts as a insulator and helps slow the cooling. I have not had any issue with these gelling after setting up the initial
process. On that day however, I tried something different, as he flasks were larger, I tried 50ml batches in the individual flasks to save on the distribution step. I had been thinking that my scale which is supposed to be accurate down to .01g wasn't as accurate under 1g, when measuring out the agar. However, the smaller batch size would likely make the heating/cooling much more rapid. When I remade the media in a larger container and the distributed it, it gelled fine. Agar, what an interesting substance.

Ben Belton wrote:
NHguy03276 wrote:
the lids have a .45um syringe filter, sealed with silicon for gas exchange.

Not sure what that is.


Now I am very confused by this statement...

At first I thought you might not know what a syringe filter was, But with your Profession is listed as Pharmacist in your profile, and years of labwork, I can't imagine you've never encountered a syringe filter, but I guess it is possible (https://www.amazon.com/Syringe-Filter-P ... 11&sr=1-55). since most molds have spores larger than 1 micron, it doesn't really matter if I use the .22 micron or the .45 micron size.

Then I thought maybe you mean you don't bother with gas exchange, but this would mean yet another major focus of almost every flasker/instructional video/talk/paper is not as important as many harp on... but... I... ...

Which only leaves what I'm actually doing here... which is to drill a hole in the lid just large enough to fit the filter, put some silicone around the hole and push the filter through the hole, sealing the filter to the lid, and adding a cheap .45 micon sized vent to the flask. I'm just noticing that the membrane of the filter on a couple of the older lids is staring to discolor, and I think it means that dust is staring to build up, and hopefully not that the membrane is starting to degrade.


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 Post subject: Re: Showing of my cloning attempt...
PostPosted: Sun Feb 10, 2019 3:49 pm 
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OK. I thought about those, but I couldn't figure out how you got them into a lid. So I decided I didn't know what you were talking about. :lol:

In the old days we used round bandaids. They worked great. I used lids with rounds bandaids for years.

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 Post subject: Re: Showing of my cloning attempt...
PostPosted: Mon Feb 11, 2019 1:47 pm 
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Thanks all for the discussion! I think I might try to experiment with stem props again :D

I hope you don't mind if I go off topic, and repeat some really basic questions... I've always had trouble sterilizing the stems -- what's your favorite method of doing this right?


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 Post subject: Re: Showing of my cloning attempt...
PostPosted: Tue Feb 12, 2019 12:03 pm 
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theshatterings wrote:
Thanks all for the discussion! I think I might try to experiment with stem props again :D

I hope you don't mind if I go off topic, and repeat some really basic questions... I've always had trouble sterilizing the stems -- what's your favorite method of doing this right?


So far I've had decent luck with the following. I remove the bract covering the node, and then soaking the stems for 10-15 minutes in a simple .5% sodium hypochloride solution, rinse/soak for 1-2 min, bath of sterile water, followed by an additional rinse in a second container of sterile water. Some people recommend a second 1% sodium hypochloride soak for about 10 mins, but I haven't done that yet.


to get the .5%, I use 90ml water to 10ml 5.25% bleach, be careful if using commercial bleaches, many are concentrated, and have higher than the 5.25%, you may need to do some math to get the .5%

Just to be clear, I'm still just a beginner myself, so I don't know how consistently reliable this is going to be. I have had a couple stem props contaminate, but there are many other vectors of contamination to consider as well.


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 Post subject: Re: Showing of my cloning attempt...
PostPosted: Tue Feb 12, 2019 7:54 pm 
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theshatterings wrote:
I hope you don't mind if I go off topic, and repeat some really basic questions

Seems appropriate to the topic to me.

theshatterings wrote:
I've always had trouble sterilizing the stems

It's not you. It really is hard and no matter how good your protocol and how careful you are, it happens. Sometimes it happens disastrously.

theshatterings wrote:
What's your favorite method of doing this right?

I have used multiple methods over the years. I learned everything before bleach was concentrated. I still do it without re-calculating. Probably a bad idea on my part. I think you have to try a few different things and figure out what works for you. When I grew under lights my plants stayed fairly dry, and I had very little issues with contamination. Since moving to a greenhouse I have had more problems. Under lights my protocol was fairly light and easy. Now it is stronger.

HP Norton, one of the greatest orchid people ever, and someone who taught several of us about lab work would gently brush stems in 25% (by volume) bleach and then soak 15-20 minutes in 10% (again by volume) bleach. He would then rinse with sterile water. Someone commented to me since that they don't like brushing the stems because theoretically you could push a contaminate down into the tissue of the stem where bleach would never reach it. Also here 11 years later I feel like 20 minutes in 10% bleach is a bit long. Also HP propped a lot of standards and polyploids which usually have thicker tougher spikes. They might stand the bleach a little longer than a soft novelty.

I found THIS site years ago. I thought I had lost it, but was lucky to find a link tonight I had forgotten I saved. I like sterilizing, at least in the last step, with peroxide. I don't like using sterile water because you can never know if you really got it sterile or if it has become contaminated. Also in the end the author mentions poly-propping. Something I have never tried, but I'd like to. Unfortunately his description is a little thin on detail.

THIS protocol from Pat at Kingfisher takes a long time, but is really good. I had great success with it in the past.

Lastly, we talked a lot about making stem props years back here on this forum. I had THIS link saved and I know there were several other threads. Somehow find Dean Stock, Robert Bedard, and/or Rob Sheppard's forum names and search for posts by them with search terms like prop and bleach maybe. I was the learner back then asking all the question, so searching on my forum name which is obviously over to the left, might work some too.

Don't forget to add a little soap to whatever you are sterilizing with!

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 Post subject: Re: Showing of my cloning attempt...
PostPosted: Tue Feb 12, 2019 7:55 pm 
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P.S. If you find any old threads, please post links! :BIG:

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 Post subject: Re: Showing of my cloning attempt...
PostPosted: Wed Feb 13, 2019 6:03 am 
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Thank you both for your tips and links! There's a lot of advice out there it seems..

Ben Belton wrote:
P.S. If you find any old threads, please post links! :BIG:


This one I think?

viewtopic.php?f=49&t=4153

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 Post subject: Re: Showing of my cloning attempt...
PostPosted: Wed Feb 13, 2019 6:10 am 
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I suspect there will be multiples.

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 Post subject: Re: Showing of my cloning attempt...
PostPosted: Wed Feb 13, 2019 12:32 pm 
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Ben Belton wrote:
I like sterilizing, at least in the last step, with peroxide. I don't like using sterile water because you can never know if you really got it sterile or if it has become contaminated.



True, it is hard to be certain the water is sterile. When I make up my sterile water, I use 1 pint canning jars with dome lids, and put them in the pressure cooker for about 3 hrs. I doubt anything that can survive that is going to be found in my home...

As long as those lids seal, I feel they are safe to use as sterile. I grew up trusting those lids, having a mother who home canned, and we would sometimes grab a jar that was 2-3 years old, at it was still safe to eat.

Once opened, I discard any left over water after the first use. Simply not worth the risk of contamination... I will probably add the peroxide step to my next batch. I've used 3% peroxide successfully to decontaminate a couple flasks, when I caught the contamination early enough. Thankfully, as I get more experience with the hood, the incidents of contamination are going down.


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 Post subject: Re: Showing of my cloning attempt...
PostPosted: Wed Feb 13, 2019 4:40 pm 
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NHguy03276 wrote:
When I make up my sterile water, I use 1 pint canning jars with dome lids, and put them in the pressure cooker for about 3 hrs

If you search around on the Phytotech web page there are some tips on this type thing. It will tell you how long you need to cook them for the volume. Would save you some time.

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