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One MS inoculated LC split into two


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#81 jkdeth

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Posted 19 April 2019 - 09:19 AM

A contaminated plate you transfer out of and dispose of.
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#82 RutgerHauer

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Posted 19 April 2019 - 10:21 AM

Thanks. I know that is an option and might do that one of these days. If I'm correct it will be a very medium but neat looking clone so it would be nice to have some more plates of that anyway.

What is the appropriate time to get it into storage? Would you need to leave some clean agar so it call still grow while in cooler temperatures?

#83 jkdeth

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Posted 19 April 2019 - 10:39 AM

Storage should slow it enough. Ideally your storing a plate your going to transfer from. I'd want a well colonized plate in good condition. If I plan on storing I make a nice thick plate. It seems more about available moisture than nutrients or surface space.
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#84 RutgerHauer

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Posted 19 April 2019 - 10:51 AM

Good info. Had read that about thicker pouring for storage but it hadn't stuck!

Ideally then I will pour some thicker ones and do a transfer from the best three, beginning with the cleanest ending with the contaminated and then get rid of that.


Just picked up a proper scalpel for agar work. Had used the same ones, but disposables with a plastic handle. Had sterilized those before but the last time I was lazy and wrapped them in foil together. They got stuck together, had this slanted double scalpel going.. Ha!

Edited by RutgerHauer, 19 April 2019 - 10:52 AM.


#85 jkdeth

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Posted 19 April 2019 - 11:13 AM

First time I sterilized those the plastic caps melted. I wrap singly, but the handles go down hill after a couple runs.
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#86 RutgerHauer

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Posted 19 April 2019 - 11:26 AM

Just another one of those mistakes one makes doing this..! I can laugh about it all though.

#87 RutgerHauer

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Posted 25 April 2019 - 08:22 PM

Ahhh, to have nice new tools and tricks.. I had trouble with the leveling of my SAB setup, since its bottom has a bulge, no pun intended. Well, it always looks silly because if I leave it as a comment it is always intended, the ability to edit implies that.

Anyway, back to SAB and AGAR. I couldn't stack for shit, as you have seen earlier in this thread. I tried last time with and oven tray upside down and on top of that a mat, but the mat was wobbly and when I touched the tub things went shaky.

Also, my positioning, my posture while doing SAB work made my back and neck uncomfortable. I put it up higher so the armholes are at height of my belly. I can hold things in front of my face much easier now, that's important. You can see!

The working area I fixed with a rack that's used to cool down (hash) cakes and other stuff. It is made of stainless steel rods in a grid. Pretty ideal, since it makes working with glass plates easier, they feel heavier and won't slide around as easily, especially since I have also fixed the problem of a stable and higher underground for my SAB: another bin of the same dimensions with all my lab stuff in it. Like, why didn't I think of that before..

Pictures: last agar pour and stack, off - SAB setup - one of my clones getting ready for storage on agar.


Some of the plates were poured too thick and the last plates have unclear agar going, like that nasty last bit of an otherwise perfectly good beer, lol.. Had counted on some percentage of failed or less ideal plates, so didn't matter. I needed six plates to do two transfers from three clones. This is the first of those clones depicted. It is showing much better and healthier growth than on the previous plates that I poured with Potato Dextrose Agar. I used light malt extract, yeast and agar now. They seem to prefer this.

I coded them simply with one and two spots and in order of color: red, yellow and blue. That makes three sets of two. You can see the one red spot if you have a good eye. (besides the lid of my pitcher, which is also just a big red spot, not a contamination, calm down.)


The new setup is pretty neat. I can fit the tripod that comes with the little basket I use to sterilize my plates just in between the grid of that grill. That pitcher work great, but is a bit bulky, it measures one liter. I make it work with putting my whole hand under that handle instead of holding it like you would with a smaller pitcher. I can move it up pretty high up to the lid, under my nose, since my hand isn't in the way.

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Edited by RutgerHauer, 25 April 2019 - 09:47 PM.


#88 Billcoz

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Posted 26 April 2019 - 12:15 AM

Yes, the bulge in the SAB is a problem for stacking plates, my shit bounces around in there when I bump it, and it's slick. I've broken glass jars just bumping it hard. Plus the bin is so light, I glued a rack from a wire shelf on the bottom, and I cut one down and set it inside, but I have to put cardboard down on it so my exacto(yeah, I'm cheap, not like your fancy-assed "proper scalpels" ha) or syringes don't fall through to the bottom while working, it does the trick. Your setup sounds cool.

 

One thing I hate about using new exacto blades is that they are so damn pointy. I have stuck my hands/fingers so many times while doing agar, and it bleeds like crazy in the glove, and out into my agar plate. One time I had it up on a high shelf above my head while I was right underneath it, and I was putting something up on the shelf and knocked the exacto knife off STRAIGHT DOWN INTO MY BIG TOE KNUCKLE!!! It went "flpdpddd" and stuck straight up and stayed there. It took a while to get it out, I had to really pull, it was in bone, scraped when I yanked it, that shit hurt like hell.


Edited by Billcoz, 26 April 2019 - 12:17 AM.

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#89 RutgerHauer

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Posted 28 April 2019 - 12:03 PM

Heavy, bill! Be careful.




Update on the clone test grow. See you in a few days when I case it. Didn't expect all the vermiculite to disappear in between those corn kernels, but it seems it almost would fit all together in two jars. Figured all jars are about 0.4 liters - Result is about 1 liter of substrate in a 2.1 liter tray.

The 'master plates' of my clones are not doing all so well, two or three contaminated. Better do another few transfers soon.

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Edited by RutgerHauer, 28 April 2019 - 04:48 PM.

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#90 RutgerHauer

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Posted 09 May 2019 - 04:38 AM

Just some pictures of the process I have been saving up to post. I have rejoined the working life recently and have less time to get on it..

First four pictures are from around the time the site was down, the last ones are from today. Looks like it's pretty much colonized though the sub is not as white as I was expecting from my previous grows: that's probably because all of the vermiculite mixed in which gives it a darker color.


I'm very unsure about the 'master' agar plates I made, the growth is so extremely fluffy, thick and white in comparison to my previous plates that looked very clean and the mycelium was hard to spot since it only grew thinly on the surface of the agar. Afraid it might be some other fungus over growing things, but I have seen thick white growth on other plates online that are supposed to be healthy so I'm not sure. Lots of condensation, so a decent picture is hard to make: there is thick fluffy growth growing from the center, with thinner growth growing in a circle around it that is hard to see.

A lot of my plates contaminated so it could be they are all fucked, but I hope to hear from someone that cubensis can also grow very thick and fluffy on agar, maybe in certain conditions like high humidity..

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