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First Agar Attempt


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#21 rockyfungus

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Posted 30 December 2020 - 11:20 PM

I've just had bad luck with standing water on my plates.

So if the syringe left a puddle usually slime mold or bacteria takes over. I've had better luck with a loop to truly get less then a drop on the agar. Sounds like you just got bad syringes though


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#22 HrVanker

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Posted 01 January 2021 - 06:23 PM

So I took a couple transfers from the most advanced GT cultures. I tried to get the most rhizo looking stuff, but I'll probably need a few more transfers to get everything clean and isolated. I also took some gills from a B+ (IIRC) isolate I had laying around and put them to agar.

 

Will post pics once there is something to see.


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#23 HrVanker

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Posted 06 January 2021 - 09:03 PM

Here are the "good" transfers from the other day. They pretty much all had some bacterial hitchhikers but I see a few good spots for transfers.

On this first one, the myc has been having a difficult time getting past the bacteria on the transfer. But the section I marked is where it made it to the fresh agar and will grow out far enough to transfer from.
4052552247702236275bd5c98557b70c.jpg

This one is similar to the first, but the myc is having more trouble. I'm mostly keeping it to see how it fares. Hope is not high.
b2619338de25d35975f9f01171ceeff6.jpg

The myc on this transfer seems pretty aggressive and I'm excited to see how it does on the next cleaner transfer.
bcaa90d1c8eae43017f395ef586f7f3c.jpg

This is the cleanest of all the transfers. It's got a nice mat going with plenty of great looking sectors.
2cce4ce3c1155c8ca814ffd060ca12b0.jpg

And lastly, this is the jar I threw a few pieces of gill from a B+ isolate from a FOAF. Within 3 days there was visible myc, and these things have been in a jar w/ silica gel for 6mo!
c258cc19616be1f2c6fc417a051ab624.jpg


Edited by HrVanker, 06 January 2021 - 09:21 PM.

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#24 rockyfungus

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Posted 07 January 2021 - 11:47 AM

Always super difficult to tell from pictures. Your first 2 jars I'd toss though, both I can't discern any healthy myc.

When you look at pic 3 it has healthy obvious myc, but right at your transfer is that same slime/bacterial presentation.

Great pictures, just so hard to discern without seeing from top/bottom/side.


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#25 HrVanker

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Posted 07 January 2021 - 12:57 PM

The jar in the first pic will be fine once the marked edge grows out a little. There's the tiniest bit of myc that's finally made it to fresh agar and should grow out nicely.

I definitely don't have high hopes for the second jar.

The third jar has a bunch of slime on the left side, and a bit on the upper left outer edge. But everything to the right is clear and free.

The downside to jars is the lack of good photos, other than that I do like them. I may try pouring some proper plates, but not right now.

#26 HrVanker

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Posted 08 January 2021 - 07:20 PM

Yesterday I made a few more transfers. So far all but one are showing signs of recovery, puffing up and making visible progress towards the new agar! I have a few different agar recipes floating around: chunky WBS agar, LME (dark blue), and a new 50/50 WBS/LME (light teal/green). So for each of the transfers from yesterday I tried to make sure that each one was on a new type agar, just to see how they do.

 

I made T1s from 4/6 of my Redspore germ plates:

RS1.jpg

RS2.jpg

RS3.jpg

RS4.jpg

 

Each of these are T2s from my GT plates:

GT1.jpg

GT2.jpg

I made 6 of these, but these are the only two worth showing at the moment.

 

And lastly, 3 T1s from the germinated B+ gills:

BP1.jpg

BP2.jpg

BP3.jpg

 

I also made up 10 4oz jars of WBS to test some of these cultures (I plan to make mini tubs with some or 10oz containers). And since the PC was running, I made 3 plates of plain distilled water agar. Planning on testing them out with some bacterial transfers to see how it goes.

20210108_1043361.jpg


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#27 HrVanker

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Posted 21 January 2021 - 06:09 PM

Last week I made some transfers from some of the jars in my last post. Most of them are doing okay, nothing to write home about. But I will say that the culture from the B+ gill is looking really good.

 

I made two transfers from a T1 B+ jar (WBS/LME). One went to my last chunky WBS-agar and the other went to a WBS/LME agar. The chunky WBS specimen is looking remarkably rhizomorphic:

20210121_1435001.jpg 20210121_1435521.jpg

The WBS/LME is looking good, but not nearly as thick:

20210121_1434381.jpg

 

Each of the cultures I transferred from last week also went to grain. I used five 4oz grain jars and picked the best remaining sections. So far, 4 are showing some nice looking growth. The grain does look a bit dry, so I am slightly concerned about that. I may end up adding some distilled water if things start to slow down.

20210121_1431531.jpg

 

Being concerned about the grain jars moisture, and having a few plates that were being overgrown I decided to do the rest of the jars on Tuesday night. One plate had a bunch of knots and a tiny pins, most were so-so looking. There was one particularly stand-out plate that was a GT culture that grew out in thick straight rays, so I'm curious to see what happens.

20210121_1433391.jpg

 

My plan is to fruit in 8oz containers inside a few sterilites. Then take a couple of clones and prints from each jar and continue working with the genetics.


Edited by HrVanker, 21 January 2021 - 06:34 PM.

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#28 HrVanker

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Posted 21 February 2021 - 01:50 AM

Okay, so here are a few of my test jars spawned to coir:

20210220_223548.jpg

Since I started this thread, I have been saying that I had GT spores going. However I see that I still have my GT syringe in the fridge, so by process of elimination the strain is EQ.

 

The container closest to the camera is fruiting slowly, showing a couple blobs in the upper left corner, and some very PE fatboy looking traits. It will be interesting to see how they develop. This culture had been transferred a couple times.

 

The middle container was an MS plate that started pinning, so I threw the entire thing into a test jar. All 3 containers were spawned at the same time, and this one had full on pins when I put them in the sterilite. I've already pulled and printed 6 caps, and took clone from the first mature fruit.

 

The far container is also fruiting a little slower, so far nothing too remarkable.

 

I have some Rustspore and more EQ test jars spawned, but two of the three RS seem to be going bacterial. I lost my one B+ test jar because it went bacterial after spawning as well. I am chipping bits of coir off of a 10lb block, and I'm having a lil bit of trouble getting the water content right, I suspect this to be the primary reason for the bacterial losses. All the jars were fully colonized, most of them starting to knot up a bit.


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#29 HrVanker

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Posted 29 March 2021 - 05:19 PM

So I figured I'd start updating my own thread rather than continue hijacking the mycogone thread that I posted in previously.

 

I harvested a couple of the Redspore/Rustspore "eyeball" fruits last night, and they seem to be pretty dense. Today there were one or two more ready for harvest, and it looks as though one of them started dropping a small amount of spores! That cap is currently on foil, while the body is in the dehydrator. It would be really cool to isolate this mutation and see if I can do anything fun with it. I have some EQ clones and prints which had very nipple-y caps, and I'm thinking it might be fun to try and stabilize both the eyeballs and nipples, and see if I can cross them to get nipple-y eyeballs! But that's getting way ahead of myself.

 

As I mentioned in the mycogone thread, these fruits are the 3rd or 4th xfer from a germ plate. Am I correct in thinking that if I were to clone any of these fruits that they would still be 4th/5th xfers and near senescence?

 

20210329_1506541.jpg


Edited by HrVanker, 29 March 2021 - 05:26 PM.


#30 Oldpunk

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Posted 30 March 2021 - 05:37 AM

Ha! I like your write up. It sounds a lot like my process of only sorta following a recipe and learning from the mistakes. I hope you have luck with these.

I'm going to keep watching and taking mental notes as I have just purchased my supplies to try my own first agar adventure.
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#31 Oldpunk

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Posted 30 March 2021 - 05:43 AM

I also REALLY wanna see nipply eyeballs.

#32 HrVanker

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Posted 31 March 2021 - 03:20 AM

I also REALLY wanna see nipply eyeballs.

We'll see if I ever get to that point. It's going to be lots of agar, flushes, and prints before I get close. Lol

It appears that these guys are dropping spores. The first fruit had a very lite print, the second is a bit better.

I double checked the order that all these spores came from, and there were no KSSS in the order. And I'm like 95% sure that these are from prints I made from normal looking cubes.

#33 HrVanker

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Posted 29 October 2021 - 10:17 PM

Now that the weather is cooling off, I decided to do up some plates and quarts of WBS.

WBS was put on the stove at medium heat and boiled for 20min w/ some gypsum. I drained it in a flour-sack cloth, but it still seemed too wet, so I tossed in some dry seed. I shook after the pressure subsided, and they look fine. There's a bit of extra moisture, but barely.

The agar is more interesting: I used 500ml distilled water, 3.5g blended WBS, 1.5g LME, 4.25g P668 orchid germination med, and 4g agar.

The germination medium has a lot of charcoal, some nutrients, and agar. The PH of pure P668 is between 4.75-5.75, which is my main concern, but it's diluted so I don't think it's the biggest deal. The charcoal was the main reason I used it, plus I just wanted to see if it would work.

I put some spores on 8 of the plates tonight. 2ea of: Print from an EQ isolate, a print from a little tiny EQ, a leucistic EQ, and the weird KSSS looking "Redspore". All prints were from my last run about 6mo ago.

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#34 rockyfungus

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Posted 29 October 2021 - 10:24 PM

Is your agar composed of blended bird seeds and orchid mix? Sounds awful LOL. Excited to see what happens.

I just use the grain water left over from boiling grain.

 

"As I mentioned in the mycogone thread, these fruits are the 3rd or 4th xfer from a germ plate. Am I correct in thinking that if I were to clone any of these fruits that they would still be 4th/5th xfers and near senescence?"

 

Got sidetracked...When I saw blended seeds. Not knocking it just nothing I would of thought it. I am dying of laughter. Pictures PLEASE.

 

Senescence is nothing you will ever have to worry about I believe. I really think it's only gourmet cultures. You are working from spores? It's all new genetics.
Gourmet is like strains that have been passed around for who knows how long. They aren't spores they are LCs and clones so that shit is 1000s of transfers out if not further?
I've hit maybe 30+ on cubes when I was new and unsure. I think you weed out a lot of possible genetics when you get past 4-5 x-fers.
Also if it's cloned from 3 or 4 it's still 3 or 4 isn't it?


Edited by rockyfungus, 29 October 2021 - 10:36 PM.

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#35 HrVanker

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Posted 29 October 2021 - 10:32 PM

Yeah, that's pretty much it. I had some good success w/ the blended WBS last year. If you go up a few posts you can see just the plain WBS and water agar.

I suspect that the orchid mix will be fine. Orchid seeds are pretty delicate and germinate in similar conditions as mushroom spores. In fact, wild orchids tend to be mycorrhyzal. But that's far from a guarantee .

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#36 rockyfungus

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Posted 29 October 2021 - 10:40 PM

Whenever I've looked into mycorrh. I've realized how little about fungi I know. All I know about mycor. is good luck pronouncing them, they are super selective, and we have utterly destroyed them with pesticide/fungicide.

 

What's the purpose is all I ask myself. All I need is leftover grain water from boiling grain and some agar. It does it all. Remove the agar and LC!


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#37 HrVanker

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Posted 29 October 2021 - 11:05 PM

Oh yeah, the grain water does work really well. I believe part of my thinking was that the bits of grain would provide some pockets of 3D space and extra food for the culture. Which could be helpful if I need to store them longer.

I think I tried the blended WBS before grain water occurred to me. So I have a small container of the blended stuff to use, and I wanted to start the agar before I started the grain.

I believe I've heard claims that senescence can set in within 7 transfers. But if you've had good performance out to 30, I'm far less worried.

Side note: I need to build a new SAB. Working in my current one is like being put in some sort of medieval torture device. I put the holes on the shorter panels of the storage container, and it forces my arms and neck into awkward positions.

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Edited by HrVanker, 29 October 2021 - 11:06 PM.

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#38 rockyfungus

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Posted 29 October 2021 - 11:52 PM

Who said anything about performance, LOL. I was new and not sure if it was my fruiting conditions or I isolated out all the "fruiting". It consolidated and ran quickly just needed to be scraped and patched and forced to fruit.

I'd consider the ground WBS for "PF style cakes". Best bang for the buck I'd imagine. I've seen people play with thicker LC's almost borderline agar for longer storage. Now it's coming together.

 

Good luck on the SAB. My back was so pissed at me. Impossible to get good clarity and comfort for the back/neck. Almost makes more sense to just SAB yourself in somehow...(maybe snorkle gear out LOL). But then the flame and any movements...


Edited by rockyfungus, 29 October 2021 - 11:55 PM.

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#39 HrVanker

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Posted 30 October 2021 - 12:50 AM

I still haven't done an LC yet. After I did agar, I felt like there was too much contam risk when re-using a jar of LC. But I may one day put some ports on some agar jars so that I can make a slurry. Sort of a best of both worlds situation.

Good to know that they get pretty stubborn after that many transfers. Based on that, I might set 10 as a rule of thumb.

I don't use a flame in my SAB. I line the bottom with paper towel, and get it pretty damp w/ ISO. I wipe down the top w/ ISO, spray all the jars and tools, lightly mist a "dry" stack of towels, assemble the box w/ everything inside, then spray some more ISO inside. Then I spray two paper towels w/ even more ISO and cover the arm holes. I let it sit for 10min or so and then spray my washed hands and get to work.

Tonight I definitely overdid it on the alcohol. Halfway through I thought I was going to pass out.

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#40 rockyfungus

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Posted 30 October 2021 - 02:25 AM

Why you going out that far? I like to fridge whatever looks clean hopefully T1-T2. That's the master and you keep hitting that and hopefully only go another plate to two to confirm clean. Or if you used all the agar and got clean spawn. Take 1 grain back to agar and now that's your master. Honestly I have a slant that was just labeled cube. 5 years old, wonder how many transfers that is. 

 

LC is one and done for me and hardly used. Unmodded baby jars I just pour or use a syringe. Prefer agar to grain.

Can always take a sterile syringe (1ml or so of water) poke a culture once and then spray out the agar to LC.

 

I always sprayed my SAB with soapy water. Waited a few. Flamed my blade out of the box went in slowly and went to town. That's too much if you're doing a ton of diff. genetics. 

I try and avoid bleach and alcohol for that reason. Probably a rite of passage to set your hands on fire too. 
 






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