38 replies to this topic

[IncredibleEdible]

Hey guys, since I've recently started dabbling in g2g, I thought I would make a post about it. I will include plenty of pictures, and the colonization times.

First, I'll include what I did. I do not have a flow hood, but extreme caution was used. I did this in a closet with a HEPA filter running for the last several months. I washed my hands thoroughly, rubbed my hands and the spoon with alcohol, then flamed the spoon. I was wearing clean clothes, and had just gotten out of the shower.

I had 3 "mother jars".

One with WBS that had stalled, completely, only a small splotch of myc. Noc'd this up quite some time ago, it was my very first attempt at WBS. It was from a pint jar. WAY too dry in there. (MALABAR strain from Sporeworks). The jar was about 10% colonized.

Second jar was another WBS jar that had completely stalled. (ECUADOR from Sporeworks) Now - I know they were viable syringes, because I'd fruited from these using BRF cakes, so it's not Sporeworks fault - it was my WBS tek. This one had maybe 3% colonization in a pint jar. Literally just one splotch of myc to take from. I was not expecting any success here.

Third jar was a fully colonized quart jar of popcorn (Burma strain from TGS). I spawned this to a a pint jar, and a pair quart jars. I used the rest of this quart to spawn a rez tray. (See my grow logs in my sig). The pint jar I'm just making to do more g2g transfers if all turns out well.
So here are the pictures.

We'll start with the Ecuador. The jar I thought LEAST likely to succeed.
http://mycotopia.net...=1&d=1268912332
This was done less than 48 hours ago, and it's already come back to life. :pirate: It's taking over the corn in some places, and looks to be making quite a recovery in it's new - more suitable environment.

Next, this is the Malabar jar. Again, this is less than 48 hours after the transfer from WBS to popcorn.
http://mycotopia.net...=1&d=1268912332
I'm extremely impressed with this strain. Malabar has crazy colonization times, and has shown itself to be very resilient in my experience.

Here is the first quart of the burma g2g. This was from healty mycelium, and had a good spoonful put into it. This was less than 72 hours ago.
http://mycotopia.net...=1&d=1268912332

Here is the second quart of the burma g2g.
http://mycotopia.net...=1&d=1268912332

And finally, here is the pint jar. This is going to be several jars "mother", as I plan on using this only for g2g transfers.
http://mycotopia.net...=1&d=1268912332

So far the conclusion I've drawn is this will be the way I proceed from now on. I'm loving g2g so far. :) I plan on just ordering a lot of different strains, and keeping a "mother jar" for all of my quart jars. It's pretty wild how fast it's colonizing, especially considering a lot of it came form myc that I had written off for dead!

Attached Thumbnails

• 
• 
• 
• 
• 

Edited by IncredibleEdible, 18 March 2010 - 06:55 AM.

[SisterMagpie]

Looking really good IE. That WBS was probably gasping for air... now that it got some it is thinking... FOOD!!! Best of luck, can't wait to see some fruits. Also are you using tyvek lids? I had tons of stalled jars when using tyvek, have never had a problem since i switched to polyfil.

[IncredibleEdible]

Looking really good IE. That WBS was probably gasping for air... now that it got some it is thinking... FOOD!!! Best of luck, can't wait to see some fruits. Also are you using tyvek lids? I had tons of stalled jars when using tyvek, have never had a problem since i switched to polyfil.

The WBS had probably everything going against them. They were just in a lid with 4 holes, and a dry verm layer on top of them. I noc'd these up before I had ever even fruited anything, so I was probably jumping the gun by moving to a new tek before I had mastered BRF. I'll admit it was completely my tek. LoL! I'm just glad to see I was able to save the life that I started - and almost killed immediately after.

[MLBjammer]

That's great results for certain. I would keep an eye on the jars originating from the stalled myc. The stalling could be due to the grain being too dry, but there could have been competitors hiding.

Grain-to-grain transfers are killer, such an easy way to expand your spawn. But anytime you start with live tissue, the process can be incredibly fast. Just remember not to do too many generations of transfers, else your mycelium will slow down and yield poor results. Fresher is always better.

I believe you have a blue thumb, my friend.

[IncredibleEdible]

MLB, that's what I was wondering about. How many generations do you think it's viable to do g2g? I thought since the myc I would be taking would always be freshly grown, that I could in theory do it forever?

Another question would be.. After a couple of generations, would taking a live tissue culture and starting a spawn that way "renew" it? I'm genuinely interested in how the genetics will work when doing a constant g2g or live tissue spawn. And any advice would be very much appreciated.

[SisterMagpie]

G2G is technically "cloning" with live tissue, just the same way starting a spawn jar from live tissue is cloning. Every time you clone that tissue, you are adding a generation. I could not tell you how long they last, seeing as i have never carried out g2g more than three or four gens, coming up on five though, i'll let you know how it works. I don't know too much about it, but when you clone that many generations the DNA wears thin, i believe, and eventually the myc won't fruit anymore. I'm pretty sure the only way to start first generation spawn is from the spore though my friend.... so make sure and print those bad boys....

[IncredibleEdible]

Here are the jars, 24 hours later.

All of the jars are self labeled, so I'm just going to throw the pics up here. :eusa_booh

http://mycotopia.net...=1&d=1268998073
http://mycotopia.net...=1&d=1268998073
http://mycotopia.net...=1&d=1268998073
http://mycotopia.net...=1&d=1268998073
http://mycotopia.net...=1&d=1268998073

Going to give the burma g2g's a good shaking today, and possibly the malabar a little later. Going to wait to shake up the Ec's for probably another 24 hours, though.

Attached Thumbnails

• 
• 
• 
• 
• 

[director of sound]

G-DAMN THATS FAST!!!! i gotta give that a try soon, mabey with some z's or taz i got left over. i wonder how wbs to cracked corn/coir would do it allready colonizes like a bat outta hell with lc, looks like G2G might be faster yet!!

(P.S. allmost seems backwords wbs to pcorn i would think pcorn to wbs but what the hell it looks like it works damn good!)

[IncredibleEdible]

The reason I had gone wbs to popcorn, is that the two wbs jars had completely stalled. the pints of wbs jars I used were probably around 6 weeks old with only tiny spots of myc, that had not grown in a loooong time.

I'm just happy to see the myc is now regaining it's health.

[IncredibleEdible]

Ok guys, so.. I shook up the jars shortly after my last post. :) Ended up shaking all of them.
So here are the results, thus far. This is only 5 and 6 days after the g2g noc up! :loveeyes:
Here is the Malabar. It seems to have been the roughest on the malabar, but they're still coming along nicely. Finally starting to see what appears to be some strong mycelium, instead of the weak stuff spreading around. Very cool.
http://mycotopia.net...=1&d=1269172863
Here are the ecuador, the other transfer from wbs that was completely stalled. These are doing much better than expected!
http://mycotopia.net...=1&d=1269172863
Here is one of the burma jars.
http://mycotopia.net...=1&d=1269172863
(What appears to be a dark spot, is where there are gaps between the corn kernels, when I saw this pic I immediately went back and re-inspected, and noticed that's what was going on. I about had a heart attack!!)
Here is the second of the burma quarts.
http://mycotopia.net...=1&d=1269172863
And finally, here is the pint jar. This thing is almost 100% colonized in a touch under 6 days!!
http://mycotopia.net...=1&d=1269172863
So wow.. between 5 and 6 days on all these jars, and they're doing WONDERFULLY! I dont think I'll ever go back to BRF cakes. :) (Other than the ones I currently have noc'd up)

Attached Thumbnails

• 
• 
• 
• 
• 

[MLBjammer]

Dude, those look great--so healthy.

As far as senescence goes, it can happen within a few generations. I never do more than 1 gtg or clone. You could get away with more, but I figure it is not worth the risk when fresh myc. and/or spores are readily available.

The best way to explain why not to go too many generations with clones or grain transfers is that it's like making a copy of a copy of a copy; each time the copy fades a bit more from the original.

I am anxious to see that myc. of yours in fruiting mode, IE!

[IncredibleEdible]

MLB, here is what I'm not understanding about it, I guess.

When you noc up a jar, what is happening, is mycelium is spreading (in this case) from one kernel to the next. Would that constitute a clone?

All doing g2g really is, I thought, is like giving myc fresh nutrition to grow to. Not really much different then adding more nutes to the jar.. Or even spawning it to a bulk.

So.. If I were to spawn half a jar, then refill the jar - would this be considered cloning? Or if I were to even add more corn to a jar, is this considered cloning for all intents and purposes? Because in a sense, that's all a g2g is.

If so - is it considered cloning when I add this jar to poo? Since I'm adding myc to a new place to grow?

Just trying to get this all straight in my head. :)

Edited by IncredibleEdible, 21 March 2010 - 07:46 AM.

[IncredibleEdible]

I guess this is the way I'm thinking of it in my head.
http://mycotopia.net...=1&d=1269174699

So... spawning a jar to bulk is considered cloning?

So, if step 1 is not considered a clone...then...


http://mycotopia.net...=1&d=1269175303

Then you would have a master jar, still.. Maybe I'm just REALLY confused. Just trying to understand how this works.

Attached Thumbnails

• 
• 

Edited by IncredibleEdible, 21 March 2010 - 07:41 AM.

[plantman21]

I have wonderd that my self. But I guess I just never tried to ask why. Thanks for asking this ? it is a good one to know.

Plantman

[MLBjammer]

Of course, a grain jar could be started from an isolate (clone). But the idea is to expand that culture; and you might experience senescence with this is if you do a transfer from a master to 1 set of jars, then expand those jars to more jars, and so on.

A clone is simply a copy of an isolate, whether from a fruit or isolated substrain (via agar or grain). If you cloned from a fruit that was cloned, then you could run into senescence again.

It is all more similar than different. Just be careful expanding a culture too many generations. It can be disappointing.

I hope that makes some sense, lol.

[ggod]

IE.... Looks great man!
Some time today I will dig out my Zoology book and give you the exact reason you do not want to clone too many times.

The cliff notes of it is basically like this..... The mushroom has ton's of genetics and the traits that show are phenotypes.
In successive generations (generations of clones), These phenotypes change ever so slightly (micro-evolution). These changes tend to be NOT an advantage for survival (e.g., less aggressive fruiting, smaller fruit size(less spores), resistance to contam's).

Paul Stament has stated in his book (can't remember which one), the Black Morel for instance is stable for 6 or 7 generations before it starts to degrade. Other strains should be fairly similar and 7 is what I use as a guide.

A fairly easy way to work around this issue is to create a master culture (slant). A slant is a good choice for a master culture because of the size and long term store-ability. When you take the wedge and transfer it to a slant you can take all those other wedges you can cut out and transfer them to grain jars. These would be generation#1. Of all those jars you can transfer to other jars..... and so on.

There will be a time when either contams take over, or you just notice it is not as voracious as it was in previous generations. Now is the time to take the slant from the fridge and transfer a bit of mycelium to another petri dish. When this dish colonizes, transfer wedges to new grain jars. I am not certain if this is still a first generation or second generation culture at this point, but how I understand it, it is first generation.

So as you can see, a single slant of mycelium can last almost indefinitely. When the time comes to make another slant, change your medium, to give it some diversity.

You are doing well with G2G, there is no reason you can not master agar work. Honestly I find it easier than G2G because contams can be "fixed" by transferring out the good mycelium to a clean dish. I also prefer to use my glove box and have not built a hood. After all, if working with a contaminated dish, you don't want the contams blowing around your study space.

Btw.... most all the fancy things people buy for agar work can be made at home.
Inoculation loops = picture hanging wire, individual stand
Scalpel = razor blade or knife with plastic handle
alky burner = baby food jar and a oil lamp wick

good luck and hope this helps,
ggod

[justweed]

This thread first had me wanting to start up some g2g....now I want to start up some agar. Good thing I have 2 months till I sign the lease on my new place! Plenty of time for research!!

Those jars look amazing though, that mother jar colonized so fast!! Keep us posted :)

[IncredibleEdible]

I would have to say this:

I'm becoming a very strong advocate of g2g with every passing day. :thumbup:

[SharkieJones]

Looking good, look forward to the fruits