Cloning Z ---> 1st flush, your help needed [Picture Log]

[pihop]

This will be my first real attempt at cloning.
As it it my first real try I want to do it properly, that is why I need all the help I can get.

I'm going to update this thread as soon as some real progress is made and plenty of pictures will be uploaded.

I've managed to get the first step going on my own. I took a rather fat pin and sliced it into six bits and put on cardboard.
No care was taken to ensure sterility, I just grabbed the closest piece of cardboard I could find, soaked it, washed my hands and put the mushroom pieces onto it.

This is what it looks like after 3 days:


The pieces to the left are between the sheets of cardboard. The closest one to the right is the hat.


So where do I go from here?
I assume I leave it to grow some more before I try to transfer it to something else.
Any suggestions?

I decided to put this thread here as the answers are pretty slow in the pic section.

[Om shanti]

Nice idea to use a ziplock bag for the cardboard!
Don't have anything sensible suggestions for the further procedure. So will keep my mouth shot about that.

[MLBjammer]

Since you didn't sterilize the cardboard, you are likely gonna have contam issues. With that method you would transfer the cardboard clone (once it has colonized all the way through the cardboard) to grain or agar or some medium to expand it.

Here's the original cardboard tek: http://forums.mycotopia.net/cloning/...oning-tek.html (cardboard cloning tek)

Good luck!

[Shamsu]

Keep the pics coming i really want to see how this turns out for you. looks like good start to colonization, best of luck man.

[Hippie3]

next you snag a tiny fragment of thick ropey tissue
and transfer to yet another piece of cardboard,
might take a few transfers to get the substrains to sector out
into pure isolates
which will then needed to be tested to see how well they pin.

[pihop]

This is how it looks today.


It's pretty hard taking good pictures through the bag.


hippie3 --->
I took your advice and transferred the thickest myc strand I could find.



Hopefully it starts growing and I'm hoping I didn't damage the original to much as most myc seen to the right in the first pic got scraped off when I took the piece out of the bag.

I will now try to isolate a sub strain AND try to colonize some grain or whatnot with the original and hopefully I can compare the two later on.

[Om shanti]

If you have cloned a mushroom you should already have a substrain, and if it's the same mushroom on all pieces of cardboard then it's the same substrain, so I would try to transfer to whatever media you like to grow in, and then cross fingers there are no contams due to the open air procedure. Otherwise you may have to transfer some back to cardboard, perhaps under some more sterile conditions.. like a on the fly cardboard glovebox.

[Leftover Salmon]

Interesting. Subscribing.

[teesus]

good job! you could dip pieces of cardboarg in h2o2 solution and tranfer them to grain jars

[pihop]

Originally Posted by Om shanti

If you have cloned a mushroom you should already have a substrain, and if it's the same mushroom on all pieces of cardboard then it's the same substrain,

That's what I thought to. But remember the first commandment "Thau shalt never doubt Hippie3".

At least I've got two cultures going now if one contaminates.

teesus---> Will probably do a transfer on to popcorn in a week or two. Depending on how lazy I am at the moment I might use H2O2.

[GadgetGuy]

Thats awesome man! Good work. Why not transfer a chunk of that into an LC and let that grow out? You could do a few popcorn jars and a few LC's with what you have upping your chances of success and maybe cutting back your chances of contams if you are careful. They don't look contaminated but I don't know shit about cardboard either....

[pihop]

Gadget guy I did as you suggested and transferred a few small pieces into LC made with pure dextrose. Hopefully it works.


From now on I'm going to leave the original be so it can recover and colonize the whole cardboard.
The transfer is coming along nicely. You can see in between the sheets that the small pieces has fluffed up a nice bit already.

[BuckarooBanzai]

Pretty cool, man. Nice amateur mycology!

I would def test that LC on a few jars/cakes before using it to inoculate a large volume of stuff.

[pihop]

Thanks!
Well first I want growth!
The last 5 LC's ive made have all failed for some reason, they have been made with 5% honey and PCd for 30 min, after a few days all of them had a thick layer of sediment.

This will be the first LC i make with Dextrose so it's going to be interesting I made it using 2 teaspoons of dextrose for 500ml of water (measurements from a tek in the vaults). Doesn't 2 teaspoons to 500ml seem a bit to little?

[Sideshow]

Originally Posted by pihop

The last 5 LC's ive made have all failed for some reason, they have been made with 5% honey and PCd for 30 min, after a few days all of them had a thick layer of sediment.

Some sediment in an LC is no problem - Honey will cause that.
But you did say a THICK layer, which does not sound good.....
Did you innoculate them with spores?
30 min @ 15 psi is a bit on the high side for an LC

[pihop]

Originally Posted by Sideshow

Some sediment in an LC is no problem - Honey will cause that.
But you did say a THICK layer, which does not sound good.....
Did you innoculate them with spores?
30 min @ 15 psi is a bit on the high side for an LC

I dropped a piece of inner stem tissue in them, not a single sign of growth.
I don't know how many psi my PC runs at but it's supposed to be ~120C

[Sideshow]

Hmmm.
Not sure what the problem is.
the ratio of honey to water sounds ok.
Try a shorer PC run next time, 15min @ 15psi or 20min @ 10psi.

[pihop]

Well I tried PCing the last LC try for 15 minutes but with the same result.
I can't control how many PSI my PC run at since it is from like mid 70s or even older.

Doesn't two teaspoons of dextrose to 500ml of water sound too little?
The TEK suggested either 2 teaspoons or 4 grams, if i use 4 grams that would only be 0,8% sugar.
THATS FREKKING NOTHING!
Ohh why the hell didn't I do the math before I made this batch.

I saw a TEK by hippie3 where he suggested 1 teaspoon of dextrose to 75 ml of water. Using the same volume to weight conversion as the TEK i followed that would be 2g dextrose to 75ml water (2,6%). I'd say Hippie3's measurement sounds a lot better.

Relevant cloning question:
Well, assume I got the LC the way it should be. When transferring myc to the LC should I just rip a small strand or take a piece of well colonized cardboard and drop in?

[BuckarooBanzai]

Firstly, you know you can boil sterilize LCs, right? No carmelization issues when you boil sterilize.

Secondly, don't measure your sugars by volume, measure them by weight. You want about 4% sugar (by weight). For 500mL of water, you would want 20gm of sugar.

Thirdly - just add myc to that LC. Dropping in cardboard means also dropping in any contaminants that have settled on the cardboard.

[pihop]

Alright.
Then I will do another round of LC with the right conc. of dextrose.
I'm just going to let the myc. recover for a few days before I go in and ravage it again.
Will also make a small jar of popcorn to make a slurry syringe of.

[BuckarooBanzai]

A popcorn jar would probably be a better choice.

You can see contams growing on popcorn - you have to test an LC.

[pihop]

Then I'l probably go with a couple small popcorn jars. This was my original thought.
I've never had popcorn go bad on me (except for contams) while I haven't rally gotten the hang of LC yet.

[pihop]

I think i messed to much with the myc. It stalled on the original cardboard so in a desperate attempt to save it I cut the original up into strips and transferred to 7 new pieces of cardboard.

I did this a week or so ago and haven't seen any real growth yet


Seeing it come to an end like this is a disappointment.
In my frustration I took another rather large pin and cut it up and put it into 3 small popcorn jars. It's fluffing up nicely and starting to take over some kernels.

EDIT:
Since the pin is from the same tray as the earlier one I will just continue this thread with the popcorn jars instead.