Cloning Fruits via Inner Cap Tissue Needle Biopsy
Posted 02 May 2023 - 10:14 PM
So some of you know that I’ve used oven bags (https://mycotopia.ne...ne/#entry168440) to clone and it’s treated me well since I’ve done it but it’s got its limitations. I can only do a couple clones per bag.. it’s bulky and there’s a lot of moving parts. Prep is much easier with this newer version as well
Easy cloning was kind of my last hurdle without using agar.
It’s not really a new idea or anything but there’s a couple things that make this extremely easy and reliable that I think it’ll help a decent amount of ppl.
One of the big factors that make this go off well is that I eliminated the need to wipe the SHIP before poking. More on the in a second.
The other big problem when trying to poke fruit material is that the stipe tissue is usually really stringy. The reason that’s a problem with this method is because even if you do get a “plug” of myc in the needle barrel, it’ll just get pulled out again because it’s still attached to the rest of the “string”
The fix for this is to use inner cap tissue instead of stipe material. It’s not stringy at all and is more like tiny orbs of myc stuck to each other and there’s no problem getting a myc plug to stay in the barrel.
Another downside to poking cap material with a normal hypodermic needle is that it’s too long from the sharp tip to where the actual opening to the needle barrel is… more times than not, if you try poking far enough into the cap to actually retrieve inner cap material, you’re probably poking through the outside of the cap on the other side and possibly contaminating the very tip.
The fix for this is to use a small very sharp wire cutters and cut most of the tip off while still keeping it sharp at the end. You can use a blunt needle to inject through a SHIP but it takes a decent amount of pressure and you risk picking up a plug of silicone (in my case). Keeping the end still sharp and pointed keeps that from happening. Cut like so..
Now that you have your modified needle, onto the modified lid… this is really simple. Basically I zip tie a piece of foil over the lids and SHIP. The foil cover has a little lip of extra foil positioned in front of the SHIP so that when I’ve got my needle tip with a myc plug, I can easily pull that tab to briefly expose the SHIP and immediately inject the needle into the ship without having to wipe it with anything and possibly contaminate it. The zip tie is to keep any air out so the SHIP remains clean after the pc cycle until you’re ready to clone
So that’s pretty much the prep that’s needed to do one clone (minus the grain prep—in my case—or LC if you’re going that route). The cool thing is that one syringe filled with sterile water can do many clones.. all you do is flame the needle between them… sometimes the plug gets a little stuck and you end up squirting out a bit more than other times but I can easily do 5+ clones per filled syringe and it’s fast.
I guess I should show the lids I use for this… I use a large DIY SHIP but any secure lid with a SHIP will work. Here’s my lid set up https://mycotopia.ne...he-test-of-time
Let’s get to the procedure, yeah?
Preparing your materials
1. I use 4oz grain jars (wheat is my preferred grain). Prepare them as you would any grain jar (2hrs @ 15+psi).
It’s hard to tell but they’re filled 2/3 the way so there’s room for shaking. Any less grain and they can dry out too fast before the plug has a chance to really take off and you get full colonization (although, it’s not necessary for them to be fully colonized… in my case, these jars are only for myc expansion. Even if they are only half colonized, that’s plenty of myc to GLC to a couple qts and another 4oz clone extension jar. Those will colonize much faster because of you’re using LC and not waiting on a tiny myc plug to expand)
The cool thing about these 4oz jars is that I can make a bunch of them in one pc run and afterwards, just wrap them in cling wrap (to slow/stop them from drying out) and just grab one when I need to clone. I’ve used them 3 weeks later and saw no visible drying out of the grains even on the top.
When I do use them later, I just attach the foil and pc again for 30mins.. this is only to sterilize the SHIP under the foil again. You could probably even get away with bringing the pc up to pressure (15psi) and then just turning the burner off… you’re not sterilizing anything but the outside of the jar (ie. SHIP and maybe a syringe with water)
2. Attach your foil cover to the lid using zip ties. Depending on how big they are, you may have to put a couple/few together so it can reach all the way around your lid. Make the foil a decent amount bigger than the lid size so that on one side of the foil piece, you can fold it enough to make a tab that you can pull with your fingers to rip the foil back and expose the SHIP right before you inject the myc plug. Make the tab right in front of your SHIP
**obviously, if your using the jar right away, attach the foil before you pc for the 2 hours… don’t forget to loosen the lid a 1/4 turn and tighten when you take it out of the pc
That’s it for the jar prep. I’ve also used the steam pen (https://mycotopia.ne...er-an-iso-wipe/) instead of the foil (especially useful if I’m using jars that were presterilized) but I worry that the heated up SHIP will damage the myc plug but it does work… it’s more of what I feel like doing at the time
3. Filling the syringe. With the modified needle/syringe I fill them by setting the syringe in a 4oz jar of water (plunger all the way in) and cover with foil like so..
I have a 16qt presto and it isn’t tall enough to set a filled syringe in a qt jar of water. So the first thing I do when I open the pc is hold onto the syringe barrel and pull the plunger up to fill the syringe with the sterile water.
Now that you have your filled sterile syringe and foil covered sterilized jar, you’re ready to clone.
Cloning your fruits
I’ll skip the part where I tell you to iso gloves and wear a mask etc. to me those things are a given
1. This part happens very quickly. Gather your materials on a clean surface.. you may want to work in a SAB, I just make sure there’s no moving air and do it in open air. I have a very clean house and feel comfortable doing that but your situation may be different. I like to hit the jar against my hand towards the opposite side of the SHIP.. this moves grains away from the SHIP area so that, A. The grains aren’t in the way of the needle and you don’t stab into a kernel plugging it up and B. It allows the myc plug to get to the bottom of the jar where it will be safe from drying out
2. Before anything else, I use a knife to cut a slit in the foil right above the zip tie in front of the SHIP. This allows the foil to tear away very easily when I pull the tab.
2. I cut the cap off most of the stem. I leave a couple inches of stem attached so I can pull it apart towards the cap to reveal the inner cap tissue. You can also just break off one side of the cap.. however you do it, you just need to expose a decent amount of tissue to poke the needle into, but don’t do that quite yet.
3. Flame your modified needle red hot and set it down.
4. Now you can tear the cap in half and expose the inner tissue.
5. Your needle should still be hot, so squirt a little water out to cool it
6. Now poke the inner cap tissue to get the plug. You may have to poke a couple times to get enough tissue. If you quickly look down the barrel, you can see whether there’s a plug or not
7. Grab that tab and pull the foil away from the SHIP and quickly inject your myc plug into the jar.
That’s it! Clone done
With the modified needle, you can use pretty small pieces of cap. The piece in the bottom is the one I used
If you want to do another clone, just cut the foil on the next jar, reflame the needle and your set to do the next one.
Here’s a couple already starting to recover from their myc plugs
Some colonizing after a shake.. I shake them a few times just to make sure I didn’t pick up any contamination during the procedure. It takes a few days to see any growth and a few more after that to get to a shake point. If there’s any contamination, that’ll be evident with the recovery.. especially mold
Now I can use these to GLC to qts and extension jars to preserve the clone or I can dry them out to use later.
This has made cloning so much easier for me. The only problem I have now is that I have too many clones to grow out
Come this fall, I’ll have a bunch of dried out cultures I can immediately get rocking in some qts. I’m very excited for that and will give me lots to look forward to this summer while I’m taking a break
I’ll update this with any clones I grow out using this method
Here’s PE clone I got using this method the first time I tried it last year (inner cap needle biopsy)
Mak x yeti f1 clone (this was pre-needle modification.. I poked through the other side of the cap on this one so not sure what caused the first flush to grow like it did—could also have been lack of water to support the flush because the second was nice)
Since the needle mod, all the clones have recovered/grown beautifully
- hyphaenation, Arathu, bezevo and 1 other like this
Posted 07 May 2023 - 01:00 AM
wouldnt a 16 g needle avoid having to trim the tip and give you are larger core sample (clone)? Also I have found the rubber injection ports work better than the silicone which can pull through at any time with multiple uses. I like the idea of a closed system with the zip ties and foil .
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Posted 07 May 2023 - 08:46 AM
The reason I like the diy silicone is because I can make them big enough that I don’t have to inject in the same spot ever. If you look at the pic of the uncovered lid (first one below the pics of the needles), there’s lots of room to inject without going through the same hole. That’s the thing I don’t like about the orange stopper types… their injection site can get pretty chewed up
To each their own tho.. like the op says, use whatever SHIP works for ya
But you hit on the two things that make this worth posting (because it isn’t really anything new as a whole cloning process):
The modded needle so you can clone smaller caps safely and the close system to eliminate the need to iso wipe (which could leave behind iso resistant bacteria)
Edited by fahtster, 07 May 2023 - 08:52 AM.
Posted 07 May 2023 - 01:07 PM
Having had a lot of problems with contams in the PNW I have gone back to as much closed system (not exposed to air )as much as possible despite flow hood, air purifier, alcohol, clorox wipes and other clean tek apparel. The PF tek in the past really hit home the use of a closed system to avoid contams. Ive been on that with Lc and LI in closed bottle inoculations. Off topic. I like the biopsy method. Thanks, Seeker
Edited by Seeker2be, 07 May 2023 - 01:08 PM.
Posted 10 May 2023 - 11:47 AM
Having had a lot of problems with contams in the PNW I have gone back to as much closed system
I hear you on this ... I also live in the PNW and have been longtime battling the green meanie. It is literally everywhere , on everything! All of my last several projects have been badly plagued by Trich. Now I don't want to sidetrack from this awesome thread ... but I will say I've often thought we should have a thread "Things psilocybes will grow on that trichoderma will not". I'm almost certain there are certain subs and sub-conditions that will either favor or retard trich. As an example PH of the sub with trich favoring acidic conditions and not liking alkalinity. Also cubensis can tolerate quite wet sub conditions whereas trich seems to stall in a wet environment. The closest I have come to finding something was trying tree leaves (maple) with cubensis. From my observations cube myc loved the leafs (which were quite wet in the jar) and trich that popped up on a piece of grain in the same jar didn't expand and just sat there. I've been meaning to explore further and test other materials. Anyhow it would be cool to discuss (elsewhere) the possibilities of trich-unfriendly substrates.
Sorry for the diversion.
Loving this thread and will give it a try when I get a chance! Thanks F.
Edited by hyphaenation, 10 May 2023 - 12:01 PM.
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Posted 12 May 2023 - 01:17 AM
Trich problems ': When I lived in Nevada never had a problem but as you say Trich is everywhere here in PNW no matter what I do (frustrating and wasteful of materials) and that is why I decided to double down on sterile technique, PC ing for 3 hours, using SHIP tops to the max to avoid any open air contact even in front of a flow hood and in a designated clean room. Not to highjack the punch biopsy cloning method but it is right along the lines of a closed system that I am using
- fahtster likes this
Posted 12 May 2023 - 01:38 AM
Clone coming in on 16qt tub with a clone I got using this method.
I think it’ll be be a nice tub all said and done.
It’s an F7 of a PE/RW cross. This was the f6 ms in a 2.5 gallon zipblock
So the clone should be pretty nice. Also have a fp+ f8 clone probably 3-4 days out from knots that I got using this method. I’ll keep this updated. Here’s the f7 ms tub I took the clone out of
Edited by fahtster, 12 May 2023 - 01:41 AM.
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Posted 17 May 2023 - 02:02 AM
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