35 replies to this topic

[fahtster]

before you put it in the bag with the H2O2, you hold it under H2O2 with a tweezers until it stops fizzing (probably less than a minute). then put it in the bag with the H2O2 for a couple minutes then go ahead and open your jar and cut the first zip tie and put the tissue in the jar. screw the lid back on. then you can go ahead and cut the other zip tie and retrieve your jar. :) the whole process from out of the pc to the clone tissue being in the jar is less than 5 min. :)

fahtster

[night_ryder]

did you use the thin inside stem or did you use a cross section?

i'm worried about the myc being killed off

[fahtster]

used an inside piece... what % H2O2 are you using? with the 3%, i left it in there for a good few min. it takes a couple days for the myc. to start 'fuzzing' up again. if you shake the jar vigorously right after the innoc. then the jar should be colonized shortly after that first couple days. :)

fahtster

[night_ryder]

jar colonised in days? how many days roughly.

seeming as though hippie says 9% is to strong im going to buy some 3%solution.

[fahtster]

five days roughly. :) shake the hell out of it. ;)

fahtster

[night_ryder]

when you say shake the hell out out it'

1. for how long

and

2.like your life depended on it.

am i right in assuming fragments from the inside stem creates multiple innoc points

[fahtster]

you aren't going to hurt anything if you shake it for five min. and that seems sufficient. so go ahead and do that. ;) you are correct in assuming that. :) tiny bits break off and innoc. all over the jar. it's extremely effective.

fahtster

[night_ryder]

will shake it like i am trying to get a dog off my hand

[fahtster]

update- the TEX from this thread... http://mycotopia.net...ead.php?t=12695 are a clone that were done with this method. :)

fahtster

[fahtster]

hey yall. I'm finally getting around to putting this thread into actual picture form instead of a drawing... I do it a little different nowadays anyhow. it's the same tek but instead of using the leftover part of the bag as the quarantine section i'm installing a small one using a clear piece of tubing about 3/4 in. in Dia. and 2 in long.

Now heres the basic idea. An oven bag (reynolds) is altered to account for air exchange after sealing. a small quarantine section is attached to the bag with one end covered with tin foil. a jar with hydrated whole grains is put into the bag and sealed. then the bag is pc'd and a small piece of clone tissue is taken and put into the quarantine section along with 3 % H2O2 til all the air in the quarantine section is displaced. Then foil is sealed tightly over the quaran. section. the jar is unscrewed while still in the bag. then (THRU THE BAG, WITHOUT TEARING THE BAG) the bottom piece of foil is broken and the clone piece and H2O2 flow into the bag where it is promptly moved into the open jar of grains. this is ALL done so far while the jar is still in the bag and the bag is sealed. it's very easy to move things around with your hands from outside the bag. then you can screw the lid back on. THEN and only then can you tear the bag and remove your grain jar with clone tissue safely inside.

ok, now heres how you do it. :)
first the bag prep.

1. here are the items you need to acquire
- an oven bag large kind... comes 5 to a box
- scissors
- jelly jar with lid (i'll show you how to make a kickass lid in a bit)
- hydrated grain... i use popcorn for these 'Master' jars.
- Three pieces of 2 in. 3/4 Dia. tubing (two stuffed with polyfil)
- small cable ties (make sure they are a 4 or above plastic rating)
- two 2 in square pieces of foil

http://mycotopia.net...=1&d=1164773526

here are the three tubes up close... the two with polyfil are going to be used for the air exchange in the corners of the bag. This assures that the bag doesn't pop in the pc. since the bag is sealed shut, there needs to be some way for the pressure to even out. the clear one is what we are going to be using for the 'quarantine section'. thise quarantine section is how we are getting the clone tissue from outside of the bag to inside of the bag without contaminating the contents of the bag.

http://mycotopia.net...=1&d=1164773654

2. Now cut both corners of your oven bag like so:

http://mycotopia.net...=1&d=1164773654

3. Take one polyfil tube for each corner and attach it to the bag via cable ties. you can probably get away with using tape if you want, but for ME, this is where i don't skimp on my sterile tek.... when i'm getting my 'masters'. I use two cable ties per bag attachment. there is a small gap, even when you tighten the cable tie as tight as you possible can, where the head of the tie and the tail of the tie meet. so i use two where the 'heads' of the ties are opposite of each other like so:

http://mycotopia.net...=1&d=1164773871

4. like i said, i don't take chances with these... i'm afraid the leftover part of the zip tie is giong to poke a hole in the bag so i cut them and fold a little piece of duct tape over the cut ends... nice and soft then. ;)

http://mycotopia.net...=1&d=1164773871

5. Now we're going to make the quarantine section. heres about where you place this QS (from here on out i'll refer to the quarantine section as QS) on the bag compared to the air vents... make sure it's in the bottom half of the bag.

http://mycotopia.net...=1&d=1164773871

5b. place the tubing in the middle of the 2 in square foil piece. carefully bend the foil up on the tube. make sure the foil goes all the way up at least half way. you are going to attach the QS to the bag with cable ties much like air vents so you have to make sure that both the bag and foil are covered by the two cable ties all the way around.

http://mycotopia.net...=1&d=1164773871
http://mycotopia.net...=1&d=1164773871

this bottom piece of foil is what we're going to break to allow the clone piece and H2O2 to flow into the bag.

http://mycotopia.net...=1&d=1164773871

6. cut a hole the size of the tubing in the bag and attach the QS... notice how the bag and foil are both covered by the two zip ties... this creates a sterile seal between the tube, the foil, and the bag... so if something wants out of the bag it has to go thru the opening, the air vents, or this new tube (which is blocked off by foil)

http://mycotopia.net...=1&d=1164767872

http://mycotopia.net...=1&d=1164767872

heres the QS with the tie tails cut and taped...

http://mycotopia.net...=1&d=1164767872

7. now use your other piece of 2 in square foil to cover the QS... BUT, keep the cable tie just tight enough to keep the foil cap on... you are going to need to take this off after you pc but you don't want it falling off before you need it to come off letting contam'd air inside you QS.

http://mycotopia.net...=1&d=1164768234

heres the bag with everything attached...

http://mycotopia.net...=1&d=1164768234

8. Making your kickass jar lid. I happen to think that the lid you use is one of if not the most important tool you use.. so it's nice to have a really safe design... and i feel i have one. first you need to bend that inner edge that cuts thru everyones tyvek when they tighten the jar too much. all you do is flip the band over and crimp it down all the way around until it's flush... it really doesn't take much...

http://mycotopia.net...=1&d=1164768567

Now that you have a safe band, you can make the rest of your lid... I use both postal tyvek and kite makers tyvek... one postal on top, a layer of kite makers, and a postal layer on the bottom. just screw your band on these three layers tightly. then one strip of duct tape down the middle. the remaining sections are the selfhealing port and the air exchange side. the air exchange side is covered up about 90 % while the master jars are colonizing... after they are about 95 % colonized, the air exchange sides are uncovered and the grain and myc. are allowed to dry. the reason i uncover at 95 % is so that the myc. doesn't go into pinning phase by the time it drys out. keeps the myc. nice and 'young'. put about a 1/4 in of aquarium silicon on the other side of the tape and smooth out with wetnap. this is your self-healing injection port.

http://mycotopia.net...=1&d=1164768567

cut off all but about 1/8 in of the extra tyvek hanging off the side. now bend that up and tape it to the band. this makes it nice and easy to screw the lid on and off... you don't have tyvek getting in yer way.
now this is where the magic happens for these lids... flip the lid over and put another layer of aquarium silicon on the exact spot it is on the top layer of tyvek.. let dry.

when you look at the pic, you will see that there is a gap between the bottom silicon and the edge of the inside band... this is for the jar rim. make sure you leave that gap silicon free otherwise you might not get a tight seal when you screw the lid on.

now for anything to get thru your lid to your very precious sterilized grain, it has to not only go thru one layer of tyvek w/self heal port and one layer of kite tyvek, but it also has to get thru ANOTHER layer of tyvek with self healing port. these lids are the shit and would not use anything else for my masters. I can pull myc. from these jars to start more jars just like it and keep that first master on a shelf to dry out and use months and months later without the worry of them getting contam'd.. unless my syringe tek is flawed (which it isn't ;) ). now remember these are jelly jars not 1/2 pints... 1/2 pints will probably work but i like the smaller jars for masters.

http://mycotopia.net...=1&d=1164768567

heres the jar with just about a 1/2 in. worth of grain (popcorn) in the bottom... this is what you are cloning to.

http://mycotopia.net...=1&d=1164769778

9. now put the jar with the lid screwed on loosely inside the bag all the way to the bottom.

http://mycotopia.net...=1&d=1164769969

10. pinch the bag about 2 in. above the QS and twist tightly like so:

http://mycotopia.net...=1&d=1164770249
http://mycotopia.net...=1&d=1164770249

11. now while still keeping the twist tight, fold the twist back on itself a couple times and zip tie the middle of the folds as tight as you can (it should be noted that all zip ties, except the QS foil cover prior to pc'ing, should be pulled as tight as possible.) this makes sure that NOTHING is getting in or out thru the oven bag opening anymore. just make sure the grain jar is in the bag first. lol

http://mycotopia.net...=1&d=1164770249

http://mycotopia.net...=1&d=1164770249

12. now you can gently squeeze the bag to get excess air out thru the air vents. fold up the air vents making sure they aren't blocked in any way and you have a nice little ball... this is what i like to call a "Cloning Machine" it's got a nice ring to it. :)

http://mycotopia.net...=1&d=1164770898

13. now take a 1.5 ft. x 1.5 ft. sheet of foil and put the cloning machine in the middle. now bring up each corner to the middle over the bag. then just put a piece of foil over the top to keep dripping water out..

http://mycotopia.net...=1&d=1164770898
http://mycotopia.net...=1&d=1164770898
http://mycotopia.net...=1&d=1164770898
http://mycotopia.net...=1&d=1164770898

now just pc this for 45 min. at 15 psi. and let cool. i usually use a few 1/2 jars to keep the 'foilball' out of the water and off the bottom.

ok... that was making the 'cloning machine'... it seems like a lot, but when you get it down it takes about 10 min. tops to prepare the bag... minus the lid prep and grain prep. and pc times of course.

fahtster

Attached Thumbnails

• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 

[fahtster]

ok, now for the actually cloning procedure.
heres what you need:

- 3% H2O2
- 70% rubbing alcohol
- cloning machine
- tissue specimen
- small dish
- tweezers
- paper towel soaked with alcohol
- a couple cable ties
- 2 in. square tin foil

1. take your cloning machine out of the pc and unwrap. get rest of materials ready

http://mycotopia.net...=1&d=1164776035

make sure you clean your small dish with alcohol and let dry before you pour your H2O2 into the dish.. fill dish about half way with the H2O2. flame sterilize your tweezers til red hot and stick straight into the H2O2 slowly. And leave in the H2O2 until you need it.

2. Now take your tissue specimen and pop the cap off. starting at the top of the stem... pull the stem apart in half. this keeps you from introducing contams that are on the outside of the stem.

3. retrieve your tweezers from the H2O2 and pick a piece out of the middle of the lower stem... I don't even use a knife to cut the chunk, just pinch a nice chunk using the end of the tweezers... works fine. and one less factor for contam's.

http://mycotopia.net...=1&d=1164776035

http://mycotopia.net...=1&d=1164776035

4. Now put the tissue in the H2O2 and swoosh around with tweezers.

http://mycotopia.net...=1&d=1164776035

5. The 3% H2O2 is strong enough to sterilize the tissue but also weak enough that you don't need to rinse it after putting it in the H2O2. I use the tweezers to hold the tissue submerged in the H2O2 while i work on the next steps.

http://mycotopia.net...=1&d=1164776035

6. With the tissue soaking i remove the loosely zip tied foil cover on the QS.

http://mycotopia.net...=1&d=1164776035

7. Now take your tissue with your tweezers and put it in the empty QS.

http://mycotopia.net...=1&d=1164776035

8. Take H2O2 and fill the inside of the QS (the tissue is also inside this chamber) to the brim... the idea is to displace all the air inside the chamber so that there is only H2O2 and the clone tissue present.

http://mycotopia.net...=1&d=1164776035

heres the chamber filled to the brim with H2O2 and tissue.

http://mycotopia.net...=1&d=1164776035

9. Take a look at the first pic in #3... see that foil that has the alcohol soaked paper towel on it? that is the top lid to your QS. I keep the paper towel on it to keep it sterile while do all the steps leading up to using it. Take this piece of foil and put it over the top of the QS that is filled to the brim with H2O2/tissue. zip tie this lid on TIGHT this time... you don't want it to leak ideally.

http://mycotopia.net...=1&d=1164776035

10. Now with your tissue safely secure in your QS, you can unscrew the lid on your grain jar inside the bag. DO NOT REMOVE THE JAR FROM THE BAG. Unscrew carefully with your hands from outside the bag... it's not that difficult actually... the bag is very workable.

http://mycotopia.net...=1&d=1164776035

11. ok, from the time you unscrew the lid to the time that the tissue is inside the jar should only take a minute at the most... it's very quick. After you unscrew the lid, take the back end (which is blunt) or something similar and (thru the bag) break the bottom piece of foil on your QS. it's very important that your don't tear the bag at all. it's just a piece of tin foil so it's not very hard to break.

http://mycotopia.net...=1&d=1164776035

notice the little white blob in the middle of the bag... thats the clone tissue now safely inside the bag... up in the top right corner of the bag you can see the broken piece of foil on the bottom of the QS.

http://mycotopia.net...=1&d=1164776035

12. Now quickly move the clone tissue into the grain jar and screw the lid back on... once the lid is back on, THEN you can tear open the bag and retrieve your grain jar with the tissue inside.

http://mycotopia.net...=1&d=1164776035

don't forget to tape up the air exchange section on your lid about 90 % so that your grain doesn't dry out before your clone tissue has a chance to colonize it. :) be sure to remove it at about 95 % colonization as well.

I have done this tek more than 15 times. I would do this over even using a flowhood as there is no air movement at all. I have done both fruit tissue tranfers and myc. transfers from pf cakes. both have worked great. I would suggest the fruit tissue over the pf cake of course cuz then you know you have a fruiting substrain.
heres the last nine cloning machines i've done...

http://mycotopia.net...66&d=1164775157

Once you have the colonized grain jar, you can let it dry out or use it right away.. i usually do either 6 qts. or 24 1/2 pints with the myc. that i harvest from each grain jar. i just break the kernels up, then shoot up with water from sterile syringe... shake the water and myc. covered kernels around until most is broken up into the water. then i suck the water back up. If i'm noccin' up qts i just go straight from there to the qts. but if i'm doing the 24 cakes i make another transfer to another sterilized jelly jar (with same lid design) filled with the amount of water that it takes to innoc. 24 1/2 pints. (about 30ml.) then i pull from that jar to refill my innoculation syringe.

the only time this has not worked is when i tried transfering myc. from a contaminated cake. there was some mold myc. present in the transfer (H2O2 will kill spores but not myc.) so it went south... but other than that i have had 100% success with this. i've even cleaned up old faht jars that i didn't know if the lid had been breeched... just ran a couple dry kernels thru a cloning machine and vola.

ALL pics in this thread... http://mycotopia.net...ead.php?t=14674 were sub strains i aquired using either this tek presented or the old oven bag tek that i drew out.

heres the bin that used to get clones.. all but one are multispored jelly jars of Haw, TC, GT, Chitwan, Koh Sui and costa rica (which turned out to be a lame tired strain that only put out cottony myc. and a couple very small fruits.

http://mycotopia.net...=1&d=1164780103

And heres a pic of some GT multispore on jelly jars that i'm really excited to start a couple bins of soon. :)

http://mycotopia.net...=1&d=1164780103

if done right... this tek is, imo, 99.9 % effective when a fruit tissue is used. there not a single speck of doubt in my mind after i do this tek that that grain jar is going to go south... none. sure you can probably pretty safely do a transfer by opening the jar in a semi sterile room and 4 out of 5 jars are going to work great... but it's not knowing where that one jar is that really gets my goat. lol so thats why i developed this tek. i'm tired of losing jars.

Another thing i was thinkin is that the new fly paper tek could be of use here also... if you don't want to do whole grains, you can just stick some fly paper on the bottom of the jar with a piece of glass to help break the myc. off the sticky paper. that thread is here: http://mycotopia.net...ead.php?t=16330 cardboard would probably work in much the same way also. :) I'm stickin with the grains, but it may be useful to some folks. ;)



One last thing... I'd like to give thanks to golly for his inspiration. I saw his work with the H2O2 that he was doing (I believe he was using 30% H2O2) using my faht grain jars and it immediately got my noodle twistin and turnin. I was working with the oven bags at the time that i saw his work and it just fell into place. :) soo.... thanks buddy! That is why this place is soooo fuckin great. ideas bounce around like thrown snowballs and accumulate to become.... i don't know, maybe that wasn't the best anology but you know what i mean. lol they turn into robotic snowmen that rule the planet...... or something comparable. thats why i get so damn mad when peeps like pf come in and say that they are the "man" or it's all because of them and that we are all growing mushrooms because of them or some shit. cuz it's all about and has always been about getting together to grow any which way we can, imo... that is. ;)

:) fahtster

Attached Thumbnails

• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 

[bb4]

hellz yeah,
you the man,
i've been reading your post for a while now,
really dig it, man!!!

:eusa_clap

[fahtster]

thanks bb4! :) does it look ok? can you understand it clearly?

fahtster

[TheJackal]

Very kewl, well written, and illustrated teks you have there fahtster! Can't wait to try them out. First, the wifey needs a PC for Xmas, as suggested by llamabox :)

Cheers!

Jackal

[AlsahrGlib]

very nice! clear as crystal:bow:
I'd call it Faht's Hymen Popping Oven Bag Clone Tek

[dial8]

Very nice, faht. Hell, I can understand that by just looking at the pics.

Archive Material > Cloning and Sterility