Buckaroo's Cloning Tek...{merged}

[BuckarooBanzai]

A friend of mine in Amsterdam sent me this. I thought you all might enjoy it. This is my first post with pictures. If someone could explain to me how to make them full size without posting to a third party server I would greatly appreciate it.

My intent: a liquid culture solution of cloned tissue, in sterile distilled water, for inoculation and/or storage purposes.
My initial considerations:

• I hate mixing/pouring agar and dealing with Petri dishes (pre existing neurotic condition based on past work experience – best not to ask). Also, I have an irrational fear of Parafilm.
• Every time source material touches air, even HEPA filtered air, there is a contamination vector introduced. I want access to my master culture via a flamed syringe passing through a self healing injection port. Petri dishes/slants involve multiple opening/closing rituals and the dreaded Parafilm.
• IME, throwing chunks of tissue straight into a liquid culture produces very unpredictable results and is not a satisfactory solution.
• Mycelia (in distilled water) has been shown to remain viable after extended periods of storage. Ending up with macerated mycelia in distilled water is very much desirable.

This is what I came up with…

Begin by mixing up a liquid culture broth of your favorite sugars and/or additives. I like light malt extract and dextrose (1:1 by weight) at a %4 dilution in tap water with just a pinch of bee pollen. Any LC recipe should work (honey, Karo or Lazlo TEKs should be fine). All you need is sugar and water in the right percentages. I have seen pure Karo and pure dextrose both work adequately. The sugar(s) you use are less important than the dilution ratio. Not sweet enough results in slow growth; too sweet results in no growth whatsoever. Note: a proper liquid culture broth is just barely sweet to the taste by human standards. An LC will have almost no smell.

Add 9mL of your LC broth (syringes work great for measuring small volumes) to the bottom of a pint or ½ pint jar. I’m using ½ pints for these pictures. Put three glass marbles in the jar to aid in swirling (and later macerating) the mycelium. Shards of broken glass work well for shredding the tissue later, but tend to get in the way of gentle swirling. IME, shards result in slightly slower colonization but also slightly less tendency for syringes to clog later. To me, marbles are superior because I cut my clumsy ass fingertip while cleaning out a “shard jar” (clogged syringes are way better than infected index fingers, IMHO).

IMG_0016.jpg

Don’t add more than 9mL of LC! The idea is to have a dry island in the center with nutrient pooled all around. It’s kind of a castle/moat situation. Once your jar is filled put it in the PC for no longer than 25 minutes at 15psi (if you PC for too long the sugars will caramelize and the LC will not work).

You’ll notice from the pictures that my jar lids all have tyvek vents in the center. I always use lids with tyvek vents to avoid a vacuum forming in the PC. Vacuums can suck in contaminants.

In your most sterile environment, tear a mushroom stem in half. If you let the stem air dry for 18-24 hours before tearing, the inner materials will shrink away/differentiate from the outer stem and make tissue extraction easier. You can see this shrinking/pulling away in the picture below. I should have taken a closer shot.

IMG_0018.JPG

Using a brand new sterile blade, excise a pencil eraser sized piece of tissue from the center of the stem. Dull blades make this difficult, time consuming and more prone to contamination. To make your shroom surgery quick and efficient you should use a brand new blade. This is a total non-sequitur, but can anybody out there give me a dime store explanation as to why flaming blades makes them dull?

Spear the tissue chunk lightly with the blade and place it in the jar. Open the lid as little as possible and for as short a time as possible. A few practice runs will make this FAR easier. Ideally, you want to open the jar, dunk the tissue in the LC then tap/drop it into the center of the jar (in one smooth motion) while opening the lid only about ¾ of an inch. In less than three seconds. Easier said than done! The neophyte will probably find it easiest to spear the chunk, open the jar just a little and scrape the chunk off on the inside (sterile) edge of the jar.

Try to end up with the mushroom chunk in the center (dry) area of the jar. If the chunk starts over at the edge, just shake and/or tap it back to the center. The process will not fail if you can’t get the chunk back to the center (it just colonizes a little slower). Do NOT open the jar to reposition the chunk, as it isn’t that important! Please note, the chunk below has been stained blue to make it easier to see. That size chunk seems to work quicker than smaller pieces, by the way.

IMG_0019.JPG

Put your sealed jar in the incubator and swirl (GENTLY) once every day for 2 weeks. This will also work (just slower) with no swirling at all. When swirling, the idea is to lap nutrient up around the edges of the growing mycelia, not to drench it. Overly wetting it by shaking violently will make it grow MUCH slower. A cool ring pattern, like rings in a tree trunk, will be visible on the bottom of the jar after a while. During the growth process, any contaminants are easily identified by carefully examining the jar from the sides and bottom (a flashlight helps). Anything that looks remotely non-mushroom like should be disposed of immediately. Note that some darkening/browning/bluing will happen to the bottom of the clone tissue chunk after a while.

Below is pictured a clone jar after 2 weeks in the incubator. You will note that almost all the food source is gone. This jar actually had three chunks of starter tissue placed in it. Three little pieces didn’t seem to expand nearly as quickly as one big piece, however. The biggest chunk of starter tissue is still recognizable as the oblong beige lump just off center. The two smaller pieces dissolved almost unrecognizably into the cloudy structures to the right and top of center.

You should NEVER open your clone jar to look at it like this! I have done so to take a picture (this culture will be disposed of). I removed the marbles because my camera’s auto focus kept centering on them instead of the mycelia.

IMG_0023.JPG

It is redundant to repeat it over and over, so from here on assume that syringes are flame sterilized red hot before insertion into any sterile jar. Also, they syringes are NOT fully inserted. Only the ¼ inch of metal that gets red hot goes inside the jar.

If you are clumsy, like me, “blowtorch” type refillable butane lighters make a great substitute for alcohol lamps when flaming syringes. I will invariably turn over and/or burn my forearms on alcohol lamps.

Using Hippie3’s airport syringe to equalize the pressure, a 60mL syringe is filled with sterilized water (use distilled for long term storage). It takes a second or two after you finish pulling up the plunger for the pressure to equalize completely (submerge the airport needle and wait for the bubbles to subside). If you pull out the 60mL syringe too soon, some unfiltered air might get sucked in through the hole in the silicone injection port. As always in this hobby, patience is a virtue.

IMG_0024.JPG

Again using the airport syringe to equalize pressure, the 60mL of sterile water is injected into the clone jar. Do this a second time (120mL total). If you are using a pint, inject 240mL of water. You will have more than enough mycelium for a pint of clone water after 2 weeks of healthy growth.

IMG_0029.JPG

The jar is now shaken VIOLENTLY for 8-10 minutes (the longer the better) until it looks like the picture below. The longer/harder you shake, the less easily your syringe will clog. Don’t worry about damaging the mycelia (tearing it to tiny shreds is your objective).

IMG_0030.JPG

A syringe (again using the airport to equalize pressure) is now filled with clone water from the jar. Once filled, the syringe will look like the picture below.

IMG_0033.JPG

The syringe will clog with mycelia as you fill it. Just squirt a little back into the jar, shake, and begin drawing again. Sometimes you can fill the syringe in one clean pull. Sometimes it takes 19 tries. Just be patient and keep working with it until you get the results pictured below. This is a close up of a clone water syringe with great potential:

IMG_0036.JPG

This syringe of clone water can now be used to inoculate whatever your heart desires. A small amount goes a long way, especially if you use the clone water to start a liquid culture. Don’t clone clones, though. Go back to spores at least every third or fourth generation. We aren’t growing orchids. Our mushroom of choice drops (in most cases) abundant spores that are easily collected, stored and germinated. There is no reason to toy with the possibility of senescence. Also, over the years, going back to spore will encourage a strain to adapt to your grains and fruiting environment. They will form a relationship with you…

If your objective is long term storage of the culture, leave the jar in your incubator for 3-5 weeks until all the nutrients are consumed (other than some condensation, the jar will be pretty dry inside). Please note that clone water from jars which have eaten all their food seems to colonize jars much slower than clone water drawn while mycelium is actively growing.

If fast colonization is your objective, shoot/shake before all the food is gone (2-3 weeks). If storage is the objective, wait for ALL the food to be gone before you shoot/shake (3-5 weeks). Storage should be in the fridge (not freezer) in an air tight and light proof container.

I can’t personally testify to the long term efficiency of sterile water storage but the work of other mycologists indicates that cultures in sterile distilled water should remain viable for 5+ years. If all the food is gone before you shoot/shake, your culture should go into suspended animation and stay viable for at least 2 or 3 years (in the fridge).

Your mileage may vary and reports of your experiences are infinitely appreciated!

[shobimono]

Nice writeup! Thanks for sharing it.

[spacecowboy]

Thanks for the info.

I was thinking of trying something kinda similar, but using a small amount of jello in the bottom instead of liquid around the edges (jello liquifies when colonized by mushroom). I really like your use of marbles, I wasn't sure they would work, but now I know they do (rather use marbles than glass too). This thread helps me out alot.

My original plan was to use a blender to cut up the mycelium, after adding water once the small amount of growth medium was consumed, but it was going to be my biggest source of contam in my experiment.

Very nice, bro. Nice twist to an old method.

Great write up!!!! And great pictures!!!


I do this for my cloning also, only slightly different. It works well for me using liquid cultures for cloning.

thnx for the info..

[Elf Salvation]

[InfectedMushroom420]

Thank you very much for the post. I find it very well written and the pics do inlarge. This is just what I was looking for. What a wonderful post to wake up to! This will be my next experiment

Questions:

1. Will using liquid culture speed up my colanization of cake jars?


2. Creating cloned liquid culture, does this mean that the tissue I use to produce this liquid culture will give me close to identical resulting shrooms as in size for instance?

Thank you

IM420

1. Yes it will speed up clonization by about 2 days. After innoculation with liquid culture you can usually see growth after about 24 hours of incubation.

2. Yes the mushrooms produced from a clone in an lc will be genetically identical. You will prolly have a better pinset too.

[BuckarooBanzai]

Spacecowboy: Personally, I would avoid the blender (contams + excessive mycelia damage). Shaking the crap out of the jar with the marbles inside works great. Also, you might consider fruit pectin over Jello. Pectin dissolves more completely and is a food source (dextrose) unto itself (don't use sugar free pectin).

Infected: Clone tissue should give you nearly identical results with nearly identical environments. If you change substrates or fruiting conditions, your results will likely change dramatically. I'm not at all certain about the uniformity of size question, though I suspect you will still have some variance in size.

Thanks, everybody, for the nice words!

[BuckarooBanzai]

Originally Posted by freakachino

I do this for my cloning also, only slightly different. It works well for me using liquid cultures for cloning.

Freakachino - do you mind if I ask what you do differently? Do you grow your clone tissue out in an LC, or do you use an intermediary step as well? I'm always looking for improvements...that's how I came up with this TEK in the first place.

It kind of got buried in my post, but does anybody out there know why flaming scalpels makes them so dull?

Here are two pics I forgot to include in the orignial post. The first is a pic of my favorite kind of scalpel blade (#12). I find it much easier to dig/cut internal tissues than a straight blade.

IMG_0003.JPG

This is a pic of the bottom of a clone water jar. The brown chunk in the center is what is left of the original piece of clone tissue. First the tissue turns blue, then off white, then beige and finally that gnarly brown. It's not contaminated, I swear! You can also see the concentric rings formed by swirling the LC.

IMG_0010.JPG

[TVCasualty]

I've used a few small stainless steel nuts (1/4" to 1/2") in LC jars in place of marbles. Sort of a compromise between the swirling and cutting functions of marbles vs. glass shards. Be careful about shaking intensity if using 1/2" or greater nuts, larger sizes make me nervous about busting the jar...Hasn't happened yet but I'd hate to explain those gashes on my hands to the ER doc.

TVC

http://mycotopia.net/discus/messages...tml?1081303707

This is how I do my cloning with lc's. It is quick and easy and simple for me. I add a marble also for swirling, but have not added marbles before, just harder to suck the mycelium up of course. Blunt ended needles I usually use to suck up my lc clone mycelium so I don't have to worry so much about mycelium clumps.

I use plastic lids also. I'm sure covering with a sterile lid after microwaving could work, but I like to just lay the plastic lid on to cover it and allow for steam to release, then I just tighten it down when its cooled slightly in the microwave.

I don't use any knives, I tear with my clean hands, and use sterile tweezers to pluck the inner tissue and drop in the jars.

I like lc clone jars, they work great for me. Thanks for the write up!

[BuckarooBanzai]

Wow, Freakachino, no PC for your LCs at all, eh? Interesting. Contam rates are low, I take it? Any problems with carmelization?

TVC - I like the stainless steel nuts. Makes me think also maybe a stainles sheet metal screw or similar. That would give me better shredding without the risk of cutting myself. I'll have to take a look next time I'm in the hardware store.

[Hippie3]

very nice.
my only criticism would be the use of tap water-
quality of tap water varies widely from place to place
so one might be well advised to use bottled water instead.

[Hippie3]

archive material

[BuckarooBanzai]

I agree on the water. Because of other considerations, I already know what the TDS and pH of my tap water are. If my numbers weren't good, I wouldn't use it. Distilled (or at least bottled spring) would give much more dependable results.

Previous places I have lived with crappy water (Oakland, for one), I used an RO unit. RO water kicks much ass for many things (including drinking), but I sold that unit when I moved. Wanted to avoid temptation in my new location.

That worked out well...

I use my tap water because I know its okay, but spring water works very well too. Distilled water gives me no carmelization usually when using karo. The microwave will carmelize if I microwave for over 5 minutes. I clean my microwave out very very well with lysol, then a bleach wipe and let it sit for a few hours. Then I do the lc jars. I haven't had any contam problems from using Hippies tek for microwaving lc's. I just prefer for clone tissue rather than spores. But I have success with it over pc'ing my lc's. Every lc I've ever pc'd has not worked for me lol. The only thing that I find annoying is the steam build up causes a lot of water to form on my microwave plate, but I just use a paper towel under the jars now.

[Hippie3]

so how do you 'wave yours,
a few 3 minute bursts or ?

[suckerfree]

Originally Posted by BuckarooBanzai

It kind of got buried in my post, but does anybody out there know why flaming scalpels makes them so dull?

It has to do with the temper of the metal. The steel will lose some carbon due to oxidation while it's red hot. That means the thin steel at the cutting edge would be lower carbon and thus softer and less capable of holding a cutting edge.

[BuckarooBanzai]

DUDE...temper...I remember something about that from the wayback machine. Something about quenching and crystaline structures and hardness. Doesn't that also have something to do with the "rainbows" that you get in tempered metal that is heated red hot? Or is that hardened metals?

Ah, heck, I'm mixing ideas. I gotta go read some stuff about tempering and oxidation and hardness.

[suckerfree]

an electrical current is used in the temper/coating process to get the rainbows.

Hippie, I had to test it a few times for my microwave power, and I found a straight 5 minutes on high works like a charm every time. Different power levels on microwaves do make a difference so I just found the 5 minute mark to work well for me.

[BuckarooBanzai]

So, basically, you clean the microwave, put in your lightly capped LC containers and run it on high for 5 minutes?

Suckerfree - could you elaborate on the electrical/rainbow process? Or just give me a technique or link...

[suckerfree]

http://www.bmfinishers.com/stainless_steel.html

here's a company that does it...

Basically, I clean the microwave. Get my jar(s) cleaned and ready with the karo/water and marble. I like to really stir it up so the karo gets mixed well with the water. I use plastic lids for these so I can microwave. Lay the cap on top, then stick in and nuke for five minutes. After a few minutes of cool down, I slowly open the door, and tighten the lid down. Sometimes I've tightened the lid too much and when I'm ready to drop the clone tissue in I've had trouble getting the lid open to drop it, so now I make sure its tight enough but not so much I can't open it to add the tissue. The plastic lids have been a great addition to my mycology hobby because they are one piece and microwaveable. I just like cloning in lc's like your tek, easier for me and quickly gives me a lot of clone mycelium in 2-3 weeks time. I really think Hippies Microwave tek for lc's is best for me. Pressure Cooking my lc jars never seemed to work for me. Hippies tek has always worked for me, but then all of Hippies teks have worked for me when I've tried them! Rock On Hippie!

Do you like half pints or pints for your liquid culture clone jars? I've only used half pints but am thinking pints might work a little better and give more mycelium. Might add a bit more time though.........

[BuckarooBanzai]

I am going to try the microwave thing. Firing up the pressure cooker just to do two or three clone jars is annoying (and wasteful).

I started with half pints, but I only use pints now. I fill the jar about 1/2 full so I have plenty of air for growth without needing any kind of venting. How fast your LC will colonize seems to be more a factor of how much tissue you start with and how often you swirl the jar than size. The pint vs. 1/2 pint question seems more a matter of how much LC you want to end up with (his eyes glaze over as he imagines using 1/2 gallon jars for LCs).

10mL of thick clone water swirled 3-5 times a day will colonize a pint in a week or so. Bluehelix has show that with a mechanical stirrer, that time frame can be shortened to 2-3 days.

About 5-6 days in, when the jar is %50-%60 colonized, I shake the living CRAP out of it. This aerates the water and breaks up the mycelium for better "syringe suck up" later on.

Once I have a good container of clone water in distilled water, I use a small amount of it to start LCs and toss the clone original in the refrigerator. The LC starts the next generation and I maintain a "young" master culture to work from.

How do you handle long term culture storage? What's the oldest culture you have successfully worked from?

[pcubie58]

OK. I've read a good deal of the archives on cloning TEK's (first-timer). Please direct me to the most widely used and beloved nowadays.
I am a rather simple person with no agar in the cupboards, what would you recommend? Karo? Marbles? Blending? Praying? Cap cuttings? Stem cuttings?
I'm utterly confused

I like using karo and water at a mix of 1 teaspoon karo to 100ml of water. I pc mine at 15 psi for 20 minutes some use a microwave on high for about 3-5 minutes with great success.

After it has cooled and you have selected the fruit you want to clone you need to do the rest of the work in a glove box. With fruit, karo jar, small dish for hydrogen peroxide, scapel, and tweezers inside the clean glove box you can begin work. With latex gloves on I like to pull the base of the stem in half long ways to reveal the inner tissue. I then take my scapel and cut a small portion of the inner tissue near the base of the stem. Then with tweezers I take the tissue drop it in the dish with hydrogen peroxide for 5 seconds and then transfer to the karo jar (no need to rinse the h2o2 off). I usually make at least 3 jars just to be on the safe side.

[pcubie58]

Excellent. This is what I thought may be best for me but I just wasn't sure after reading so much info. A person just gets frazzled after a while.
Thanks so much for your help.

No problem, man.
Here is a link that may help. http://www.mycotopia.net/discus/mess...tml?1004627146

I do not cut and peel like nan describes in this link. Like I said, I pull the stem open along the mushroom's long axis to reveal the inner tissue. Not only is this approach cleaner, imo, but it also leaves the inner tissue frazzled so more mycelium will come into contact with the karo water.

[pcubie58]

While you're on a roll......
can you then take some of this solution after its' incubation period (how many days do you let it sit?) and inject a syringe-full into another Karo jar or do you always start with a fresh piece of innards for each jar of solution?
Again, thank you-thank you

Innitially the 3 to 4 jars I do are all done with tissue, but if you are wanting to expand your mycelial mass into more lc's then yes you can take some from your original karo jar/jars and transfer via syringe to more karo jars.

I usually let mine incubate for 14 to 20 days. If at that time I am not ready t use it i stick it in the fridge. They can stay good in the fridge for quite a while.

[pcubie58]

Does a person ever get "bored" cloning the same strain, same "big" shroom over and over again? Same flushes, same size, same same same? Do you ever revert to multi-spore just to see what you may come up with as a new host? Just curious... my mind usually can't stay focused in just one direction for any length of time, so I think I'll find myself enjoying this cloning thing for a while but will end up back from where I first started, if for nothing else but new visuals in the chamber

Multispore is very important as kind of a reset button. Too much cloning can cause many problems. I do not clone the fruit produced by a cloned fruit. And expanding the mycelial mass especially cloned mycelium to far away from the original sample can sause degeneration. So yes, it is very important to still do ms inoculations.

[pcubie58]

Awesome. Now I fully understand how to go through the process. I'm really looking forward to a bit of experimentation of my very own! Thank you again.

[python]

flowhood/glovebox, interior tissue, agar/karo................cloning

[Hippie3]

see
http://forums.mycotopia.net/showthread.php?t=5290 (cardboard cloning tek)
for a good contam-resistant method

[igetit]

im not sure if hippie posted a link to his hydrogen peroxide cloning, but I tried it and it is totally sucessful.

Inner tissue blended in a hydrogen peroxide / water solution -----> innoculation of sterilized jars -------> easiest way to clone that I've found.

[pcubie58]

This has all been very interesting reading, especially Python's link on the reproductive cycle. Just can't get enough of that!
I have 26 casings in fruiting chambers at the moment and so came the question about cloning for my next project...I'm wanting to try the Straw Log.
I've always multi-spored it and so it's time to do something different. I can't wait to see which strain comes up first and how aggressively.
I may try both the Karo and Cardboard teks for comparison. Either one seems easy enough!
I appreciate all your inputs. It's great to come home from work and have your hobby questions answered

[Hippie3]

long term LC store i use the fridge so metabolism slows.
i've heard of cultures 2 years old being revived
but they grew out with diminished vigor.

archive material

[viraljimmy]

Has anybody tried dehydrating cultures for
long-term storage?

I was thinking with a little liquid or substrate,
allowed to dry in a jar with a poly or tyvek filter,
that it might last indefinitely.

Then it could be rehydrated and used to
innoculate. Would drying the culture damage
its future potential?

[Hippie3]

dry tissue can indeed be re-animated later
by re-hydrating under growth conditions.

sounds like a good way to mail things?

[Hippie3]

here's why cloning makes good sense-
http://mycotopia.net/discus/messages...tml?1034628376