Cloning with Brf/verm

[Mass Mush]

I've read two tutorials about cloning and my brother in law insists that I can remove a piece of tissue from the stem and place it straight into a brown rice/vermiculite mix. I've also read that It can be done using a syringe but I don't feel comfortable buying them. Keep in my mind that this is not a "magic" mushrooms. I don't really do them my husband does.:p

[AzPengi]

I've read two tutorials about cloning and my brother in law insists that I can remove a piece of tissue from the stem and place it straight into a brown rice/vermiculite mix. I've also read that It can be done using a syringe but I don't feel comfortable buying them. Keep in my mind that this is not a "magic" mushrooms. I don't really do them my husband does.:p

You could do that, however it would be much much better to create a Liquid culture from the tissue. It would colonize a lot faster and greatly reduce the possibility of contamination.

It doesn't matter what type of mushroom your growing as this will work for almost any species:)

If your uncomfortable purchasing syringes you can buy a marinade injector from any large retail store. However just about all mushroom spores and cultures are available in a syringe form and you can reuse these thereby bypassing the need to personally go to a medical supply store, just something to think about.

materials:
rubbing alcohol
cotton balls
metal marinade injector (I'll refer to this as a syringe throughout the rest of this tek)
aluminum foil
micro poor tape
a half pint jar
some organic honey, karo syrup or dextrose/light malt
distilled water

step 1. Prepare a small liquid culture.

The liquid culture containers are simple jars, with a regular old metal lid and ring, with a single hole punched at the top. The hole should be big enough to accommodate your needle, with a little room to spare so that a vacuum is not created later when aspirating solution. The hole is covered with a piece of micro-pore tape, and then covered again with a piece of aluminum foil loosely crumpled, to act as a dust cover.

Recipe:
You can use any of the standard LC media, organic honey, karo and dextrose/light malt will all work. Water quality is important to LC, distilled works best, and gives the clearest solution. Honey quality also varies widely You may need to experiment with brands and types of honey to find one that works. For a sure thing, you can definitely use karo, and if you're industrious and willing to hunt down dextrose/malt, by all means use that.Honey seems to outperform karo, however dex/malt seems to work best. Just remember that ratio is the same for any liquid culture, 4% media(honey, karo dex/malt, ect) by weight

Paste:
mushroomgeeks.com/forum/showthread.php?p=258972#post258972
into your browser to see Liquid Culture Basics for more detailed information on Liquid Cultures

I'd of imbeded the link but for some bizarre reason you can't do that until you have posted 15 or more posts :(

Start with a half pint jar. Add about 4 grams of honey. Water weighs 1 gram per 1ml, so add 100ml. Microwave for about 30 seconds, just to warm the water, allowing the honey to dissolve easier, and stir it up. Obviously, when making larger LC's, just do the math to stay at that 4% mark.

This will be the "master" culture. From this culture, small samples can be taken to start larger liquid cultures in quart jars that are your "working" cultures used for inoculation. The remainder of the master culture can be refrigerated, where it will keep for several months. When you run out of working culture, it only takes a few drops of the the master to start a whole quart of working culture all over again. Its important to take from the master each time, rather than go from working culture to working culture, so that each of your working cultures are "second generation."

Step 2. Sterilization.

There are two acceptable ways to sterilize LC for consistent results, steaming or pressure cooking. This gives you an option if you don't own a pressure cooker.

In both cases, wrap the top of your jar in tinfoil as usual, and take a syringe about half full of water, and also wrap it in tinfoil then sterilize them both at the same time.

Steam sterilization:
Place a cloth at the bottom of a pot with a tight fitting lid. Use an inch or two of water. Place your jar and syringe inside. Bring to a slow rolling boil. Once a boil has been achieved, start your timer for 30 minutes. At the end of 30 minutes, turn off the heat, walk away and leave it for a few hours to let it cool.


PC sterilization:
Use your PC as per its instructions with something to keep the items off the bottom of the pot (like a cloth, or a rack if you got one.) Slowly bring up to pressure,15psi. Do not exceed 15psi. Once at desired pressure, set your timer for 15 minutes. DO NOT exceed 15 minutes. When the time is up, cut your heat and walk away until its cooled.

Step 3. Select specimen.

Find a nice looking fruit! You want to clone desirable qualities. There are different ideas as to which fruit to clone; ie. choosing the first one up might give you a speedy isolate to work with later, choosing a big one might give you a genetic predisposition towards large fruits; and so on. Just take a nice looking fruit and hope for the best. You'll find out in time if it was a good choice or not. If it didn't turn out as good as you were hoping, don't fret, you can try again from a different specimen from a different multispore grow. If it did turn out for you, treat that master culture like gold!

Step 4. The biopsy

Here's where you'll want to practice your sterile procedures. Ensure there's no drafts, clean your work surface, wash your hands with antibacterial soap, and hit them a second time with some hand sanitizer. Try spraying Oust or Lysol into the air and wash everything with a 10% bleach solution .

Take your fruit, and clean the stem thoroughly with an alcohol soaked cotton swab. Clean it all the way around. Do not touch any areas with your hand near the site of the biopsy. Quickly wipe your needle with alcohol and flame if desired (if you flame push a bit of water through to cool the needle) then stab it straight through the stem to the other side.

Remove the foil from your prepared LC jar. Using a dry cotton swab, clean up any condensation/moisture on the lid. Then using an alcohol soaked one, clean the lid again. Give the needle another wipe, insert into hole, and push the plunger. The water in the syringe will force the cross section of the stem out into your LC. Quickly cover the hole with a piece of micro-pore tape (if your lid is still wet with alcohol, that's fine, the tape will stick just fine once it dries) and cover with a aluminum foil.

Incubate at around 84 degrees F, do not exceed 86 degrees F

In 7-10 days you should have a nice looking cloud of mycelium.

Use your new LC to inoculate your brf substrate jars.


Some additional LC tips:
-Follow the measurements carefully. You'll get no growth or poor growth if you use too much media. When in doubt, use a little less than 4%.
-If your myc cloud is thick and can't be sucked up easily, suck up what you can, and squirt it back down into the cloud. It will break right up.
-To aspirate from your single-hole jars, Just tilt them on their sides, being very careful not to spill any out the hole. The needle reaches in just fine. Gently swirl it around and capture as much mycelium in the syringe as possible. The thicker the better!
-People often ask, "when is my LC done?" Well, its done when you want to use it! If there's enough mycelium floating around that you can capture a bunch in your syringe, go for it. The rest will continue to grow until the nutrients have run out. When it doesn't seem to be growing any more, you can refrigerate it, and it lasts many months. (6 or more.)
-When you aspirate, Take an alcohol soaked cotton swab, and swab right over the piece of micro-pore tape. Then stick the needle right through the tape, and fill up your syringe. Then quickly cap the syringe, and quickly replace the soggy piece of micro-pore tape with a fresh piece. You can certainly use any number of the fancy LC container teks out there, but it doesn't get much simpler than a hole with a piece of tape on it.
-A perfectly prepared karo or honey LC should be nearly crystal clear. Dex/malt will produce a yellowish solution. If your karo or honey solution turns yellow, you may have overcooked. Did you follow the sterilization directions to the T? Either way, a little overcooked will still work, its not the end of the world. Likewise, sediment from honey, or from malt/dex will not harm a thing - it just makes spotting possible contamination a little more difficult. Some take some pretty extreme measures to filter their solutions before sterilizing to get the sediments out, I'll leave it up to you.
-Unless you want cracked jars, allow them to cool gradually and naturally.
*excerpts from creamcorn

[The Truth]

This is what I was doing without this tek, wish I would have seen this as it is an awesome way to clone.
Very nice AzPengi.

[skygear]

i agree, great write-up !! this is what i was hoping to find when i found this forum...

note to mods and forum owner "Thanks Button"

[mouclininly]

Hi Alison,

Thanks very much for your response. Your second answer is more the sort of thing I was looking for, although I was really looking for something like the infinite cloner that dare I say it SMART offers.

Cheers

Carl