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BB does LCs

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#1 Hippie3



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Posted 20 August 2006 - 10:09 AM

The abbreviation for liquid culture is LC. When you see LC, the author is talking about a liquid culture.
You don’t have to use LCs. You can use a spore syringe to inoculate jars directly and omit this step. The question is one of simple economics. A 10mL spore syringe will inoculate roughly 8-10 half pint jars. If you use 1mL of spores to create 300mL of LC, you can inoculate 250-300 jars and still have 9mL of spores left in the syringe.
The process of turning a few mL of spore water into a few hundred mL of liquid culture is referred to as “expanding” the culture. LC is the three dimensional equivalent of expanding cultures on a Petri dish.
Expanding cultures through LC is an important technique to master. LCs will save you a lot of money on spores and open the door to cloning your best fruits later on. If you add agar to the recipe below, you can pour your own Petri dishes and slants.
Parts/ingredients list:

  • 500mL DISTILLED water
  • 2 Teaspoons of Karo Syrup
  • Pinch (less than .1g) of bee pollen
  • *TINY* amount of unscented dish washing detergent or Jet Dry
  • 6 mg of Light Malt Extract (from here on, LME; I like the Sporeworks brand)
  • 6 mg of Dextrose (I like the Sporeworks brand)
  • 6 clear glass marbles OR a small pile of broken glass.
  • 2 pint jars
  • 2 jar lids with silicone injector ports (but no Tyvek vents)
  • 2 jar bands to secure lids
  • Pressure cooker capable of maintaining 5psi
  • Incubator capable of maintaining 84F
  • Scale capable of measuring in .1 gm increments
I’ve never tried it personally, but I am told that sucrose (table sugar) will work. ANY sugar (in the right proportion) should work. Don’t lock yourself into these ingredients, just the magic ratio of 4% sugar (by weight) to water. Honey works. Pure dextrose works. I’m pretty certain glucose would work. Lactose might even work. If you get the percentages right ANY sugar should work. A mixture of sugars seems to work a *little* better than just one sugar. If you only use one sugar, use Karo syrup (1 teaspoon per 100mL distilled water).
The magic ratio of 4% is achieved by mixing 4g of sugar with 96g of water. Remember that 96g of water is also 96mL of water. Different sugars are different densities, so it is hard to give a volume (tablespoon) measurement of how much to add. Get a cheap digital .1 gram scale. They are immensely helpful for many aspects of this hobby. Measuring doses is much easier with a .1 gram scale, for instance.
BE AWARE: a good LC will taste just barely sweet by human standards. If your “sugar water” tastes sweet, it is too strong. If your LC is too weak, growth will be sluggish. If your LC is too strong, growth will be prevented completely. DON’T add extra sugar to speed things up – it doesn’t work that way. Too much sugar will prevent growth.
Start with 500mL of distilled (not tap) water heated in the microwave or on the stove. If you use a microwave to heat distilled water, either use a rough sided container or insert a chopstick, to prevent the possibility of superheating and the resultant nasty burn. Hot water dissolves sugars MUCH better. Take the 500mL of hot distilled water and add your LME, dextrose and Karo. Stir until all the sugars dissolve completely. There should be no sediment remaining when you stop stirring.
Now you need to add the detergent/Jet Dry. You want about 1/12-1/16 of a drop. A *VERY* tiny amount is all you need. The correct amount can be acquired by putting a drop on a plate and then touching the drop with the tip of a toothpick. After a few seconds, stir the toothpick in the hot sugar water. This will add the “proper” amount of detergent. DON’T USE ANY FORM OF ANTIBACTERIAL/ANTIFUNGICIDAL DETERGENT. Detergents with emollients and perfumes should also be avoided.
Now you have 500mL of hot “soapy” sugar water. Pour about 250mL of this solution into each pint jar. An LC jar should only be ½ to 1/3 full. Do NOT fill an LC jar completely. You need plenty of air in that sealed container for healthy mycelia growth. Add the marbles at this point. I prefer marbles because I’m a clumsy putz. If you feel safe working with shards of broken glass, they WILL work a little better. You do not absolutely have to use marbles or glass, they just make things a little easier.
Now PC your LC jars for 15 minutes at 5psi. Since these jars have no tyvek vent, the band must not be screwed on tight. Just barely finger tight is perfect. Let the jars cool inside the PC. As SOON as the PC is cool enough to open, tighten the bands. Wait 24hrs before using these jars. If you can’t wait that long, wait until they are cool enough to hold against your face (comfortably) for more than 30 seconds. A hot LC will kill your spores.
Your LC jars may have a little bit of sandy particulate stuff in them after the PC run. This is from the LME. Don’t worry about it unless there is a whole lot of it. If there is a bunch of sandy stuff in the jar, it has probably “caramelized.” Badly caramelized jars won’t work very well and should be discarded. Remember, a small amount of sandy stuff is OK.
Once the jar cools, it is ready for use. Shake it violently for 5 minutes until it is nice and frothy on the inside. This puts oxygen back in the water. The PC run will drive most of the oxygen out of the water. Mycelia needs oxygen to grow. This is why you shake and also why you leave the air space in the LC jar. If you leave air inside the jar, you don’t need to worry about any kind of fresh air vent.
Your LC jar will have a vacuum in it after pressure cooking. You need to release that vacuum before injecting spores. Simply inject the LC jar with a flamed airport syringe to release the vacuum BEFORE injecting spores. A vacuum jar will suck a spore syringe dry almost instantly. After inserting the airport syringe, wait 10 seconds to allow the vacuum to be fully released.
Shake your spore syringe very thoroughly before each use. Flame the needle red hot, let it cool and then inject 2 mL of spore water into the LC jar. You can cool the needle quickly by dribbling a tiny bit of spore water out of the syringe.
Recap the spore syringe immediately after use and put it into the refrigerator (not freezer – spores shouldn’t be frozen).
Shake the jar for another 30 seconds after injecting the spores. Injecting spores is known as inoculating the liquid culture.
Mark the date and strain on tape on the top of the LC jar and put it in the incubator. Once or twice a day you want to take the LC out of the incubator and swirl it for 20-30 seconds. The more often you swirl, the faster it will grow. Don’t shake violently every day or growth will be retarded.
You should see growth in the jar within 3-7 days. The first thing you see will be tiny little clouds of mycelia. The jar will be finished in 10-20 days. It doesn’t have to get thick with mycelium, just cloudy. The thicker the jar is, the more likely it is to clog your needle. Don’t let the jar get thick.
Finished liquid culture jar.
A finished LC jar should be put in the fridge until it is to be used. An LC in the fridge should stay “hot” for 3 months or so and valid for 5 to 7 months. Once an LC hits 9+ months, it should be evaluated for viability and/or moved to a different nutrient source.

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