Liquid Culture – How and Why…

[BuckarooBanzai]

Firstly, the abbreviation for Liquid Culture is LC. When you see somebody talking about shooting up their jars/bags with a/an LC, this is what they are talking about.

Second, why am I doing a primer on stuff that has already been covered ad nauseum by members far more qualified than myself? Answer: I’m home sick and this is distracting me nicely from the shooting pains in my abdomen.

Thirdly, why screw around with LCs at all? This answer is a multi-parter:

1. Using LCs will make your spore syringes go a lot farther. One good 10mL syringe, used only to inoculate LCs, can easily shoot up two to five THOUSAND jars. If you really want to pinch your pennies, the last of your LC can inoculate the next jar of LC without ever taking the spore syringe out of cold storage, and so on. Be aware, cultures start slowing down at the 2-5 month point, depending on the strain. LCs are NOT a “golden bullet” for avoiding germinating spores.
2. LC inoculations grow faster. Since you are shooting up with live mycelium, you don’t have to wait for germination. This gets you that much further ahead of any “dormant” contaminants and can shave 1-7 days off your incubation time. 1-2 days for ½ pints, 5-7 days for bags/half gallon jars.
3. LCs will allow you to expand mycelium far faster than agar because growth occurs in three dimensions instead of just two.
4. Mastering LCs is the easiest/best way to do clones successfully.

Well, you are asking, what are the down sides?
1. It is all but impossible to tell, from appearance alone, if a/an LC is contaminated.
2. If you are counting total time to fruit, using an LC actually increases your time frame by 12-16 days.
3. If you are not careful, your LC “master” can become contaminated and subsequently contaminate every jar you shoot with it.
4. You’ll need an extra jar to do the LC in.
5. You’ll need an extra syringe/needle to create an airport.

Okay, the first thing to know is that there are MANY successful recipes for LC broth. Please see:

Mycotopia LC vault: http://mycotopia.net/discus/messages...tml?1108528324

That said, this is my recipe for making two LC pint jars. Please note, each jar only gets 250mL of broth (leaving plenty of air space):

1. 500mL DISTILLED water (your tap water might work, but distilled from the grocery store is far most dependable). I have read many, many horror stories of people failing to make successful LCs with tap water. Tap will work, but your chances for failure are MUCH higher.
2. 1 Teaspoon of Karo Syrup.
3. OPTIONAL: pinch (less than .1mg) of bee pollen
4. OPTIONAL: *TINY* amount of dish washing detergent (no scent and no emollients, use an organic brand if you can find it)
5. 4 mg of Light Malt Extract (from here on, LME; I use the Sporeworks brand)
6. 4 mg of Dextrose (I use the Sporeworks brand)
7. 6 clear glass marbles OR a small pile of broken glass.
8. 2 pint jars with ported lids and bands.
9. 1 pressure cooker capable of containing your jars and maintaining 10psi. That’s not a misprint. LC jars should be done at 10 (not 15) psi.
10. 25 minutes in the PC at 10psi.

Please note, other members (Freakachino comes to mind) have had great success with the microwave method (see link above). Personally, I’m a lazy bastard with a filthy microwave that I don’t intend to clean, so I’ve never tried that method. I have, however, heard VERY good things about said TEK. If it’s good enough for Hippie3, it should be good enough for you.

I’ve never tried it personally, but I am told that sucrose (table sugar) will also work. ANY sugar (in the right proportion) should work. Don’t lock yourself into these ingredients, just these percentages. Honey works. Pure dextrose works. I’m pretty certain glucose would work. If you get the percentages right ANY sugar should work. BE AWARE: a good LC will taste just barely sweet by human standards. If your “sugar water” tastes sweet, it’s probably too strong. Too weak and growth is stymied. Too strong and growth is prevented. It’s a percentage thing.

All that said, we will start with 500mL of distilled water heated in the microwave. Hot water dissolves sugars MUCH better. Be smart about it, however. Don’t microwave in Pyrex and, if you do, put a wooden chopstick in beforehand. Superheated water from the microwave sends many people to the hospital every year. Hot liquid burns SUCK. They take forever to heal and tend to scar badly. The most painful burn you will ever receive is from steam (trust me, I know). Forewarned, don’cha know…

Water superheating in a microwave (and the results):
http://forums.mycotopia.net/attachme...8&d=1137178542

Okay, so you’ve got your 500mL of hot distilled water. Now you add your LME, dextrose and Karo syrup.

YES, Karo is basically just dextrose with some additives. One of those additives, however, is Vanilla. Our friend Lazlo has already demonstrated the inherent value of that additive in an LC. Using Karo is NOT gospel, however, just what I’ve decided upon.

Can’t find dextrose? Use 8g of LME. Can’t find LME or dextrose? Use 5 teaspoons of Karo. Can’t find LME, dextrose or Karo syrup? Get thy ass on-line or to a local homebrew shop and purchase some (you want LIGHT malt extract, by the way)…you have got to give to receive in this hobby. I recommend Sporeworks.com as a primary source. They have everything you will need and then some, spores included. They even take PayPal. If you dig my technique, mention my name in your order. You won’t get any kind of discount…but, if enough of you order, I might!

DON’T use antibiotic additives in LCs. IME, they simply don’t work unless you put in a boat-load. Antibiotic cultures also tend to be “weaker” than their counterparts. Antibiotic additives are for agar clean up work, not LCs. This is an opinion.

Peptone does work but, IMHO, isn’t worth the extra expenditure. Your mileage may vary. This is also only an opinion.

So, take the 500mL of very hot distilled water and add your LME, dextrose and Karo. Stir until no particulates are visible. If you still have particles after 30-40 seconds of stirring, filter through a coffee filter. You want clear water for your LC so you can identify growth easily.

If you are using detergent (highly recommended) use about 1/10-1/16 of a drop at a maximum. The correct amount can be achieved by putting a drop of detergent on a plate and then touching the drop with the tip of a toothpick. After a few seconds, stir the toothpick in your LC. This will add the “proper” amount of detergent. DON’T USE ANY FORM OF ANTIBACTERIAL/ANTIFUNGICIDAL DETERGENT.

So, now you’ve got 500mL of hot “soapy” water with sugar added. Pour about 250mL of solution into each pint jar. DON’T forget to add the marbles/broken glass 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 better.

Now, the hardest part: PC your jars for 25 minutes at 10psi. As SOON as they are cooled enough to open the PC, tighten the lids. Wait at least 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.

Why is this part hardest? Well, sugars have a tendency to “carmelize” and drop out of solution. The chemistry is way complicated, but it boils down to this: if you see clouds of stuff in your LC after cooking it isn’t going to grow good mycelium. If your LC comes out of the PC looking almost the same as when it went in, you are in good shape.

DON’T USE CARMELIZED JARS. You will get growth but you will also drastically increase your chance of getting mutations. If it comes out looking weird, start again. It’s just sugar water, after all…

The only additional consideration is inoculation. A good LC jar will do fine with only .5-1mL of spore water. To be certain, I use 2mL of spore water, but far less will work. All you really need is 2 compatible spores out of the billions that are in your syringe.

Once knocked up, your LC should be shaken VIOLENTLY until it is nice and frothy. Remember that PCing will remove most of the oxygen in the water and your mycelium needs oxygen to grow. Now, put the jar in the incubator. A “proper” LC jar will have 1-3 inches of “air space” above the water. The jar should be swirled (gently) at least once a day. If you can swirl 8-10 times per day, growth will be sped up even more. Bluehelix has demonstrated that under constant mechanical stirring, the jar can be done in as little as 3-4 days. At about %10-%20 colonization and again at %70-%80, I will shake the jar violently (the detergent additive will make a nice foam on top). This aerates the water and breaks up the growing mycelium.

Once your new LC jar is cloudy with mycelium, suck it up into a syringe and inoculate the substrate of your choice (use an airport to equalize pressure). I would suggest doing one test jar to check for contamination. Nothing sucks worse than shooting 24 jars with a contaminated LC. Once you are done, put the LC jar in the ‘fridge (just above freezing). I’ve gotten good results from 2 month old LC that was stored at around 40F.

[~Hippie3]

shampoo or jet-dri for dish soap
i don't like to pressure cook LCs.
the extra temperature makes carmelization more likely
and it's just not needed
since it's just water and sugars there, not grains
hence no endospores to kill therefore no pc needed.
simple boiling point of 212*F/100*C is enough.

I also don't pressure cook my LC's.
Distilled water keeps carmelization minimal, but doesn't give as good of growth as spring water or tap water.


Great write up Buckaroo!

[BuckarooBanzai]

I also prefer tap water, but I realize that everybody's tap isn't going to be on par with mine. Also, everybody out there isn't going to know the precise PH and EC levels coming out of their taps. I just *happen* to know because I use my tap water for growing *other* things...I think distilled will give folks the best chance of getting reproducable results. Also, I knew fine folks like you two would chime in with other opinions! Personally, I think the best TEK is the one you "hybridize" out of other's ideas to fit your needs and resources.

Freaky, do you use bottled spring water or are you lucky enough to have a well of your own?

Hippie, I never even considered just plain boiling! D'OH! I bet I could get away with just "steaming" the jars with lids almost closed. Experiments, I believe, are in order...

[xxanxx]

So, put 1-2" of water in a pot, set your LC jars in, cover, and boil? For how long??

Buckaroo, yes, I have my own well. My tap water germinates the spores most times too. I use it for all myco work, except when I make spore syringes to mail to friends.

I also think the best results come out of trial and error of mixing different teks. Finding what works best for personal environment, situation, equipment, etc.

[agentstone]

Originally Posted by Hippie3

shampoo or jet-dri for dish soap
i don't like to pressure cook LCs.
the extra temperature makes carmelization more likely
and it's just not needed
since it's just water and sugars there, not grains
hence no endospores to kill therefore no pc needed.
simple boiling point of 212*F/100*C is enough.

I missed the purpose of the soap somewhere. Whats it for again?

Also when you nuke your LC, do you just wait till it boils for a minute then its done or for longer than that?

thanks in advance.

-Stone

[blackout]

Great write up. Just a few points.

Originally Posted by BuckarooBanzai

2. If you are counting total time to fruit, using an LC actually increases your time frame by 12-16 days.

Can potentially increase, but can reduce. I got spores and started an LC, after 5 days it was colonised (I did wait longer due to no jars being ready but no more growth was apparent) I injected a lot of LC into grains that were calculatedly below their optimal moisture content. This resulted in the jars being well on their way after 3 days. The jars in the pic are 3 days old.
No shaking was needed at all, this also allows you to put more grains in the jars since "shake space" is not needed. So after 8 days my jars could have been looking like that. I find spores germinate faster in an LC, and not having to shake by using lots can actually increase the time to fruit.
I say CAN, but the way many people use it is does slow things down.

Originally Posted by BuckarooBanzai

5. You’ll need an extra syringe/needle to create an airport.

The lid can be loosened and put in a glove box, or spray the air well with disinfectant. There is a physical barrier to airborne spores in the fact that they have to go under up and over the cap and lip of the jar.

[BuckarooBanzai]

The dish soap/jet dry significantly lowers the surface tension of the water, allowing for much smaller air bubbles when you do your violent shakes. Also, you can tell for certain when you have shaken enough by looking at the nice layer of foam that forms on top of the jar.

Blackout - I agree that sometimes an LC is actually faster than straight spores. But other times it isn't. My FOAF wants to give people the most wide open time frames possible. If a newbie gets full colonization 5 days before they expected it, it's much beter (IMHO), than if they still don't have it 5 days after they were expecting it.

The primary purpose of the airport is to safely vent the vacuum which forms in the PC. If you open them without venting, even in front of a laminar hood, they will suck in a bunch of air (and any contaminants present). It doesn't take but 1 bad spore to wreck an LC. That sugar water will feed just about anything.

[negativezer0]

Excellent write-up BuckarooBanzai!

This document cleared up a few minor uncertainties I had about liquid culture; it was a big help.

I figure this document will be included in mycotopia's new vaults section; But, I noticed one minor detail that you might have not intended to leave out:

The purpose, details and the construction of the "air port"

[BuckarooBanzai]

Here is fearless leader's airport thread:

http://forums.mycotopia.net/showthread.php?t=5245 (airport re-deux)

The airport (and it's purpose) are two fold.

1. The silicone injector site gives you the ability to get a needle into a jar with no fear of any outside air leaking in, as they are self healing. Since silicone is viable to temperatures far above what the PC can reach, you can cook them many times over without them deteriorating in the slightest.

2. The poly filled syringe body gives you the ability to easily vent off a jar's vacuum with filtered air (again, no risk of outside air contamination). Using the airport syringe, you can cook jars sealed and not have the vacuum suck your syringe dry when you inject. The poly filled syringe also lets you suck all the LC out of the jar by equalizing pressure outside/insided.

I'm glad my write up helps! If you have any other questions, don't hesitate to ask. The "old pros" around here are all well versed in the use of liquid cultures and will be happy to answer any question I can't.

[BuckarooBanzai]

Forgot to mention, silicone injector sites work very nicely with spawn bags, as well...

[Mycolangelo]

You've insired me to give LC a try.
Made up a couple with just Karo, water and marbles. Put in a piece of agar with Ps. Cyan 3 days ago and there are lots of floating bits now.

Would it be best to innoculate PF, grain, WBS or hard wood saw dust?

[BuckarooBanzai]

Sorry, dude, but I've got no experience with the Cyans. I would go a questing for some work by WayLitJim. He seems to be the undisputed master of Cyans...