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Offlineghiajake
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Liquid Culture De-Mystified: Ghia Style * 3
    #19541843 - 02/08/14 09:47 PM (9 years, 11 months ago)

De-vilifying an extremely useful and easy tool: by GhiaJake


    Whether you are new or old to the debate between agar and LC (liquid culture), everyone can agree that LC has a bad rep here. Opponents of LC always state that it just isn't worth it and you would be spending your time better just using agar instead. I don't see how so many people can just blatantly dismiss such a valuable tool in mycology, other than people giving up after too many failed attempts.

    Anyone that has ever bought a culture syringe from a sponsor is using LC. If agar was SO much better than LC, then why don't the sponsors all sell wedges instead? There is no better way to grow, and store, mass amounts of usable culture in such a small area as with LC. Period. Yes, agar is great for germinating spores and isolating genetics. But how many dishes would it take to inoculate over 200 jars of grain. One quart jar with only 600mL of LC can do it easily. Also, when storing agar dishes in the fridge the moisture will separate from the agar, leaving pools of water lying on your culture. The LC culture is always hydrated, never get opened to the air (sterile or otherwise), and just continues to grow while being stored.

    I went through a patch of not wanting to mess with agar, since I got reports of contaminated wedges I had traded. Again, to anyone that received a contaminated wedge, I'm sorry. To be honest, I still haven't done any new agar work since, even since I built my flow hood. I decided instead to spend my time mastering the "dreaded" LC. I have to give credit to Claybuddy for showing me that LC is viable and should be looked into more (How else are you going to grow non-sporulating Purple Mystics, right? :wink:). I can't remember the link he gave me to where he learned his LC recipe, and I by no means claim it to be mine, but below I will describe how I now do my LC jars. To start off, here's a few pics of some of the LCs I've made using my method. The first is Shiitake (S75) inoculated with sterile dowel spawn. The second is King Oyster inoculated with a few colonized milo grains. The third is Pearl Oyster (or pseudo Hyp Ulm) noc'd with dowel spawn. The forth is Ps galindoi, noc'd with 5cc of GLC. The fifth is Pan cyan noc'd the same as the ATL#7.



    Now, enough bragging, let's get down to business making some LC! Keep in mind, LC is meant for live culture transfers only!!! Trying to germinate spores in an LC solution will end in failure most of the time.


    There are many ways people attempt to do the whole LC thing. I can say I've tried most of them by now and this is the easiest method, with the best results. There are a few things you are going to need to pick up to do this right. Keep in mind that if you half-ass things you will only ever get half-ass results. Spending the extra time to do things right will be well worth it in the end.

    Here's what you will need to make your "no-tilt" jar lids:



Plastic jar lids
Self-healing inoculation ports
"Whatman-style" syringe filters (PVDF or PTFE, 0.2 micron)
Rubber grommets (tattoo gun grommets work fine, but I like the thicker ones)
1/4" stainless tubing
1/2" drill bit
1/4" drill bit (3/8" if you use the thicker grommets)
High-temp silicone (optional, not really needed as the tubing seals the noc port)

Step 1:

:gethigh:

Step 2:


  Start off by drilling two holes (carefully) in each lid with your two drill bit sizes. Clean the edges of the holes with a knife, or whatever.

Step 3:


    Insert the noc port into the 1/2" hole, it should snap into the retention slot on the noc port. The grommet fits in the 1/4" hole. You can see the difference between the two grommets in the above picture.

Step 4:


    Carefully insert the filter syringe through the grommet. If you use the tattoo gun grommets, you will want to watch that you don't push the grommet through the hole. Make sure the filter and grommet are properly seated to the lid.

    You can use your high-temp silicone for added protection at this time, but it isn't really needed when making your lids this way.

Step 5:


    It is important to get stainless steal tubing for your "downspout". You don't want rust in your LC! For quart sized jars cut your length to 6" from base to tip, 4" for pint jars. Cut one end square, and the other at a drastic angle. This allows for a much larger area of myc to be drawn into the tubing. I also sharpen the edges of the tips to help with cutting up the mycelium when swirling (see section on swirling) Smooth the edge on the other end, and slip it into the bottom of noc port. It will fit quite snugly, and will keep the noc port from pulling back out of the lid (see above pic)

    Make sure to turn the tip of your downspout into the direction of your swirl. You want the sharpened tip to cut the oncoming mycelium when it is spinning in the jar

Step 6:


    Now that you have your lids made, you're almost ready to start on the LC itself. If you don't have a magnetic stirrer, just toss in a couple clean stones into each of your jars.

Step 7:

:gethigh:

BTW, from here on I'll have to update later with photos. I have a batch to make in the next couple of days, so I'll update the OP once I get all that done. For now, we continue on...

Step 8:

    Now that all your jars are ready, it's time to mix up your liquid. This is one of those widely debated subjects, which nutrient base to use in your LC. I have tried many different ways: honey and/or karo, potato water, grain soak water, etc. The best results I've found are from just extra light malt extract. It's cheap, has great results, and you barely need any for it to work. I use 0.6g of ME(got it by mistake) for every 100mL of DISTILLED water. Don't use tap water folks! Miss a beer and go buy a gallon of distilled water for Jebus' sake! Or use water collected in a house dehumidifier like I do. :wink:

    What I usually do is pour all the water into a big pot and bring it to a boil. Shut off the stove, then stir in your malt extract. Make sure it all dissolves. I then pour the brew through a double-layered clean t-shirt to filter out the particulates. Do this until your liquid is clear. Now fill up your jars and get the PC ready. Double check that you did both Step 5 and Step 6 before closing your lids, since Step 7 sometimes makes you forget things.

Step 9:

    Pressure cook your LC jars at 15-18 PSI for 30 minutes. There is no need for aluminum foil over the lids. Make sure you don't quickly release the pressure at any point or your jars will expel your LC liquid due to the pressure equalization. Just let them cool down in the PC until the next morning. Your jar temps need to be below 80F before inoculating anyways. If you correctly filtered the liquid before pressure cooking, you shouldn't have any particulates in your jars after PC'ing. If you have some, don't worry. The mycelium should eat it. You may notice a color change if you use ME instead of ELME, but again don't worry. It's fine.

Step 10:

    Now we're onto the meat of the issue. Inoculation. The whole agar thing and I can't see eye-to-eye most of the time (fucking agar), so I do my LC a little different than most. If you have no problems with agar then just noc up your LC with it if you like. But this way you aren't "touching" the culture in any way, so you are eliminating that chance of contamination

All inoculation methods should be done in a SAB, or in front of a laminar hood, and all sterile procedures should be followed!!!


    1. LC or GLC transfer: If you already have an LC syringe, or just made some GLC from a clean jar of colonized grain, all it takes is 2-3cc of solution to get your jars going. Make sure you have clean culture first!

    2. Grain transfer: The simplest way to inoculate your LC is to sprinkle in a couple colonized grains from a clean, fully colonized master grain jar.

    3. Dowel spawn: I had made up a bunch of dowels of various species. Having never opened the sterile jars, I decided to inoculate a couple LC jars with colonized dowel plugs. The mycelium grew in a dense cluster off of the dowels and required vigorous swirling to break up the mass. This is only a viable option if you have sterilized dowels colonized, do not attempt with pasteurized dowels.

Step 11:

    Sit back and watch you mycelium grow! Let it get a good foot-hold in the jar before you start swirling. When you swirl try to direct the mycelium across the tip of your downspout to slice it on the sharpened edges. Do this every couple of days until the myc has grow out enough to store the jars in the fridge.

    Keep an eye out for cloudiness of the liquid, or discolored spots growing on the sides of the jar or surface of the liquid. These are all signs of contaminated culture. Even without these signs, you should run a few test jars every now-and-then to make sure your culture is still clean. I usually G2G those clean test jars for a run of spawnings.


    Well folks, that's about it! Hope this helps to dispel the bad rep LC has gotten. Please try for yourselves and post your results here.


Edited by ghiajake (10/04/14 01:22 AM)


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InvisibleEastBayRay
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Re: Liquid Culture De-Mystified: Ghia Style [Re: ghiajake]
    #19541877 - 02/08/14 09:57 PM (9 years, 11 months ago)

Is your other handle "Morelman"?


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Offlineghiajake
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Re: Liquid Culture De-Mystified: Ghia Style [Re: EastBayRay]
    #19541913 - 02/08/14 10:09 PM (9 years, 11 months ago)

Nope. No other handle.


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Offlineghiajake
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Re: Liquid Culture De-Mystified: Ghia Style [Re: ghiajake]
    #19541982 - 02/08/14 10:30 PM (9 years, 11 months ago)

Note to mods: If this thread would be better suited in the "Mushroom Cultivation" forum, or any other forum, please feel free to move it.


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InvisibleLustyLocks
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Re: Liquid Culture De-Mystified: Ghia Style [Re: ghiajake]
    #19542092 - 02/08/14 10:58 PM (9 years, 11 months ago)

Wow. Very nice write up. Well done sir! :raisemyglass:

To be honest. I cannot remember where I got my recipe. I looked at many to get ideas and ended up doing the same as you.

One thing I would like to add. When it comes time to add the ME, instead of just putting it in the pot I get two coffee filters and put my ME in there and then tie it off with a rubber band. That eliminates the whole pouring through a filter. You just have to wait for the ME to dissolve in the filter and then pour into jars. I've never had any sediment in my jars by doing it this way. I am pretty certain that I found that idea from monstermitch.
:2cents:


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OfflineSaint
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Re: Liquid Culture De-Mystified: Ghia Style [Re: ghiajake]
    #19542106 - 02/08/14 11:02 PM (9 years, 11 months ago)

like your write up on making your LC I need to ask you about cloudiness of  a  grain LC does it need to be clear also. Before doing the LC I have a photo  This is after grow in grain. 500ml of water use to pull. Was thinking I could expand with kero? In a new jar or inject sterile water/kero? not sure if i am on right track.


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Offlineghiajake
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Re: Liquid Culture De-Mystified: Ghia Style [Re: Saint]
    #19542142 - 02/08/14 11:12 PM (9 years, 11 months ago)

I was doing it like that Clay, like you suggested before, but I was still getting the sediment. Weird, probably the filters I used.

Quote:

Saint said:
like your write up on making your LC I need to ask you about cloudiness of  a  grain LC does it need to be clear also. Before doing the LC I have a photo  This is after grow in grain. 500ml of water use to pull. Was thinking I could expand with kero? In a new jar or inject sterile water/kero? not sure if i am on right track.




GLC will usually be cloudy, but I don't know if I'd use what you have there. I suggest inoculating your LC straight from the syringe when you do your pull. Otherwise you're badly risking contamination. And you only wanna use a couple cc of GLC, more doesn't mean better. Your LC will be too cloudy to see through if you use anymore GLC than that.

Also, as I stated in the OP, don't even bother with karo or honey. It sucks, no matter what you've read. Trust me!


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OfflineSaint
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Re: Liquid Culture De-Mystified: Ghia Style [Re: ghiajake]
    #19542174 - 02/08/14 11:23 PM (9 years, 11 months ago)

Quote:

ghiajake said:

GLC will usually be cloudy, but I don't know if I'd use what you have there. I suggest inoculating your LC straight from the syringe when you do your pull. Otherwise you're badly risking contamination. And you only wanna use a couple cc of GLC, more doesn't mean better. Your LC will be too cloudy to see through if you use anymore GLC than that.

Also, as I stated in the OP, don't even bother with karo or honey. It sucks, no matter what you've read. Trust me!



Thanks for leting me know about the grain looking cloudy i thought it would. Some one said not. I'll read some more to find what works the best for the shittake GLC  I do like  your write up here looks great.


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Offlineghiajake
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Re: Liquid Culture De-Mystified: Ghia Style [Re: Saint]
    #19542199 - 02/08/14 11:32 PM (9 years, 11 months ago)

Just use the malt extract, it works for every thing. The first pic at the top is Shiitake LC made my way.


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OfflineMrcloudy
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Re: Liquid Culture De-Mystified: Ghia Style [Re: ghiajake]
    #19542283 - 02/09/14 12:29 AM (9 years, 11 months ago)

I don't understand the bad rep that liquid culture has gotten. I mean if your starting from spore, then yes it is a terrible idea. And I do understand that generally speaking you can't tell at a glance whether it is contaminated or not. But I have made several liquid cultures with several different species and have noticed that each one has a distinctive look and behaviour, and not all mycelium in LC looks the same. However if you spin it up every day it will mix it up and it will be a homogeneous blob which if you have some bad things in there they might be hard to distinguish.

I think it is similar to grain really. If you see anything that looks off, toss it. So far I have had no issues with properly made and inoculated LCs I have seen some spore LCs do some pretty disturbing things. If you start from spore, you're going to have a bad time. Inoculating with living mycelium is the way to go, I think a lot of people have issues because they start from spore. Not saying it is perfect as any lapse in sterile procedure and something could still get in but I did all my work in front of a impromptu "flowhood" and had no issues.

You should however always test your LCs on agar or a single jar before using it on a larger scale, just in case there are problems. I think a properly done LC can be a very useful tool in this hobby.


Excelent write up BTW! :thumbup:


--------------------

10 different Ganoderma species from across the USA

AMU

MrCloudys guide to North American GanodermaUpdated A rough guide to North American Ganoderma species, with an emphasis on the laccate species.


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Offlineghiajake
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Re: Liquid Culture De-Mystified: Ghia Style [Re: Mrcloudy]
    #19542382 - 02/09/14 01:48 AM (9 years, 11 months ago)

Good to see another fan of LC. :wink:

I give each LC jar a test once a month at least. It is these test jars that I G2G from to expand my grain spawn


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OfflineForresterM
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Re: Liquid Culture De-Mystified: Ghia Style [Re: ghiajake]
    #19542548 - 02/09/14 04:03 AM (9 years, 11 months ago)

I fucking love it, thanks for posting!


--------------------
Repugnant is a creature who would squander the ability to lift an eye to heaven, conscious of his fleeting time here.
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Have some medicinal mushrooms and want to get the most out of them?  Try this double extraction method.


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Offlineghiajake
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Re: Liquid Culture De-Mystified: Ghia Style [Re: Forrester]
    #19543116 - 02/09/14 10:01 AM (9 years, 11 months ago)

No prob Forrester, thanks. :smile:


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Re: Liquid Culture De-Mystified: Ghia Style [Re: ghiajake]
    #19543393 - 02/09/14 11:07 AM (9 years, 11 months ago)

This is a cube i started 3 days ago in a kero jar. It started out with the spore looking like black specks on the very bottom. Should of took a pic then. last night. This morning they formed in to this. I am going to try some of your incite to doing LC. I just like the grain LC and going to keep testing. Wish I could find more info on grain lc.


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Re: Liquid Culture De-Mystified: Ghia Style [Re: Saint]
    #19543420 - 02/09/14 11:12 AM (9 years, 11 months ago)

Looks like good myc, but if you started from spores don't get your hopes up too soon.

What do you wanna know about GLC? There are some good teks on the subject floating around, but I can answer what you wanna know.


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Offlinecubenpete
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Re: Liquid Culture De-Mystified: Ghia Style [Re: ghiajake]
    #19554116 - 02/11/14 07:29 PM (9 years, 11 months ago)

Very nice write-up, I like the straining suggestion, not sure why I havent done that before but I will now.


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OfflineRogerRabbitM
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Re: Liquid Culture De-Mystified: Ghia Style (moved) [Re: ghiajake]
    #19554257 - 02/11/14 08:02 PM (9 years, 11 months ago)

This thread was moved from Advanced Mycology.

Reason:
Quote:

ghiajake said:
Anyone that has ever bought a culture syringe from a sponsor is using LC. If agar was SO much better than LC, then why don't the sponsors all sell wedges instead?




Because they're selling to noobs without a flowhood or skills to use agar.

Not an advanced mycology topic.  Moved to cultivation.
RR


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Offlineghiajake
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Re: Liquid Culture De-Mystified: Ghia Style (moved) [Re: RogerRabbit]
    #19554307 - 02/11/14 08:14 PM (9 years, 11 months ago)

Quote:

Because they're selling to noobs without a flowhood or skills to use agar.




That may true, but it doesn't invalidate its usefulness. Merely casting LC aside once you increase you skills and equipment is a waste of a perfectly good tool.

Thanks for moving it to the appropriate forum RR.


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OfflineRogerRabbitM
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Re: Liquid Culture De-Mystified: Ghia Style (moved) [Re: ghiajake]
    #19554319 - 02/11/14 08:19 PM (9 years, 11 months ago)

Also, the reason new growers have trouble with water separating from the agar in petri dishes is due to improper storage.  You're supposed to keep the petri dishes in an insulated cooler in the refrigerator to prevent the temperature swings from reaching the dishes.  The insulated cooler does just that.
RR


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Offlineghiajake
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Re: Liquid Culture De-Mystified: Ghia Style (moved) [Re: RogerRabbit]
    #19554816 - 02/11/14 10:15 PM (9 years, 11 months ago)

Thanks for that bit of info RR. I'll be sure to do that on the next batch to go in the fridge. I use a dorm fridge for my dishes. Any thing I could use for that, or do I need to get bigger fridge?


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