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InvisibleStipe-n CapMDiscord
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How I make crystal clear LC * 16
    #28104670 - 12/19/22 10:46 AM (1 year, 1 month ago)

Liquid Culture






Liquid culture broth is much like agar, just without the agar :lol:

Noob disclaimer:

When first starting out I advise you resist the urge to buy or use liquid culture for the following reasons:

Don't use LC until you have all of the lead up techniques including the ability to produce reliably clean spawn nailed down. Stay as far from LC as possible until then, otherwise you'll be chasing variables and failures forever.

Each skill builds upon the next.

1. Spores to agar to produce culture; cleaning to produce pure culture, making transfers, and agar to grain.

This step will familiarize you with the fungal life cycle and introduce you to the basics of identification for both contamination and healthy mycelium. During this phase you'll learn how to reliably produce clean cultures and sterilize both grains and media.

Once these skills are mastered, move to skillset #2

2. Grain to grain expansion.

This step expands upon the previous production phase by introducing how to expand your pure culture exponentially. This is a more advanced stage dealing with inoculation techniques and aseptic techniques which will prepare you for the next step:

3. Liquid inoculant/liquid culture.

Liquids will not work for you if you haven't learned to identify pure culture, contamination, or prepare sterile media which was learned way back in step one. Liquids won't work for you if you don't understand/master the aseptic techniques required for expansion.

Walk before you run, otherwise there's too many variables to troubleshoot. Each phase carries it's own set of criteria for troubleshooting problems, if you skip them, you're going to be fucked.


Now onto liquid culture:


I prefer to use low nutrient concentration in the broth (.1%-.2%) for maximum clarity, clarity will allow you to easily visually identify bacterial contamination by the presence of turbidity (cloudiness). Turbidity is directly correlated to the severity of contamination by bacteria:



By utilizing a greater nutrient concentration in your broth (greater than .5%) you will end up with discolored broth and sedimentation, both of these will make visual inspection for turbidity difficult if not impossible.

Broth made @ 1% concentration compared to broth @ .1% (some folks are running broth @ 4+% which looks something like swamp water :rofl:)



Broths made with higher concentrations will grow perfectly fine, higher nutrient ratios will not harm your mycelium, higher concentrations simply reduce visibility. Reduced visibility reduces the likelihood of visual identification of bacteria which will require testing on agar, by testing on agar you're now unnecessarily exposing your media to potential contamination via handling. Not a huge deal, just something to consider if you're experiencing failure.

To ensure that your liquid media comes out of the pressure cooker crystal clear there are a few considerations:

1. Distilled h20 versus tap: Tap water often contains high concentrations of dissolved solids, these minerals will undergo a chemical reaction during sterilization which will result in the yellowing/darkening of your broth;

2. Nutrient source: I recommend using light malt extract with a low lovibond value, 2 lovibond is what you're after.



"On the Lovibond scale, a pale golden lager might have a color of 2˚ or 3˚, a pale ale 10˚– 13˚, a brown ale or dark lager 17˚–20˚, all the way through to the near black of imperial stout at 70˚."

The lower the number the greater the clarity. This is what I use for both LC and agar.

Sterilization times are volume dependent, adjust accordingly. I cycle all liquids for 45 mins @ 15psi to ensure all solids evenly dissolve, and as a redundancy measure for sterility:




Sugar as a nutrient source:

Karo:




SITR:



Honey, slightly better than the previous 2:



Although these "nutrient" options "work", take a look and decide for yourself....



https://www.foodbeverageinsider.com/sweeteners/malt-extract-can-do-it-all-clean-label-snacks


The result of that IP broth:



7 day's after inoculation:





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InvisibleShrimps
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Re: How I make crystal clear LC [Re: Stipe-n Cap] * 1
    #28104674 - 12/19/22 10:49 AM (1 year, 1 month ago)

Awesome writeup p9hu7! Thanks alot for all your effort writing this down for us, really appreciate everything you're doing for all of us!

May I ask, have you also tried LC with just Dextrose?


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InvisibleStipe-n CapMDiscord
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Re: How I make crystal clear LC [Re: Shrimps]
    #28104679 - 12/19/22 10:51 AM (1 year, 1 month ago)

No, I prefer LME because for the reasons listed above and because I have it already in hand for agar.


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OfflineDave Bowman
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Re: How I make crystal clear LC [Re: Stipe-n Cap]
    #28104762 - 12/19/22 11:55 AM (1 year, 1 month ago)

Quote:

p9hu7 said:





How did you get your jar to spin but your stir bar to stay stationary?

EDIT: I'm just stoned and realized you probably just turned off the stir bar and let it keep spinning to record :stoned:

Anyways, awesome post and sweet write up, love seeing the clear LC's. :mushroom2:


Edited by Dave Bowman (12/19/22 11:57 AM)


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Invisiblebigfootscreepyuncl
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Re: How I make crystal clear LC [Re: Dave Bowman] * 3
    #28104766 - 12/19/22 12:03 PM (1 year, 1 month ago)

Here we go again, another invaluable thread for the archives :rockon:

My LC always fails me during inoculation so I know it's me, not my LC :lol: a2g is more my speed..


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OfflineShebaNoguera
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Re: How I make crystal clear LC [Re: bigfootscreepyuncl] * 1
    #28104802 - 12/19/22 12:37 PM (1 year, 1 month ago)

I allways do LC with malt extract. It is so much better then everything else. Mycelium grows faster and there is basically no delay when inoculating grain like you would have with Caro or honey e.g.
I guess, the malt extract and grains are a very similar source of food because in the end malt extract is made from grains. So the mycelium doesn't need to switch its metabolism from one kind of carbohydrates to another but immediatly starts growing on the grains.

I like to use higer concentrations because I find the light brown color useful as it gives a bit of contrast against the white mycelium.
With higher concentrations of course you get sedimentation und maybe cloudiness. But this can be solved quite easily by COOKING TWICE:

I put around 300ml of water (tap water will get brownish, destilled water slightly greenish with the malt extract I use) and add a teaspoon of malt extract (ca.3g) to the jar. I do not put a lid on but just cover with aluminium foil to keep additional water out while cooking.
I then pressure cook for around 30-40 minutes. After that the content of the jar is very cloudy and would be useless if used directly.

So I just leave the pressure cooker closed with the jar inside and let it stand over night for about 12 hours. Then I carefully open the pc and take out the jar. All sediment is now at the bottom of the jar. I decant the clear liquid, put it in a fresh jar with self healing injection port and syringe filter. This will then be pressure cooked for another 40 minutes and will not produce any sediment again.


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InvisibleBaba Yaga
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Re: How I make crystal clear LC [Re: ShebaNoguera] * 3
    #28104839 - 12/19/22 01:12 PM (1 year, 1 month ago)

Also using LME at 0.1% and distilled water for a while now and it can't get any better. When you see this 4 days after inoculation then you know what's up.




As mentioned in OP distilled is superior to tap water if you want absolute clarity. The difference in the following photo comes just from using bottled water instead of tap. This was before I switched to distilled and at a LME concentration of 0.5% LME IIRC, it shows the effect it has on the color of the end product quite well.




Our tap is also very hard and running it through the PC will not only cause darkening of the broth but also sediment because of lime precipitating out of the solution. Distilled H2O FTW.


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InvisibleStipe-n CapMDiscord
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Re: How I make crystal clear LC [Re: Dave Bowman]
    #28104841 - 12/19/22 01:14 PM (1 year, 1 month ago)

I turned off the stir plate to allow the broth and myc to swirl :cheers:


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OfflineGreg
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Re: How I make crystal clear LC [Re: Stipe-n Cap]
    #28166091 - 01/31/23 02:49 PM (11 months, 21 days ago)

I switched to ~.15% LME LC vs my usual 2% and the difference is night and day.
Even though I'm using well water instead of the recommended distilled water these things are nearly crystal clear and colorless.

Some tampanensis on day 7 right after a stir plate beatdown:


After settling for a bit, next to a blank from the same batch:


Edited by Greg (01/31/23 03:00 PM)


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InvisibleTucky
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Re: How I make crystal clear LC [Re: Greg]
    #28166174 - 01/31/23 04:01 PM (11 months, 20 days ago)

Check my math, 25mg/.25g LME to 250 ml of water?


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OfflineGreg
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Re: How I make crystal clear LC [Re: Tucky]
    #28166190 - 01/31/23 04:12 PM (11 months, 20 days ago)

Quote:

Drunken Lullabies1 said:
Check my math, 25mg/.25g LME to 250 ml of water?



:thumbup:


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Offlinepinehurst
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Re: How I make crystal clear LC [Re: Greg]
    #28167282 - 02/01/23 09:16 AM (11 months, 20 days ago)

Why not give it preferred amount of nutrients and just test on agar, like you'll probably do anyway? Either, if it's bad you'll throw it out so not like it's a possible increase in waste


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OfflineGreg
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Re: How I make crystal clear LC [Re: pinehurst]
    #28167466 - 02/01/23 11:49 AM (11 months, 20 days ago)

Why not save some LME and have LC broth with less denatured junk floating in it?
The growth is the same with any amount of nutrients in the .1-2% range as far as I can tell.

You're correct that I still test LC sometimes, though I'm doing so less lately.
Point is I wont even waste a petri or a microscope slide on LC broth that's visibly cloudy.
This method makes those turbidity-based sanity checks much easier.


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OfflineHindsight
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Re: How I make crystal clear LC [Re: Greg]
    #28167611 - 02/01/23 01:37 PM (11 months, 20 days ago)

LC has taught me that plates I would have sworn were clean, were actually not. I use LC now for everything - even MS grows where I will only use it once, because it is the only way to know 100% for sure that you have a clean culture. Agar as a first step, LC as a final verification. LME is cheap and LC jars are reusable. Really no waste. Just an extra week of time.

Great write-up. I have been running 0.8% lately and while it's not crystal clear, I can still detect cloudiness. I would like to run 0.2% but the last few times I tried it, I was only getting about 25% colonization of the broth. You guys having similar colonization levels?


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InvisibleBaba Yaga
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Re: How I make crystal clear LC [Re: Hindsight] * 2
    #28167670 - 02/01/23 02:16 PM (11 months, 20 days ago)

This is how I'm using LC as well, got 20 100ml and 20 250ml media bottles and basically use them the same way I use plates. Sometimes only inoculating a couple of jars. Also mostly making 3 LCs from a plates and using only the best looking one and also having extras for long term storage. Almost feel more confident to judge a culture in liquid form then on a plate, at least with pans that is.

I get less colonization volume as well , also around 25% of the total volume for a non shaken culture, but I have no filters or ports and can shake the hell out of it which breaks it down really well and gives the same number of inoculation points. Well, I'm also doing this extra step where I put PF substrate on top of the grain and let that colonize before I shake cause I had problems with slow recovery sometimes but I do it with everything now cause it I like seeing the PF sub getting colonized and once you give t a shake it finishes fast.

LC is my jam and so nice to work with in a SAB instead of cutting wedges. Inoculating 10 jars doesn't take as long as setting up the SAB and cleaning up afterwards. Worth while the extra step for the gained comfort alone.


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InvisibleStipe-n CapMDiscord
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Re: How I make crystal clear LC [Re: pinehurst] * 2
    #28167765 - 02/01/23 03:42 PM (11 months, 19 days ago)

Quote:

pinehurst said:
Why not give it preferred amount of nutrients and just test on agar, like you'll probably do anyway? Either, if it's bad you'll throw it out so not like it's a possible increase in waste




Why open your LC and risk contamination via handling? Also, what's the "preferred" amount of nutrients? The preferred amount is what will result in healthy expansion of the colony, not some noobs over zealous desire to drown mycelium in "nutez".


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Offlinec01h
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Re: How I make crystal clear LC [Re: Stipe-n Cap]
    #28582899 - 12/14/23 05:25 PM (1 month, 13 days ago)

This should be stickied

:takingnotes:


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Invisiblemeta_mmxxii

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Re: How I make crystal clear LC [Re: c01h] * 1
    #28583028 - 12/14/23 06:59 PM (1 month, 13 days ago)

Awesome write up Stipe, I have avoided LC because of all the reasons you listed. But I will find this very helpful when I reach that stage in my journey.
Thanks for taking the time to share this with us.


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InvisibleStipe-n CapMDiscord
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Re: How I make crystal clear LC [Re: meta_mmxxii]
    #28583034 - 12/14/23 07:02 PM (1 month, 13 days ago)

Glad you guys found it helpful.


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OfflineGuyOnaBuffalo
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Re: How I make crystal clear LC [Re: Stipe-n Cap]
    #28583197 - 12/14/23 09:10 PM (1 month, 13 days ago)

Quote:

Stipe-n Cap said:
Glad you guys found it helpful.




Does the lower percentage of nutrient do anything to speed up/slow down/ or leave unchanged, the growth rate of the myc? I'd expect that low nutrients would make the myc reach out farther faster but more likely to run out of energy as well.


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