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Well geez - absolute LC failure? And dry grain jars :(


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#1 SelfTransformingMachineElf

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Posted 21 April 2021 - 10:15 AM

Once upon a time, I had *really* good luck with LC - I think I was using light corn syrup then.

 

I got my ratio right (half jars w/ LME, half jars with honey), but after 8 days, there's not a bit of growth!  Same spore syringes have all germinated on other media - some PF, some rye grain, some agar.  My temperature was really low - I bumped it up a few days ago.  The only other thing I can think of is that I'm using 2 SHIPs and no air - although I believe there should be plenty in the jar to get the LC started.

 

The only LC that DID work is from another LC (purchased).  The growth in the LC looks good, but there's also a cottony white something growing on the top.  I suppose it's a contaminant - maybe I can get some pictures.

 

I put a heater and humidifier in the room, but the humidifier quickly ran out of water, and the heater got the room significantly too hot.  So I'm a tad worried about my grain jars.

 

I have material enough to rebuild, I think.  But I sure do like the ease of LC.  I guess I'll just try some of my agar into LC as a back-up, and then if I get nice sectoring in a few agar transfers I'll try another LC.


Edited by SelfTransformingMachineElf, 21 April 2021 - 10:16 AM.


#2 Auhron

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Posted 21 April 2021 - 11:00 AM

Cottony white growth on the top of your LC could be your target organism, my B+ and Oyster LCs do that readily if I let them sit without stirring for a while. Did you remember to stir the hell out of the LC after sterilization? PCing a liquid drives dissolved gases from it, which could lead to your mycelium growing on the top of your broth (where it can breathe).

It's not recommended to start spores in a LC as it is difficult to determine if you have a contam present until you move out of the LC onto another media (grain for example), I have rolled those dice before with limited success but after a few failures I no longer think it's worth the risk and have ceased that practice. sometimes spores take their time producing visible mycelium, maybe wait a bit more?

How long did you PC your broth for? Too much caramelization of the sugars can be a deterrent for mycelium growth, as can too much sugar (recommended not to exceed 4% sugar content). What's your LC recipe? 

 

I too like the ease of using liquid cultures, once you have a clean LC it makes grain inoculation really quick. I look forward to your success!


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#3 Oldpunk

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Posted 21 April 2021 - 01:31 PM

With my very limited experience I'm really digging the LC route. I just shook mine up and the ones from spore print seemed to float on top more than the one from clone material which clung to the tissue in the middle of the jar.

The grain jar (my first) that I used spore print LC in (also my first) is almost half colonized after a week. I love this stuff.

I'm using light corn syrup.
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#4 SelfTransformingMachineElf

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Posted 21 April 2021 - 06:10 PM

Auhron - King Stropharia is what's growing the white on top.  It looked pretty healthy to me, so maybe that's what it is!

 

My LC recipe was 2 tsp (either LME or honey) and 250 mL water, PCed @ 15 psi for 30 minutes.  I suppose that's nearer to 5% than 4% - I'm an excellent calculator, so not sure how I did funny math there.  I'll try scant tsps next time.   I shook vigorously after the PC run

 

I'm definitely going to wait it out - who knows!  Once I get nice sectoring, if I run out of my remaining pints jars, I may dump them then.  That's probably a month - at least.

 

Thanks Oldpunk - it is fun watching the spawn jars just absolutely explode in days, isn't it?


Edited by SelfTransformingMachineElf, 21 April 2021 - 06:11 PM.

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#5 coorsmikey

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Posted 21 April 2021 - 06:17 PM

You ever see a moldy candy bar? Me neither, thats because after the 4% mark the sugars start to work like preservatives. Try a side by side and see what performs better, 2% or 4% and you will see that even 4% is a bit high but still possible to grow a culture. IME 4% is the upper limits  and even though it can work it should not be a standard target. Personally I would rather 0.5 % over 4% any day cuz it just works "Better" Than the OMC 4% standards.


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#6 SelfTransformingMachineElf

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Posted 21 April 2021 - 06:20 PM

coorsmikey - I have indeed seen a moldy candybar!  But your point is taken.  I'll back off the sugar ratio moving forward.  Thanks.


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#7 Auhron

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Posted 21 April 2021 - 09:03 PM

You ever see a moldy candy bar? Me neither, thats because after the 4% mark the sugars start to work like preservatives. Try a side by side and see what performs better, 2% or 4% and you will see that even 4% is a bit high but still possible to grow a culture. IME 4% is the upper limits  and even though it can work it should not be a standard target. Personally I would rather 0.5 % over 4% any day cuz it just works "Better" Than the OMC 4% standards.

 I like that insight, I haven't had any issues aiming for the 4% mark ( I always add a little splash of extra water after measuring so I'm probably more around 3.5ish with my LC's), but I think with my next round of LC's I'll aim for 2%. Definitely worth trying.


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#8 SelfTransformingMachineElf

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Posted 22 April 2021 - 11:25 AM

I'm getting ready to make some up.  Going to go 3/2 tsp sugar, 250 mL water for nearly 3%.

 

When I have a reliable isolate, I'm going to fiddle with this and try to tune in the number.



#9 SelfTransformingMachineElf

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Posted 30 April 2021 - 07:35 AM

As an update - two of my initial LCs (Mexicana-Jalisco and ATL7) absolutely blew up.  Really nice healthy looking growth.  I'll grow them out to make sure I'm not looking at contaminant and create some new LCs.  The original LC was from MS, so I know I've probably got many different viable strains in there - so next I'll put just a tiny bit of LC into a second set of LCs and test all of those to see which seems to be the best.

 

I used to go three LC layers deep (1: from MS) -> (2: from 1) -> (3: from 2).  Always had really good luck that way.  Obviously an LC can inoculate MANY more LCs, so once I find my target in 2, I can really ramp up the LCs and even store some so I have a nice supply into the future.



#10 sandman

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Posted 30 April 2021 - 08:23 AM

what the fuck is 3/2 tsp? Do you mean 1.5 tsp? 



#11 SelfTransformingMachineElf

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Posted 30 April 2021 - 02:51 PM

They're.... the same.


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#12 sandman

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Posted 30 April 2021 - 07:13 PM

I'm curious, are you French? I've never heard anyone say an amount of tablespoons in such a interesting way. 


Edited by sandman, 30 April 2021 - 07:13 PM.


#13 SelfTransformingMachineElf

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Posted 01 May 2021 - 12:43 PM

No, but I did stay at a Holiday Inn Express last night.  Actually I didn't.  But I'm an engineer.


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#14 Sidestreet

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Posted 01 May 2021 - 03:00 PM

 

are you French?

 

 

No, but I did stay at a Holiday Inn Express last night.

 

 

That's the best exchange I've heard all day.


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#15 Jrotten

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Posted 01 May 2021 - 04:07 PM

I tare my jar. Add my water and and then add my nutrients usually light corn syrup for the clarity. 2% is what I aim for but I’m working that down. I’m curious to see if I cut the nutrition of my inoculant (grain or LC) if I can improve contamination rates. Using weight is easier math.
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#16 Oldpunk

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Posted 01 May 2021 - 06:09 PM

I tare my jar.

Using weight is easier math.


When I tried that I passed the limit on my pocket scale and had to try and transfer everything to a lighter cup.....hell of a mess.
This time I used a disposable Dixie cup.
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