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OfflineHiLIFE
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Fully colonized grain Spawn to Liquid Culture
    #25389985 - 08/15/18 11:26 AM (5 years, 5 months ago)

So I went from a clone to lc, then used some of the lc to make grain spawn. Can I got from grain spawn and make a new lc? Pretty sure I can just break up the fully colonized grain spawn and fill it with water and then suck it back out and have a new LC right?


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OfflineLtLurker
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Re: Fully colonized grain Spawn to Liquid Culture [Re: HiLIFE]
    #25390000 - 08/15/18 11:31 AM (5 years, 5 months ago)

clean it up on agar first.


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Re: Fully colonized grain Spawn to Liquid Culture [Re: LtLurker]
    #25390012 - 08/15/18 11:36 AM (5 years, 5 months ago)

I mean if it's clean? There'd be the whole thing of having grain in the lc... You could inoculate a petri, make one transfer, and use that for an LI... Why not G2G?


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OfflineMrSturgill
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Re: Fully colonized grain Spawn to Liquid Culture [Re: Caps McGee]
    #25390021 - 08/15/18 11:39 AM (5 years, 5 months ago)

:whathesaid:
G2g would be better, why add extra steps for the same result?


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OfflineLtLurker
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Re: Fully colonized grain Spawn to Liquid Culture [Re: Caps McGee]
    #25390024 - 08/15/18 11:39 AM (5 years, 5 months ago)

Quote:

Caps McGee said:
Why not G2G?



That's a way better response. Why add the extra step(and contam vector) at all unless you're gonna clean it up a bit first?


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OfflineHiLIFE
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Re: Fully colonized grain Spawn to Liquid Culture [Re: LtLurker]
    #25390467 - 08/15/18 03:38 PM (5 years, 5 months ago)

Im not going G2G because I use bags with injection ports and I use LC. I'm not doing agar because its a clone. I either use a clone or a isolate. Not both. Clone vs Isolate is basically the same thing just from different ends but lets stay on topic. The clone performs very well, I dont need to grow it out on agar. I also use LC because I can inoculate each 6 quart bag of rye grain with 20cc's of LC and its done in less than a week. My simple question was could I go from colonized grain to LC!?!?! LOL I didn't pull this idea out of my ass either. Somebody had posted about it a long time ago. Somebody knowledgeable in the mycology field. I guess i'll see if I can find it.


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Re: Fully colonized grain Spawn to Liquid Culture [Re: HiLIFE]
    #25390478 - 08/15/18 03:41 PM (5 years, 5 months ago)

What you're referring to is called a grain liquid culture or GLC.

Search engine should help you from there.


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OfflineLtLurker
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Re: Fully colonized grain Spawn to Liquid Culture [Re: HiLIFE]
    #25390488 - 08/15/18 03:45 PM (5 years, 5 months ago)

What do you think the definitions for clones and isolates are? I think you're confused. You use agar for both of those things.

Clean culture on agar(whether it's ms, clone or isolate) is used to make lc. I think you're just gonna make a mess trying to make an LI like you described.


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Re: Fully colonized grain Spawn to Liquid Culture [Re: filthyknees]
    #25390499 - 08/15/18 03:49 PM (5 years, 5 months ago)

I pour soft, 60mm dishes, and use a full dish cut into sixteen wedges (3 line tic tac toe pattern) for each jar... these could be inoculated with a single grain, stretching your culture to thousands of dishes... and  as stated above,agar can be mixed with sterile water to make LI... Hundred ways to skin a cat


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InvisibleCaps McGee
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Re: Fully colonized grain Spawn to Liquid Culture [Re: Caps McGee]
    #25390505 - 08/15/18 03:52 PM (5 years, 5 months ago)

If you're confident that the grain is clean, drop a spoonful to sterile water,shake,and draw out the resulting fluid with your syringe... Grain Liquid Innoculant, or GLI


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OfflineGeinstein
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Re: Fully colonized grain Spawn to Liquid Culture [Re: Caps McGee] * 1
    #25390517 - 08/15/18 04:00 PM (5 years, 5 months ago)

Do this get your self a blender change things up to get the blade attached to a pint jar PC water in that jar (don't add anything to it grains will already have to much nutrients) place some grains in the jar place blender on wal-la that's everything
Side note you can do G2G with bags apparently and that will be much faster then your LC
PS I have know idea if anyone can make out what I'm saying.


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InvisibleCaps McGee
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Re: Fully colonized grain Spawn to Liquid Culture [Re: Geinstein]
    #25390526 - 08/15/18 04:03 PM (5 years, 5 months ago)

No idea*, and grammar is off a little... but no worse than some, I'd've never guessed, but english a second language?


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OfflineSpikeSpiegel
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Re: Fully colonized grain Spawn to Liquid Culture [Re: HiLIFE]
    #25390700 - 08/15/18 05:33 PM (5 years, 5 months ago)

Quote:

HiLIFE said:
Im not going G2G because I use bags with injection ports and I use LC. I'm not doing agar because its a clone. I either use a clone or a isolate. Not both. Clone vs Isolate is basically the same thing just from different ends but lets stay on topic. The clone performs very well, I dont need to grow it out on agar.




:okiapprove:

Personally I would't do what you're thinking (and a good argument for agar) because IMO there's something sketchy about grain floating around in nutrient-rich liquid for days. I'm sure it can be and has been done. If you've lost spawn to dirty LC before though you'll appreciate others' words of warning, that's all.

I think I get what you're trying to say about a clone not needing agar but I don't think that's the point people are making. The point is that agar is a good route to arrive at clean LC, not because you're after an isolate or trying to find better performing myc.

And what does LC and injection ports have to with G2G? You can still do it. Or you don't want to?


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OfflineGeinstein
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Re: Fully colonized grain Spawn to Liquid Culture [Re: Caps McGee]
    #25391450 - 08/16/18 12:45 AM (5 years, 5 months ago)

Quote:

Caps McGee said:
No idea*, and grammar is off a little... but no worse than some, I'd've never guessed, but english a second language?



I live in Africa its my 4th language and almost no one speaks it here by me.:lol:
So sorry for any other mistakes I make in the future.


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InvisibleCaps McGee
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Re: Fully colonized grain Spawn to Liquid Culture [Re: Geinstein]
    #25391609 - 08/16/18 04:59 AM (5 years, 5 months ago)

Quote:

Geinstein said:
Quote:

Caps McGee said:
No idea*, and grammar is off a little... but no worse than some, I'd've never guessed, but english a second language?



I live in Africa its my 4th language and almost no one speaks it here by me.:lol:
So sorry for any other mistakes I make in the future.



Again, had you not mentioned it, no one would've known I don't think lol... Just curious,but how does one decide what order "their" languages go in? Is it like, order learned?


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Offlinescifipirate
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Re: Fully colonized grain Spawn to Liquid Culture [Re: HiLIFE]
    #25391670 - 08/16/18 05:40 AM (5 years, 5 months ago)

Quote:

HiLIFE said: Pretty sure I can just break up the fully colonized grain spawn and fill it with water and then suck it back out and have a new LC right?



This is correct, go for it. Used to be a pretty standard way of making LCs, guess it's lost popularity...


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Re: Fully colonized grain Spawn to Liquid Culture [Re: scifipirate]
    #25391682 - 08/16/18 05:53 AM (5 years, 5 months ago)

I would advise against the GLC. You just can't tell how clean the grain spawn is, even minute amounts of bacteria will expand in the LC.

Start LC from clean cultures on agar. You can end up with more LC than you know what to do with, so just do it right.

Or you cab inoculate a grain master from the culture, you can go 1 to 10, 10 to 100 if you needed. Its just as fast.


But OP, if you have clean LC to begin with why do you need to make a new one anyways?


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Edited by mushpunx (08/16/18 05:56 AM)


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InvisibleCaps McGee
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Re: Fully colonized grain Spawn to Liquid Culture [Re: mushpunx]
    #25391733 - 08/16/18 06:40 AM (5 years, 5 months ago)

LC>LC?


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OfflineHiLIFE
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Re: Fully colonized grain Spawn to Liquid Culture [Re: SpikeSpiegel]
    #25392028 - 08/16/18 10:09 AM (5 years, 5 months ago)

Quote:

SpikeSpiegel said:
Quote:

HiLIFE said:
Im not going G2G because I use bags with injection ports and I use LC. I'm not doing agar because its a clone. I either use a clone or a isolate. Not both. Clone vs Isolate is basically the same thing just from different ends but lets stay on topic. The clone performs very well, I dont need to grow it out on agar.




:okiapprove:

Personally I would't do what you're thinking (and a good argument for agar) because IMO there's something sketchy about grain floating around in nutrient-rich liquid for days. I'm sure it can be and has been done. If you've lost spawn to dirty LC before though you'll appreciate others' words of warning, that's all.

I think I get what you're trying to say about a clone not needing agar but I don't think that's the point people are making. The point is that agar is a good route to arrive at clean LC, not because you're after an isolate or trying to find better performing myc.

And what does LC and injection ports have to with G2G? You can still do it. Or you don't want to?



I dont have a flowhood so I dont do g2g!


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OfflineHiLIFE
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Re: Fully colonized grain Spawn to Liquid Culture [Re: scifipirate]
    #25392032 - 08/16/18 10:12 AM (5 years, 5 months ago)

Quote:

scifipirate said:
Quote:

HiLIFE said: Pretty sure I can just break up the fully colonized grain spawn and fill it with water and then suck it back out and have a new LC right?



This is correct, go for it. Used to be a pretty standard way of making LCs, guess it's lost popularity...




So basically what I ended up doing was putting 60ml of sterilized LC into the jar, and then sucked up as much as I could back out and added back into the LC jar where it should grow to a new LC that i'm sure is clean. As soon as I get a flowhood i'll be doing g2g but until then. I feel like LC is just so easy though.


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OfflineMtez44
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Re: Fully colonized grain Spawn to Liquid Culture [Re: HiLIFE]
    #25392034 - 08/16/18 10:13 AM (5 years, 5 months ago)

Quote:

HiLIFE said:
I dont have a flowhood so I dont do g2g!


Quote:

SpikeSpiegel said:
Quote:

HiLIFE said:
Im not going G2G because I use bags with injection ports and I use LC. I'm not doing agar because its a clone. I either use a clone or a isolate. Not both. Clone vs Isolate is basically the same thing just from different ends but lets stay on topic. The clone performs very well, I dont need to grow it out on agar.




:okiapprove:

Personally I would't do what you're thinking (and a good argument for agar) because IMO there's something sketchy about grain floating around in nutrient-rich liquid for days. I'm sure it can be and has been done. If you've lost spawn to dirty LC before though you'll appreciate others' words of warning, that's all.

I think I get what you're trying to say about a clone not needing agar but I don't think that's the point people are making. The point is that agar is a good route to arrive at clean LC, not because you're after an isolate or trying to find better performing myc.

And what does LC and injection ports have to with G2G? You can still do it. Or you don't want to?








You dont need a flowhood to do G2G. Also you can do G2G with bags as well. its not just for jars


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Re: Fully colonized grain Spawn to Liquid Culture [Re: HiLIFE]
    #25392038 - 08/16/18 10:15 AM (5 years, 5 months ago)

You can g2g in a SAB just fine. Same for agar, which is way cleaner as we all keep telling you.

:whiteflag: on the rest. Can't make heads or tails of half what you're saying and dumping water into a grain bag then sucking it up doesn't sound clean at all. GL hope it works out for ya.


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Re: Fully colonized grain Spawn to Liquid Culture [Re: Mtez44] * 1
    #25392053 - 08/16/18 10:22 AM (5 years, 5 months ago)

if you want to stick to LC only you should do this method for most exponential use of mycelium while keeping "generations" or "transfers" low. This may not be the fastest method but is best long term.

Clean up clone or MS or whatver on agar.

Inoculate a large batch of LC, this will be the "master"- at least 600 mL can easily fit in a quart jar and have room for mixing. Store in fridge in a ziplock baggie.

Use 0.5-1 mL of fully colonized LC to inoculate your "working batch" of LC. You could do another 600 mL in a quart jar or less. This really depends on your volume of output and your need for speed.
0.5 mL of LC will still colonize a quart of grain faster than a wedge, but 10 mL (if you account of the extra water) will increase how fast it colonized considerably.

Once you are nearing the end of your working batch of LC, use a small amount of the master LC to inoculate a new working batch of LC. This will take 7-14 days to colonize so plan accordingly. Or use more LC so its faster as above... you know what you want to do.

This will allow you to inoculate more grain than you could probably use in years without exhausting the culture via G2G and excessive transfers from LC to grain and back.

Take a few weeks to set up your master and working solution and you're off to the races in terms of production as long as you keep up with doing things ahead of time.


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OfflineFishLevelMidnight
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Re: Fully colonized grain Spawn to Liquid Culture [Re: LtLurker]
    #25392066 - 08/16/18 10:27 AM (5 years, 5 months ago)

Quote:

LtLurker said:
You can g2g in a SAB just fine. Same for agar, which is way cleaner as we all keep telling you.

:whiteflag: on the rest. Can't make heads or tails of half what you're saying and dumping water into a grain bag then sucking it up doesn't sound clean at all. GL hope it works out for ya.




For what it is worth, this was a technique used a few years ago. I remember reading up about it on here. If done correctly (PC water in a syringe to add to the bags, mix well then draw out, you will lose a good portion of it, like recover 100 mL from 150 or somethng) it is viable. As long as your grains are prepped right and the inoculate was clean, there is no reason for this to not work.

Basically you are just sheering off bits of mycelium into the water and making a sterile suspension of mycelium (its not a true liquid culutre).

I personally don't like the contam vector risk and would rather use an LC i know is clean, but its not a horrible idea.

Some people even soak their grains in water after shaking and before spawning (like 10-20 minutes, to rehydrate them) so you could do that (sterile addition of sterile water ofc) and then draw off the water and use it as 'fuck it" inoculate- like for a project you weren't counting on


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Re: Fully colonized grain Spawn to Liquid Culture [Re: FishLevelMidnight]
    #25392091 - 08/16/18 10:36 AM (5 years, 5 months ago)

I get it used to be more common, but lots of things have fallen by the way side cause they just aren't as good as the methods more common now.

Most grain jars/bags that i've seen aren't 100% clean. Seems like adding/drawing water is going to practically guarantee picking up some contams, then you're gonna put them into an lc where contams thrive.

Now, there doesn't seems to be a good reason to do it that way since g2g (or a2lc) is cleaner, faster, and doesn't require different equipment. Unless you're just having some fun and failure is ok.

but of course, it's just :2cents: and he seems intent on going this route, so I give and hope it works out alright for him.


Edited by LtLurker (08/16/18 10:37 AM)


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Re: Fully colonized grain Spawn to Liquid Culture [Re: HiLIFE] * 1
    #25392109 - 08/16/18 10:46 AM (5 years, 5 months ago)

Quote:

HiLIFE said:
Quote:

scifipirate said:
Quote:

HiLIFE said: Pretty sure I can just break up the fully colonized grain spawn and fill it with water and then suck it back out and have a new LC right?



This is correct, go for it. Used to be a pretty standard way of making LCs, guess it's lost popularity...




So basically what I ended up doing was putting 60ml of sterilized LC into the jar, and then sucked up as much as I could back out and added back into the LC jar where it should grow to a new LC that i'm sure is clean. As soon as I get a flowhood i'll be doing g2g but until then. I feel like LC is just so easy though.





You mean 60ml sterilized LC *broth* I take it?

This was extremely unnecessary man. No need to use LC broth, you simply squirt water into the spawn jar and then suck it back out, that's it.
No need to grow a new culture.

But honestly, anything you get from going this route is most likely gunna be gross. Any minute amount of bacteria that was in the grain jar is going to expand in the LC broth.


If you absolutely must go this way using GLC , just squirt water in and pull it out.


No need for a flow hood for G2G at all!! Please tell me you are working with a still air box right now though? And are doing agar work?


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Re: Fully colonized grain Spawn to Liquid Culture [Re: LtLurker]
    #25392112 - 08/16/18 10:48 AM (5 years, 5 months ago)

now that im thinking about it wouldn't there be the exact same vectors involved as a grain to grain though?


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Re: Fully colonized grain Spawn to Liquid Culture [Re: Mtez44]
    #25392452 - 08/16/18 01:34 PM (5 years, 5 months ago)

G2G can be done in a SAB too? Granted,might be awkward... I'd say for LI, clean grain culture would be fine, but as mentioned, would avoid having a liquid prepped in this fashion sitting around to stew up nasties


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Re: Fully colonized grain Spawn to Liquid Culture [Re: Caps McGee]
    #25393181 - 08/16/18 07:07 PM (5 years, 5 months ago)

Granted it will be hard in a tiny SAB, that's why as far as still boxes go the bigger the better.

But yea , of course. Anything that can be done in front of a flow hood can be done in an SAB, even spawn bags although you can't blow a plenum of sterile air in them before sealing.

In a still air box, contams will be settling/falling down , and stirred up by the air. That's why its important to make controlled, decisive hand motions, and to never move anything unsterile (such as your hands) over top any open sterile media. You don't want to leave sterile media open long either.

With a flow hood, sterile air is blown towards you in laminar flow pattern, which means if I were to drop spores above open media they would get trapped in the laminar flow and blow straight outwards instead of settling down into it. The difference here is that you never move anything unsterile *in front* of open media as contams might get blown in.

Flow hoods require better sterile technique than SABs. They are more about elbow room and convenience.


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