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Refilling LC Jar through syringe filter?


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#1 Jrotten

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Posted 26 July 2020 - 06:25 PM

Has anyone tried this to refill an active liquid culture? The water should be sterile you would need to ventilate the SHIP which you could do under positive pressure or with a second syringe filter on a needle.

Edited by Jrotten, 26 July 2020 - 06:27 PM.


#2 FunG

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Posted 26 July 2020 - 10:46 PM

I've heard of people doing exactly as you mentioned to maintain a clone lc for years.

Never done it myself however
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#3 PJammer24

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Posted 27 July 2020 - 12:08 PM

Has anyone tried this to refill an active liquid culture? The water should be sterile you would need to ventilate the SHIP which you could do under positive pressure or with a second syringe filter on a needle.

I think you would be better off making an entirely new LC solution and simply taking a few CC's out of the original LC and using it to start the new LC... What you are suggesting can likely be done, however, LC's can easily be contaminated during normal use and over time can often get contaminated. For best results, I would suggest just making a new one and using the same solution to get it started...

 

Even better would be to fruit the old LC and then take another clone making a new generation which will further isolate your genetics.

 

Starting fresh is almost always a better option for any of the stages of the growing process.


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#4 Jrotten

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Posted 27 July 2020 - 01:18 PM

Right, I wasn’t thinking it was something to do on a regular basis. What my thinking was that it could progressively create something similar to a liquid inoculant than a liquid culture if that was desired. As in there might be a point where a less nutritive liquid would be more desirable. Just thinking out loud

#5 FunG

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Posted 27 July 2020 - 07:32 PM

I think the concept was to use 2cc's of a isolate lc to a gallon of nutrient solution to create a batch for mass propigation and the author of the thread even had a foot pedal operated aspirator that pumped in LC from a pump that was attached to the drum of innoculent.... lol

Which makes more sense then refilling the used lc. I guess that's why they're called primary masters and secondarys.

#6 Jrotten

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Posted 27 July 2020 - 11:10 PM

But follow me:

If I have an LC I like and I’ve got good mileage out of, I’ve got my master already, I just have 25% left in a particular jar. But I want to do a very large inoculation to a pasteurized substrate where the sugars and nutrients in the LC may not be ideal.

My options then would be filter off the broth or I could try to dilute the LC in increments to get as much culture to the least amount of substrate.

Does anyone else have a simpler way to get a non-nutritious liquid inoculant from LC?

#7 PJammer24

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Posted 28 July 2020 - 08:53 AM

But follow me:

If I have an LC I like and I’ve got good mileage out of, I’ve got my master already, I just have 25% left in a particular jar. But I want to do a very large inoculation to a pasteurized substrate where the sugars and nutrients in the LC may not be ideal.

My options then would be filter off the broth or I could try to dilute the LC in increments to get as much culture to the least amount of substrate.

Does anyone else have a simpler way to get a non-nutritious liquid inoculant from LC?

I don't know why you would want to attempt to colonize a substrate directly from LC rather than using the LC to start a spawn jar and then using that spawn jar to colonize the substrate... I don't know of many people who have had good results inoculating substrate directly from LC or MS syringe...

 

I don't think I fully understand what you are trying to do...


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#8 Jrotten

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Posted 28 July 2020 - 08:03 PM

Right now I'm doing LC direct inoculation for pans in bags and it is ridiculously fast.  I can create an LC so thick I'm not sure it could grow anymore from an agar wedge in about 3-4 days.  From there I'm injecting sterilized substrate that has been hydrated just below field capacity to allow for 60-120ml of LC.  I have yet to get to fruiting, but I'm getting there fast.  Starting from a culture on agar I think I can reasonably have a tray ready for casing in under 21 days and maybe 14.  It's also pretty forgiving and easy math to make sub. But if it's not sterile there will be issues because the LC nutrient water is basically a contamination magnet.  I first saw the technique used by Blue Helix, but it is also how Asura outlines his grows in his thread https://mycotopia.ne...s/#entry1426988

 

What I'm wondering about in this particular post is converting a Liquid Culture into a Liquid Inoculant so it it could be utilized without the the sterile closed conditions of a sealed myco bag.  I'm not really trying to do anything with any of it at the moment, but once I make more jars I'm thinking of experimenting with some LC's that get low in the jar.  

 

My thinking is something like this; I start with a 4% LC and use 70-80% of it or have some left over that just doesn't work with the math of the bags I'm working with.  Could I then add say double the existing volume, and get a 2% LC and let that grow out, do it again and get 1%... let that grow... then potentially cut that further, and be able to use that somewhere like a monotub and use the liquid inoculant as the final hydration part and essentially have a many more inoculation points than I could get with say grain, but with less contamination risk of grain or LC.?


Edited by Jrotten, 28 July 2020 - 08:05 PM.


#9 PJammer24

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Posted 29 July 2020 - 08:47 AM

Right now I'm doing LC direct inoculation for pans in bags and it is ridiculously fast.  I can create an LC so thick I'm not sure it could grow anymore from an agar wedge in about 3-4 days.  From there I'm injecting sterilized substrate that has been hydrated just below field capacity to allow for 60-120ml of LC.  I have yet to get to fruiting, but I'm getting there fast.  Starting from a culture on agar I think I can reasonably have a tray ready for casing in under 21 days and maybe 14.  It's also pretty forgiving and easy math to make sub. But if it's not sterile there will be issues because the LC nutrient water is basically a contamination magnet.  I first saw the technique used by Blue Helix, but it is also how Asura outlines his grows in his thread https://mycotopia.ne...s/#entry1426988

 

What I'm wondering about in this particular post is converting a Liquid Culture into a Liquid Inoculant so it it could be utilized without the the sterile closed conditions of a sealed myco bag.  I'm not really trying to do anything with any of it at the moment, but once I make more jars I'm thinking of experimenting with some LC's that get low in the jar.  

 

My thinking is something like this; I start with a 4% LC and use 70-80% of it or have some left over that just doesn't work with the math of the bags I'm working with.  Could I then add say double the existing volume, and get a 2% LC and let that grow out, do it again and get 1%... let that grow... then potentially cut that further, and be able to use that somewhere like a monotub and use the liquid inoculant as the final hydration part and essentially have a many more inoculation points than I could get with say grain, but with less contamination risk of grain or LC.?

You are going to lose out on yield... The substrate is considered "semi"-nutritious... The grain is really where you get the bang for your buck in terms of nutrition... If you want it to colonize more quickly, use more spawn...



#10 sandman

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Posted 29 July 2020 - 11:56 AM

IMG 20170420 173711892   Copy
 
That's an older picture I do it differently now. I use JB weld instead of silicone and found better luer fittings and use stainless onepiece regular mouth lids instead of plastic wide mouth lids.
 
One luer fitting accepts a luer plug and the other accepts a syringe filter. Remove plug to access the female luer and attach a syringe and you can withdraw or inoculate.
 
For what it's worth I will often use an older LC to inoculate a bunch of new lc with these lids. It is the fastest way to colonize an LC it's CRAZY on a 24/7 stir how fast you can finish an LC from LC.

Edited by sandman, 29 July 2020 - 12:01 PM.

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#11 PJammer24

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Posted 29 July 2020 - 12:08 PM

 

 
 
That's an older picture I do it differently now. I use JB weld instead of silicone and found better luer fittings and use stainless onepiece regular mouth lids instead of plastic wide mouth lids.
 
One luer fitting accepts a luer plug and the other accepts a syringe filter. Remove plug to access the female luer and attach a syringe and you can withdraw or inoculate.
 
For what it's worth I will often use an older LC to inoculate a bunch of new lc with these lids. It is the fastest way to colonize an LC it's CRAZY on a 24/7 stir how fast you can finish an LC from LC.

 

 

 

Well shit... Good to know!!
 



#12 sandman

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Posted 29 July 2020 - 12:14 PM

Just so no confusion,

 

I will make fresh bottles of LC broth and inject 5-10cc of LC, with a no needle syringe through my female luer  port, into the fresh LC.

 

The syringe that I injected the LC with was withdrawn from a finished LC.

 

Not refilling the same jar by refilling with sterile LC.


Edited by sandman, 29 July 2020 - 12:20 PM.

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#13 PJammer24

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Posted 29 July 2020 - 12:50 PM

Just so no confusion,

 

I will make fresh bottles of LC broth and inject 5-10cc of LC, with a no needle syringe through my female luer  port, into the fresh LC.

 

The syringe that I injected the LC with was withdrawn from a finished LC.

 

Not refilling the same jar by refilling with sterile LC.

Ok... This makes more sense to me... Injecting fresh LC solution into a finished LC didn't make a lot of sense... I have jump started fresh LC from a finished one many times...

 

maybe the ole sandman can interject on the other point of contention... Unless I misread OP's post or there is a miscommunication due to the vocabulary used, OP intends to colonize substrate directly from LC rather than LC to spawn prior to mixing with substrate. I see multiple problems with this... The main one being that a significant part of the nutrients used by the mushrooms comes from the grain spawn... From my perspective there would be a significant loss in yield by colonizing substrate directly from LC... The closest I would consider to doing this would be creating a slurry from my spawn (most likely brown rice flour) and pouring it over the substrate... I will do this outdoors to mitigate the risk of my spawn being devoured by varmints...

 

If enough LC is used, I suspect the substrate will colonize adequately but the performance of that particular substrate would pale in comparison to what one would see using grain spawn due to the increase in nutrients provided by the spawn...



#14 sandman

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Posted 29 July 2020 - 01:15 PM

Substrate without grain just doesn't seem to have what it takes to get a liquid culture to jumpstart growing.  It will just not do anything or spread out, is most likely.

 

You are right that it would be missing much nutrients even if it did work.

 

There are teks of LC/LI/Slurry to a STERILIZED substrate that also has grain(s) in the mix to give the LC a place to grow and more nutes too.


Edited by sandman, 29 July 2020 - 01:15 PM.

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#15 p2p

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Posted 29 July 2020 - 10:25 PM

I have done a number of LC to substrate and can say that it takes about 70 days to colonize the substrate.

Colonization was successful every time, but 70 days is not worth it.

 

Despite the great idea of eliminating the grain step, I did not find any benefit to do LC to Sub.

 

I have switched back to G2Sub and have much better and consistent results.


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#16 Jrotten

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Posted 29 July 2020 - 11:07 PM

I haven't tried liquid inoculant other than an actual LC, but 120ml has colonized roughly 2 quarts completely extremely fast. I forgot to date the bag, but the T1 from the germ plate was done 18 days ago. Since then I created a maximum density LC, did direct inoculation and have 99% colonization. I'm not sure you could go any faster? That one has a small amount of grain in the sub. I have the same LC matured on the stir plate a few more days, direct injected to essentially the same sub, but more of it and lacking grain because I ran out and it is definitely going. I have no idea how the grow will go, but I'm getting visible recovery in under 24 hours with this LC on sterilized straw/horse poo/verm +/-WBS

The completed one is maybe 5 days ahead of the other one which was injected 4 days ago kneaded again yesterday to mix it up. There are visible spots of expansion from yesterday.

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Edited by Jrotten, 29 July 2020 - 11:09 PM.


#17 Jrotten

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Posted 30 July 2020 - 12:44 AM

@Sandman are you using luer fittings as injection ports? Are you using syringe filters as well? Like serviceable LC lids? If so do you just leave filters in the fittings as FAE and exhaust/ventilation?

Edited by Jrotten, 30 July 2020 - 12:48 AM.


#18 sandman

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Posted 30 July 2020 - 05:11 PM

totally serviceable lc lids. One is male one is female. One holds a syringe filter the other holds a plug untul you want to remove the plug and remove the lc with syringes. Or inoculate through the port too.


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#19 Jrotten

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Posted 30 July 2020 - 09:27 PM

That's actually awesome.  Did you find the ports off the shelf or did you MacGyver those?  I have a box a bunch of lids to make pretty quick.  I was planning on the standard SHIP with permanent filter, but there's just no way those can last as long as ports you can just hook up to.  Are you using just jars as well or are you drawing from the luer port on the jar and then putting a needle on the filled syringe to inject bags, etc?  Can you get away with smaller gauge needles if that's what you're doing?


Edited by Jrotten, 30 July 2020 - 09:28 PM.


#20 sandman

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Posted 31 July 2020 - 06:29 PM

Yes the ports can be found here https://componentsup...er-fittings.php

 

Man you really nailed me bro I am drawing from the quart jar into syringes in front of hood. I remove needle, connect syringe to luer on lid, tilt, draw. Put needle back on. Lay on shelf until I shoot bags later that day

 

(open top bags in hood, needle is mainly used as a plug but also gives you a longer reach to keep your hand and syringe out of bag when you shoot)

 

((i talk like this now))


Edited by sandman, 31 July 2020 - 06:29 PM.

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