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OfflineAjm
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Lc colonization
    #14491795 - 05/21/11 10:32 PM (12 years, 9 months ago)

Tried using the Search Function, but couldn't find anything about: How long it takes for an LC, which is inoculated by MS, to colonize. Or, When can you tell that it, LC, is ready to be used. I plan on doing the Sugar in the Raw LC.


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OfflineBuckeye Oysters
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Re: Lc colonization [Re: Ajm]
    #14491842 - 05/21/11 10:43 PM (12 years, 9 months ago)

LC's shouldn't be used with spore inoculations so you know as it is impossible to distinguish contams from mycellium in a LC.  LC's should be made with a wedge of colonized agar or another proven clean LC.  Also the best recipe is using malt sugar bought at party supply / beer supply.

If you are a beginner you should stick with grain to grain transfers until you have a laminar flow hood and can do more advanced techniques such as LC.


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Evolution is Lamarckism in disguise.  Adaptation never creates a new species or trait, but rather the new species/trait always existed within the parent DNA until circumstances allowed it to be activated.  For instance, every wolf has the DNA for poodles, but that DNA would never be revealed without man selectively breeding for it.

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Re: Lc colonization [Re: Buckeye Oysters]
    #14491996 - 05/21/11 11:26 PM (12 years, 9 months ago)

I'm a noob & am doing LC's with great success & in no way as sterile as I should be.
First I'm using 1/2 pint jars with approx. 1/2 of a jar of water.
1 tea spoon of Light Karo. Mixed until dissolved, (warm tap water helps dissolve).
Then I stick it in the nuker until it starts to boil.
As I have metal lids with a hole in it I can't nuke them so I wipe with alcohol and place on the jar.
Then I place a piece from the middle of the stem into the LC & put lid on with filter.
My hands are clean & knife is flamed but that really is all I do.
I have had failures, which is why you test B4 knocking up a whole bunch, but I would guess my success rate is about 90%

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OfflineAjm
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Re: Lc colonization [Re: mabachko]
    #14492099 - 05/22/11 12:01 AM (12 years, 9 months ago)

Quote:

Buckeye Oysters said:
LC's shouldn't be used with spore inoculations so you know as it is impossible to distinguish contams from mycellium in a LC.  LC's should be made with a wedge of colonized agar or another proven clean LC.  Also the best recipe is using malt sugar bought at party supply / beer supply.

If you are a beginner you should stick with grain to grain transfers until you have a laminar flow hood and can do more advanced techniques such as LC.



I have ingredients for the PF Tek and have done it before. So, in this context what you said will not work because I probably do not have the ingredients used for what you say, but if I had other ingredients and knew the procedures that are used for what you are talking about, I would probably have done what you seem to suggest. Could just knock up the PF jars with the MS itself, but from what I have read it seems that LC is faster at colonizing a substrate then the MS itself. Just wondering how long the LC would take to colonize once inoculated with MS, and if there were no contams.


Quote:

mabachko said:
I'm a noob & am doing LC's with great success & in no way as sterile as I should be.
First I'm using 1/2 pint jars with approx. 1/2 of a jar of water.
1 tea spoon of Light Karo. Mixed until dissolved, (warm tap water helps dissolve).
Then I stick it in the nuker until it starts to boil.
As I have metal lids with a hole in it I can't nuke them so I wipe with alcohol and place on the jar.
Then I place a piece from the middle of the stem into the LC & put lid on with filter.
My hands are clean & knife is flamed but that really is all I do.
I have had failures, which is why you test B4 knocking up a whole bunch, but I would guess my success rate is about 90%



I plan on using the same thing, in terms of the nuker. Just add water to a jar and nuke until it starts boiling, and then add SITR, stir, put lid, with Silicone inoculation port and equalizer port, on jar and PC for 12 min. I like how you say you use alcohol on your lids.  Probably do that. How long does it usually take for the LC to colonize or be ready for use? Like the liquid that is being inoculated with spores from a print or using MS.


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Offlinemabachko
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Re: Lc colonization [Re: Ajm]
    #14493433 - 05/22/11 10:49 AM (12 years, 9 months ago)

I would mix the karo prior to nuking.
And it takes a couple of week for the myc to grow. After that it just keeps growing.
Don't know how long it will last as I have not been at it that long.

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OfflineAjm
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Re: Lc colonization [Re: mabachko]
    #14493466 - 05/22/11 10:56 AM (12 years, 9 months ago)

So, it takes about two weeks and then I can break up the mycelium and aspirate into a syringe and inoculate jars. Why mix before nuking?


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Offlinemabachko
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Re: Lc colonization [Re: Ajm]
    #14493485 - 05/22/11 11:01 AM (12 years, 9 months ago)

Thats how long I waited.
After you take out what you need it will still keep growing, assuming you left some.
When it breaks up it really is all over the place.
I generally will fill the syringe & fire it back into the jar to break up the myc. Works pretty good, but sometimes I have to do it a couple of times.
It's like breaking up a quarrt of rye.

Edited by mabachko (05/22/11 11:02 AM)

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Offlinejimmy8
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Re: Lc colonization [Re: Ajm]
    #14493515 - 05/22/11 11:10 AM (12 years, 9 months ago)


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OfflineAjm
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Re: Lc colonization [Re: jimmy8]
    #14493649 - 05/22/11 11:41 AM (12 years, 9 months ago)

Quote:

mabachko said:
Thats how long I waited.
After you take out what you need it will still keep growing, assuming you left some.
When it breaks up it really is all over the place.
I generally will fill the syringe & fire it back into the jar to break up the myc. Works pretty good, but sometimes I have to do it a couple of times.
It's like breaking up a quarrt of rye.



Alright cool, thankyou dude for all the tips:thumbup:
Quote:

jimmy8 said:
You have ingredients for the PF tek?
http://www.shroomery.org/67/Poor-Mans-Mycelium-Syringe-Tek



This seems cool, but I'm starting from scratch meaning I have no colonized PF tek jar to do this. I might give this a shot once a cake is colonized though. Why not?  I'm trying to figure out whether a PF cake inoculated with MS is faster or slower then a PF tek jar inoculated with LC, including the time to colonize the LC itself. In other words if I inoculate a PF tek jar with MS and and LC with MS, which jar would finish colonizing first. And then the LC once colonized needs to be inoculated into a PF Tek jar and while this is happening the PF jar with the MS will still be growing, if no contams. I dont know if I'm explaining this clearly, but I will probably get my spores Monday so I will run a little experiment.


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Offlinemabachko
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Re: Lc colonization [Re: Ajm]
    #14493677 - 05/22/11 11:51 AM (12 years, 9 months ago)

If you start everything on Monday the MS would probably be faster. But, the second and later batch's would go to the LC, as it is already growing and doesn't need to germinate.
If your like me you just need to do it yourself so that you know for sure.
Not a prob.
If someone says I can't, you bet your ass I gonna give it a try anyway.
Good Luck

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OfflineAjm
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Re: Lc colonization [Re: mabachko]
    #14493727 - 05/22/11 12:03 PM (12 years, 9 months ago)

Quote:

mabachko said:
If you start everything on Monday the MS would probably be faster. But, the second and later batch's would go to the LC, as it is already growing and doesn't need to germinate.
If your like me you just need to do it yourself so that you know for sure.
Not a prob.
If someone says I can't, you bet your ass I gonna give it a try anyway.
Good Luck



Haha.Yea it might. I agree with what you said about the second batch. Once the LC and BRF/VERM jars are inoculated, I will post a few pics with details and then update from there.


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OfflineBuckeye Oysters
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Re: Lc colonization [Re: Ajm]
    #14494821 - 05/22/11 04:29 PM (12 years, 9 months ago)

It will take longer to get results with LC at start because you will have to prove clean before doing a large batch.  LC's should be pressure cooked for 45min, not microwave nuked.  I really don't know how you have had success doing that Mabachko other than you have a very very clean house.


--------------------
Evolution is Lamarckism in disguise.  Adaptation never creates a new species or trait, but rather the new species/trait always existed within the parent DNA until circumstances allowed it to be activated.  For instance, every wolf has the DNA for poodles, but that DNA would never be revealed without man selectively breeding for it.

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Re: Lc colonization [Re: Buckeye Oysters]
    #14494945 - 05/22/11 04:55 PM (12 years, 9 months ago)

When I started cloning I figured that the shroom had been exposed to the elements for around a month so what was the use of worrying about contams.
I try to make more than one in case of contams and I guess I'm just lucky.
But, if it works, don't fix it.

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Invisiblet23

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Re: Lc colonization *DELETED* [Re: mabachko]
    #14495126 - 05/22/11 05:24 PM (12 years, 9 months ago)

Post deleted by t23

Reason for deletion: .


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OfflineAjm
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Re: Lc colonization [Re: t23]
    #14498151 - 05/23/11 09:09 AM (12 years, 9 months ago)

Quote:

t23 said:
Yeah I'm with mabachko, you don't need to be so anal about using the proven hardcore sterility methods, and the microwave has been scientifically approved to kill all bacteria. Flowhood not necessary at all. Glove box isn't even necessary but I wouldn't try it without one.

I'll for sure be using mabachko's methods for my first LC cloning.

Here's a good link for LC comparisons:
http://www.shroomery.org/forums/showflat.php/Number/9591904



Thanks for the Link:thumbup: Might be a few days before pics are up


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OfflineBuckeye Oysters
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Re: Lc colonization [Re: t23]
    #14498968 - 05/23/11 12:58 PM (12 years, 9 months ago)

Quote:

t23 said:
Yeah I'm with mabachko, you don't need to be so anal about using the proven hardcore sterility methods, and the microwave has been scientifically approved to kill all bacteria. Flowhood not necessary at all. Glove box isn't even necessary but I wouldn't try it without one.

I'll for sure be using mabachko's methods for my first LC cloning.

Here's a good link for LC comparisons:
http://www.shroomery.org/forums/showflat.php/Number/9591904




Its not about killing live bacteria, it is about killing bacterial endospores which a very tough and only die in high pressure high heat.  Im telling you all, if you don't have good sterile conditions and a flowhood you are paving a way to disaster in the future.


--------------------
Evolution is Lamarckism in disguise.  Adaptation never creates a new species or trait, but rather the new species/trait always existed within the parent DNA until circumstances allowed it to be activated.  For instance, every wolf has the DNA for poodles, but that DNA would never be revealed without man selectively breeding for it.

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OfflineAjm
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Re: Lc colonization [Re: Buckeye Oysters]
    #14499819 - 05/23/11 04:05 PM (12 years, 9 months ago)

7 jars are going to be inoculated with MS B+ and 1 jar with LC, once colonized enough to do so or if it even colonizes. This way if the LC is no good then there will not be many jars lost to contam, but if it is good then we will see which jar seems to grow the fastest. I will do a LC to LC, so it is proven clean buckeye, if this LC works haha.
:jamming:


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OfflineApple core
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Re: Lc colonization [Re: Ajm]
    #14499948 - 05/23/11 04:35 PM (12 years, 9 months ago)

I'm all about LC. It turns 2 ml of spore solution into about 500ml of liquid culture. That's done me about 25-30 cakes so far and I've still got about 150ml left.

I don't think LC is any more 'advanced' than PF-tek, if you can inoculate a cake w/out contamination then your technique is fine. I did LC for my first ever project and always have since.

I use 4% organic honey by weight. So for every 94g of water you want 4g of honey. I make sure the water's warm before-hand as the honey dissolves more easily. I wouldn't microwave it, but only because I know my method works fine. If people here say it's good then whatever, just research it a bit. I pressure cook mine for up to an hour, then inoculate once cool. 

If you want to speed things up, make yourself a magnetic stirrer. They're really easy (and fun!) to make from recycled household odds and ends, and will both speed up your colonisation time and help your culture stay homogeneous for when you're ready to inoculate.

Edited by Apple core (05/23/11 04:39 PM)

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OfflineAjm
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Re: Lc colonization [Re: Apple core]
    #14500192 - 05/23/11 05:28 PM (12 years, 9 months ago)

http://www.shroomery.org/forums/showflat.php/Number/6378527/fpart
This is the Tek I plan on doing
/1/vc/1
Quote:

Apple core said:
I'm all about LC. It turns 2 ml of spore solution into about 500ml of liquid culture. That's done me about 25-30 cakes so far and I've still got about 150ml left.

I don't think LC is any more 'advanced' than PF-tek, if you can inoculate a cake w/out contamination then your technique is fine. I did LC for my first ever project and always have since.

I use 4% organic honey by weight. So for every 94g of water you want 4g of honey. I make sure the water's warm before-hand as the honey dissolves more easily. I wouldn't microwave it, but only because I know my method works fine. If people here say it's good then whatever, just research it a bit. I pressure cook mine for up to an hour, then inoculate once cool. 

If you want to speed things up, make yourself a magnetic stirrer. They're really easy (and fun!) to make from recycled household odds and ends, and will both speed up your colonisation time and help your culture stay homogeneous for when you're ready to inoculate.



I was looking at the honey tek but was concerned about carmelization. I wouldn't nuke either if doing honey, but for the tek linked it should be fine to nuke. I've read of the magnetic stirrer being mentioned in other threads, but haven't looked it up. It might be a good idea to do so. Thanks for the suggestions :cheers:


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OfflineAjm
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Re: Lc colonization [Re: Ajm]
    #14506049 - 05/24/11 06:43 PM (12 years, 9 months ago)

Just got done steaming the PF Tek jars and LC; The jars are now in the cooling process. After the inoculation of all jars I will try to post a pic of the jars, PF and LC, tomorrow, as well as the magnetic stirrer, which is still in the process of being built. Peace :smoker:


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