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Invisibleskyjohnny
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LC over agar...Hmmm.
    #14474770 - 05/18/11 06:38 PM (12 years, 8 months ago)

I got a Shittake 75 plate, transferred it to agar in sterile specimen containers (UA cups) as well as to grain. The grain is going VERY slow. The agar transfers have grown out and began climbing the walls of the containers. I just took 24 cc of LC solution and placed in one of the colonized agar dishes. Does anyone think this will work to form a viable LC? I left a whole master culture to use if it turns to garbage, this is an experiment.
Questions? Comments? Ideas? Has this been done. I could find nothing on the search.


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"Man is that he might have joy. Joy is the "true meaning" of life. May you all experience "true joy". -CJM


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Offlinefungus_tao
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Re: LC over agar...Hmmm. [Re: skyjohnny]
    #14474982 - 05/18/11 07:25 PM (12 years, 8 months ago)

I don't see why that wouldn't work. In fact, I think it's :brilliant:


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OfflineRogerRabbitM
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Re: LC over agar...Hmmm. [Re: fungus_tao]
    #14475002 - 05/18/11 07:29 PM (12 years, 8 months ago)

I've made mycelium syringes from agar plates before.  Hopefully you had a 14 gauge or larger needle.  You would have been miles ahead to inoculate grains with agar though.
RR


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InvisibleMonkeyKnifeFight
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Re: LC over agar...Hmmm. [Re: RogerRabbit]
    #14475131 - 05/18/11 07:58 PM (12 years, 8 months ago)

Wait are you saying you put a sugar solution in the actual petri dish and left it there?


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Offlinetrout
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Re: LC over agar...Hmmm. [Re: MonkeyKnifeFight]
    #14475155 - 05/18/11 08:04 PM (12 years, 8 months ago)

RR are you saying to use agar transfer is better than LC for grains?


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Well things don't always look as they are and things can be misread and mistaken for what they realy are so don't read too much into what I say since I might be mistaken myself. And remember I rarely ever give a definate answer.


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OfflineRogerRabbitM
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Re: LC over agar...Hmmm. [Re: trout]
    #14475545 - 05/18/11 09:12 PM (12 years, 8 months ago)

Yes.
RR


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semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
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Invisibleskyjohnny
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Re: LC over agar...Hmmm. [Re: RogerRabbit]
    #14477538 - 05/19/11 09:16 AM (12 years, 8 months ago)

Why is agar transfer better than LC? My thinking was that injecting LC would spread myc over more area than a few pieces of agar (i.e. more inoculation points = faster colonization)


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"Man is that he might have joy. Joy is the "true meaning" of life. May you all experience "true joy". -CJM


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OfflineRogerRabbitM
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Re: LC over agar...Hmmm. [Re: skyjohnny]
    #14477554 - 05/19/11 09:20 AM (12 years, 8 months ago)

Making an LC adds another vector of contamination with zero benefit.  Mycelium is mycelium.  Diluting it in water does not increase the amount of cells.  Simply inoculate your grains with an agar wedge and then shake well after it begins to colonize.
RR


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semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
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Offlinetrout
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Re: LC over agar...Hmmm. [Re: RogerRabbit]
    #14477743 - 05/19/11 10:03 AM (12 years, 8 months ago)

I will not question RR information, but I think that I can be more sterile making a grain lc in a glove box and then inoculating jars through an injection port in the open air than I can using wedges in the open.  If I had a flow hood I would use wedges.  But alas I do not and working with jars in a glove box is maddening to me.


--------------------
I need tropical cultures, ABM, V.v....!!!!

Well things don't always look as they are and things can be misread and mistaken for what they realy are so don't read too much into what I say since I might be mistaken myself. And remember I rarely ever give a definate answer.


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InvisibleMonkeyKnifeFight
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Re: LC over agar...Hmmm. [Re: RogerRabbit]
    #14477763 - 05/19/11 10:09 AM (12 years, 8 months ago)

Also think about the speed gain.  If you have an agar plate ready to go you can take some of it, make an LC and wait for that to grow.  Then since contams are basically invisible in LC you have to grow out ANOTHER plate from the LC just to make sure it's clean. 

In that amount of time you could have taken that first plate and started 10 more plates and still had plenty of agar left to inoculate half a dozen grain jars.  And at every step contams are clearly visible so you don't waste your time wondering.


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InvisibleMonkeyKnifeFight
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Re: LC over agar...Hmmm. [Re: trout]
    #14477774 - 05/19/11 10:12 AM (12 years, 8 months ago)

Quote:

trout said:
I will not question RR information, but I think that I can be more sterile making a grain lc in a glove box and then inoculating jars through an injection port in the open air than I can using wedges in the open.  If I had a flow hood I would use wedges.  But alas I do not and working with jars in a glove box is maddening to me.




I guess that's a matter of taste.  I don't have a flow hood and do everything in my janky home made gloveless box.  Overall I find precise tasks like transferring agar the most annoying.  Opening grain jars and plopping in a wedge has become pretty automatic.


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Invisibleskyjohnny
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Re: LC over agar...Hmmm. [Re: RogerRabbit]
    #14477847 - 05/19/11 10:34 AM (12 years, 8 months ago)

I see. Shaking would effectively accomplish the same end without the added risk of contamination. Thanks RR.
Since LC is an accepted means of expanding cultures, it may prove valuable for those wanting to do an LC. Though it wouldn't be much different than injecting LC solution into colonized grains, per another LC tek. I just figured I would eliminate one avenue of possible contam while making an LC. 
Instead of opening any containers I did the no-tilt LC tek and merely inserted a sterilized needle to suck up solution, re-sterilized and injected into the culture jar.
I feel good about the technique's sterility, where I have not yet had any luck at all making good LCs. That would be an accomplishment of its own for me!


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"Man is that he might have joy. Joy is the "true meaning" of life. May you all experience "true joy". -CJM


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Invisibleskyjohnny
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Re: LC over agar...Hmmm. [Re: skyjohnny]
    #14477856 - 05/19/11 10:37 AM (12 years, 8 months ago)

Even in front of my flowhood, I ended up with a spot of trich in one jar using the agar transfer. Hence the act of opening the jars and petris is an avenue of possible contam I was hoping to eliminate.
You're not ahead if you make 200 agar tranfers and they get contam'd cuz they were opened in the first place
Granted it was only one jar out of 10, but had that trich landed in the petri, it could have been catastrophic.


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"Man is that he might have joy. Joy is the "true meaning" of life. May you all experience "true joy". -CJM


Edited by skyjohnny (05/19/11 10:42 AM)


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InvisibleMonkeyKnifeFight
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Re: LC over agar...Hmmm. [Re: skyjohnny]
    #14477983 - 05/19/11 11:07 AM (12 years, 8 months ago)

If you're working in front of a flow hood and getting mold contamination then you need to work on your practices.  It doesn't matter if you're using agar or LC or a grain jar if you let mold blow around things will contaminate.  I've never used a flow hood but I can tell you that I get very low contams in my box.  And when I let someone else try working in the box basically everything contaminates even though visually they're following the same steps I do.  Little things like economy of movement and working smoothly make all the difference.


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Invisibleskyjohnny
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Re: LC over agar...Hmmm. [Re: MonkeyKnifeFight]
    #14478208 - 05/19/11 11:55 AM (12 years, 8 months ago)

Ya. I have only done a couple grows using MS syringes to grain. So I definitely have lots of practice to do. I appear to have gotten 90% success with my A2G transfers, so that's not bad, and it was only a tiny spot of trich. I'm pretty impressed with myself.
These were also my first ever agar transfers. The A2As I did went 4/6, not ideal by any means, but still my personal best. No mold on the agar, I had some bacteria (I think. Little yellowish spots) but the agar was poured hot and ended up with lots of condensation which eventually gathered and ran onto the agar forming a puddle. I don't want to use antibiotic agar, as I would have no way of gauging the sterility of my technique.
Thank you for the advice MKF. I will work to improve my movements and fluidity. I am also hoping to build a cleanroom soon.
My inexperience was another reason for hoping to find better ways of making LC and such.


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"Man is that he might have joy. Joy is the "true meaning" of life. May you all experience "true joy". -CJM


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Offlinekotter
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Re: LC over agar...Hmmm. [Re: skyjohnny]
    #14480611 - 05/19/11 07:49 PM (12 years, 8 months ago)

I'm really new at growing shiitake mycelium but have been taking a number of different approaches to see what works best for me. Doing agar right into grain jars has consistently colonized the jars faster for me than has inoculating grain jars at the same time with liquid culture of the same strain (Aloha 75). They both have worked fine for me though.
For whatever reason when I first got the Aloha 75 going from the master slant it was really slow growing but now I can barely keep up with it.
I really enjoy growing mycelium in liquid culture. The best success I've had has been from carefully teasing bits of mycelium loose from agar plates and dropping the pieces into LC jars with lids that have homemade injector ports as I found described on this site. I use those for pulling inoculant out of later but have had no trouble with contamination opening up the lids and dropping in small pieces of mycelium (in front of a flowhood).  I did have some incidence of contaminants doing this with agar included.
Thanks to advice also encountered here I've been using a luerlok needle designed for injecting flavor into meat intended for jerky making (from the grocery store). Its got two holes and a pointed tip. The two holes work similarly to a handheld homogenizer when material is drawn in and nicely break up the mycelium. Stubborns bits can be assisted by sucking it back and forth gently. I have no idea what gauge it is but its fat. It makes big holes in the injector ports so I've been using yet more advice encountered here suggesting smearing on more high temperature silicone to seal the ports again as I don't how big is too big.


Edited by kotter (05/19/11 07:55 PM)


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Invisibleskyjohnny
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Re: LC over agar...Hmmm. [Re: kotter]
    #14487636 - 05/21/11 12:13 AM (12 years, 8 months ago)

So, it appears to be growing into the solution well. I could see a "mat," of myc rising from the bottom today. If it happens to be a good clean LC I might as well use it.:shrug:


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"Man is that he might have joy. Joy is the "true meaning" of life. May you all experience "true joy". -CJM


Edited by skyjohnny (05/21/11 12:15 AM)


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OfflineRogerRabbitM
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Re: LC over agar...Hmmm. [Re: skyjohnny]
    #14488506 - 05/21/11 06:15 AM (12 years, 8 months ago)

Quote:

skyjohnny said:

Since LC is an accepted means of expanding cultures, it may prove valuable for those wanting to do an LC.




Not exactly.

LC isn't for expanding cultures because it's so horribly slow.  I could colonize 20 gallons of grain spawn in the time it takes a pint jar of LC to get 'fuzzy'.  With grain to grain transfers, you expand the mycelium much faster and you always know if it's contaminated.  With liquid culture, you never know until you use it. . . .after all that wait.

If you're moving to agar work and edibles, you need to pony up the dough for a flowhood.  Using syringes is a way for noobs to grow cubes with some degree of success, but it teaches bad habits that are hard to break, such as working in open air instead of utilizing proper sterile technique.
RR


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semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
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OfflineMGS
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Re: LC over agar...Hmmm. [Re: skyjohnny]
    #14490163 - 05/21/11 03:31 PM (12 years, 8 months ago)

In my experience LC performs faster than agar wedges once transfered to grain. As long as you use sterile techniques and use blanks to pinpoint vectors of contimnation LC is better in commercial applications due to the volume. When you have to pour x number of agar plates that in itself is a vector, you can get the same volume of mycelium from one liter LC then you could from pouring 50 agar plates.


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Invisibleskyjohnny
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Re: LC over agar...Hmmm. [Re: MGS]
    #14490537 - 05/21/11 05:01 PM (12 years, 8 months ago)

So far the only growth is rising from the bottom. I think that because I used a flowhood, it is a clean culture. With any luck I will have made my first clean LC, though I guess we'll see when I 'noc a test jar.
It seems like LC could be a more convenient means of inoculating many containers. I have trouble juggling two lids and a tool most days. I also like that the only exposure this myc will have had to the outside world is when I transferred agar to agar (Which went well, 'cuz it colonized 100%) and then when I inoculate.
Thank you RR, I appreciate your input. Though the boat for deciding whether to do it or not, sailed before I posted this. Worst case scenario, I chalk it up as experiential.


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