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MS to Agar


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#1 NotFadeAway

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Posted 25 February 2019 - 11:01 PM

This is my first time shooting spores on to agar and I am curious as to what my objective is. This may be a dumb? Am I trying to Isolate a strain like I would do with a clone or LC on agar? I have done some tissue samples on agar and successfully isolated an ape strain after many attempts. Is that the idea here? Or am I rather growing the spores out just to make sure there is no contam? I would think the latter because I was always told to clone from your biggest fruit. With that theory I would think you wouldn't want to isolate until you have a good strong gene pool. Just trying to get an understanding of the next level of this hobby. I've kind of been stuck in all of this. 9bf82432711990b1434305c2897ce0b7.jpg
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#2 deemesis

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Posted 25 February 2019 - 11:28 PM

I've only ever scraped a print over agar, other than cloning with fruit body. But yea, MS on agar was always just to make sure things were clean and if not, make more transfers.


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#3 raymycoto

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Posted 26 February 2019 - 12:13 AM

 

This is my first time shooting spores on to agar and I am curious as to what my objective is. This may be a dumb? Am I trying to Isolate a strain like I would do with a clone or LC on agar? I have done some tissue samples on agar and successfully isolated an ape strain after many attempts. Is that the idea here? Or am I rather growing the spores out just to make sure there is no contam? I would think the latter because I was always told to clone from your biggest fruit. With that theory I would think you wouldn't want to isolate until you have a good strong gene pool. Just trying to get an understanding of the next level of this hobby. I've kind of been stuck in all of this.

 

You could do any or all of those, even concurrently. For ex, keep taking samples or plugs from clean looking areas on your plates and replate on new agar. Look for sectors that you like and eventually isolates that favor more rapid and appealing (rhizomorphic, I suppose) growth. Meanwhile, if your original plate(s) appear uncontaminated, you may use those originals or any subsequent generation plate(s) for blended agar to liquid nutrient culture (LC).

 

This way you are using the old plates and also breeding some new ones in search of isolates. If you run out of bandwidth to proceed with the many weeks of growing and transferring, at least then you have made a MS LC with earlier plates.

 

You might, in addition, save a small piece or two at one or more points along the way to a 'slant' that you label as a particular plate generation in case something ends up totally contaminated. You can then go back to your slant even if it might be MS from the first or second plate, for ex.

 

This is one way you can proceed with some MS spawn for a couple of weeks before you have an isolate. You may also do other means of spawn colonization such as agar wedge of any plate to sterile grain jar.

 

BTW, your plates look pretty good! Store them upside down and stacked and they will have less condensation on the top.


Edited by raymycoto, 26 February 2019 - 12:41 AM.

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#4 Billcoz

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Posted 26 February 2019 - 12:41 AM

Exactly what those guys said, raymycoto laid out really good options, agar makes things so nice!

 

I take transfers, then if the donor plates are still clean I just fruit those to use em up, plus I do "no pour" in jars, so I like to get the jars back for new batches.



#5 jkdeth

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Posted 26 February 2019 - 02:33 AM

Clean up, after that its up to you. You can isolate from there, or grow out a clean plate and clone. Lot of folks do a little of both.
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#6 Deleena24

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Posted 26 February 2019 - 10:50 AM

Nobody mentioned this yet, but isolating on plates is good, but you'll still have to grow each isolate out to see if it even fruits well...

So I'd recommend using those plates just to get a clean MS culture. Grow that out, and pick the fruits you want to play with. You can do this while concurrently isolating on plates so you have at least some fruits while youre looking for the perfect isolate.
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#7 peacefrog

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Posted 26 February 2019 - 07:01 PM

Good advice above.

Agar is a tool for us as cultivators. It will aid in cleaning up/isolating clean growth from contaminates, allow one to select and isolate cultures, cloning mushroom tissue etc. It’s not a “must do” in our world as amateur cultivators, but it’s a damn good tool in the tool belt IMO.

I started with agar and I’m glad I did and I always take spores to it first no matter what. It’s just what I am used to and comfortable with. I’ve just used agar for so many years, I depend on its 2 deminsional plane to help me spot out contamination or sectors easily.

As said above, if you want to just a quick grow, transfer until you have clean growth. Don’t worry about sectoring or isolating strains.

But if you want to play with it and find yourself many cultures to test, then by all means transfer until you have a “pure culture”. Just have as many as possible and test as many as possible in between transfers. There is no way to tell how a culture will grow from looking at the mycelium. You have to grow out and test them.This is the part of cultivation I love best. I don’t mind spending weeks or months trying to find that awesome culture.

Good vibes.
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#8 jkdeth

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Posted 26 February 2019 - 07:08 PM

You can do both as well. Once you have that clean plate, transfer a few sectors, take the rest to grain. Best of both worlds.

#9 WalkingCatfish

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Posted 26 February 2019 - 09:02 PM

Great comments, above. I've used agar mainly to produce inoculum for grain (usually, starting from a print). Agar lets me see at a glance that a culture is clean, so I don't waste a bunch of grain on questionable inoculum. I don't isolate and test monocultures, but I think I'd enjoy that. For now, I prefer to work the other way around, and clone specimens that have already proven themselves. When I have a spore syringe on hand, I just squirt it into a bunch of BRF jars and hope for the best. :biggrin:


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#10 Seeker2be

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Posted 26 February 2019 - 09:26 PM

How about this also? Put some agar in the bottom of a 1/2 pt jar to cover it, sterilize (may want to do more that one jar or a bunch of jars.) then transfer the sectors you like with rhizomorphic growth from your petri dish ..  IOnce the agar  is colonized,, scratch it  multiple times with a sterile needle or use a scalpel to cut in 4 pieces, add sterile water then close.  Shake like hell then suck up the water through your injection port and you have instant L/C or LI to inoculate many jars of grain or  maybe  outside inoculation of wood chips (if you are growing wood lovers)..   You can do this also on a smaller scale by scratching a colonized petri dish and injecting and aspirating sterile water to create a syringe of LI.. So agar has many routes to cultivation and that is the reason to learn it but not fear it.


Edited by Seeker2be, 26 February 2019 - 09:31 PM.

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#11 Billcoz

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Posted 27 February 2019 - 01:55 AM

How about this also? Put some agar in the bottom of a 1/2 pt jar to cover it, sterilize (may want to do more that one jar or a bunch of jars.) then transfer the sectors you like with rhizomorphic growth from your petri dish ..  IOnce the agar  is colonized,, scratch it  multiple times with a sterile needle or use a scalpel to cut in 4 pieces, add sterile water then close.  Shake like hell then suck up the water through your injection port and you have instant L/C or LI to inoculate many jars of grain or  maybe  outside inoculation of wood chips (if you are growing wood lovers)..   You can do this also on a smaller scale by scratching a colonized petri dish and injecting and aspirating sterile water to create a syringe of LI.. So agar has many routes to cultivation and that is the reason to learn it but not fear it.

That's what I use for agar plates anyways, 1/2 pint jars, and after I inoculate them I slpi the whole jar into a ziplock bag and seal, trapping in some air, then put the lid ring on, rather than putting the lid disc on, so I can see straight onto the sectors. A non see through lid makes it a bitch to see them-IMG_20190115_180740.jpg IMG_20190115_181007.jpg

 

Some call what you described a "slurry" or LI, I made some syringes like that, only I just squirted and aspirated it repeatedly and sucked it back up. It colonizes grains in 7-11 days, and are more resistant to contams.



#12 NotFadeAway

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Posted 27 February 2019 - 09:30 AM


This is my first time shooting spores on to agar and I am curious as to what my objective is. This may be a dumb? Am I trying to Isolate a strain like I would do with a clone or LC on agar? I have done some tissue samples on agar and successfully isolated an ape strain after many attempts. Is that the idea here? Or am I rather growing the spores out just to make sure there is no contam? I would think the latter because I was always told to clone from your biggest fruit. With that theory I would think you wouldn't want to isolate until you have a good strong gene pool. Just trying to get an understanding of the next level of this hobby. I've kind of been stuck in all of this.


You could do any or all of those, even concurrently. For ex, keep taking samples or plugs from clean looking areas on your plates and replate on new agar. Look for sectors that you like and eventually isolates that favor more rapid and appealing (rhizomorphic, I suppose) growth. Meanwhile, if your original plate(s) appear uncontaminated, you may use those originals or any subsequent generation plate(s) for blended agar to liquid nutrient culture (LC).

This way you are using the old plates and also breeding some new ones in search of isolates. If you run out of bandwidth to proceed with the many weeks of growing and transferring, at least then you have made a MS LC with earlier plates.

You might, in addition, save a small piece or two at one or more points along the way to a 'slant' that you label as a particular plate generation in case something ends up totally contaminated. You can then go back to your slant even if it might be MS from the first or second plate, for ex.

This is one way you can proceed with some MS spawn for a couple of weeks before you have an isolate. You may also do other means of spawn colonization such as agar wedge of any plate to sterile grain jar.

BTW, your plates look pretty good! Store them upside down and stacked and they will have less condensation on the top.
Thanks for the tip man. These ones had alot more air bubbles than usual.

#13 NotFadeAway

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Posted 27 February 2019 - 09:32 AM

Nobody mentioned this yet, but isolating on plates is good, but you'll still have to grow each isolate out to see if it even fruits well...

So I'd recommend using those plates just to get a clean MS culture. Grow that out, and pick the fruits you want to play with. You can do this while concurrently isolating on plates so you have at least some fruits while youre looking for the perfect isolate.

In a nutshell that's exactly what I was thinking the most logical process would be. Thanks for that awesome explanation!

#14 NotFadeAway

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Posted 27 February 2019 - 09:37 AM

Great comments, above. I've used agar mainly to produce inoculum for grain (usually, starting from a print). Agar lets me see at a glance that a culture is clean, so I don't waste a bunch of grain on questionable inoculum. I don't isolate and test monocultures, but I think I'd enjoy that. For now, I prefer to work the other way around, and clone specimens that have already proven themselves. When I have a spore syringe on hand, I just squirt it into a bunch of BRF jars and hope for the best.

So you just clone your large fruits transfer it to a clean plate and then knock your grains up? No isolating the tissue transfer if it contains multiple sectors? Or do you take the extra step and make some LC?

#15 SharkieJones

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Posted 27 February 2019 - 10:02 AM

I used to isolate the best looking mycothreads on to another disk, when I had my best looking strain of mycelium I'd create live tissue spore syringes to knock up my grain jars. Then I would transfer to my straw logs.

#16 raymycoto

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Posted 27 February 2019 - 10:06 AM

 

So you just clone your large fruits transfer it to a clean plate and then knock your grains up? No isolating the tissue transfer if it contains multiple sectors? Or do you take the extra step and make some LC?

 

You could do either. Place sterile material from a good fruit onto agar. I do multiple plates if I really want success. Sometimes you end up with contamination on a plate or two. Once you have success with one or more of these plates, then you can do whatever you like - agar wedge to grain or make an LC. If you have duplicate plates, then they should be clones so just mix them if you like or save one. Perhaps make a slant from one of the good plates in case you want to go back for a do over on that one some day. I have not had good luck with agar straight to grain but must just be me. It's commonly used.

 

I have also read about doing blended stipe, for ex to a liquid nutrient to make an LC or taking this blended fruit and going directly to grain. I tried both of those one time and they must have had some contam on board bc my LC from blended sterile internal stipe material went bad as did my immediate noc of a grain jar with that stipe slurry  (LC before incubation).

 

But I have read of success doing both those things but just did not work for me on my attempt.


Edited by raymycoto, 27 February 2019 - 10:08 AM.

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#17 Billcoz

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Posted 27 February 2019 - 10:19 AM

I read about cloning DRIED tissue in this old archive thread-http://archives.myco...html?1106008656 from the man himself, Hippie3.


Edited by Billcoz, 27 February 2019 - 10:20 AM.


#18 Seeker2be

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Posted 27 February 2019 - 10:19 AM

Try adding grain water to your agar. It aids epigenesis of enzymes then the agar specimen takes better to grain.


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#19 raymycoto

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Posted 27 February 2019 - 01:21 PM

 

Try adding grain water to your agar. It aids epigenesis of enzymes then the agar specimen takes better to grain.

 

I have done that a few times. IME, unfortunately I have found that it can reduce the hardening of the agar. Even when adding the correct agar gm per total volume or even a bit of extra agar. I have still have had messy agar that won't harden or stick to the glass resulting in a sheet of slimy agar that falls off the plate if you invert. It seems to develop this problem at PC temps when the agar is PCd with the grain water already in the mix. It does something to the agar at high temps. I have tried once adding the sterilized grain water later when both the grain water and agar mix had cooled to around 55C. That seemed to solve the problem but I have not repeated it and I'm really cautious now about autoclaving agar with the grain water in place. In my case it was WBS water and I now use mainly whole oats and I'm not sure if that does the same thing.

 

But I agree, it's a nice way to add nutrients that the fungus will see again soon.


Edited by raymycoto, 27 February 2019 - 01:22 PM.


#20 Seeker2be

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Posted 27 February 2019 - 04:18 PM

As with all additives I wouldnt make agar entirely with grain water. The mycelia just needs to get exposed to its future environment.  Never had the problem with Oats, Rye, Wheat , or Pop corn but WBS is a different animal . It appears to have a lot more starch and stickiness even in processed jars.


Edited by Seeker2be, 27 February 2019 - 04:19 PM.

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