Isolating On Agar


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Posted by: Fungusmaximus Nov 30 02, 08:21 AM GMT
All right, Ive go a load of 2 pint jars full of karo/h2o growing clone samples, that are contamed w/ green mold. I'm waiting on some agar, dextrose, and petri dishes, right now. When they arrive, what/how do I need to get the clone isolated? I really really want this clone! Its a biggun, over a foot tall and thick! Can someone give me some advice? Thanks smile.gif unsure.gif

Posted by: DirtyWOP Nov 30 02, 08:34 AM GMT
Your gonna need your nice glovebox.....

What is the liquid culture like?
A chunk floating in water or is it blended?
Try to get the nicest substantial piece of mycelium or mushroom tissue out of that soggy mess and drop it in a jar of 3% peroxide to rinse it, and then transfer to a freshly made plate....

You might want to just leave the dish right there in the glovebox...
Agar contams easily....
And be sure to add H2o2 to you agar, since the mycelium is already diggin it.

Posted by: Fungusmaximus Nov 30 02, 08:38 AM GMT
I got chunks of original samples w/ new growing clouds of myc. wink.gif

Posted by: DirtyWOP Nov 30 02, 11:12 AM GMT

try to get the myc that is the farthest away from the green mold. Use the mush chunk if that isn't whats moldy....and don't disturb the contaminant spores

you know.....sometimes I've tossed liquid clone cultures because I thought the mushroom chunk had trich....but I dumped it, and it was just extremely bruised. Peroxide will do that.

Posted by: Nanook Nov 30 02, 12:56 PM GMT
What you are going to need to do is go in with a sterile needle into the cleanest portion of the culture and draw up a few drops of the cleanest stuff you can.

Shoot Peroxidated Agar, about 20 of them. Have another 10-20 clean plates standing by.

Shoot 20 plates with a drop of inoculum each. If you get any clean plates, culture them out. If you do not get clean plates you must perform transfers: take a wedge of peroxidated agar and place it upside down on a clean plate... Hopefully clean mycelia will grow out, while contam spores remain trapped in the sanitizing agar.

See: Dirty Prints

Posted by: Fungusmaximus Dec 02 02, 05:39 AM GMT
No wop, this is trich. I shot a load of jars and its greener than a leprachaun on st patty's day, smells like ass too bleah.gif I wish it was, only bruising.

I cant see the mold spores in the karo, or the mold. It looks like a great culture, but Ive shot it in a few different syringes and the same results.
Unless its the glovebox!? unsure.gif but there is always so much lysol floating around in there it chokes me to open it. I even keep lysol wipes laid inside to set my tools on.

Can I shoot some peroxide into the karo solution before I drop on peroxidated plates?


And would a uv light fish tank water filter work in sterilizing liquid cultures??


Posted by: DirtyWOP Dec 02 02, 08:15 AM GMT
How the hell would we know?
try it man!

probably no point in shooting H202 in there now. I thought it was already in the liquid culture when you wrote karo/h20....I misunderstood you.

Try not to get the thing all stirred up in you can. The mold spores are in there somewhere, you just got to find out where.


Posted by: Nanook Dec 02 02, 04:54 PM GMT
UV is going to cause some genetic damage so that is pretty much out for this application. You can squirt some peroxide right into the karo, but mind you... peroxide does little against Trich

Posted by: Fungusmaximus Dec 02 02, 07:01 PM GMT
Wont it at least kill the spores??

Posted by: Fungusmaximus Dec 04 02, 10:11 PM GMT
How do I mix agar agar?

I have a lb of dextrose also do I need other additives to grow a good culture?

Posted by: czen Dec 04 02, 10:19 PM GMT
Here's the method I used. Worked pretty good.


Agar for Dummies

Posted by: Fungusmaximus Dec 05 02, 10:05 AM GMT
Thanks CZEN wub.gif

Posted by: czen Dec 05 02, 10:49 AM GMT
Fungusmaximus...

Here's Workman's method using instant potato flakes. I'm going to give it a shot next:


Here is my suggested formula:
18-25 grams of agar/dextrose premix
5 grams of instant potatos
500 ml of water
This should do around 20 standard petri dishes if you pour them fairly thin. Good luck.

Have a good one,
czen

Posted by: Fungusmaximus Dec 05 02, 02:53 PM GMT
I found these fat round little spice jars 8 I think, for 3$. They seem like the glass will hold up in the pc, so I can process the agar. And that way I have a bunch of smaller jars of agar so I dont contam the whole batch at once, IMO this will make the odds a little more in my favor. Ill let you know how they hold up.

Posted by: Fungusmaximus Dec 06 02, 07:29 PM GMT
How do I sterilize those plastic petri's from sporeworks?? They feel thin, way too thin to PC... unsure.gif

Posted by: czen Dec 06 02, 07:35 PM GMT
Hello again,

I got some of the presterilized plastic ones, too. I asked Nan and he said
to spray the outside of the sleeve they come in with lysol and then pour the agar inside a glove box. Haven't done that yet.
I'm going to get some glass dishes that can be pc'd.

czen cool.gif

Posted by: Fungusmaximus Dec 06 02, 08:22 PM GMT
They arent in the sleeve anymore.? unsure.gif I took them out. OOPs!

Posted by: czen Dec 06 02, 08:34 PM GMT
If you haven't opened them I'd guess you could spray them individually. Although that'll be a pita.


Posted by: Mycota Jan 29 03, 12:00 AM GMT

Popular belief has it that when you pick an isolate (off agar - usually). That you should pick a strong rhizomorphic type, over a thin cotton like hyphae type of mycelium.

IMHO, that is not true. "Hyphae" & "rhizomorphs" are actually one & the same, in differing form. If healthy, either will serve as an effective isolate. As - one can change into the other, as circumstance requires.

Rhizomorphs serve varied purposes, specifcally for exploratory or migratory needs of fungus. For example, suitable food sources for saprotrophic, wood-decay fungi can be widely separated from each other. To you the distances between bits of old wood on a forest floor may not seem great, perhaps a foot or two. To a fungus the intervening areas of bare wood free soil is like the distance across an barren ocean.

If a fungus has exhausted all the nutrients in one area, it faces a challenge getting to another. Rhizomorphs serve as itís exploratory tool. The outer skin protects the inner hyphae in the traversal of an inhospitable region. Additionally, rhizomorphs grow much more quickly than isolated hyphae, thereby speeding up the exploration for fresh food sources.

Rhizomorphs also have considerable advantages when a fresh nutrient source is found. As there are typically numerous hyphae making up a rhizomorph & when it finds a new nutrient source, the numerous hyphal tips within it will fan out and rapidly colonize the nutrient mass as a diffuse, branched mycelium.

A well-structured rhizomorph is able to transport large amounts of water, nutrients or oxygen back to the mycelium mass, and will easily fuel the rapid expansion of the fungus into new territory. The contrast between a rhizomorph and an individual hypha is similar to the difference between a 8 lane freeway and a narrow, winding road. Or, big teeth, verses tiny teeth.

Mycota (aka 6T)

BIG TEETH


Posted by: Mycota Jan 29 03, 12:02 AM GMT

Little teeth................Mycota (aka 6T) wink.gif


Posted by: OneDiaDem Jan 29 03, 12:08 AM GMT
*I bow to your wisdom Mycota*. I love learning new things about our fungi. This really made alot of things clearer. Thank you! biggrin.gif


Posted by: phillinwierd Jan 29 03, 12:23 AM GMT
Interesting metaphor Six Tango. Gonna have to see if that plays out. Empirical knowledge with me, ya dig?


Posted by: phillinwierd Jan 29 03, 12:55 AM GMT
Why would one get both cottony and rhizomorphic growth on a plate then? Nutrients are equal throughout the plate. The culture isn't searching for anything. Therefore, fungus capable of rhizomorphic growth may be more suitable for culture that those that are not.....survival of the fittest. What difference this has on fruit size or quality is debatable though I guess. Like I say, empirical knowledge, I'll have to experiment some more. Or did I miss the point?


Posted by: Mycota Jan 29 03, 01:15 AM GMT
Have you ever been munching some chips? But, it was not quite enough, to satisfy your hunger. Then, head towards the fridge, to get some salsa, to. Same thing going on in an agar plate, sometimes.

I have had fluffy plates (no rizo's) & when spawned into a substrate, they shot out rizo's like arms, heading every which way.

Mycota (aka 6T)


Posted by: Subgen1us Jan 29 03, 01:17 AM GMT
I think he was talking about better as in speed wise.
Am i right?


Posted by: Mycota Jan 29 03, 01:24 AM GMT
Rizo will move faster. As that is inpart what it is designed to do.

But, fluff can change to rizo & head out, just as well. Or, rizo can turn to fluff.

Same thing -- in differing form.

Mycota


Posted by: phillinwierd Jan 29 03, 01:33 AM GMT
So, what's the point in isolating strains?


Posted by: Mycota Jan 29 03, 01:48 AM GMT
QUOTE (phillinwierd @ Jan 29 03, 06:33 AM GMT)
So, what's the point in isolating strains?

To get one you know is clean, healthy & better than mulyispore mix. Mycota


Posted by: phillinwierd Jan 29 03, 01:53 AM GMT
Yes, but now you've got me thinking maybe I shouldn't be showing preference to rhizomorphic growth. huh.gif


Posted by: Mycota Jan 29 03, 01:59 AM GMT
Rizo is the more aggressive of the two. One time I isolated some strong rizo to a differing plate. Only diff between plates was 1 = PDA.... 2 = PMDA.

After the transfer, the isolate turned fluffy. Hmmmmmmmm

Got me to thinking.

Nutes were better in the #2 plate & it fluffed up -- instead of staying rizo.

Mycota


Posted by: phillinwierd Jan 29 03, 02:06 AM GMT
Hmmmmmmmmmmmm. Now I see where you're going with this. Plate 2 = more nutes mycellium got lazy..........what happened to plate 1? Stayed aggressive?


Posted by: Mycota Jan 29 03, 02:14 AM GMT
Yup... not lazy -- just easy street..... I think. So, I reversed the transfer & it changed back -- again.

Mycota


Posted by: DirtyWOP Jan 29 03, 11:14 AM GMT
I still think it's better to isolate the rhizomorphic growth.....
I mean,
Would stamets bullshit you?


Posted by: Fungusmaximus Jan 29 03, 01:03 PM GMT
QUOTE (DirtyWOP @ Jan 29 03, 10:14 AM GMT)
I still think it's better to isolate the rhizomorphic growth.....
I mean,
Would stamets bullshit you?


No, but new findings and better understandings of research happen all the time.

I agree Mycota, I too have witnessed and wondered about it.
Available air plays a factor too! If you have a plate w/ great rhizo and seal it up or limit the air to very little the myc will fuzz up, give it some air again back to rhizomorph. If your sub sucks and has little nute value so will your myc, give it some good sub ( dung/straw compost high nutrient agar w/ proper air and you will see the same fluff turn to solid fingers climbing the walls. my 2


Posted by: Mycota Jan 29 03, 01:27 PM GMT
Stamets is not known for bullshiting anyone about fungi. But - you might note the date of whatevet you are reading -- authored by him. Since that time -- some thinking has changed. And, no -- I'm not going against anything he advises.

The point is, if you have a poor nutrient selection in any agar mix. Myc is prone develope rizo's enabling it to move further -- faster in a hunt for more or better nutrients. As that is what rizo's are better suited for.

Alternativly, if the agar mix is rich in nutrients, myc is less prone to develope strong rizo characteristics -- as there is no need to move further -- faster in a hunt for more or better nutrients.

The viability of the isolated strain, it's characteristics, fruiting ability & the overall yeilds it will produce, has nothing to do with the fact you isolated one or the other. Simply because one can turn into the other, dependent on indiviual circumstance.

IMHO, you want any strain of myc that has the ability to change, from one to the other, as needed. Because it is better suited to survive & thrive.

As an example, I once received a B+ spore strain that, once the spores germinated did not demonstrate any rizo capacity -- at all -- ever. Fluffy myc was it. The strain colonized bulk substrates very slowly & did not fruit out -- worth beans -- on those bulk substrates.

My assumption with those particular spores was/is; it had been grown out on some substrate -- most probobly -- verm/brf cakes repeatedly -- over many -- many -- many generations. In doing so -- it lost some genetic traits that allowed it to develope rizo type growth to rapidly colonize either a dung/straw combo, or compost type substrate.

For instance, if you ate nothing but candy bars -- all your life. You would develope side effects & suffer various damaging consiquences. I believe that example to hold true with fungi. As, if you cultivate them on any single type -- limited nutrient -- substrate -- such as verm/bfr over numerious generations, without change. The genetics of the strain may become damaged. Some problems PF suffered, may be an example of that.


Psilocybe Cubensis are habitat specific. Meaning, they cannot grow in the wild, unless their habitat provides a suitable environment, along with sufficient natural nutrients. Over the millennia, they have evolved inherent genetic traits best suited for their continuous survival in specific geographic area's they successfully inhabit.

All fungi feed by absorption of nutrients. Because of the huge range of potential nutrient sources, fungi evolved enzymes suitable for the specific environments in which they are generally found. The range of enzymes, though wide in may species, is not sufficient for survival in all environments.

Psilocybe Cubensis excrete a complex array of genetically predetermined enzymes for digestion. The enzymes are present in multiple forms, based on a single inherent genetic sequence, and include a range of isoenzymes, which arise from different inherent genetic sequences.

Simply stated, Psilocybe Cubensis excrete enzymes into the organic material in which their underground mycelia (root) system naturally grow. Those enzymes degrade nutrients there, into simple soluble forms of sugars and amino acids, which are then easily absorbed into the mycelia network. Resulting in them acquiring all essential elements with which to grow fruit bodies, and spores (seed) by which they propagate their species.

It is common knowledge that most strains of Psilocybe Cubensis flourish in select warm moist habitats worldwide, associated where horses, cattle and water buffalo naturally spread bovine type manure. Consequently, Psilocybe Cubensis developed inherent genetic traits, enabling then to excrete specific enzymes best suited to enable them to specifically dissolve, digest and take up nutrients available from bovine type manure, and/or soil enriched with it.

Therefore, Psilocybe Cubensis own inherent genetic traits attest that bovine type manure alone, or soils highly enriched with it, is best suited to their nutrient needs, in the wild.

Taking that fact, one step further. Aged leached dry bovine type manure, when aerobically composted together with a small percent of other select fruits, vegetables, grains and straw provides an even more enriched super nutrient source for cultivation of Psilocybe Cubensis . Moreover, a compost of this type provides an ideal moist subsurface habitat (substrate) that, Psilocybe Cubensis mycelia will colonize faster than any other.

Mycota (aka 6T) wink.gif


Posted by: el.jefe Jan 29 03, 02:02 PM GMT
QUOTE (Mycota @ Jan 29 03, 01:27 PM GMT)
Mycota (aka 6T) wink.gif

aka stamets? cool.gif


Posted by: DirtyWOP Jan 29 03, 02:10 PM GMT
I agree that diversifying substrate and agar mix is good.....
But I don't think the idea that isolating rhzomorphic mycelium,
to better the fruitings, is obsolete or ineffective at all. I mean look at that maz plate of Joseph's.....it is purely rhizo, and then look at the fruiting.....


Posted by: sinoptik Jan 29 03, 02:11 PM GMT
When's the book due out?

"Mycota's Guide to Mushroom Cultivation" biggrin.gif


Posted by: Mycota Jan 29 03, 02:56 PM GMT
QUOTE (DirtyWOP @ Jan 29 03, 07:10 PM GMT)
I agree that diversifying substrate and agar mix is good.....
But I don't think the idea that isolating rhzomorphic mycelium, to better the fruitings, is obsolete or ineffective at all. I mean look at that maz plate of Joseph's.....it is purely rhizo, and then look at the fruiting.....

I am not saying the practice is obsolete or ineffective at all.

I chose rizo type growth for transfers, myself. Simply because I can see that they are aggressive.

Knowing that. The strain is IMHO better suited to colonize large volume straw/dung combo, or compost type substrates. As it has it's traverling shoes on & is ready to go.

The point of the whole thing is, if the myc is healthy & without any inherant genetic defects. It will change from fluffy, to rizo type, as it's enviro & nutrient needs dictate.

Mycota (aka 6T)


Posted by: Mycolaureat Jan 29 03, 03:25 PM GMT
it make sense to me ...

I want to know more about it,
what's you information sources?
is it based on your own experience?

thanks


Posted by: Mycota Jan 29 03, 04:33 PM GMT
QUOTE (Mycolaureat @ Jan 29 03, 08:25 PM GMT)
I want to know more about it, what's you information sources? is it based on your own experience?

My own feeble experience.

Of course, after smoking a bowl of N. Cal Bubbleberry & drinking some J/D & coke, my notes become more like messy doodles, that are difficult to work with -- when I try to decrypt them -- the next day. All in fun, anyway. Life is better with a goodly amount of laughter involved on a daily basis.

Mycota (6T)


Posted by: phillinwierd Jan 29 03, 06:56 PM GMT
One of the best threads ever. Especially Myc's hypothesis of strain mutation/degeneration of the B+ resulting in it's inability to adapt to a straw dung environment after being grown of cakes for succesive generations. No more big teeth!
Also, I'm going to try different agar mixes with some surplus cultures to change the myc. from cotton to rhizo and back again. See what happens. It's all just so damn interesting.


Posted by: OneDiaDem Jan 29 03, 08:08 PM GMT
This is a great thread!


Posted by: Zoom Jan 29 03, 08:55 PM GMT
Excellent thread Mycota. The very nature of mycelium is to change to meet it's eviroment. Really enjoyed the way you explained the process.


Posted by: Fungusmaximus Jan 30 03, 01:35 AM GMT
Not all that new of a concept really, actually heard it before thats why I agreed. But one thing that puzzles me but makes since is the nutrient rich sub comment.
QUOTE
Alternativly, if the agar mix is rich in nutrients, myc is less prone to develope strong rizo characteristics -- as there is no need to move further -- faster in a hunt for more or better nutrients.

So my ultra rich nutrient agar should produce fuzzy growth even in ideal conditions, right?
So if it was amazingly rhizomorphic before on regular agar the same culture transfered to the ultra rich formula will begin slower more fuzzy growth, due to its lack of need to search for nutrients? Would the opposite be true?Rhizo ultra= fuzzy

transfered to regular it should begin to form the fingers or strands of thick growth?

I think that maybe thick strands or rhizo fingers arent just for the purpose of finding food, but for protection... serving dual purposes for the colony. Fuzzy growth is easily injured, a thick strand can take allot more abuse and moves more quickly. Thus it has a greater chance to colonize all the substrate and begin fruiting until maturity is reached and spores carry on the trait. Explaning why more rhizos = more fruit. I have NEVER seen a highly rhizomorphic cubie mycelium not fruit, however I have seen cottony mycelium never fruit .
I believe there is truth in the rhizo isolation, but, rhizo is highly dependent on environmental factors as well.
If you study the growth of mycelium, you will notice that the "fingers" pave the way. then the fuzzies fill in the gaps. example a strong leader is more likely be able to blaze the way AND find food for the colony while expanding. sorta roping it off until the rest of the colony gets there. MY 2


Posted by: Subgen1us Jan 31 03, 03:15 PM GMT
One comment i have.
everyone keeps saying rhizo = more fruit. I think u may be jumping the gun because from wat i have read is that rhizo = more primordia.
The reason people isolate the rhizo is because it is faster growth and produces more primordia but still doesnt mean the will be more fruit. i think its just u will have an even growth instead of 1 big one here and one small one here.


Posted by: Fungusmaximus Jan 31 03, 04:10 PM GMT
Well primordia turn into fruit right? Most usually do, some abhort, or just stop growing, but most mature into fruits.


Posted by: Subgen1us Jan 31 03, 05:50 PM GMT
the reason we isolate the rhizo is because we want the faster, primordia producing myc. with this we get more fruits(wich leads to more yields. I wasnt trying to say that more primordia doesnt lead to more fruits, just that u were jumping the gun. because it is better to have more primordia, and some aborts than matted myc that only produces a few pins. Didnt mean to say in erlier post that ist doesnt mean there will be more fruit. my mistake.
the more primordia u get, the more even the pinset is.
even though some may abort they are all starting at the same time witch should lead to an even distribution of nutrients to each mushroom.
If u use the fuzzy myc then it becomes matted wich leads to less primordia giving u uneven pins.

What would be considered better then, an even pinset resulting in all ur fruit from 1 maybe 2 flushes or a uneven pinset resulting in more flushes but all in all coming up with the same amount when weight is considered?


Posted by: Fungusmaximus Jan 31 03, 06:46 PM GMT
What book are you reading?

QUOTE
the more primordia u get, the more even the pinset is.
even though some may abort they are all starting at the same time witch should lead to an even distribution of nutrients to each mushroom.
If u use the fuzzy myc then it becomes matted wich leads to less primordia giving u uneven pins.

Well first off I dont agree more primordia =more even pinset.
You can have a shit ton of primordia all pop up in one spot.
Even pinset is attributed by an even substrate and casing layers, isolating merely helps produce more primordia to fill the gaps in your pinset making a full tub of mushies instead of patchy mushie clusters.

Fuzzy myccelium will matt yes, so will rhizomorph, both leads to less fruit
and uneven pinset, what is your point? unsure.gif


Posted by: Subgen1us Jan 31 03, 07:09 PM GMT
change where i said more even pinset to more chances for fruits then. im still new to this and need people to put me in check.
what does it matter if u get more fruits when in actuality its the yield that everyone wants.

or does more fruits mean more yield to everyone?

i have read tests of people taking a cake and removing all but one mushroom wich makes it grow larger than it should.

also i thought with more primordia it gives it (number wise) more chances to produce more fruit.
that was my point. wich should be true because we are discussing it using optimum factors.
also i think when i argue i tend to come out with an angry tone but i dont want u to think its anything personal its a flaw i have.


Posted by: Subgen1us Jan 31 03, 07:20 PM GMT
yes i went back and read it all, i did mistakinly use even pinset instead of when i should have said gives more chances for fruits.

in reply to:Whats ur point?

I was saying why people picked out the rhizo and isolated them instead of the fuzzy crap wich was the whole topic of this thread.

of course rhizo will mat if u dont fruit it, but why wouldnt u?


Posted by: Fungusmaximus Jan 31 03, 11:32 PM GMT
QUOTE
or does more fruits mean more yield to everyone?

Ummmmm, yea, yes it does. just cause you only get a few clusters from a casing dosent mean its gonna weigh the same as a casing full of mushie clusters. It doesnt compenstate for lack of fruit by growing less but bigger mushies, unless you only have a small surface you allow to fruit, and below that is a huge mycelium colony like this. Fruits will come from the jar opening, this should make for fewer but larger mushies.
The black area is colonizing straw dung, the lid w/mini hepa is fruiting surface


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