Isolating On Agar
|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|
|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.|
|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
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!? 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|
|Posted by: czen Dec 05 02, 10:49 AM GMT|
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,
|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...|
|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.
|Posted by: Fungusmaximus Dec 06 02, 08:22 PM GMT|
|They arent in the sleeve anymore.? 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.
Mycota (aka 6T)
|Posted by: Mycota Jan 29 03, 12:02 AM GMT|
Little teeth................Mycota (aka 6T)
|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!|
|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.
|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|
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.|
|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.
|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.
|Posted by: DirtyWOP Jan 29 03, 11:14 AM GMT|
| I still think it's better to isolate the rhizomorphic growth.....
Would stamets bullshit you?
|Posted by: Fungusmaximus Jan 29 03, 01:03 PM GMT|
|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)
|Posted by: el.jefe Jan 29 03, 02:02 PM GMT|
|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"
|Posted by: Mycota Jan 29 03, 02:56 PM GMT|
I am not saying the practice is obsolete or ineffective at all.
|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?
|Posted by: Mycota Jan 29 03, 04:33 PM GMT|
My own feeble experience.
|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.
So my ultra rich nutrient agar should produce fuzzy growth even in ideal conditions, right?
|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?
Well first off I dont agree more primordia =more even pinset.
|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|
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.
More Strain Isolation Procedures : Mushroom Documents & Papers : Shroom Glossary