Panaeolus Subbalteatus on Agar

[smartshop]

I have been looking around on this site and elsewhere for information on growing panaeolus subbalteatus. There is some information but little personal growlogs, so i thought it might be interesting to share my experiences in trying to grow this one. (since this site doen't have a spelling checker you might find some "dutchisms" in my written english).
I had previously inoculated about 6 agar plates with some sporeworks spores. Just one grew mycelium (and some mold..). Then on may 22 (day 0) I transferred some pieces of agar from that plate to 6 new plates. The picures show first two pictures of the original plate and then the six isolate plates at day one.

[smartshop]

Well the pictures are a bit poor quality but this is day three. The transfer did not get rid of the mold. Plate F and B have clear spots of green mold. Plate A and B have some tiny blackish mold but grow quite vigorously. Plate E has no sign of mold. C and E have a strange fenomenon in that there is a center of mycelium and then a circle nothing and then a "halo" of myciul around this. The myceium is not very rhizomorphic , but Stamets says it is normally "cottony" myceium. So i am hoping i can clean up plate A and B with antibiotic and maybe salt?

[crazy1]

You can also try a "grow through" cleaning. Inoculate the bottom of the agar, and most likely the mycelium will grow through it and the contam will grow at the bottom. Then you must only transfer the top part of the agar to your next substrate.

[the_chosen_one]

it's true. you don't see a lot in regards to these. probably the mild potency is to blame. PF always used to ask me "why the hell do ya wanna grow those for?" Pan Sub is one of my favorite shrooms to grow from wild spores though. once you get them cleaned up, you'll actually find them quite easy to grow. much of the standard cubie teks can be used and fruiting temps are only slightly lower.

that myc is crazy huh? you may even get to a point where you will wonder if your not cultivating cobweb mold.

not to tread your thread, but here's a pic of what you have to look forward to. this was simply some left over millet innoculant that was cased in 50/50 mix.

many good vibes your way! thanks for posting this!

[Hippie3]

this site doen't have a spelling checker

au contrair, mon frere.
see the little above the quick reply box ?
that's the spell checker.

cleaning on h2o2 agar would be an easy way..
i'd recommend that.. just transfer your mycelium to the new h2o2 agar dishes..
good luck

[Bobcat]

You can combine teks as well. Make your h202 agar and pour it hot (after some cooling time) over the contam'd plate. You can use Lazlo's salt tek as well here. I've combined all three for great results. For any plates that have mold on them, you'll want to transfer the good mycelium, which should pop through first, as soon as you get a nice little spot. After a very shallow transfer, you ought to have a clean culture.

[smartshop]

Well i oversaw that hippy
For the rest i would be a little worried that the mycelium would have a hard time growing though the agar top. Does this take a long time? And do you first take a small piece of agar wich you transfer to a new plate wich you cover with a new layer of agar, or does one pour some agar over the plate as it is now?
The chosen one; nice to hear that you have successfully grown this species. What medium would you recommend? I had thought of casing some rye berries or popcorn.
I hope my glass petri dishes arrive soon enough to go on working on this. Until then i will have to pour my agar in small jars (wich lend themselves less well for taking pictures).
Thank you guys for responding to this thread

[the_chosen_one]

Originally Posted by smartshop

Well i oversaw that hippy
The chosen one; nice to hear that you have successfully grown this species. What medium would you recommend? I had thought of casing some rye berries or popcorn.

well, most of my success with it has been outdoors, but once in a while i get an urge to do an indoor grow. usually small.
the only drawback to indoors is that the thin myc leaves the grain very exposed to contams. much like any Pan species. if you case grain, be prepared to fight some trich at some point.
as far as what grain, rye works well. millet...everything fruits on millet for me, one of my favorites! never tried them on popcorn. may work well though.
another trick with these is to case them right on top of a bed of wet perlite. spread a thin layer of casing on that, then spawn, then more casing. give them time too. could be a month before you see any signs of fruiting.
spawning to horse poo works great for outdoors.
i'm pretty excited to see this. i've always wondered about Workman's strain, just never gotten around to it.

[smartshop]

OK. So its 28 may. Six days after inoculation. I made some pictures of the plates (really need a better camera).
Plate A is doing well but has some blackish circle contamination just as plate B.
Plate B is growing just as vigorously as plate A, you can see that there is some blackish contamination (seen as a circle in the counterlighted picture).
Plate C looks about as much growth as one would find pubic hair on a porn actress...
Plate D grows reasonably fast but has some black and green growth.
Plate E is growing half as fast as A and B. It does have a circle of lesser growth but no visible black stuff.
F has some green growth just above the G in "Groen" (green).

I just made four flasks of agar:
1.
150 ml H2o
6 grams rice meal (because the pressure gauge on my pc is broken and i don't want caramellisation)
3 grams of agar
2 1/4 teaspoons of salt
10 mg of gentamicin (i hope, i stored too warm and it became a sticky goo).
bit of a B vitamin pill

My little paper indicator strip tells me the above is PH 7

2.
Same without the gentamicin

3.
Same as 1 but with 2,2 grams of agar (for top layer sandwich)

4.
Same as 2 but with 2,2 grams of agar.

3 and 4 are for a salt sandwich test on green mold in a cubensis agar culture

Wish me luck... this is the first time i will be trying gentamicin and sandwiching.

[the_chosen_one]

from what i can see, this one looks pretty normal.

in one of the other pics, that ringing effect where there seems to be almost a dead zone in the middle is fairly normal too. often the myc will fill that space in with age.

this is great! many good vibes to you!

[smartshop]

Well that plate has some green mold just under the little circle i made on the lid. But i could still try to work with it. Yesterday i only used plate B to do some sandwich and non-sandwich transfers on antibiotic salt agar. The sandwich tops came out pretty thick though (probably 4 millimeters). I am worried that there will not be enough oxygen for the mycelium.
I also worked with some cubensis mycelium with green mold. I am curious to see if simple salt agar will actually select this contam out. I did take some heavily infested pieces to transfer and turned of the flowhood for the procedure, so i am not too shure how realistic this is.
Hope that my glass plates come soon, now i have to work with make-shift plates (small jars).

[Hippie3]

good luck

[Lazlo]

Moldy mushroom mycelium can really be a pain to clean up with certain species. When using the sandwich technique, I apply a tiny drop of agar to a new plate, allow it to harden and then I apply the contaminated transfer. The drop doesn't even have to cover much of the bottom of the plate. Just enough agar so the transfer can grow. Then as soon as I see growth from the transfer to the agar, I zap the transfer with piping hot agar. Just enough to lightly cover the transfer. Then after a day or so mushroom mycelium will start to appear on the surface of the new agar coating. Then hit it again with hot agar and again until the dish is nearly full with agar. See what I mean? This way you can hit the moldy transfer with piping hot agar around 8 or so times in one dish, without having to transfer one single time to a new plate. The salt only seems to be a help when using the sandwich technique when molds are the problem. Bacteria absolutely hates the salted agar anyway it's used. Good luck.

[Orchidman]

I grew subbalteatus a few years ago and tried to establish it in my garden in Toronto as it can withstand hard freezing. I had trouble with contams and I am not as dedicated as you are and I just threw the whole thing out into the garden bed but it never survived.

I did find out that it loves hay and so I found "Timothy's Hay"rabbit food. It comes whole like straw or ground and compacted into pellets and mixed with alfalfa which it loves. The subbalteatus loved the soaked pellets which break down into a finely ground meal about 1/8". However it contaminates easily so if you can be sterile you should try grass for the subbalteatus. Maybe soaking the pellets and hay in reverand tryp's Sunlight Antibacterial dish soap with acetasalacylic acid could keep it sterile. I also composted the soaked pellets with potting soil to use as a casing. The meal composts in only a week or two and will give you a great casing loaded with grass nutrients. Composted cattle manurer from garden centers is good to add to the casing as well.

Also for your contaminated agar I will suggest an experiment. It seems like according to reverand trip the mycellium of cubensis can be exposed to the antibacterial dish soap and not only withstand it but thrive on it. So why don't you soak your plate in soapy water, to destroy the mold and leave a residual coating of acetasalacylic acid to prevent regrowth of the mold. Maybe soak one of the contaminated plates after you have transferred everything off. See if the dish soap will kill the mold. I have no idea if this would work or if the subbalteatus could withstand the dish soap technique. It's just one more thing to try.

[smartshop]

Well my worries have turned out to be uneccesary. The plates look pretty good.

Plate 1 Looks healthy (salt gentamycin sandwich)
Plate 2 Looks healthy (same)
Plate 3 Still has the black contamination (no sandwich on salt gentam.) (three pics)
Plate 4 Looks healthy, but the growth isn't very fluffy as the others (sandwich salt/genta).
Plate 5 Looks healthy (agar wedge in stead of just the mycelium in salt/genta sandwich).
Plate 6 Looks pretty good but some of the black contam is still visible (only on the agar transfer wedge) (just salt agar no sandwich).
Plate 7 Looks pretty good, but i am not sure if i can still see some contam in the middle (no sandwich on salt/genta).

Well this seems to go well. Now i just really need some new agar dishes to transfer to.
Also the rice meal agar seems to be working just fine.
Paneolus mycelium is pretty fast; seems faster then cubensis (and no need for an incubator, this was done in roomtemperatures of about 20 celsius).

[smartshop]

And here two pics of cubesis on salt agar and salt/Gentamycin agar just to show that green mold/trich isn't stopped if there is no sandwich (and probably not even if there is).

[crazy1]

Have you thought about trying some H2O2 in your agar? It does have an adverse effect on trich.

[Hippie3]

pix are not so great.
direct sunlight kills trich,
might test it just fyi .

[the_chosen_one]

Originally Posted by smartshop

Well my worries have turned out to be uneccesary. The plates look pretty good.

Paneolus mycelium is pretty fast; seems faster then cubensis (and no need for an incubator, this was done in roomtemperatures of about 20 celsius).

you'll get it

yes, very fast. it likes to run fast and thin, then backfill. that's why contams are such a problem with it. at least in the agar and grain stages.
as far as temps it's very forgiving. however, fruiting is typically a bit less so. although my last indoor grow was done using a standard cubie fruiting tek. that particular strain seemed very heat resistant.

[smartshop]

Well only number 6, 7, and 5 don't look contaminated today. All the original plates and all the other salt/gentamycin plates had bad blackish mold and i threw them out.
I think i will try Lazlo's idea to pile layer on layer until a clean culture emerges.
The cubensis with trich was just a test of the salt tek against trich. I have clean cultures to work with so i threw the diseased plates out.
Of course if i do get a clean culture of Pan Subb i still have a small chance of it being a fruiting strain. But i think i am learning quite a bit on the way.

[the_chosen_one]

Originally Posted by smartshop

Of course if i do get a clean culture of Pan Subb i still have a small chance of it being a fruiting strain. But i think i am learning quite a bit on the way.

very small chance. i've never seen one personally.

pan sub is a good teacher. once you get it clean, it'll treat ya right

[smartshop]

Well i think i am getting somewhere.
Of the first transfers only number 5,6 and 7 didn't contaminate. So i made a second transfer from 6 and 7 to some nice and handy sterile urine containers for drug screens (monday i will finally get some petri dishes). I was planning to do some hot agar pours on these. They look uncontaminated now, but that happened with the first transfer also, so i don't now if i should still do that. One container looks pretty good. I am hoping i can transfer that one to rye or popcorn at some point.
When i have my petri dishes i think i should also go back to the sporeprint and start right away with hot pouring agar until the mold gets left behind. I guess i probably need to try 10 different multi strain grows before i can hope to see a fruiting strain.
The chosen one:
"very small chance. i've never seen one personally"
Do you mean that any grows you now of have been done with clones from wild specimens, not started from spores?

[smartshop]

..and some pics..

[the_chosen_one]

Originally Posted by smartshop

The chosen one:
"very small chance. i've never seen one personally"
Do you mean that any grows you now of have been done with clones from wild specimens, not started from spores?

nah. i'd say that about 98% of the work i've done with these has been from wild spores and it's fruited every time. even after agar work.

your chances of getting a non-fruiting strain are a bit higher on agar because as you jump clean samples to new dishes you will be at least partially isolating, but i'd be suprised if you corner a non-fruiting strain. i think the lack of clear sectoring like psilocybes may have some role to play in this.

[smartshop]

Well i think the hot agar pour on the four urine drug screen containers has more or less killed the mycelium. There are two containers that show some very weak questionable growth now, but it has taken them very long (possibly because of too thick a top layer? caramellized sugar?). I will give them some more time.
Finally my petri dishes have arrived so i transferred from my last healthy plate (nr. 5, wich is descendant from plate B) to ten new plates. If this grows out healthy i am going to go to transfer to grains.
I also put some spores on two new plates.

[smartshop]

So far the spores have done nothing at room temperature. Might try a little warmer.
The ten agar plates with the transfer from plate 5 are doing just fine. I will transfer them to rye today.
When i did my last transfer i also put some mycelium from plate 5 on some bird seed, just to see if it would grow without contamination. It does! This is fast growing mycelium. Below is a pic after just four (or was it five...) days of growing.
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