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Pan-Pan-Panaeolus


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#1 425nm

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Posted 29 August 2018 - 05:25 PM

As is my habit I am starting a thread after I am several steps into a grow

 

As the title makes quite clear I'm growing some Pans. What Pans you ask? I am growing: Panaeolus copelandia cyanescens 'Hawaiian'

 

Obligatory aside regarding naming:
I'm honestly super confused as to what the proper name for the species. It seems to be a bit of a mess. mjshroomer insists that they are Copelandia cyanescens not Panaeolus. See here. But that thread is a year old and taxonomy can change super suddenly. I know PinkMenace did some sleuthing a few months back regarding proper naming but I cannot find the thread at the moment.
Wikipedia has them listed as Panaeolus cyanescens with only two references one of which is a Gaston Guzman paper WHICH is goddamn downloadable right off of the wikipage. Cannot say how nice it is to not have to chase it down separately (downside: its a scan so you can't ctrl + F the 92 page monstrosity). That paper however is from 1998 which is a million years ago as far as taxonomy is concerned at the moment (DNA sequencing has flipped the table on everything). Regardless Guzman lists:
 

C. cyanescens (Berk. & Broome) Singer [=Panaeolus cyanescens (Berk. &Broome) Sacc.;...

 

Which, I think is him sighting Singer who was sighting Berk. & Broome as the original people to name the species. Annoyingly Guzmans references section is not numbered not is a date specified in the citation sooooo I have no idea which publication of Singer he's refering to. He cites over 10 of Singer's works and none of the titles mention Panaeolus so honestly I have no fucking idea.\

 

Stamets also lists them as Panaeolus cyanescens with Copelandia cyanescens as synonym. He also cites Berk. & Broome but also fails to have a clear reference for them in his literature cited which leads me to believe these two are old old old mycologists and their original work is hard to get hold of.

 

TL;DR - Taxonomy is a mess and I have no idea. I also tried the  Integrated Taxonomic Information System only to discover its terrible for mushrooms. God awful. I am disappointed.

 

IMG 20180829 133306
 
IMG 20180829 133322

Spore print courtesy of our own feline chiropteran who I believe told me they originally got the strain from Peacefrog (I could me misremembering that).

 

I already have spores to agar and three isolates taken:

 

IMG 20180829 123519
 
IMG 20180829 123512

 

Spore germination plate seen above. If anyone thinks I've neglected a choice sector by all means please speak your piece.

 

IMG 20180829 123548
 
IMG 20180829 123542
 
A2 shown above.
 
IMG 20180829 123530
 
IMG 20180829 123525
 
A3 shown above.

Why no A1? Well that's because it contaminated and I forgot to take pictures of it before I composted the contents ¯\_(ツ)_/¯
 
I'd say A3 is looking like the favorite, given that growing at roughly twice the rate of A2.

SO! The question now is: How do I proceed? Usually with cubes I generate LC from my agar isolates and then use that LC to inoculate grain. Having perused some other folks threads I know that Pans are a little more on the delicate side of things.
Peacefrog mixes manure in with grain, wedges a master, and G2Gs it from there. See here. Considering I am working in a SAB I'm not a huge fan of doing G2G (hence my preference for LC).

Now of course I wouldn't be me if I hadn't already sort of done both of these things with a previous attempt at growing the same strain that I didn't document here and tragically fell prey to Trich. I have been sitting on the LC (standard 5% corn syrup solution with a drop of kelp fertilizer) I made for that grow for some reason. For clarity: This LC was generated from the same spore print as seen above but a different scraping of it.
 
IMG 20180829 123615
 
IMG 20180829 123611

 

Four months old at this point. Just been sitting at room temp. By no means ideal but out of curiosity I shot some into a little 250mL jar of sterile grain and it colonized! Did I remember to photograph that proof jar for y'all? Nope, sure didn't but what I did do is G2G (just grain no added manure) that business into larger jars just last night.
 
IMG 20180829 123634

 

Can't located the masking tape I like to use for labels so I was forced to use micropore.

 

In addition to that G2G with the old culture I also inoculated six 1L jars of grain (just grain no added manure). Why? Well I've been teaching a friend to grow and they bailed on me super last minute when we were meant to go through how to sterilize grain. So I had to deal with it all myself (had already been set to soak the night before) and by the time I got to it the grain was a touch ferment-y. So I had six liters of sterile grain just sitting around, wasn't sure when friend would reschedule, and I had proofed the old Pan LC.
 

IMG 20180829 133352

 

I inoculated those jars on the 22. Exactly a week ago and none of them have shown any growth. Does this match with the little proof I did? Honestly, I cannot remember. I should keep notes! Why am I so bad at keeping notes? Ugh, I am my own worst enemy. So maybe they're just being slow or maybe the fermentation produces some metabolites that are inhibiting mycelia growth. Only time shall tell I suppose.

Hopping back to what to do with my plates What are people's feels regarding LC vs wedging grain masters for Pans?


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#2 Sidestreet

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Posted 29 August 2018 - 06:44 PM

I'm impressed!  I'm pulling up a chair.

 

You and I have similar experiences trying to school friends.  They have been interested but don't seem to have the follow-through even when it's spoon-fed to them.  They just don't have the FIRE in their bellies.  I guess that's why we come here to teach people instead.



#3 onediadem

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Posted 29 August 2018 - 08:16 PM

That LC is delicious! I too am pulling up a chair.


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#4 Da-1

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Posted 30 August 2018 - 12:54 AM

I am here too can`t wait to see what you do bud :thumbs_up:

 

subbed and ready to learn.

 

 

 

Regards, Da-1   



#5 peacefrog

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Posted 30 August 2018 - 03:58 AM

I personally like wedges to agar better than lc to grain, but it should work just fine if the lc is clean and the grains have been hydrated and sterilized properly.

Pulling up a chairs well. Good luck!
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#6 PolarDBN

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Posted 30 August 2018 - 04:52 AM

Panaeolus colonize really quick. If you where so inclined, agar to bulk is completely possible. Spawning to grain is probably still preferable as always but if you get your bulk recipe right you could skip the step all together.

For bulk I used horsepoo, vermiculite, wbs and calcium hydroxide @ 3;3;1;0.25, brought upto capsity and nocked up with a wedge. Colonized 80% in 6 days.

7 days for agar, 10 for bulk... Could have fruit in under a month.

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#7 PolarDBN

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Posted 30 August 2018 - 04:58 AM

Would you mind expanding a bit how you got such healthy looking LC. Just carrowater stood somewhere out the way or was there stirring shaking involved?

Not sure what I'm doing wrong but, my LC doesn't C. ;)


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#8 ducatidave

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Posted 30 August 2018 - 10:37 AM

Panaeolus colonize really quick. If you where so inclined, agar to bulk is completely possible. Spawning to grain is probably still preferable as always but if you get your bulk recipe right you could skip the step all together.
For bulk I used horsepoo, vermiculite, wbs and calcium hydroxide @ 3;3;1;0.25, brought upto capsity and nocked up with a wedge. Colonized 80% in 6 days.
7 days for agar, 10 for bulk... Could have fruit in under a month.
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Quick question for clarification; Is the bulk sub ratio you posted volume or gravimetric?
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#9 PolarDBN

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Posted 30 August 2018 - 12:34 PM

That's volume in cups and a close approximation since I basically just wing it. PH is at around 8 when I'm reddy to sterilize.



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#10 425nm

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Posted 31 August 2018 - 01:01 AM

My goodness, I was not expecting so much interest. Well here's hoping I don't screw this up!

 

I'm impressed!  I'm pulling up a chair.

 

You and I have similar experiences trying to school friends.  They have been interested but don't seem to have the follow-through even when it's spoon-fed to them.  They just don't have the FIRE in their bellies.  I guess that's why we come here to teach people instead.

 

I should say, despite my friend bailing on me last minute it is the only time they have ever done so and have actually be a very diligent student : P They're something much closer to a metal worker than I so the flow of knowledge goes both ways.

Learning from your friends is the best.

 

That LC is delicious! I too am pulling up a chair.

 

Thanks Diadem! Its oooold but surprisingly still active.

 

I personally like wedges to agar better than lc to grain, but it should work just fine if the lc is clean and the grains have been hydrated and sterilized properly.

Pulling up a chairs well. Good luck!

 

Speak of the Frog and they shall appear!
Good to know. I have not yet decided upon my overall plan of attack.
 

Panaeolus colonize really quick. If you where so inclined, agar to bulk is completely possible. Spawning to grain is probably still preferable as always but if you get your bulk recipe right you could skip the step all together.

For bulk I used horsepoo, vermiculite, wbs and calcium hydroxide @ 3;3;1;0.25, brought upto capsity and nocked up with a wedge. Colonized 80% in 6 days.

7 days for agar, 10 for bulk... Could have fruit in under a month.

Sent from my SM-G955F using Tapatalk

 

Would you mind expanding a bit how you got such healthy looking LC. Just carrowater stood somewhere out the way or was there stirring shaking involved?

Not sure what I'm doing wrong but, my LC doesn't C. ;)


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My go to LC recipe is a 5% volume/volume solution white corn syrup with 2-4 drops of kelp fertilizer. I do however have a stir plate/stir bar. Which is wonderful, you can buy them for brewing or make one yourself (I believe a computer fan is what is often used) if your so inclined. More on that below!

Been a busy-ish day. Managed to get myself some fresh high temp RTV silicone so that I can finally affix some nice new synthetic filter discs I got two weeks ago:

IMG 20180830 162954


I also picked up a thrift blender that I am hoping to turn into some sort of DIY Eberbach blender but that may get its own thread depending on how complicated the re-engineering gets.

It is fortuitous that PolarDBN should ask about LC as I was planning on doing some tinkering with my recipe. I've been doing some casual reference mining of Paul Stamets's "Growing Gourcmet and Medicinal Mushrooms" and came across a paper on optimizing liquid culture for shiitake (quite different from Pans I know but bare with me):
 
Yee How Tan and David Moore. 1992. "Convenient and effective methods for in vitro cultivation of mycelium and fruiting bodies of Lentinus edodes" Mycol. Res. 96 (12): 1077-1084
 
The authors compare chemically five different media: 2X defined growth media (all nutrients from chemically pure sources; One media autoclaved vs filter sterilized), potato dextrose broth, the yeast extract/malt extract/glucose/peptone mixture  and molasses media (3% w/v).
At the 14 day mark the molasses media had the highest biomass however by the 28 day it had the second lowest (the chemically defined media did the best at 28 days).
 
This got me wondering. My corn syrup LC isn't altogether that different from the molasses only the molasses should actually have more in the way of minerals and vitamins as it is less processed. So I decided to give it a try.

IMG 20180830 193251

 

I usually make 300mL LCs in 500mL jars. I tried the 3% w/v but it yielded an incredibly dark solution.

 

IMG 20180830 191903
 
IMG 20180830 194721

 

So then I tried a 5% v/v solution that was 3/4 corn syrup and 1/4 molasses and it was still super dark.
 

IMG 20180830 194814
 
IMG 20180830 195441

 

So I finally settled on a 5% v/v solution where ~14mL of corn syrup and ~1mL of molasses in order to get the desired clarity.
 
IMG 20180830 195751

 

How much benefit am I getting from only 1mL of molasses? Hard to say but probably not a huge amount. Perhaps I'll try a potato dextrose broth next  go round.
 

IMG 20180830 201006

 

Either way I ended up with one LC of my standard recipe (5% corn syrup with a few drops of kelp; on the right) and 5% v/v corn syrup plus a little molasses (left).
A note on the silicone gaskets: I really only use the things for LC. They're so thick that you have to crank the lids down pretty tight otherwise they are liable to make lids pop off when you're shaking grain :/
I also have some syringe filters I've been meaning to fit to some LC only jars.

While I was in the cooking mood I decided to whip up some agar too. More so for other grows than the Pans but there is a sector I pan to pull off the germination plate that looks like it might be forming primordia. I tried to get a picture but the combination of glare and condensation on the lid foiled me.
 
IMG 20180830 203955
 
IMG 20180830 204425
 
Just standard PDYA. Nothing fancy, though I do my glass petris as no pours because no one wants to pour hot agar in a SAB while also trying not to drop glass dishes on one another.
 
IMG 20180830 205214

All this being said I still don't have a plan of attack regarding the Pans. I will probably make another LC but I may also wedge some grain. Stay tuned!

 


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#11 Sidestreet

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Posted 31 August 2018 - 05:39 AM

I do my glass petris as no pours

 

Cool, I hadn't considered that I could do my glass petris as no pours.  That makes them a lot less annoying.  :biggrin:


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#12 Cuboid

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Posted 31 August 2018 - 08:53 AM

I do my glass petris as no pours


Cool, I hadn't considered that I could do my glass petris as no pours. That makes them a lot less annoying. :biggrin:
When I've done this in the past I had a lot of trouble with them boiling over and getting agar all over them. Any tips on how to do it without the agar rebelling and escaping the Petri's? Are yours the usual 15mm high or extra deep?

#13 PolarDBN

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Posted 31 August 2018 - 01:19 PM

I do my glass petris as no pours

Cool, I hadn't considered that I could do my glass petris as no pours. That makes them a lot less annoying.
When I've done this in the past I had a lot of trouble with them boiling over and getting agar all over them. Any tips on how to do it without the agar rebelling and escaping the Petri's? Are yours the usual 15mm high or extra deep?
Found that the internal pressure in the pressure cooker needs to decrease slowly and naturally to atmospheric. I cover mine with insulation.

When it hits atmospheric pressure (need a pressure gauge to verify) it's best to open the valve so the pc doesn't create a vacuum... And let it cool naturally from there.

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Edited by PolarDBN, 31 August 2018 - 02:39 PM.

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#14 PolarDBN

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Posted 31 August 2018 - 01:24 PM

Appreciate the detailed response 425nm. Would love to get some LC going purely for experimental reasons. Previously built a stir plate for home brewing.

Pans thus far ate everything I threw at it... Seemed to colonize wbs really fast, might wanna give that a try as the grains.

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#15 onediadem

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Posted 31 August 2018 - 02:25 PM

I am glad you are cooling that down slowly. It has the potential for quite a mess. You can actually lower your burner to warm to keep it from cooling down too fast also and cover the top with a towel. It usually takes about an hour to zero out the pressure (depending on your stove) and from there you can turn off your burner. It is a much slower cool down and avoids a vacuum scenario. Doing this also keeps your bags from blowing out sidewalls.


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#16 PolarDBN

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Posted 31 August 2018 - 02:36 PM

That's a rather eloquent solution ODD.

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#17 Cuboid

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Posted 31 August 2018 - 02:50 PM

Thank you both. I'll try cooling slower next time. Burner to low until pressure equalised it will be.
:)
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#18 bezevo

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Posted 31 August 2018 - 04:11 PM

i was told  i could not PC  my glass plates with AGAR  ,as i don't have a flow hood i  would poor AGAR into sterile plates in SAB ,

Doing some plates  this way would remove one more opportunity  for Contams   .  

It just adds extra hours with real slow cool down .......  but isn't this hobby about patience ...?

 

i think will  do both ways next time see  if there's a difference   on contamination  rate ....

All pours transfers/inoculation will be done in a SAB


Edited by bezevo, 31 August 2018 - 04:15 PM.

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#19 425nm

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Posted 31 August 2018 - 05:15 PM

Mmmm my strategy of multi-quote is not going to work here.

Regarding no pours and sterilizing agar in general. Definitely letting your PC return to atmospheric pressure gradually makes a difference. I really wouldn't advise bringing it down rapidly at any pressure above 5psi regardless of what you're doing. Then again I don't know how prestos or AAs with a toggle valve work visa via depressurization.

This is what I'm working with:
 

IMG 20180831 144251

 

Rocker weight and gauge. It actually came with a toggle valve (which I still have) but I swapped it out because I like to be able to hear that its running (helpful when roommates accidentally turn the wrong burner off).

As for letting it come down in pressure I just turn the burner off completely and let the pressure drop on its own. A lot of the time I let it cool over night. If I've got a lot of shit to PC I will occasionally vent it once its under 5psi (not something I do with plates though since lifting out still liquid plates of agar is no fun). Mine will hold a vacuum for about 12 hours after its cooled but not passed that. Maybe because its old? Maybe because I don't lubricate the metal to metal seal as often as I should.

 

That is only part of the equation though: Don't fill your dishes past half way
Technically the upper limit is around 3/4 full but that's dependent on the geometry of the vessel you're using. Like you can definitely autoclave 700mL of agar in a 1L conical flask without it boiling over.
This being said you really don't need a super thick layer of agar in a petri. The fungi are only going to be growing on the horizontal axis so no real need for excessive depth.

You can usually bank on needing about 25mL/90mm plate (your standard size of petri).

I wish I had a wire caddy for my dishes so that they could just all live in a stack and be carried more easily. As is I just lift them out (assuming they've set) carefully being sure to get a firm grip on top and bottom. Then I just store them in a iso'd plastic tote in a cupboard. I don't even put a lid on it so that some of the condensation will dry out (how I wish I had a flow box. One day!). Its not an ideal way to store them by any means but the fridge is a no go. Occasionally some will contaminate before i get a chance to use them so I try to just make as many as I need at a time.

 

 

Appreciate the detailed response 425nm. Would love to get some LC going purely for experimental reasons. Previously built a stir plate for home brewing.

Pans thus far ate everything I threw at it... Seemed to colonize wbs really fast, might wanna give that a try as the grains.

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Interesting, how fast are we talking? As the isolates I've taken thus far seem to be a bit on the slow side. Maybe that's just because I'm used to cubes? The cultures cabinet is a little on the cool side at the moment, around 22C (I'm sure they'd appreciate another 2C at least). I do believe the grain I have at the moment is in fact WBS.


Edited by 425nm, 31 August 2018 - 05:18 PM.

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#20 PolarDBN

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Posted 31 August 2018 - 05:22 PM

Pan pan panaeolus indeed. Thought we might all enjoy this information.

Again in this particular sample Panaeolus cyanescens shown to contain more active alkoloids than P. azurescens.

Share its greatly varied, still that's impressive!

https://www.semantic...7c8876b72da1ef93b2c47ae994cbf6a9e353c6ccf3f92b9.jpg

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