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Offlinespitzenkorper
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pan cambo agar- myc turned black?
    #24366883 - 06/01/17 03:07 AM (6 years, 9 months ago)

I am cleaning up some old spore syringes on agar. I have worked in some plant pathology and microbiology labs over the years and have some experience with agar, but this is my first time attempting to get psilly about it.
I'm actually cleaning up a bunch of stuff, but what i am curious about right now is Panaeolus cambogenensis "Suphanburi Goliath", on agar. i has a nice fluffy bit of myc pop up, and got excited and did some transfers. a few days later, i looked at the original plate, and the epicenter of the mycellium was turning dark. not on the surface, but in the agar. its kind of green, then goes pretty much black. it forms in strands, and looks like it is the same myc, maybe dying? i do not see any sporulation happening. I am cleaning up like 20 different sets of spores right now, and have all kinds of contaminates going on, but this is unique.
on of the tansfers never grew, i dont remember if i was using flame or alcohol sterilization, but im guessing that one the tool was too hot or had excess alcohol and killed the myc. a second transfer looks awesome, im so excited looks like some thick fluffy healthy myc. the third transfer is dark like the first. the dark starts from the center, and grows out following the myc. the edges of the mycellial ring are still fluffy. i just took more transfers this morning, from all three with myc, we'll see what happens.
is this some green/black contaminate? or is it my myc dying? what would cause the myc to die so quickly? Multi spore competitive strains killing each other off? how do i avoid this dark fate on these new transfers i just made, how do i keep the one healthy looking one looking healthy?? how do i even know this is pan myc lol??

wish my camera was better, these pics dont really show it all that well. the first 2 pics are of the original plate, then 3 more dark ones are of one transfer from the original, and 2 cleaner looking pics of the other transfer also from the same original plate. i took 3 more from each of these this morning, will post pics as i see growth.





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OfflineChefButtes
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Re: pan cambo agar- myc turned black? [Re: spitzenkorper]
    #24367295 - 06/01/17 09:03 AM (6 years, 9 months ago)

To me, it looks like there is a fuzz going over the mycelium that is sporulating black spores all over those plates. Maybe wait for someone elses opinion, but pretty sure those are fucked.

Just to be clear, mycelium isn't unique to these mushrooms we love. There are a bunch of types of creatures that also grow with mycelium, but then it starts sporulating and it's clearly different.

Edited by ChefButtes (06/01/17 09:08 AM)

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Offlinespitzenkorper
dabberwook
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Re: pan cambo agar- myc turned black? [Re: ChefButtes]
    #24367672 - 06/01/17 11:50 AM (6 years, 9 months ago)

Word thanks for your input.
These pics really suck, looking at them again.
I agree that there deff is potential that it's all contaminated, but if that's the case, Id like to know a genus for the contam and try to learn more, and see if it is isolatable for confirmation.
I'm not completely convinced though. If it is contamination, it is unique to this set of Goliath plates I am working. I have a bunch of other projects going ATM, and probably have close to 200 other plates. Most are pretty contaminated, but there's some growth n such I'm working around. Common contain I encounter in my workspace CONSISTENTLY are what looks like Penicilium spp., Aspergillius spp., Trichoderma spp., and Rhizopus spp., as well as some creamy bacterial goos, sometimes tinted yellow, sometimes tinted pink. Sorry, I don't know bacteria too well. But all of these contams have characteristic morphology, and and I can ID much of it before it sporulates. And although it is prevalent, I am able to clean around it. Again, I'm no expert or anything, but I do have some lab experience, and know how to make educated observations, etc. this line of Goliath plates is doing something different.
I tried inoculating several plates from this same syringe of "Goliaths" initially, but so far this line is the only set to show growth. So I do not have any other comparisons. I am also cleaning up some pan tropicalis, and have made one set of transfers, and waiting for it to grow out. When it gets a lil bigger, I can compare morphology and see how that goes. But no signs of this blackness showing up so far on tripicalis. Only on the Goliath.
So my thought on the black Goliath growth are either:
  1.it is imtimitqtely imbedded with some other mold that has this coloration, and the black myc eventually outcompetes and dominates. But there is no obvious signs of sporulation, at least not in the same way as the other contams listed above.
2. There is some dark/blackish bacteria growing with the myc. I'm not as familiar with bacteria, I don't know of any that appear this dark, but I'm sure some exist. The black spreads outward, through the Ava, but doesn't goop up into clear colonies on the surface. It just seems more fungal than bacterial, in its habit and how it's spreading, etc.
3. It could be dying myc. I'll admit, I did not have any potatoes on hand when prepping my plates, so I just kind of winged it. I also intentially slacked slightly on the sugar and agar ratios and added a bit extra water because a I have several old spore prints I'm cleaning as well, so I figured I'd prep things on the wet side so that spores would rehydrate effieciently. I boiled some wheat berries and amaranth seed together for a bit till things got starchy, measured out about 600-700ml of this liquid, added about 8g agar powder, and oh idk about 5-7 g sugar (evaporated cane juice), I've dont this 2 or three times, not too strict on measurements. So old if there's just something about those potatoes that makes them great for culturing, or if any source of starch should suffice. But maybe my plates are lacking in so me nites.  The other thing is that these Petri dishes are like super tight fitting, practically seal shut. I can casually pick them up and drop in on the ground and chances are it will stay shut. So there might be an issue of limited oxygen that is causing these plates to crash out. Temps have a bit low too for pans I believe, beetween 65 and 75 F generally. Would low temps cause the myc to crash, or would it just go dormant? I thought one could store plates and culture slants for years in refrigerated dormant state.... does this depend on the species? Are pan Cambos just intolerant of temps below 65F?
I'm trying to figure out if I should keep transferring, if I should prep plates differently, if I should put these dark plates in the fridge try and stall growth, or just let it all keep growing and see how it develops.
One final note, those last 2 pics don't show it well, but there was not any of the dark/blackness that I am concerned about on that plate yesterday, looked like nice fluffy myc. Where I took some myc from this plate for transfers, it now appears to be turning dark. Which also leads me to think it has something to do with damaged myc.

I'll see if I can't take some better quality pictures. Maybe I'll sacrifice one plate and take some pics with the lid off.

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Offlinespitzenkorper
dabberwook
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Re: pan cambo agar- myc turned black? [Re: spitzenkorper]
    #24377044 - 06/04/17 03:25 PM (6 years, 9 months ago)

Sooo, I think it may be something to do with UV, in case anyone's following and curious. I was not incubating in the dark, just in a SAC with natural day/night cycles, in a darker room with curtains, but certainly still light.
I put the more established plates that had turned dark in the fridge for the past 2 days or however long it's been; those look like there's possibly new growth, growing over the dark, so it it's white on black looking grayish. Fairly uniform, tho, not like some new colony is competing for the space.  Bringing them back into warmth.
I left the newer transfers with little to no growth on them in a dark cabinet higher temps between 75-80 F or so, and they have all taken off, almost whole plates are covered, and no blackening of mycelium as had been seeing. You can see where it did almost start to blacken before I put in the dark, but all looks robust white n fluffy now.
Gonna make few more transfers on to some better quality media, and will post pics.

I mean it's not completely conclusive... but I did not realize the myc was so sensitive to UV. Has anyone else ever experienced this before? Is this specific to pans maybe cambos or even just Goliath's? I have psilly plates going, not experiencing this blackening problem. Noticing a definite increase in growth rate when in dark conditions,  but not noticing explicit tissue damage from light with the psillys.

Edited by spitzenkorper (06/04/17 03:50 PM)

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Offlinespitzenkorper
dabberwook
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Re: pan cambo agar- myc turned black? [Re: spitzenkorper]
    #24404991 - 06/14/17 11:17 AM (6 years, 9 months ago)

Ok folks, heres a bunch more pics. this is the 4th generation i think on agar...

the 4 generations:
1. the original sample,
2. then i made 5 new plates from this (one never grew).
3. from each of these 5 plates, i made 3 transfers
4. from the best of each set of 3, i made one transfer.

the pictures below of gens 3 & 4.
the pan myc moves quickly. generation 4 is at about 30 hrs from transfer, and i can check the dates to be more exact, but id say the myc colonies the whole plate in about 5 days.
i am still experiencing this darkening effect of the myc. i searched around a bit and found some posts from folks saying that this can be common to pans on agar.
i was thinking about adding some manure to my agar mix, and see if that can produce a more robust myc. im not sure if the darkening is a negative sign, or if it is just part of its phenotype/ result of environmental conditions. most of the dark is blackish, but check the flipside pic- not sure if the pic does it justice, but the whole agar media is stained sapphire blue... pretty beautiful i must say.

soo im considering whippin up some manure agar. my biggest issue with this is that i have not be PC'ing my agar media, just doing extended boils on the stovetop. i get a little bit of contams, but less than 10%, and since i am doing such dirty cleaning right now, its not even the biggest issue. everything gets dirty as soon as i open a dirty sample, so its really just a series of races until start getting more clean samples. these pans are my only clean batch going for the moment. i feel like boiling manure agar mix is not going to kill all the bacteria present. but then pasteurization is often used for manure anywhere, not sterilization, so might boiling suffice? i know stovetop boiling is not ideal, but it is working for me right now with standard PDA... dont patatoes have a bunch of bacteria living on the skins and such? i scrub them before i boil, but i mean its bacteria, shits persistent. but i am not experience serious contams from potatoes alone.

will manure agar make a noticeable diff in my plate growth?

how many generations should i go with this? i didnt realize i was actually on the 4th. the 4th generation is actually what i would consider the fist nearly "isolate" generation. some of the 3rd generation looks pretty uniform, but not all, and its all overgrown anyway, so where going from the 4th gen.

my plan was to take 2 more generations from this isolate, meaning let 4th gen plates grow for another day, take a sample to innoc a 5th gen, let that grow for 3 days, thake a sample from gen 5 and start gen 6. so going from 4-6, i would have 3 clean isolated generations. i can keep gens 4 and 5 to pull from later and start new lineages. gen 6, i plan to take a sample from, start a gen 7, and store gen 7 until next time. i then plan to cut up gen 6 and throw in a jar rye/wheat berries. this jar will be be a mini master, and will be used for grain to grain transfers, one of which will be an official master. master goes into storage, the rest go to fruit.



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Offlinespitzenkorper
dabberwook
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Re: pan cambo agar- myc turned black? [Re: spitzenkorper]
    #24405041 - 06/14/17 11:43 AM (6 years, 9 months ago)

I have worked in microbio and plant path labs, but i have actually never done agar work for this hobby before, or fruited pans before, so i would appreciate any tips for what to do next. i had some nice jars of tropicalis going a few years back, but long story short, they didnt fruit. i have done a few batches of cubes in the past, but am really fascinated by all the other species, so thats really where i commit all my efforts..
i did a bunch of atl7 and tampanensis sclerotia, and got the leftover spawn to fruit outdoors, which was super cool. but im not gonna lie, i dont really know what im doing with these pans.

i know they need manure, looks like they might want it in the agar. when i make grain jars, should i supplement them with manure? say ii fill a jar halfway with hydrated grains, then toss in a tablespoon or two of manure, will that affect growth? can pans be started on soley grains, then spawned to manure for fruiting? that is what i tried with the tropicalis a few years back, and jars were nicely colonized, i dont think i manured them. when i fruited, my bulk was too dense, manure was too composted. plus my house was old and totally infested with like 7 different colors of mold that would strait grow on the walls in the winter. could not fruit anything there.

i just collected some field dried horse paddies, and this looks like wayyy better consistency. i wouldve liked get some cow manure, but the cows are lil farther away, and havent been able to coordinate that whole adventure yet. it sounds like most folks like horse manure better, but i think that is in reference to cubes. it is just a hypothesis, and i know its been discussed before, but i feel like the pans prefer bovine dung to equine. from what ive gathered, it sounds like the tropical pan species are more common in bovine dung within their native range, and i dont think it is mere coincidence, bovines digest their food more completely, which may produce a more suitable substrate for tertiary decomposers such as pans. horse dung has a bunch of hay leftover due to less stomachs and less effective digestion, leaving nice matter for primary decomposer such as cubensis to feed on.
but i have horse manure, so it will have to work for now.
The manure field dried, should i just break it up and add it to the mix, or should it be treated before put to use?

i want to move these agar plates to grain soon. should i just prep standard grain jars, and only worry with the manure in bulk sub? or do i need to mix some manure into the grain jars as well?

any good bulk sub recipes out there? i can search around the forum, but its always nice when someone gives a direct answer. i have this horde manure, cococoir, verm, coffee, gypsum, both calcium hydroxide and calcium carbonate, hort sand... im thinking something like a 1:1:1 manure:coco:verm, with a healthy dash of coffee gypsum and calcarb. case with sand verm calcarb maybe a bit of gypsum and a tiny dash of calhydo...???


biggest question right now, cause im gotta to PC today, SHOULD I ADD MANURE TO GRAIN JARS? if so, in what proportion to grain mix?

THANKS!!


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InvisibleLizardWizard
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Re: pan cambo agar- myc turned black? [Re: spitzenkorper]
    #24405843 - 06/14/17 04:55 PM (6 years, 9 months ago)

do not mix manure in grain jars, that's asking for serious trouble. Don't have time to read your whole post, sorry.


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Offlinespitzenkorper
dabberwook
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Re: pan cambo agar- myc turned black? [Re: LizardWizard]
    #24405863 - 06/14/17 05:02 PM (6 years, 9 months ago)

sweet thanks! that keeps things moving for now...


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Offlinespitzenkorper
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Re: pan cambo agar- myc turned black? [Re: spitzenkorper]
    #24405874 - 06/14/17 05:05 PM (6 years, 9 months ago)



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