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OfflineZah
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Bringing the dead alive - a PANdora's box?
    #11367235 - 11/02/09 05:56 AM (8 years, 21 days ago)

This is my first post on the board. Thanks all for the nice forum on which I have and will spend many hours of reading.


Someone I know has been eating mushrooms in the past, but hasn't done this for at least 8 years now. Recently he discovered some dried mushrooms he didn't use at the time. They must be at least 8 years old. It concerns some type of Pan cyan, called 'Hawaiian' in the Netherlands.

So here's the deal:
He now wants to have a shot at growing these shrooms, starting from this material. He has a special relation with these, and likes a challenge. Although he knows sterile technique, he's never grown any before.

First step: Getting the mycelium to grow on agar.
OK, this is what he has learned from the forum about agar: too rich means a lot of contams; too high a temperature means a lot of contams. But: too little nutrients and the 8year old tissue won't grow.

So he chose to make some dung agar and variations.

Some aged cow manure was collected from a field, cooked for 2 hour (1 part manure, three parts water) and allowed to cool overnight. Being afraid that this would not do the trick, he added some finely sliced potato to another batch (2 small potato in 1L total).

The shit was filtered through some cheesecloth and used to prepare the plates:

-manure 100%
-manure potato 100%
-manure 50%
-manure potato 50%
-manure 50% yeast extract tryptone
-manure 50% yeast extract tryptone

All media contain either ampicillin or ampicillin and kanamycin (the 2 at the end of the list).

Then, he 'buried' some dried Pan cyan material into the agar and incubated his plates in his 'tub in tub' incubator at 26C or 79F.

After three days, he saw some growth on one of the plates:



Unsure of what it is he took it from the incubator and watched it grow at 20C or 68F:




He read on the forum that a mold will get a color, but that mycelium should stay white, sightly gray or have some blue 'shine'. So now he's wondering whether he is already successful.

But one other difference between a mold and a mycelium, he read, is that a mycelium will grow through the agar. But there is no sign of that:


Does any of you pan experts recognize this growth as pan mycelium?

I will keep you posted on the advancement of this project.

Thanks for any suggestions :-)


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OfflineThemadhatter106
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Re: Bringing the dead alive - a PANdora's box? [Re: Zah]
    #11367261 - 11/02/09 06:16 AM (8 years, 21 days ago)

Good job, I'm suprised that you had an 8 year old uncontaminated mushroom.

I'm almost sure though, that you can't tell the differance between diffrent species mycelium.


--------------------
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Don't Grow mushrooms just because you want to trip. Grow them to stop drug dealers from going to jail and for the satisfaction of being self-sufficient.


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OfflineZah
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Re: Bringing the dead alive - a PANdora's box? [Re: Themadhatter106]
    #11367316 - 11/02/09 07:18 AM (8 years, 21 days ago)

Thanks ;-)

Well, we still don't know whether this is a contam or not... I will grow this one till it covers the agar and put it in the fridge. In the mean time I will see what the other plates will develop.

But the problem remains: if I take this mycelium and try to make it produce fruits, what will my conclusion be if it fails?

Does anyone know of any common contaminations that look like my mycelium?

Should I just give it a shot?


Thanks for any helpful input!


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OfflineMYSTIQUE
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Re: Bringing the dead alive - a PANdora's box? [Re: Zah]
    #11368218 - 11/02/09 12:42 PM (8 years, 20 days ago)

Id just throw a little chunk of the plate in a grain jar see what comes of it or make a quick lc out of one of the plates like a grain lc and shoot up a jar.


--------------------
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InvisibleCH HELL
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Re: Bringing the dead alive - a PANdora's box? [Re: MYSTIQUE]
    #11368579 - 11/02/09 01:37 PM (8 years, 20 days ago)

It looks Pan myc.


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OfflineLitCloset
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Re: Bringing the dead alive - a PANdora's box? [Re: CH HELL]
    #11368639 - 11/02/09 01:49 PM (8 years, 20 days ago)

you can tell the difference between species on agar.

pan is much less dense and in my experience a lighter color. no real rhizo growth either.

i have yet to have a pan can hi dig into the agar to any appreciable depth.

i would say thats pan cyan for sure. do another transfer to make sure its clean (i love h202 agar) then throw it in a grain jar.

congrats! very cool. i too love this species/variety above all others.


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OfflineZah
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Re: Bringing the dead alive - a PANdora's box? [Re: LitCloset]
    #11374767 - 11/03/09 07:20 AM (8 years, 20 days ago)

Well, this is great news! Thanks all you guys for the encouragement!

So without delay I will do an agar transfer, just to make sure the stuff is pure. Then I will try to grow it in a liquid culture.

I was planning to grow finally on a dung/straw substrate; so now the plan is to use the manure tincture I still have left as a liquid medium. This week I will try and find the bits and pieces needed to do this, while the mycelium is growing on the agar.

Ah, anyone ever tried growing this mycelium in a manure tincture culture?



Also: thanks for making clear that pan cyan mycelium doesn't penetrate the agar that much; I guess I mixed up with cubensis.


Amazing though how quick this mycelium grows! From inoculation to full plate coverage in about 10 days!



Great new hobby this, I even have the beginner's luck ;-)


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OfflineLitCloset
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Re: Bringing the dead alive - a PANdora's box? [Re: Zah]
    #11375352 - 11/03/09 10:48 AM (8 years, 20 days ago)

ive never used the dung tincture mostly because i have great success with the media i have plenty of, peptone yeastt dextrose agar.

also malt extract agar is very cheap and easy. i worry about all the contams that could grow in the dung, i realize thats what the pc is for but endospores etc... i like the nice and easy stuff mentioned. it seems to work great though!

it seems you have some micro expereince but your new to mycology right? have you tried making a lc before? a lot of people hate them because they contam so easily and its very hard to tell they are contaminated.

i love grain liquid cultures, GLC. there is a link in my sig. just put some colonized agar in a grain jar, once it colonized and healthy, fill it with sterile water, shake and remove with a syringe. then ou get very healthy liquid culture with out as much worry of contamination.

whatever you end up doing, good luck! i have my first two trays of pan cyan HI in the fruiting chamber right now. its been 3 days and 1 day, hope to have pins soon!


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OfflineZah
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Re: Bringing the dead alive - a PANdora's box? [Re: LitCloset]
    #11381748 - 11/04/09 07:15 AM (8 years, 19 days ago)

Quote:

LitCloset said:
ive never used the dung tincture mostly because i have great success with the media i have plenty of, peptone yeastt dextrose agar.

also malt extract agar is very cheap and easy. i worry about all the contams that could grow in the dung, i realize thats what the pc is for but endospores etc... i like the nice and easy stuff mentioned. it seems to work great though!




I think you have a point here: I should not get carried away and start taking unknown/lesser known routes, especially since I'm still not 100% sure what I'm dealing with here. I will go for the peptone yeast lc, and see what happens.

Quote:

LitCloset said:
it seems you have some micro expereince but your new to mycology right? have you tried making a lc before? a lot of people hate them because they contam so easily and its very hard to tell they are contaminated.




You're spot on here, I have a couple of years of experience, but never worked with mycelia.

BTW, speaking of mycelia, I did the transfer yesterday and could smell the mycelium. Is it right that Pan cyan mycelium smells like a forest in autumn? (closest description I could come up with :tongue:). It's for sure not an unpleasant smell.

Quote:

LitCloset said:
whatever you end up doing, good luck! i have my first two trays of pan cyan HI in the fruiting chamber right now. its been 3 days and 1 day, hope to have pins soon!




Cool, thanks! I hope your grow will work out nice! Post some pictures of the fruits here once they have popped up, for good vibes! :cool:


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OfflineBatista
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Re: Bringing the dead alive - a PANdora's box? [Re: Zah]
    #11381792 - 11/04/09 07:39 AM (8 years, 19 days ago)

Zah you have really done your homework to be able to do this..HATS OFF TO YOU!!! And they will grow so best of luck :smile:


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OfflineZah
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Re: Bringing the dead alive - a PANdora's box? [Re: Batista]
    #11382772 - 11/04/09 12:36 PM (8 years, 18 days ago)

Quote:

Batista said:
Zah you have really done your homework to be able to do this..HATS OFF TO YOU!!! And they will grow so best of luck :smile:




I've done a bit of homework, but I also realize that I was very lucky to get anything growing at all. Go figure:

12 agar plates in which I stuck 3 dried shrooms each. Only one of them produced this white mycelium, the others had some bacteria, but no mold.

But anyway, thanks for your nice words. Hopefully I can show some :regularshroom:shrooms:regularshroom: in a couple of weeks time.


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OfflineBatista
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Re: Bringing the dead alive - a PANdora's box? [Re: Zah]
    #11388964 - 11/05/09 05:01 AM (8 years, 18 days ago)

Hey, atleast you did your homework before asking all these friendly folk for advice :smile:

Im sure you will succeed!

Cheers


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OfflineThyrax
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Re: Bringing the dead alive - a PANdora's box? [Re: CH HELL]
    #11389006 - 11/05/09 05:28 AM (8 years, 18 days ago)

Quote:

CH HELL said:
It looks Pan myc.


:thumbup:


--------------------


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OfflineLitCloset
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Re: Bringing the dead alive - a PANdora's box? [Re: Thyrax]
    #11390661 - 11/05/09 02:38 PM (8 years, 17 days ago)

yea as soon as mine pin ill post picks, knowing my luck you may beat me...

im not sure how to describe pan cyan myc smell. i once read that some people think its fishy! i dont have that experience at all. i havent really noticed much of a smell from the pure stuff but i know the taste is different than cube. let me give that another try before posting what the taste is :stoned:

i once made armstrongs mixture back in elementry school, in my room in a quart glass jar, a total of one pipnt mixed....

i knew it was sensitive but... damn 3rd graders are stupid. anywhay it wasnt mixed to well yet so it burst into  flames while i was holding the jar, window and door closed. i eventually got the window open and out went the jar (and the back yard). since then my nose hardly works for me at all because of the terrible chlorine anf Cl compound fumes, plus the phosphorous... hand still has a nice burn on it too :cool: long story short, dont trust my nose. :crazy2:

anyone else have expereicne with pan cyan taste/smell of myc compared to cubes?


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OfflineZah
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Re: Bringing the dead alive - a PANdora's box? [Re: LitCloset]
    #11424026 - 11/10/09 04:25 AM (8 years, 13 days ago)

Quote:

LitCloset said:
yea as soon as mine pin ill post picks, knowing my luck you may beat me...




Nice, I'm sure you will beat me! After all, I'm still not even 100% sure that I'm not culturing cobweb mold:wink:


In the mean time, here are some pics of the agar transfer:


Day 1




Day 2



Day 3



Day 5


Interestingly, this time I don't see this wavy appearance of the mycelium as before. Now I think that this has to do with the fact that, the first time, I took the plate out of the incubator when I saw growth; the waves may reflect the differences in temperature during daytime and nighttime. This time the plate was left in the incubator all along.

Question: Is the speed of growth consistent with that of pan cyan? This plate was incubated at 79F/26C.

Last Sunday a lc was started (YPD medium). Here's a picture of my cheap ass setup (magnetic stirrer will be installed tonight):



Now here is another question I couldn't find the answer to: Is there a difference between a lc of pan cyan myc and a lc of cobweb mold?

I hope someone can help me out on this one. If there are no differences, could a grain jar make this difference clear?

Thanks for any helpful input!


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OfflineSillyBilly
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Re: Bringing the dead alive - a PANdora's box? [Re: Zah]
    #11424093 - 11/10/09 04:50 AM (8 years, 13 days ago)

lc's are hard to tell what you have in there
this is pan cambo which is similar to pan cyan
3 days in constant stir


this is just 24 hours before the first picture


as far as growth
yes they grow very fast
and i have also had that wavy water ripple looking thing happen and also not happen
i dont know what the deal is or which is better
id like to know as well if maybe one is better or not


--------------------
By the livin' Gawd that made you,
    You're a better man than I am, Gunga Din


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Invisiblememes
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Re: Bringing the dead alive - a PANdora's box? [Re: SillyBilly]
    #11424544 - 11/10/09 09:33 AM (8 years, 13 days ago)

Pretty dope expirament!


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Re: Bringing the dead alive - a PANdora's box? [Re: MYSTIQUE]
    #11424664 - 11/10/09 10:09 AM (8 years, 13 days ago)

Fuck I missed another QUALITY thread!

That is really sweet also the first plate pics looks like you gotta a monoculture which is heavy. Your growth will be consistent if the mono is used and you know it will fruit. i have also noticed the 'blue' on pan cyan myc.

Quote:

MYSTIQUE said:
Id just throw a little chunk of the plate in a grain jar see what comes of it or make a quick lc out of one of the plates like a grain lc and shoot up a jar.




Id nocc up some grains asap


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OfflineZah
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Re: Bringing the dead alive - a PANdora's box? [Re: SillyBilly]
    #11431135 - 11/11/09 05:24 AM (8 years, 12 days ago)

Quote:

SillyBilly said:
as far as growth
yes they grow very fast
and i have also had that wavy water ripple looking thing happen and also not happen
i dont know what the deal is or which is better
id like to know as well if maybe one is better or not




Actually, my clone had this wavy thing happening before the transfer, and not after. So, if fruiting capacity only depends on the clone you selected, then this wavy thing is no predictor of fruiting efficiency. But let's wait until we are sure of what I'm working with here :wink:

BTW. Thanks for the pics!


Quote:

badman said:
Id nocc up some grains asap




I will try and find some grains next weekend, hopefully I will have some pictures of growth next week. I found one pic of cobweb mold growth in a grain jar somewhere on the forum, it indeed looks quite different from Pan cyan myc.


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OfflineZah
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Re: Bringing the dead alive - a PANdora's box? [Re: Zah]
    #11458712 - 11/15/09 10:42 AM (8 years, 8 days ago)

So, just an update:

A couple of hours after switching on the magnetic stirrer, my culture fell over (in the dirty waterbath). So just before discarding it, I took a sample for the microscope. And surely, it was contaminated (bacteria and yeast). Here are some pics of the mycelium.


100x



200x




400x



Tonight I will re-start my cultures (LC and parakeet seeds; couldn't find grain anywhere).

To be continued...


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