Agar Pinning

Just something fun that has been happening for me a lot lately. Its just something cool I like to see if the strain on the agar will put out. All I can judge, is Psilocybe Cubensis, just wants to grow mushrooms


The first pic is a separate PF Albino clone I had. The first one to grow on the agar is pictured in this thread.:
http://forums.mycotopia.net/showthre...?t=6284&page=3 (PF Albinos)

This pic is the one I should have used lol.
4-3PFAagarpins.JPG when I first noticed pins
2006_0407Image0026.JPG

This pic is of the Malaysion Strain. I got busy with some other stuff, and shelved this for a bit because the growth on the agar just didn't look really dense or strong. So this was more of a work with in the future strain. I left it, and it grew a fun cluster Don't worry friend, its still my homework assignment
2006_0409Image0014.JPG

These are my PE isoaltes, A-F. These are the third transfer jars and can be read about in this thread:
http://forums.mycotopia.net/showthread.php?t=8303 (My first strain isolation attempt, PE)

These are my isolates, and all 6 fruited on agar. Some of you may have seen the pics of isolate E in the thread linked above. Well, it was first, the others all followed suit.
Isolate F was seperated cuz it had a contam spot. It still fruited

2006_0407Image0025.JPG 5 isolate jars
2006_0409Image0009.JPG isolate A
2006_0409Image0010.JPG isolate B
2006_0409Image0011.JPG isolate C
2006_0409Image0012.JPG isolate D
2006_0409Image0013.JPG isolate E
2006_0407Image0024.JPG isolate F


I just wanted to share these, I know they aren't as exciting to anyone else but me, but I thank you for letting me share it anyway. I love Mycotopia and my mycology hobby

[Spark]

isolate A looks very healthy

[BuckarooBanzai]

Screw that! I find this stuff very exciting as well. Personally, I've dug ALL your pics. And hey, if they'll fruit off of nothing more than hopped up sugar gel, it strongly suggests that I may be wasting a lot of time and energy with my EarthJuice habit.

That Malaysian reminds me of something I've been mulling for a while. I'm really inclined to say that a lack of strong rhizo character doesn't automatically say anything about fruiting potential. Hillbillys are very low on the stringy rhizo look, but they fruit to beat the band. B+ are crazy with the rhizos. If my FOAF hadn't worked with HBs first, he would have been very needlessly worried by the lack of strong rhizomorphic character.

Sorry to jack your thread, that picture just reminded me of that thought.

[inky]

Go Freaky! Nice pics, gonna go head off and read up on agar now.LOL Always a fan of your work.
Peace
~Inky~

[SharkieJones]

I love when that happens. Isolate E looks like a really nice fruiter. Thanks for sharing these.

[Pedestrian]

I agree sharkie. substrain E looks like it REALLY likes to fruit.

Thanks for the kind words, glad you enjoy it as well. It just does something goofy to my brain this mycology stuff. I love it!

Buckaroo, I know what you're saying about the rhizos. Something interesting to study for sure. That malaysion is going to get worked with, it pinned like crazy and the mycelium looked so weak to me. A great strain for me to watch I think!

And those PE isolates astound me. Isolate E is my favorite of course, its getting cased tonight and put to fruit. Isolates A and F are ready to pin any day, and isolate B is fruiting already. Thats all another thread though Soon


I was under the impression that PE isolates were a draw for fruiting. This is 100% isolate fruiting. Is that normal for the strain or not?

[fucgubarn]

hihihih
very cool

Fucgubarn, you should post some pics if you have any. I think I've seen some posts before with em Its cool to see em grow on such a small low nutrient medium.

[fucgubarn]

all my shit is packed,general rebuilding of the house couse a baby is comming,
some other time i promice

Good, your pics are great!!!!!

[dbzeroone]

That's f'ing cool Freakachino!!
It's very interesting how the ultimate goal for this organism it to make more spores. Give em some food and little else and they'll get busy making sure that their kind survive. Thanks for sharing!

[Killjoy]

Cool Man!
How were the Dishes Inoculated? Tissue?LC?
Special Agar recipe?
How long to fruit?

The PF Albino clones were inoculated from clone tissue.

The malaysion are from an agar transfer from a prior dish inoculated with spores. There are two transfer pieces in the malaysion plate above.

The pe isolates are 3rd transfer isolates, these were transfers for the fourth and final isolates. I just left them instead of dumping to see if they'd fruit or not.

No special recipes. All potato agar using potato flakes, karo/vanilla , distilled water.

They were incubated at 80 degrees, but then when fully colonized put on a shelf and left in room temp. Forgotten about for a while and left alone. These were all started a couple months ago. The malaysion is the youngest dish of all.

I'm testing a redboy from spores on two seperate dishes to see if the multi-spore agar will fruit too. Just for fun

[Pedestrian]

Yo freaky I got a Q for ya.


I am still a little confused.. Did you take isolates from one of those plates in the first 3 pics? Also, what was the method of extraction for each substrain? Did you remove tissue from directly underneath select pins or just certain areas of mycellium?

Thanks champ

Rock on.

-Ped

Ped, the PF Albinos, the first dish pictured, I just took a small pin outta my invitro brf jar, and grabbed a small bit of tissue, dipped it in peroxide for a few seconds, layed it on the agar. I did two different clones. The other pic is in the PF Albino thread linked above.

The Malaysion, the 3rd pic, I just took two transfer pieces from the first dish, and put them in this agar dish.

I'll try to do a pictorial of agar inoculation for you guys.

Hope that helps some

And I haven't done anything with the fruits in these pics yet. Haven't quite decided how much agar I wanna make and dishes I wanna use for any of it.

If I do, I'll probably pull the fruits and use those, just like regular cloning. The mycelium on the agar I don't really want, its pretty old now and could have some contam issues. I'd rather take the fruitbody tissue and clone it to new agar. Maybe I'll do that and do a pictorial up in a few days.

[BuckarooBanzai]

Have those jars been sealed the whole time (since inoculation, I mean)? If so, those fruit bodies should be quite clean. Even if there is some contamination on the agar, with no air circulation it should stay right there.

We all know one easy way to bump yield and predictability is by using clones, but clean clones aren't always the easiest thing to produce. The exise central tissue/peroxide dip could maybe be avoided all together with an approach like this.

I'm thinking that if you could get a multispore inoculation to pin like that, you would have two things:

1> Absolute guarantee that you had isolated a fruiting strain.
2> Pretty damned good chance of working with sterile tissue.

Man, this is why I dig your threads. Your stuff always makes me pinwheel with ideas and new considerations. Thanks much for that.

[Milton Waddams]

Very nice. I always thought that looked awesome.

[WeedyResin]

I love yer stuff Freaky! Your my hero!

[Lazlo]

This is the best way to find the peak performer from agar isolating. You can put the multispore dish under flourescent light 24/7 to speed the fruiting process up. You'll be able to see hyphal knotting in the starter dish on fruitable sub-strains. The ones with heavy knotting is the one to use. Simply acquire some of the mycelium from that particular sub-strain for a transfer to its own dish.

Thats what I like about it Lazlo. Finding a good fruiting sub strain pretty quick, without multiple isolations from the mycelium.

Buckaroo, yes, those jars and dishes have been closed. The jars were opened twice, the first time for its initial inoculation, then the 2nd time to take transfer pieces for the final isolates. They've been closed since then.

And I have Red Boys sitting out waiting, so I'm going to see what happens with them. They should be a pretty clean fruitbody.

I like that this can possibly take out transfer steps for me. Just get fruit bodies to clone off the agar and then work from there.

[BuckarooBanzai]

Pardon me if this is a totally ignorant question...

Once you have a fruit body, is it also (by definition) an isolate? Do sub-strains mix in fruit bodies, or is fruit an indication of strain isolation?

Damn that SOUNDS ignorant, but it seems like I remember reading something about clones continuing to sector. Then again, I eat a lot of drugs and just because I remember it doesn't mean it actually happened. I've read so much out of the archives in the last couple of weeks, I may be totally talking out my ass.

And I am LOVING THAT AVATAR, WEEDY!

Buckaroo, I have read Roger Rabbit say that before. I just recently laid 5 different cubensis clones on agar, and they all grew uniform, no sectoring. So I think of clones as an isolate.

I'm sure if you set some tissue and happened to have spores laying on it it could appear to be multi-strained and sector.

And that isn't an ignorant question at all imo. I often wondered also when I read what Roger said. Though I've seen total isolation with clone tissue to make me believe otherwise.

[the_chosen_one]

Oh here it is! Lol, I've been stumbling around for the last couple days trying to find this . Sorry I'm late! Very nice work freaky, very nice indeed
Makes me wanna

[~Hippie3]

that malaysian looks impressive.
nice work freaky.
pic of the month for you-

Attachment 22566

[the_chosen_one]

Make it two....Lol, I was just thinking the same thing! Thanks Hip!

[altered_states]

Originally Posted by BuckarooBanzai

Pardon me if this is a totally ignorant question...
Once you have a fruit body, is it also (by definition) an isolate?

Thanks for asking that question.
I was always under the impression that each fruitbody from a multispore culture is a substrain, and also an isolate, making a clone the same thing as an isolate.
I've been reading a lot about agar lately, and started to doubt myself.
I've cloned many times, but never isolated on agar.
I've always referred to my clones as isolates.

[~Hippie3]

a clone is an isolate, by definition.
rodger was mistaken, imo.

Thanks!!!!!

And I have to agree that a clone is an isolate. In fact, from my elementary work with agar up to this point, I'd say its better to use clone tissue as an isolate, than taking multiple isolations from agar to multiple dishes, then fruiting, etc. (Like I did with the PE.) If I can clone a couple fruitbodies that I fruit from the agar, then I'm isolating in just 2 steps. Makes it much quicker to get the isolate you want, and saves agar and dishes imo.

And from the clones I've laid on agar, they've all been isolated strains that I can tell. No sectoring has occured. I've got some more pics to take, and I'll update em soon

the only thing your missing with skiping that step freaky is when you do the macro test bathces with each isolate you get to see the fruiting potential before going to bulk . just cause a mushroom poped up in a dish doesnt mean that its going to be what your looking for when its time to go to fruitin . i noticed a long time ago that when i use the glass half/pints i get vulanter fruits way more then just using petri dishes . happy easter all
in a hurry so not even trying with the spelling
later VII

[~Hippie3]

noticed a long time ago that when i use the glass half/pints i get vulanter fruits way more then just using petri dishes

why, the extra air ?

Interesting Sev...thanks for all the info. And I'll be able to compare with the PE isolates I did and the clone isolates I take without doing all these transfers......and I'll update pics here in a few, I was super busy lol. And it will pin from spores on agar also. My redboys proved that to me, I didn't transfer anything and it was a multi-spore agar inoculation. So this may allow a clone isolate on agar to get fruiting and then allow agar transfer also to still isolate the multi-spore agar. I'm really thankful for this site and the knowledge and sharing and that you all got me over my fear of using agar. I thought it was only for advanced myco-heads. But it really has moved me into a new level using agar and learning about how substrains really differ and how many there are in just a touch of spores. Amazes me really

[doodaa man]

Na freaka, IMHO all your therds and pic's get me excited ! I always get wood when i see your stuff !

[~Hippie3]

Originally Posted by doodaa man

I always get wood when i see your stuff !

me, too.

Glad I can make ya'll feel better

[doodaa man]

Shazam !
Damn hip, i never would have thought you would be so damn beautifully voluptuous ! more curves then a roller coaster ! LOL
Is that the wife ? you lucky bastard !
Note" not to be takin personal " The bastard part.
Just being politically correct !
Peace !

[cap]

now now, hipp
you should know better than to post dirty pics like that this early in the mornin

us guys tend ta be just gettin it down by this time and now yer fuckin things up,man



these pics are a rsight for sore eyes, freak..

..and the mushrooms aint bad either

[~Hippie3]

Originally Posted by doodaa man

Shazam !
Damn hip, i never would have thought you would be so damn beautifully voluptuous ! more curves then a roller coaster ! LOL
Is that the wife ? you lucky bastard !
Note" not to be takin personal " The bastard part.
Just being politically correct !
Peace !

that ain't me, dood
that's freaky.

[doodaa man]

Opp's
sorry hip.
Dang freaka ! You got it going on !
Like i said, more curves then a roller coaster, Your old mans one lucky bastard!
Gotta luv that !

Thanks for the kind words

Well, here's a shot of a cool Dixieland clone dish I did. I gotta quit forgetting about random agar dishes lol.
2006_0418Image0010.JPG

And this is the Redboy. 2006_0418Image0013.JPGYou can tell the Redboy is multi-spore agar from all the sectoring of substrains. And I think that from seeing this, the bubbly stem issue is not a verticillium issue, rather a low oxygen issue, low oxygen circulation issue. It doesn't look sick, just needing air.....but thats from my observation, I don't know for sure. I just know this is what my PE and some other strain get without looking sick with vert, so I think its more an air issue with bubble/gnarly stems without splitting or sickly looking. I'll never know for sure probably, just my thinking

[~Hippie3]

perhaps low air exchange favors verticilium's growth,
that's true of many bacteria and molds.
however in your photo
i'd say that deformity is likely a result
of the shroom hitting the lid of the petri.

Yeah, Hippie, I'm not ruling anything out as the cause. I just would think that this dish hasn't been opened since inoculation. So, if the vert. bacterium was there, wouldn't it be showing up on other parts of the agar? Also, your thinking it could be because it doesn't have room makes just as much sense also. So many things to think about I love this place!

[~Hippie3]

if the vert. bacterium was there, wouldn't it be showing up on other parts of the agar?

not necessarily.
to see it with the unaided eye would require
very large [on bacterial scale] colony of bacteria
growing in isolation.
if verticiluim grew on the mycellia and did not
form into large concentrations
it would take a microscope to spot it.

Yeah, I hough about that after I posted........now to get a good scope to really check things out...........after the new digi though

[BuckarooBanzai]

THAT, fearless leader, is one hell of an interesting proposition.

Perhaps the vert is present in the whole plate, but only "visible" when it gets the chance to affect maturing tissues? Perhaps some intrinsic property of the maturing body "feeds" it while simultaneously making it visible?

Many common bacteria are basically omnipresent in the environment, but only obviously apparent under certain circumstances.

Regardless, I stand by my previous statement: Freakachino, you have got the MAD myco skills!

Thanks BB, your myco skillz are Great too!!!!

And I think Hippie is on to something more accurate than me. This would also explain possibly why my PE are all fruiting with this trait. Its dormant until it has the fruitbody to contaminate......that is very possible. Because Verticillium takes some days to affect the tissue, as soon as pins form it attacks.....that sounds most likely to me. I was thinking it was my compost/past. work. But other strains don't show this vert. contam.

Thanks for always keeping me thinking I love it!!!!