Invitro Agar experiment: now at 8th gen. B+ & 4th gen. Tex

[BuckarooBanzai]

FOAF has been doing some invitro experiments fruiting agar on the plate. This experiment has been going for a little over a 13 months now. Results are pretty darned interesting so far.

Agar recipe (measurements are by weight):
2% LME, 2% Dextrose, 1.5% Agar-agar (Sporeworks.com brand) in spring water.

Plates are sealed with a double layer of Parafilm and incubated at 84F.

The original idea was to try and get better multispore invitro results by collecting and germinating spores from successive generations of invitro grows. The plates below are 6th generation B+ and 3rd generation Tex.









The fruits are allowed to sporulate on the agar, without ever opening the plates. The spores are picked up by very lightly touching them with a sterile cotton swab. The swabs are put in small sterilized vials and then put in the freezer for 5 days.

5 days seems to work well to kill any errant mycelia that was picked up without killing too many of the spores. About 10% of the swabs don’t germinate at all..

After the 5 freezer days, spores are germinated by either rubbing fresh plates or dropping a swab into a 1/3 filled pint popcorn jar. Newly inoculated plates/jars are watched closely. If growth occurs before 2 days, it is probably a mycelia clone and not a germinating spore.

About six months ago, it was discovered that the invitro fruiting was more aggressive if two different strains were put on a plate simultaneously (both plates above are B+ on left, TX on right). This has not been experimented with except on agar, but it might do interesting things on a cake.

Full size invitro results with Buckaroo Bulk Nuggets (Buckaroo Bulk Nugget Tek (invitro, spawn bags and/or ½ gallon jars)) have been excellent and seem to show distinct generational improvements in performance thus far.

[fahtster]

pretty sick man, interesting indeed... quite the experiment too. keep it comin!

fahtster

[Bobcat]

Nice study, Bucky, with interesting results!

5 days seems to work well to kill any errant mycelia that was picked up without killing too many of the spores. About 10% of the swabs don’t germinate at all..

Thats cool. I wonder why 5 days is okay for cube spores? How long can they be frozen? Of course this begs the often asked question: why aren't they everywhere? Er, sorry. But fascinating.

Do you typically double your parafilm?

Its a really interesting observation that your ms plates fruit better in the company of another ms strain! I'll be taking a front seat for this one!

[BuckarooBanzai]

Thank you, sah! I haven't done any big bags lately, but here is that same generation in a spawn bag:




That bag yeilded a ton without even having to be opened until harvest time. And the nugget flushed again in a tub, so it was all good!

My FOAF is pretty psyched to see what the next generation does. This is fun for my FOAF cause invitro is sooooo neglect oriented. You can do fun stuff but also kind of "drop everything" for a week or so and not loose all your work.

The 5 day kills some of em (around 10% of those swabs do diddly). FOAF always goes with a full two layers of Parafilm between the plate and the air. One piece stretches to do it. Just cut it on the thick blue lines.

holy shit that dish looks crazy! two strains..
cool invitro! i love your tek, i wish i had those bags tho..
good luck w this experiment

[BuckarooBanzai]

Those bags are quite affordable from sporeworks.com

If you are real cheap you can even re-use them.

Your thread on the invitro dishes is what prompted me to post this!

[duncan]

Sweet looking grow bro! I might have to get some of them bags and give them a try. I am working on a straw log grow which will be my first log. I am planning on using millet, PE and straw/h/poo. I hope to get this up and going soon.

Congrats on a nice invirto bag!

[Freaky]

Bucky! Those are awesome invitro agar pictures! Very good scientific work! I like the swab idea, then the freezer, very interesting!

I love that Bag!

Originally Posted by BuckarooBanzai

Those bags are quite affordable from sporeworks.com

If you are real cheap you can even re-use them.

Your thread on the invitro dishes is what prompted me to post this!

thanks for the info.. il try to find some locally first lol it might take a while for it to ship internationally or might come out too costly..

your dishes are way cool! does it actually prove to be better when you tested it? in what ways?

[BuckarooBanzai]

Thanks, Freaky! Means a lot from a fellow Bob Ross fan!

Vinz - the bags are really quite light...but the Impulse sealer that closes them isn't! You might consider looking into some of Sandman's work with oven bags and polyfil inserts:

http://forums.mycotopia.net/showthre...highlight=oven (The new hobo-tek in progress)
http://forums.mycotopia.net/showthre...highlight=sand (Ive got the biggest sack on my block!)

I think it was Indicaz who also did some interesting bag stuff.

I don't understand the question about the dishes. Does *what* prove better? Pardon, it's early and I'm cooking MJ granola treats (some of which I've already been eating).

This is the same bag size/subsrate recipe, but from the 2nd generation:



Even with the bag torn away, it doesn't look as nice as the other. Hopefully, generation 10-15 will be superior invitro performers from multispore on every run.

MAN it is fun playing with a life form that has such a short life cycle! These kinds of experiments with green plants would take much longer. God bless those dudes who take the time on MJ genetics though. Such important work...

thanks for the links buckaroooo

i mean does the 8th gen b+ or 4th gen tex perform any different as compared to a 1st gen strain? like does it do better invitro? pin faster fruit more or anything like that..
coz i havent tested what a fruit grown on agar cloned come out to be when its used to fruit..
coz you are way ahead of me so i was just wondering what i am going to be getting myself into.. thanks!

oops, you answered it already.. thanks again buckaroobanzai!

[thepope]

excellent work, BB

One question: how many times can you reuse the bag successfully?
Every time Foaf tried to reuse a bag, they all contaminated...
Looking at the filter patch, it had degraded quite a bit compared to a fresh bag, or one that been pc'ed only once: its pores were huge, and it seems to allow way too much air exchange

This is a clear example of how improving a strain can greatly improve one's shrooming; foaf is still working with multispore EQs and Texans, as these have been sufficient so far, for the learning curve
This is an inspiring thread for sure

[lost_onabbey_rd]

awsome stuff bucky
i wonder if the reason they fruit better is b/c their are 2 different strains on the same substrait. almost like a race to fruit and spread spores before the other one gets a chance.
wonder what would happen if you injected one side of the bag with one strain and the other with another. i wonder if you'd get the same kind of results that you do on the plates
o, and maybe i missed it but what's in the bag buck?
keep up the good work
and keep the pics comin
lost

[golly]

Are these fruit pretty dense like others have reported..? Sure looks like an interesting and productive way to go..
Gooluk with your master race Bucky... There's alot to be said for finding
a strain and developing it's full potential...

[fahtster]

very nice bucky! loving the invitro bags.

fahtster

[BuckarooBanzai]

FOAF has used a spawn bag twice successfully. Be aware, FOAF uses the pre-sealable type, which obviously have slightly different filter patch material than “standard” bags. Re-using ‘em is a huge pain, though, and wholly not recommended.

The hope is that just like strain isolation gives better results in certain conditions on certain substrates, this generational isolation will do the same to later multi-spore offspring. The results have been good so far, and it has been a fun experiment to do.

Another cool thing is that there is always plenty of different well proven tissue around to expand and play with! FOAF has very anally stored samples of almost everything in distilled water in 10mL Vacutainer tubes.

The plates are very much better invitro performers when two strains are put on the same plate. Fruiting a plate and waiting for spores to drop takes a lot longer when only one strain is present. I think different strains on opposite sides of the same cake could be interesting, but my FOAF hasn’t had the time for that yet. I mention the phenomena for him in case somebody else wants to play with it…

The bags are Buckaro Bulk Nuggets (Buckaroo Bulk Nugget Tek (invitro, spawn bags and/or ½ gallon jars)), good sir Lost. Popcorn, castings, coir and poly – grain spawned and fruited in a single bag. My FOAF’s most functional idea so far (in this field).

The fruits are denser than normal. Hollow stems don’t show up, but you do get the “bread dough/willow bark” look on the stems. Looks almost exactly like vert, but they don’t crack/split. You get lots of pretty normal caps on top for printing, too.

The BB Nug (Buckaroo Bulk Nugget Tek (invitro, spawn bags and/or ½ gallon jars)) invitro is not more productive than classic bulk tub/tray scenarios. Pulling those nugs after they are fully colonized and doing a DEC makes for better harvests than fruiting them invitro.

But doing them invitro is infinitely cleaner and easier since no terrarium is required. The whole dream behind the experiment is to isolate/breed towards a strain that prefers invitro and worm poo. My FOAF wants easy bulk for the masses…

[TVCasualty]

My FOAF wants easy bulk for the masses…

As a member of the masses, I thank you for your efforts! This is a great thread, keep up the good work...

I really like the long-term nature of this project. It's inspiring me to think along those lines and design some long-term experiments of my own.

[the_chosen_one]

/me flashes back to a little over a year ago and smiles contently.

Damn Bro! Just look at you now

[Hippie3]

nice work

[python]

Have you kept some statistics Buck'.......?

First of all nice work........

I have been thinking these experiments through and have been trying to put it all together........

First, you can grow two seperate strains on the agar but if u were using a single substrate like a cake, ultimately one strain would over take the other, unless a clamp connection is formed between the two (unlikely).......

Now as far as your population experiments.........

What u are searching for is better invitro pinning generations......

----and this, by my guess would be tricky to anaylze........namely because invitro and open air growing, are still typically the same environments (rh and temp), which are necessary for the p.cubensis to grow.....So where is the factor that governs selection......IMO it would be the substrate
Now the mushroom genetic code maybe getting changed ever so slightly due to the substrate which is used (i.e. by the means of chemicals the mycelium is gathering from the substrate........one would think that transportation chemicals/enzymes or intermediate proteins, or receptors could possibly be affected (favored by natural selection) by the more abundantely available chemicals in the substrate.......therefore we might be able to assume some small % of natural selection based on the properties of the substrate........

Plus so much variation from multispore would prob be another factor on results.......

but who knows, i have to rush these posts all the time, too much stuff to do

again, Nice Work Buck