Orissa Strain Isolation

[zeegos]

ok, i have a strain isolation project underway and want to end up with about 10 isolates by the end of it. i am limited to the number of petris i have (about 15 to work with) so im trying to work out how many i'll need for each part.
there shoud be about 3 transfers from start to finish so let me know if this sounds right.

-1 petri for the spore germination
-transfer best looking mycelium to another petri
-transfer best looking mycelium to 2 petris
-transfer 5 best looking strains from each petri giving me 10 in total

then i'll fruit out each of the 10 isolates in the same conditions to find the best fruiter. for me, im looking for the bigguns, rev style

[gunji]

might be easier in the long run to do a regular multispore grow and clone the shroom you want to reproduce. i feel you on wanting to isolate. but thats going to be alot of chances for contams to interefere with what you want to do.

im assumeing you dont have clean room conditions or you wouldnt be limited to "15" petris.

be realistic, if you only have 15 petris, can you realy keep every last bacterial endospore out of the equation thru 3 or 4 transfers? ive never been able to.

im lucky to get a clean clone from a shroom.

to be honest with you, the substrate has a lot to do wit how big the boomer gets, ie the same isolate on a pf cake probly wont be as big as it would get on some of Hippie's donky dung straw tray would get.

[zeegos]

well iv just made a new glovebox which im having a good success rate with. if it comes to it, i could always use squat jars instead of petris but i doubt i would need to. i do have other petris with cultures which i could scrap and use again.

i know cloning is good, but isolation can be even better. if cloning was as good im sure no one would go to the bother of isolation

this gives a good understanding on the topic
http://archives.mycotopia.net/discus...tml?1034628376

[Hippie3]

interesting question you imply,
do more get isolates by cloning or by agar selection ?
i'll create a poll.
btw
ever heard of petris with dividers, in effect turning 1 into 3 or 4 ?

[zeegos]

yea, iv seen them on ebay but i just bought the normal ones. might consider getting myself some next time.

[gunji]

Its my understanding that if you can get a clean tissue sample from a shroom you want to clone, and cleanly put that piece of tissue on a petri dish, then you have an isolate. itll grow mycelia, then you can transfer that to grain or another petri to be sure its clean. might save you some headaches.

i was able to get a chunk of a nice boomer to grow mycelia in a squat jar with brf and peroxide(there is a tek here somewhere) mr pf was talking about it, it works) , all done in open air, with no contams. but i have so much liquid culture at my disposal, that can produce way more than i need anyway, i never bothered to use it ).

the idea of isolateing appeals to me too, but to be honest, once i figured out how to do a caseing with straw dung or even the oldtimers tek( a cake thinly sliced and bedded down in coir), i really had more shrooms than i knew what to do with. unless your trying to produce for an army i cant see the advantage of doing the isolate, UNLESS it makes you happy to tinker with it! in which case i wish you the best of luck )

[zeegos]

- Its my understanding that if you can get a clean tissue sample from a shroom you want to clone, and cleanly put that piece of tissue on a petri dish, then you have an isolate.

not meaning to be picky but that isnt really true. a clone isnt a pure strain isolate, a clone will consist of a number of substrains and will therefor have a mixture of getetic properties. this mixture will have some strains with good genetics and some with poorer genetics.
a pure strain isolate is a single substrain which has only its own genetics, obviously . this means when you isolate a very good strain, it will have only its own good genetics and no others amongst it which may hinder its performance.

i agree that cloning is much simpler and for some people, the extra work involved with isolation just isnt worth it.
for me i'd be happy to go all the way for the real deal, i mean its only a little work done in a glove box and time does the rest. besides, i love this hobby :

[Xerces]

So let me get this straight, because I was planning on isolating on my first project. You take a tissue sample from a good looking shroom and put it in substrate to colonize again and create nazi shrooms?

[zeegos]

no, you dont take tissue from a fruit or from a colonised substrate.
you germinate spores on agar, mycelium will grow out, you will see different sectors of mycelium which shows faster and more rhizomorpic growth, this is transfered to another petri, it will grow out in sectors again then you transfer the best looking mycelium again to a new petri and so on until you have 1 single strain.
read that post from the link above. once you understand that, this shows you how its done:
http://archives.mycotopia.net/discus...tml?1067230771

man, im gonna be dreaming this stuff all night

[Hippie3]

you can take tissue from a fruit,
grow it out on agar or in a liquid culture .

[Hippie3]

, a clone will consist of a number of substrains and will therefor have a mixture of getetic properties.

you state that as a fact
but i bet you cannot prove it.

by definition
a clone is genetically identical to the source.

a group of organisms or cells produced asexually from one ancestor or stock, to which they are genetically identical.

[zeegos]

i bet i cant either. i dont know much about mycology, only the basics iv gathered from archive threads

[Hippie3]

we had one person here who made the claim that tissue samples were multistrain,
he based his assertion upon his observation of tissue sectoring as it grew
but he neglected to consider other possible causes for sectoring.
as far as i know there is nothing more to it that that, one guy's opinion.

as far as science is concerned though,
clones are genetically identical
and our polls here reveal that cloning seems to indeed be a way
to get isolates,
one just should minimize any chance of multiple substrains
by using very small sections of tissue for this cloning,
a mere fragment of a mycellial thread is enough to grow out

[zeegos]

so really, cloning and isolating are the same thing but different in the sence that, when you clone you are taking a 'clone' of a strain from a fruit instead of agar transfers to get an isolated strain? IF the transfer of mycelia from a fruit is small enough.

[Hippie3]

correct.
multispore germination gives you many substrains
so you can test many at once
while cloning just gives one substrain
which will be [genetically] exactly like the shroom cloned/sampled.
you could clone several shrooms off one multispore-created cake/casing to get the
equivalent to a multispore germination.

[Xerces]

So isolating in a petri dish with a tissue sample would create a single-strain or multi-strain colony?

[Hippie3]

single substrain of a strain.
the strain would be the name brand,
such as Golden teacher.
an isolate would be a substrain of GT.

[beebopboy]

Should be single strain

[Donor]

The point on isolating strains from multispore swipes on agar, is to give a larger number of substrains to choose from. When one selects a clone, it may or may not be the best fruiting, most consistent, even pinning substrain, however, it is known to produce fruits, and is the reason why many have greater success by cloning alone.. As with anything though, the law of averages comes into play.. When one selects from 100 substrains, as opposed to 10, they have a better chance at finding the ULTIMATE substrain that gives the phenomenal results one sees in pictuures by the great mycologists. Just like the best cannabis breeders select the best plant from 1,000's of seeds, not from a bag of ten. This goes for breeders of any form, however, there is always the chance someone gets lucky and just happens upon the superstrain, so clone away!!

[shroom_seeker]

Nice thread Zeegos. This hobby is so cool, I can hardly wait to make my first isolate. I'm also dreaming of the bigguns that Rev's foaf always seems to get.

[zeegos]

After reading up on the topic of strain isolation, iv decided to give it a bash myself. It seemed complicated at first but after a bit of reading its pretty easy to understand the purpose of it and the advantages over cloning.
Iv decided to do a strain isolation with the renowned strain Orissa India. Im looking for a strain which will throw out the bigguns and from what we've seen with this strain, it seems like a good choice.

So heres a rundown of my procedure:

-Germinate spores on agar
-Transfer the best looking mycelium to another petri
-transfer two fragments from that petri to 2 petris
-transfer 5 strains from each of the 2 petris for storage



After 3 transfers i should hopefully have 10 pure isolates which i can then fruit out individualy to determine which is the best fruiting strain.

I'll be doing all of this in just a glovebox so contamination might introduce itsself once or twice but so far iv had a good success rate with it.

Heres the supplies i'll be using:



I use dilute bleach to spray the surfaces of the glovebox and the peroxide for sterilizing tools (as well as flame sterilization). For transfers im using a syringe needle which makes life easier when transfering tiny mycelia fragments.

So lets get down to business. Iv had the multispore petri growing out for a week or so to let the fastest mycelium expose itself. In this picture you can see the sectoring of the mycelium.



The agar im using is a potato/honey recipe which is a good substitue if malt extract and dextrose isnt available.
All equipment needed was placed into my glovebox along with my multispore petri to be transfered. I let the glovebox sit for long enough for the air to settle but not too long otherwise the agar would set.
After pouring the agar and letting it set, i snaged a tiney fragment of mycelium from the edge of the growth and placed it in the center of the other petri. Normally i tape it up with micropore tape but supplies are low so iv used parafilm.



now its time to wait

[Hippie3]

you know that after you germinate spores
and are transferring live tissue
you can use peroxided agar to dramatically reduce contams

great pix btw
nice diagrams too

[zeegos]

i added a small amount of peroxide to the agar, forgot to mention. this stuff is 18% conc so i only used a drop.

[reverend trips]

Nice thread Zeegos.

You picked a good strain to work with, this will be fun to watch.

I need to get pair of gloves like those for my glovebox.

[zeegos]

thanks rev, your posts have been my motivation. i dedicate this one to ya

[Hippie3]

the good reverend is an inspiration to us all.

[reverend trips]

i dedicate this one to ya

Ok, this project has been blessed hehe... I got some pull with the mycogods.

You guys are too kind

[CoyoteMesc]

anything ever come of this project?