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sudont
Stranger ThanFiction

Registered: 01/19/06
Posts: 129
Loc: Chicago
Last seen: 13 years, 11 months
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Good Way to Start LC?
#5755264 - 06/15/06 09:03 PM (17 years, 7 months ago) |
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Hi! I've been reading about beginning a liquid culture, and it's been recommended to wipe down a fresh 'shroom with alcohol, then slice it open, cut out a piece, then blend in the solution. I was thinking, why not just dip a large pin, or small 'shroom in 91% alcohol, then toss the whole thing in the solution and blend? I can PC all my parts, (blender blades, etc.), but don't have a really clean place to work, flow hood, etc., so this occurred to me as a quick way to work. The only caveat I can come up with is that the 'shroom might absorb some alcohol. But would such a small amount cause problems? What do you all think?
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creamcorn
mad scientist


Registered: 03/13/06
Posts: 2,962
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Re: Good Way to Start LC? [Re: sudont]
#5755284 - 06/15/06 09:09 PM (17 years, 7 months ago) |
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i've had success by picking a nice looking fruit, wiping the outside of the stem with a piece of cotton soaked in isopropyl, then stabbing straight through the stem with the needle. (syringe pulled back part way and filled with a little water beforehand). quickly inoculate the LC jar as you would with a spore syringe, but instead the water forces out the little cross section of the stem you trapped inside the needle into your LC. incubate and watch 'er go. voila, an isolated strain, cloned from a single fruit, in an LC... easy as pie.
i usually do a few at a time in this manner because for some reason they don't always work... but when one does, you can suck up some of it and shoot up several more LC's an expand it out.
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majlo
crazy


Registered: 04/20/06
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Re: Good Way to Start LC? [Re: creamcorn]
#5756165 - 06/16/06 02:27 AM (17 years, 7 months ago) |
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great one CREAMCORN!
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SeaOfGreen
Stoned


Registered: 03/03/06
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Loc: North Carolina
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Re: Good Way to Start LC? [Re: majlo]
#5756415 - 06/16/06 07:04 AM (17 years, 7 months ago) |
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i have a good question (to me atleast). if one was to make a LC from a very large fruit on its FOURTH flush would the clones be as large on their FIRST flush? the reason i ask this is because the fruits seem to get larger but more scarce in later flush's.
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sudont
Stranger ThanFiction

Registered: 01/19/06
Posts: 129
Loc: Chicago
Last seen: 13 years, 11 months
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Re: Good Way to Start LC? [Re: creamcorn]
#5759353 - 06/17/06 12:03 AM (17 years, 7 months ago) |
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Quote:
creamcorn said: i've had success by picking a nice looking fruit, wiping the outside of the stem with a piece of cotton soaked in isopropyl, then stabbing straight through the stem with the needle. (syringe pulled back part way and filled with a little water beforehand). quickly inoculate the LC jar as you would with a spore syringe, but instead the water forces out the little cross section of the stem you trapped inside the needle into your LC. incubate and watch 'er go. voila, an isolated strain, cloned from a single fruit, in an LC... easy as pie.
That's an interesting variation of cutting a piece out.
Quote:
i usually do a few at a time in this manner because for some reason they don't always work...
Perhaps because you don't always get enough myc with this method? Again, why not just use an entire piece? As I'm sure you know, small-mouth Mason jars are the same size as blender pitcher fittings, which can be autoclaved. Seems to me, dunking a small 'shroom in alcohol to kill any contams riding along, then adding to a nutritious solution and blending, would be a quick-n-easy way to go. Anyone tried this? Have thoughts?
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niteowl
GrandPaw


Registered: 07/01/03
Posts: 16,291
Loc:
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Re: Good Way to Start LC? [Re: sudont]
#5759672 - 06/17/06 02:42 AM (17 years, 7 months ago) |
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Sounds like a good idea to me too.
The only thing left to do now, is try it.
Thats what this site is about after all.
Let us know how it goes.
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MLBjammer
Invitro Freak


Registered: 02/24/06
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Loc: Southeastern USA
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Re: Good Way to Start LC? [Re: sudont]
#5759772 - 06/17/06 04:45 AM (17 years, 7 months ago) |
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You will most like have a lot of contams. with that approach, but there's only one way to find out . . .
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sudont
Stranger ThanFiction

Registered: 01/19/06
Posts: 129
Loc: Chicago
Last seen: 13 years, 11 months
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Re: Good Way to Start LC? [Re: MLBjammer]
#5770770 - 06/20/06 12:15 AM (17 years, 7 months ago) |
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Quote:
MLBjammer said: You will most like have a lot of contams with that approach, but there's only one way to find out . . .
Which approach in particular do you have in mind? Dunking a pin/shroom in alcohol, or pushing a needle through one? Could you say a little more about why? Thanks!
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creamcorn
mad scientist


Registered: 03/13/06
Posts: 2,962
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Re: Good Way to Start LC? [Re: sudont]
#5770845 - 06/20/06 12:34 AM (17 years, 7 months ago) |
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well i can say i've never once had a contam with this method (at least one i could observe). like i said sometimes i got no growth either, but you're probably right in that its due to such a tiny sample. it gets all soggy and and breaks up and sits there for a few days until i give up on it and dump it.
your way sounds good too if i'm picturing it right... a small container that has the blades already in it, that you fill with LC and PC as is, then can blend right in the jar without fussing around transferring things between containers?
the only cons i see with your way is 1) opening the jar enough to get your tissue sample in is a little more contam prone than being able to "inject" it with a needle... but if done quickly in a clean environment (better yet a glovebox or flowhood) its not a problem. and 2) you're limited by how many of these blending apparatuses you happen to have (again if i'm picturing this right).. i know i got at least a dozen half pint jars with different strain isolates chilling out in the refrigerator for long term storage, i use them as "masters" taking little bits to knock up fresh LC's that i work from... but maybe im just a packrat and you won't necessarily need to keep a dozen+ strains around for a rainy day. 
using a pin seems like a good idea, RogerRabbit posted not too long ago about cloning pins on agar and found that they grow mycelium very vigorously. the downside there is i like to clone fruits based on their appearance when they're fully grown, so cloning a pin can be a bit of a mystery and a gamble until you grow it out later.
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sudont
Stranger ThanFiction

Registered: 01/19/06
Posts: 129
Loc: Chicago
Last seen: 13 years, 11 months
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Re: Good Way to Start LC? [Re: creamcorn]
#5770979 - 06/20/06 01:11 AM (17 years, 7 months ago) |
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Quote:
creamcorn said: well i can say i've never once had a contam with this method (at least one i could observe). like i said sometimes i got no growth either, but you're probably right in that its due to such a tiny sample. it gets all soggy and and breaks up and sits there for a few days until i give up on it and dump it.
your way sounds good too if i'm picturing it right... a small container that has the blades already in it, that you fill with LC and PC as is, then can blend right in the jar without fussing around transferring things between containers?
the only cons i see with your way is 1) opening the jar enough to get your tissue sample in is a little more contam prone than being able to "inject" it with a needle... but if done quickly in a clean environment (better yet a glovebox or flowhood) its not a problem. and 2) you're limited by how many of these blending apparatuses you happen to have (again if i'm picturing this right).. i know i got at least a dozen half pint jars with different strain isolates chilling out in the refrigerator for long term storage, i use them as "masters" taking little bits to knock up fresh LC's that i work from... but maybe im just a packrat and you won't necessarily need to keep a dozen+ strains around for a rainy day. 
using a pin seems like a good idea, RogerRabbit posted not too long ago about cloning pins on agar and found that they grow mycelium very vigorously. the downside there is i like to clone fruits based on their appearance when they're fully grown, so cloning a pin can be a bit of a mystery and a gamble until you grow it out later.
Yes, you have it visualized correctly. And I'm only thinking of doing one LC jar, with which to inoc grain. I figure, if it's fruiting, it's probably ok, strain-wise. I intend to take it from a cake that's already putting out, so to speak. If it works with GFs, why not LCs?
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fastfred
Old Hand



Registered: 05/17/04
Posts: 6,899
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Re: Good Way to Start LC? [Re: sudont]
#5770996 - 06/20/06 01:14 AM (17 years, 7 months ago) |
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"GFs" ???
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