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InvisibleMr.PhilCybin
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When working with Isolates
    #22385755 - 10/15/15 09:22 PM (8 years, 3 months ago)

Questions Isolate gurus -


Do you think it would be better to make a master from the plate before transferring to grains, or take a tissue sample from a pin?

Ideally I would think to just get it straight from the plate, but it's been a few months of nothing but agar transfers, and I'm worried about senescence.

any input would be appreciated


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Re: When working with Isolates [Re: Mr.PhilCybin]
    #22385869 - 10/15/15 09:48 PM (8 years, 3 months ago)

If senescence is an issue why don't you just alternate agar media/nutrients?


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Re: When working with Isolates [Re: Mr.PhilCybin]
    #22385879 - 10/15/15 09:51 PM (8 years, 3 months ago)

:popcorn:

because new nutrient sources don't change the age of mycelium. I think

edit: Don't want to thread jack but does getting a pin from your isolate and cloning it avoid senescence somehow?? Very cool if so


Edited by Psilosopherr (10/15/15 10:07 PM)


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Invisibleazur
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Re: When working with Isolates [Re: Mr.PhilCybin]
    #22385937 - 10/15/15 10:02 PM (8 years, 3 months ago)

Quote:

Mr.PhilCybin said:
Questions Isolate gurus -


Do you think it would be better to make a master from the plate before transferring to grains, or take a tissue sample from a pin?

Ideally I would think to just get it straight from the plate, but it's been a few months of nothing but agar transfers, and I'm worried about senescence.

any input would be appreciated



If it's been a few months of agar transfers, i would DEFINITELY wait for a pin.


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InvisibleMr.PhilCybin
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Re: When working with Isolates [Re: azur]
    #22386050 - 10/15/15 10:21 PM (8 years, 3 months ago)

word.


just to clarify, I'm talking about a pin from the bulk sub in fruiting conditions


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Invisibleazur
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Re: When working with Isolates [Re: Mr.PhilCybin]
    #22386062 - 10/15/15 10:24 PM (8 years, 3 months ago)

Yea. You can do that. But you'll probably have to clean it up...which means more transfers. I'd try to get the plate to pin.


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InvisibleMr.PhilCybin
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Re: When working with Isolates [Re: azur]
    #22386129 - 10/15/15 10:35 PM (8 years, 3 months ago)

alright. I'll just keep the plates after I transfer to grains tonight, and hope one shoots a pin.


I made that batch really light in nutrients though so I'm not sure if it will or not.


time will tell I guess.

I got a clean plate first transfer off some oyster fruits I grew a while ago, maybe I'll get lucky


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Re: When working with Isolates [Re: Mr.PhilCybin]
    #22386138 - 10/15/15 10:37 PM (8 years, 3 months ago)

:vibin:
Keep us posted


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InvisibleMr.PhilCybin
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Re: When working with Isolates [Re: azur]
    #22386142 - 10/15/15 10:38 PM (8 years, 3 months ago)

:thumbup:

thanks for the words man.



I've done hundreds of transfers, but this is my first time making it to actual iso's.

shit's a lot of work :lol:


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Invisibleazur
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Re: When working with Isolates [Re: Mr.PhilCybin]
    #22386157 - 10/15/15 10:42 PM (8 years, 3 months ago)

Yea. It certainly can take a lot of time.
With a steady hand and good sight you can speed it up some though.


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InvisibleMr.PhilCybin
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Re: When working with Isolates [Re: azur]
    #22386161 - 10/15/15 10:43 PM (8 years, 3 months ago)

Yeah, not sure what took so long with these,

but they just would NOT  stop fucking sectoring.


chron gave me the tip of thinning out the nutrients though which went a long way.

good thing I like messing with plates


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Re: When working with Isolates [Re: Mr.PhilCybin]
    #22386521 - 10/15/15 11:59 PM (8 years, 3 months ago)

:threadmonitor:I'm trying to get my isolating skills up to par too...:goodluck: and...Let us know if you get a pin from that plate. I'm curious.:popcorn:


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InvisibleMr.PhilCybin
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Re: When working with Isolates [Re: Darkhome]
    #22386547 - 10/16/15 12:06 AM (8 years, 3 months ago)

it'll be a while so don't hold your breath.

but I'll resurrect this thread when it happens, or if the plate gets old and I toss it.


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Re: When working with Isolates [Re: Mr.PhilCybin]
    #22386571 - 10/16/15 12:12 AM (8 years, 3 months ago)

:seriousthumbsup:


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Re: When working with Isolates [Re: Mr.PhilCybin]
    #22386608 - 10/16/15 12:23 AM (8 years, 3 months ago)

ive had the plates I innoced with MS pin, and ive had transfers not pin.  For me, it seems to take a little over a month maybe a month and a half to get a plate to pin, but then I have several pins or one after another.  definitely is cool to see though.

The ones I inoculated with MS were very light with nutrients, but they pinned, and are still pinning in fact, so I think you should be ok.

:twocents:


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Edited by firstTIMER420 (10/16/15 12:24 AM)


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Re: When working with Isolates [Re: azur]
    #22386630 - 10/16/15 12:31 AM (8 years, 3 months ago)

Quote:

azur said:

If it's been a few months of agar transfers, i would DEFINITELY wait for a pin.



why? if it's already isolated what good does waiting for a pin do?a few transfers on agar will do next to nothing for myc, this happens over hundreds of jars not a few transfers and the younger the culture the better IMO., i'd go to grains first then make another plate or two to keep on hold till you test the isolate in fruiting conditions, then if it's a keeper you got a couple plates to get you some slants and more jars, but that's just my opinion


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InvisibleMr.PhilCybin
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Re: When working with Isolates [Re: cronicr]
    #22386644 - 10/16/15 12:37 AM (8 years, 3 months ago)

I have 3 isolates that I'm gonna compare fruiting capability with.

I figured I could just slant all 3, and then toss the 2 I don't want.

I wasn't sure if I'd be better off hitting the myc after it's fruited though.

sorta like how people say to clone a pin instead of an adult mushroom :shrug:


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Re: When working with Isolates [Re: Mr.PhilCybin]
    #22386646 - 10/16/15 12:38 AM (8 years, 3 months ago)

if a jar overgrows on me i'll wait for a pin but for a perfectly fine plate i just go right to grains and wait to slant it


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Re: When working with Isolates [Re: cronicr]
    #22386664 - 10/16/15 12:45 AM (8 years, 3 months ago)

i always dip pieces in H2O2 before putting on agar.


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InvisibleMr.PhilCybin
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Re: When working with Isolates [Re: cronicr]
    #22386675 - 10/16/15 12:49 AM (8 years, 3 months ago)

Quote:

cronicr said:
if a jar overgrows on me i'll wait for a pin but for a perfectly fine plate i just go right to grains and wait to slant it



so you don't bother making a slant til you see fruits?


just throw the plate in the fridge in the mean time?


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InvisibleMad Season
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Re: When working with Isolates [Re: enlightened seed]
    #22386688 - 10/16/15 12:55 AM (8 years, 3 months ago)

Quote:

enlightened seed said:
i always dip pieces in H2O2 before putting on agar.



Yeah... H2o2 doesn't belong with agar..


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Re: When working with Isolates [Re: Mr.PhilCybin]
    #22386720 - 10/16/15 01:13 AM (8 years, 3 months ago)

Quote:

Mr.PhilCybin said:
Quote:

cronicr said:
if a jar overgrows on me i'll wait for a pin but for a perfectly fine plate i just go right to grains and wait to slant it



so you don't bother making a slant til you see fruits?


just throw the plate in the fridge in the mean time?



yep


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Re: When working with Isolates [Re: cronicr]
    #22386818 - 10/16/15 02:25 AM (8 years, 3 months ago)

Quote:

cronicr said:
if a jar overgrows on me i'll wait for a pin but for a perfectly fine plate i just go right to grains and wait to slant it





So you use half the plate and save the other half to slant if you need too?


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Re: When working with Isolates [Re: firstTIMER420]
    #22386825 - 10/16/15 02:28 AM (8 years, 3 months ago)

I just toss a couple wedges on new plates after I inoculate a master grain jar


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Re: When working with Isolates [Re: cronicr]
    #22386832 - 10/16/15 02:31 AM (8 years, 3 months ago)

and then slant those new plates. gotcha.

Disregard my PM then....


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Re: When working with Isolates [Re: cronicr]
    #22387189 - 10/16/15 06:50 AM (8 years, 3 months ago)

Quote:

cronicr said:
Quote:

azur said:

If it's been a few months of agar transfers, i would DEFINITELY wait for a pin.



why? if it's already isolated what good does waiting for a pin do?a few transfers on agar will do next to nothing for myc, this happens over hundreds of jars not a few transfers and the younger the culture the better IMO., i'd go to grains first then make another plate or two to keep on hold till you test the isolate in fruiting conditions, then if it's a keeper you got a couple plates to get you some slants and more jars, but that's just my opinion



Because he said it's been months of transferring. Which could mean a lot of transfers.


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Re: When working with Isolates [Re: azur] * 1
    #22387333 - 10/16/15 07:51 AM (8 years, 3 months ago)

Quote:

azur said:
Quote:

cronicr said:
Quote:

azur said:

If it's been a few months of agar transfers, i would DEFINITELY wait for a pin.



why? if it's already isolated what good does waiting for a pin do?a few transfers on agar will do next to nothing for myc, this happens over hundreds of jars not a few transfers and the younger the culture the better IMO., i'd go to grains first then make another plate or two to keep on hold till you test the isolate in fruiting conditions, then if it's a keeper you got a couple plates to get you some slants and more jars, but that's just my opinion



Because he said it's been months of transferring. Which could mean a lot of transfers.




True but cell division is more of an issue than time is. Pinning means lots of cell division which in effect ages the culture more. Personally I would use the plate to inoculate a slant, a master grain jar or LC, and a few plates. The plates and slant go in the fridge and the master or LC are expanded to do a grow.  If the culture is determined to be a keeper then keep the slant and expand the hell out of the plates. People would be surprised at how far you can push a culture,  especially a young one you made yourself.

A quick tip for those looking to isolate is to do a transfer after spores and once clean go to grain. Then put grains from that master on plates. Sectors will show up soon, if not right off the grain. Can cut the number of transfers needed dramatically.


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Re: When working with Isolates [Re: Pastywhyte]
    #22387619 - 10/16/15 09:41 AM (8 years, 3 months ago)

Hey Pasty that is an awesome tip! I came to that realization on my own after working with stubborn spores that I could only get to germinate on grain
I use a tiny jar, like 1/4 pint so it colonizes real fast


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Edited by mushpunx (10/16/15 09:47 AM)


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Re: When working with Isolates [Re: Pastywhyte]
    #22387655 - 10/16/15 09:50 AM (8 years, 3 months ago)

Quote:

Pastywhyte said:
Quote:

azur said:
Quote:

cronicr said:
Quote:

azur said:

If it's been a few months of agar transfers, i would DEFINITELY wait for a pin.



why? if it's already isolated what good does waiting for a pin do?a few transfers on agar will do next to nothing for myc, this happens over hundreds of jars not a few transfers and the younger the culture the better IMO., i'd go to grains first then make another plate or two to keep on hold till you test the isolate in fruiting conditions, then if it's a keeper you got a couple plates to get you some slants and more jars, but that's just my opinion



Because he said it's been months of transferring. Which could mean a lot of transfers.




True but cell division is more of an issue than time is. Pinning means lots of cell division which in effect ages the culture more. Personally I would use the plate to inoculate a slant, a master grain jar or LC, and a few plates. The plates and slant go in the fridge and the master or LC are expanded to do a grow.  If the culture is determined to be a keeper then keep the slant and expand the hell out of the plates. People would be surprised at how far you can push a culture,  especially a young one you made yourself.

A quick tip for those looking to isolate is to do a transfer after spores and once clean go to grain. Then put grains from that master on plates. Sectors will show up soon, if not right off the grain. Can cut the number of transfers needed dramatically.





Definitely will do this! I for the life of me cannot get pan cyan to look good on agar, itll germinate, but its just weird.  Im planning on taking some colonized grains and putting them on GW agar plates.

Whats the time frame as far as using a plate for slants after being in the fridge? Ive had some plates in the fridge for a month or two, and I wanted to slant them


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Re: When working with Isolates [Re: firstTIMER420]
    #22387732 - 10/16/15 10:13 AM (8 years, 3 months ago)

If my plates have been in the fridge a long time I usually make a transfer to grow out a new plate before I use it just to be safe.  Id probably do that before slanting one

Depending on how you stored it it could be just fine but its not a big deal to put it to another plate :shrug:


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Re: When working with Isolates [Re: mushpunx]
    #22387766 - 10/16/15 10:21 AM (8 years, 3 months ago)

Quote:

mushpunx said:
If my plates have been in the fridge a long time I usually make a transfer to grow out a new plate before I use it just to be safe.  Id probably do that before slanting one






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Re: When working with Isolates [Re: mushpunx]
    #22387768 - 10/16/15 10:21 AM (8 years, 3 months ago)

gotcha. I was thinking about that but im worried about senescense. When I got the cultures they were on the second transfer, ive put them through two already.


How do you guys print PE? ive been trying with no luck and I don't want to go the swab route.


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Re: When working with Isolates [Re: firstTIMER420]
    #22387773 - 10/16/15 10:23 AM (8 years, 3 months ago)

Make syringes. And start your own thread


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Re: When working with Isolates [Re: firstTIMER420]
    #22387834 - 10/16/15 10:39 AM (8 years, 3 months ago)

Quote:

fiis stTIMER420 said:
gotcha. I was thinking about that but im worried about senescense. When I got the cultures they were on the second transfer, ive put them through two already.


How do you guys print PE? ive been trying with no luck and I don't want to go the swab route.




You can always clone :shrug: but other than swabbing its quite difficult I guess. The vendors have thier own tricks to making PE syringes.

Are these isolated strains?
2, 3 transfers is nothing man! When isolating a strain sometimes I don't even see sectoring proper for 8-10 transfers.
We move cultures around all the time. I guarantee you won't ever see senescence on a culture just from normal moving it around and expanding.


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Re: When working with Isolates [Re: Pastywhyte]
    #22387965 - 10/16/15 11:24 AM (8 years, 3 months ago)

:takingnotes: :themoreyouknow:


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Re: When working with Isolates [Re: mushpunx]
    #22387974 - 10/16/15 11:27 AM (8 years, 3 months ago)

Quote:

mushpunx said: I guarantee you won't ever see senescence on a culture just from normal moving it around and expanding.




I did after 9 months


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InvisiblePastywhyteMDiscord
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Re: When working with Isolates [Re: azur]
    #22388006 - 10/16/15 11:37 AM (8 years, 3 months ago)

Quote:

azur said:
Quote:

mushpunx said: I guarantee you won't ever see senescence on a culture just from normal moving it around and expanding.




I did after 9 months




Thats about right. But if you care for your masters 9 months can be stretched out for 20 years.


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Invisibleazur
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Re: When working with Isolates [Re: Pastywhyte]
    #22388045 - 10/16/15 11:55 AM (8 years, 3 months ago)

Indeed


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


A cube is NOT a cube.

FALL IN LOVE WITH LC
FOTTSE!!!
ALL NOOBS READ THIS!!!



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Offlinemushpunx
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Re: When working with Isolates [Re: azur]
    #22389087 - 10/16/15 04:03 PM (8 years, 3 months ago)

:shakefist:
Mushpunx guarantee!!

:lol:


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