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OfflineRo0k
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What am I looking for on agar plates to isolate?
    #22446623 - 10/28/15 09:02 PM (8 years, 3 months ago)

This is my first time using agar to try and isolate strains, but I'm struggling a bit.  I really don't know what I should be looking for.  Strain is B+.

Both of the plates below are the second transfer from a MS syringe, transferred on 10/20.  I just sort of rolled the dice and picked a sector from each of the original plates. 

Can anybody help me out and recommend a good area to take from on these for the third transfer?  I'm really not sure what I should be looking for.  Is there anything worth taking from these?  Should I wait a bit longer?

11:

Maybe around 6 to 7 on this?  I'm not sure, it looks like concentric rings rather than sectoring.




12:

This I can actually see some sectoring.  I want to say between 11 and 12 or 3 and 6 on this one, and exclude that bit from 7-10.



Edited by Ro0k (10/28/15 09:04 PM)


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InvisibleJosh.0
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Re: What am I looking for on agar plates to isolate? [Re: Ro0k]
    #22446829 - 10/28/15 09:41 PM (8 years, 3 months ago)

Can you get better pics
These look suspect IMO..


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Invisibleinsanemike

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Re: What am I looking for on agar plates to isolate? [Re: Josh.0]
    #22446915 - 10/28/15 09:59 PM (8 years, 3 months ago)

That mycelium looks very blotchy and not very ropey. Bacteria is most likely the cause.


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InvisibleMr. Alien
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Re: What am I looking for on agar plates to isolate? [Re: insanemike]
    #22447029 - 10/28/15 10:26 PM (8 years, 3 months ago)

looks like mold.


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Invisibleinsanemike

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Re: What am I looking for on agar plates to isolate? [Re: Mr. Alien]
    #22447049 - 10/28/15 10:33 PM (8 years, 3 months ago)

Quote:

Mr. Alien said:
looks like mold.




You think? I would have thought the mold would look like a seperate growth but those plates look like scattered cube mycelium. I've seen this a few times on my plates and just assumed it was bacteria because anytime I've ever seen mold on a plate, I could see clear seperation between the cube myc and mold myc. I could see the speration even when the cube myc grew over top of the mold.


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OfflineBoomertown
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Re: What am I looking for on agar plates to isolate? [Re: insanemike]
    #22447073 - 10/28/15 10:40 PM (8 years, 3 months ago)

I would just transfer the best looking parts you think look good. When I first started with agar, everything looked like shit. It was mostly due to lack of sterile technique, so I would look at that before your next transfer.

Transfer 1 or 2 cuts from each plate, and keep doing it. I think I did about 6 or 8 transfers before I finally found a plate that was grain worth. Also a good way to tell if you got a good plate is holding it up to the light. If it looks strandy, then you got a good one. I also had the problem of "fluffy" mycelium. So to me it always looked bad, because I was looking for the awesome Rizo looking plates I see around the site.

I also kept the plates I did transfers from, going. Just so I could see how they turned out.


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InvisibleJosh.0
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Re: What am I looking for on agar plates to isolate? [Re: insanemike]
    #22447082 - 10/28/15 10:41 PM (8 years, 3 months ago)


Theres many good agar isolation writeups and step by step pics on this site..you just have to dig a little

Even from my crappy pic you can see a second transfer close to being an iso culture

The forking sectors still have multiple genetics
You would want to transfer the leading edge of the (singular finger):finger:
lookin myc to narrow down an isolate. :thumbup:


--------------------
:trippinbawelz:                              :trippinballs::mindblown::trippinballs:                      :awecid:

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InvisibleJosh.0
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Re: What am I looking for on agar plates to isolate? [Re: Mr. Alien]
    #22447095 - 10/28/15 10:43 PM (8 years, 3 months ago)

Quote:

Mr. Alien said:
looks like mold.



That too :cookiemonster:


--------------------
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InvisibleMr. Alien
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Re: What am I looking for on agar plates to isolate? [Re: insanemike]
    #22447214 - 10/28/15 11:15 PM (8 years, 3 months ago)

Quote:

insanemike said:
Quote:

Mr. Alien said:
looks like mold.




You think? I would have thought the mold would look like a seperate growth but those plates look like scattered cube mycelium. I've seen this a few times on my plates and just assumed it was bacteria because anytime I've ever seen mold on a plate, I could see clear seperation between the cube myc and mold myc. I could see the speration even when the cube myc grew over top of the mold.




Yeah you might be right, now that i look more closely the first picture...i can see like bacteria growing on top of cube myc. No separation whatsoever, like you said. You mean like the tiny little kind of bacterial whitish droplets?


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Invisibleinsanemike

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Re: What am I looking for on agar plates to isolate? [Re: Mr. Alien]
    #22447238 - 10/28/15 11:22 PM (8 years, 3 months ago)

Quote:

Mr. Alien said:
Quote:

insanemike said:
Quote:

Mr. Alien said:
looks like mold.




You think? I would have thought the mold would look like a seperate growth but those plates look like scattered cube mycelium. I've seen this a few times on my plates and just assumed it was bacteria because anytime I've ever seen mold on a plate, I could see clear seperation between the cube myc and mold myc. I could see the speration even when the cube myc grew over top of the mold.




Yeah you might be right, now that i look more closely the first picture...i can see like bacteria growing on top of cube myc. No separation whatsoever, like you said. You mean like the tiny little kind of bacterial whitish droplets?




Yes. What I think is happening is that the bacteria is inhibiting proper cell division and that's why the cube myc looks scattered, instead of uniform.


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InvisibleMr. Alien
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Re: What am I looking for on agar plates to isolate? [Re: insanemike]
    #22447273 - 10/28/15 11:33 PM (8 years, 3 months ago)

Make sense could be. This myc surrounded by mold, you clearly see the separation lines between them. And if you see the back side of the plate against a light source you would see even more clear the separation lines. I had to clean that plate, took myc from around 6:00.



Edited by Mr. Alien (10/29/15 12:35 AM)


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Invisibleenlightened seed
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Re: What am I looking for on agar plates to isolate? [Re: Mr. Alien]
    #22447459 - 10/29/15 12:33 AM (8 years, 3 months ago)

it is best not to use MS on agar.  in my opinion it is best to isolate a good mushroom onto agar and go from there.  using MS will give you different results than a good piece from something prolific.  :cool:


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InvisibleMad Season
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Re: What am I looking for on agar plates to isolate? [Re: Ro0k]
    #22447467 - 10/29/15 12:37 AM (8 years, 3 months ago)

It is possible it is mold. Look at trich for example.

Quote:

Kizzle said:
Actually only the aerial mycelium is white. The vegetative mycelium that grows through the substrate is sort of greyish and is so thin it's to hard to see even on agar.




The aerial myc makes those blotches. Yeast can do it too, as well as bacteria :smile:

I would not trust any of those plates. You might need a bunch of transfers.. But sometimes its just easier to start over


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Edited by Mad Season (10/29/15 12:43 AM)


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OfflineMachiavelliavore
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Re: What am I looking for on agar plates to isolate? [Re: Mad Season]
    #22447531 - 10/29/15 01:07 AM (8 years, 3 months ago)

I was wondering if growth such as that might be caused by water sloshing around on the plate.  I've seen growth that looked sort of like that on my plates and never transfered from those areas.  It never exactly seemed contaminated though, just weak.


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


I spawned some popcorn casings and had double-overlay cause I didn't put enough hydrogen peroxide in my automated aquarium mister.  I only got one mushroom so I cut off the head part where the seeds fall from and put it in a jar of LC and sprayed it all over a tin of PF cakes I made with gravel, cardboard, and bisquick in my microwave.  I think it will be good cause B+ is so potent.
Triggered yet?

Only a square would say "a cube is a cube."


No, this does not look right...


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InvisibleMad Season
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Re: What am I looking for on agar plates to isolate? [Re: Machiavelliavore]
    #22447546 - 10/29/15 01:15 AM (8 years, 3 months ago)

It is possible to be weak. And I could see the water thing (tho ime unlikely. Its colonized no problem with a bit of extra water). However contaminations can be sneaky too. Its hard to say. One thing is for certain tho. It's not trusted! :P


--------------------
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How to shroomery like a pro! (Seriously, everyone read this!)
Improve your sterile techniques! (A comprehensive guide to agar)
Links upon links of literally EVERYTHING UP TO DATE

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OfflineMachiavelliavore
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Re: What am I looking for on agar plates to isolate? [Re: Mad Season]
    #22447555 - 10/29/15 01:20 AM (8 years, 3 months ago)

I should just fill a syringe with water, pc it, and squit it on some healthy agar plates and see if it produces that appearance.  I just inoculated a lot of spawn with bacterial grass seed slury so I'm sad.  Last time I get backstabbed by fucking grass seed.  Spawned to identical cakes a few days ago that smelled fine.

This cornmeal LI sounds super legit.


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


I spawned some popcorn casings and had double-overlay cause I didn't put enough hydrogen peroxide in my automated aquarium mister.  I only got one mushroom so I cut off the head part where the seeds fall from and put it in a jar of LC and sprayed it all over a tin of PF cakes I made with gravel, cardboard, and bisquick in my microwave.  I think it will be good cause B+ is so potent.
Triggered yet?

Only a square would say "a cube is a cube."


No, this does not look right...


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Invisibleenlightened seed
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Re: What am I looking for on agar plates to isolate? [Re: Ro0k]
    #22447579 - 10/29/15 01:37 AM (8 years, 3 months ago)

this is what healthy mycelium looks like


it is not MS, it is a clone.


Edited by enlightened seed (10/29/15 01:39 AM)


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OfflineRo0k
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Re: What am I looking for on agar plates to isolate? [Re: enlightened seed]
    #22448037 - 10/29/15 07:09 AM (8 years, 3 months ago)

Luckily I always take 2 (minimum) of everythimg. I still have 2 plates from the first transfer in my fridge and 2 other plates that I didn't post pics of.

Based in the feedback, those 2 are garbage. Luckily neither of the other 2 have the concentric ring pattern you see on the first plate. Gonna take a couple of transfers from those and see what happens.

AND review my SAB sterile technique. Maybe going to switch to bleach spray instead of soapy water.

I never thought growing fungus would be so dang difficult!


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InvisibleMr. Alien
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Re: What am I looking for on agar plates to isolate? [Re: enlightened seed]
    #22448384 - 10/29/15 08:58 AM (8 years, 3 months ago)

Quote:

enlightened seed said:
this is what healthy mycelium looks like


it is not MS, it is a clone.




lol a clone is actually MS.


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OfflineYaMoonSun
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Re: What am I looking for on agar plates to isolate? [Re: Mr. Alien]
    #22448395 - 10/29/15 09:02 AM (8 years, 3 months ago)

Multiple Sectors?


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InvisibleLeviticus969
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Re: What am I looking for on agar plates to isolate? [Re: Ro0k]
    #22448506 - 10/29/15 09:39 AM (8 years, 3 months ago)

Even if there is mold on the the plates u should still be able to transfer away from it...

Anyways, id try transferring to another plate from those 2 pics u posted. U may get better growth on the next transfer. The reason why your plates look like that (tomentose) is bcuz of the spores, not ur sterile technique. Some spores give that fluff n some ropey. Both styles will fruit.


U may even see rhizomorphic (ropey) mycelium on the next transfer. There's not many sectors on ur plates so just take 1 or 2 from each plate.

2 cents


Edited by Leviticus969 (10/29/15 09:42 AM)


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InvisibleMr. Alien
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Re: What am I looking for on agar plates to isolate? [Re: YaMoonSun]
    #22448582 - 10/29/15 10:10 AM (8 years, 3 months ago)

Quote:

YaMoonSun said:
Multiple Sectors?




MS do not stands for Multiple Sectors. MS stands for Multi spore in terms of shroomery. And a clone also have multiple sectors.


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OfflineMadSeasonStudent
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Re: What am I looking for on agar plates to isolate? [Re: Mr. Alien]
    #22448755 - 10/29/15 11:09 AM (8 years, 3 months ago)

That Doesn't Look contaminated. Looks wispy is all. I wouldn't toss it just yet. When it grows out some more just get a piece that isn't wispy and transfer that over and from there you should get some acceptable sectors.


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OfflineYaMoonSun
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Re: What am I looking for on agar plates to isolate? [Re: Mr. Alien]
    #22449020 - 10/29/15 12:26 PM (8 years, 3 months ago)

I know, I was just poking fun :cheers:


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Offlinespacechildo
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Re: What am I looking for on agar plates to isolate? [Re: enlightened seed]
    #22449069 - 10/29/15 12:41 PM (8 years, 3 months ago)

if you dont put "ms" or spores on agar then where the * do you put your spores? :lol:
gotta start somewhere to get that mushroom you so desperately wanna clone..


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Invisibleenlightened seed
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Re: What am I looking for on agar plates to isolate? [Re: spacechildo]
    #22449101 - 10/29/15 12:46 PM (8 years, 3 months ago)

Quote:

spacechildo said:
if you dont put "ms" or spores on agar then where the * do you put your spores? :lol:
gotta start somewhere to get that mushroom you so desperately wanna clone..



PF jars


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Offlinespacechildo
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Re: What am I looking for on agar plates to isolate? [Re: enlightened seed]
    #22449151 - 10/29/15 01:00 PM (8 years, 3 months ago)

but why?
agar ensures clean growth, you get way younger in vitro pins that are prime candidates for cloning,
you dont need to fuck around with syringes or scraping spores into shotters and that crap.


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InvisibleMr. Alien
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Re: What am I looking for on agar plates to isolate? [Re: spacechildo]
    #22449168 - 10/29/15 01:03 PM (8 years, 3 months ago)

:whathesaid:


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Invisibleenlightened seed
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Re: What am I looking for on agar plates to isolate? [Re: spacechildo]
    #22449197 - 10/29/15 01:09 PM (8 years, 3 months ago)

Quote:

spacechildo said:
but why?
agar ensures clean growth, you get way younger in vitro pins that are prime candidates for cloning,
you dont need to fuck around with syringes or scraping spores into shotters and that crap.




well there are different routes people can take to create an isolate.  i have yet to try to isolate a vitro pin but i will keep that in mind for future experiments.


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OfflineRo0k
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Re: What am I looking for on agar plates to isolate? [Re: Mr. Alien]
    #22449206 - 10/29/15 01:14 PM (8 years, 3 months ago)

But there IS myc on thosr plates right? It's not just a ton of mold I'm misreading?

I ask because the print i used is from 2012 I think, and it started germinating in 3 days from a syringe if I remember correctly.

I also made a syringe from a NEWER amazonian print (2013) and after a week there's zero growth. Not even bacteria/mold.


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Offlinespacechildo
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Re: What am I looking for on agar plates to isolate? [Re: Ro0k]
    #22449217 - 10/29/15 01:17 PM (8 years, 3 months ago)

cant really say if its mold or bacteria but either way I'd start over if those were mine.
cant really see anywhere I'd transfer from either, sorry dude..


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OfflineRo0k
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Re: What am I looking for on agar plates to isolate? [Re: spacechildo]
    #22449492 - 10/29/15 02:42 PM (8 years, 3 months ago)

Eh. There are no mistakes if you learn something. I'm going to try going directly from the print to agar using a couple of methods instead of the syringe middle-man.

If the print is more than a year old, how long do you think I should wait if I see no growth before wondering if I did something wrong?


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Offlinespacechildo
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Re: What am I looking for on agar plates to isolate? [Re: Ro0k]
    #22449509 - 10/29/15 02:45 PM (8 years, 3 months ago)

2-3 weeks no matter how old.
I dont like  syringes -> agar, high chance of getting too much water on there.
just scrape/swipe some spores with a home made loop onto the plate


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