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OfflinetubbyToLoose
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Registered: 05/26/20
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clear slow growth
    #26698031 - 05/26/20 09:13 PM (3 years, 7 months ago)

on my 12th transfer from spores all of a sudden everything has changed from rhyzo to this clear slow growth, Is this what it looks like when you get close to an isolate ? or is this potential mold or contaminate ? should i turn back and start over ? or can i go ahead with this ?



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InvisibleApples in Mono
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Registered: 09/21/17
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Re: clear slow growth [Re: tubbyToLoose]
    #26698033 - 05/26/20 09:18 PM (3 years, 7 months ago)

It can switch back and forth between rhyzo and tomentose growth. Did the agar recipe change a bit for that transfer?


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OfflineMad Hatter
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Re: clear slow growth [Re: tubbyToLoose]
    #26698075 - 05/26/20 09:45 PM (3 years, 7 months ago)

Have all the transfers been to the same agar recipe and do you let them grow out that much after each transfer?  You may be seeing a bit of senescence not necessarily degradation of the strain but possibly due to the same media being used over and over and to say in layman's terms its getting bored.

In the future make your transfers much much smaller for strain isolation you only need a very small fragment. Smaller fragments mean less transfers to obtain a monoculture and less energy and cell division means a younger more vigorous culture once a monoculture is obtained. Less petri dishes used to. Look into 3 compartment petridishes for strain isolation. They are cheap on ebay for a 500case like $50.  I  slice off the very tip of rhizomorphic growth. Literally the size of a syringe tip.

Also from personal experience if you isolate monocultures test multiple at once. You may get a nonfruiter i have before.....


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InvisibleCaps McGee
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Re: clear slow growth [Re: Mad Hatter]
    #26698077 - 05/26/20 09:47 PM (3 years, 7 months ago)

That white creamy run in the second photo is bacterial metabolites and the reason for the slow weak growth... 12 transfers is FAR from an isolate; even if you started from a clone (yes, single fruit will still contain hundreds, even THOUSANDS of individual strains)

Note also the creamy opaque halo around the majority of the cultures? Also indicative of bacterial bloom

Watch them carefully for a few days and see if you get any off shoots that you can transfer to escape


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InvisibleApples in Mono
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Re: clear slow growth [Re: Caps McGee]
    #26698084 - 05/26/20 09:51 PM (3 years, 7 months ago)

I think that's just the reflection from the white baseball cap he was wearing at the time. Lol


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InvisibleCaps McGee
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Re: clear slow growth [Re: Apples in Mono]
    #26698088 - 05/26/20 09:53 PM (3 years, 7 months ago)

No, that's liquid running on the plate and its white... second look as t the second pic you can distinctly see the bacterial colony's leading edge an inch or so out in front all the way around... that plate is likely a total loss

Is that a hat? Lmao... either or: doesnt explain the halos, or why the growth is very weak... something fucky is going on


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InvisibleApples in Mono
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Re: clear slow growth [Re: Caps McGee]
    #26698098 - 05/26/20 09:57 PM (3 years, 7 months ago)

Yeah, I just see some condensation and a baseball cap


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InvisibleCaps McGee
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Re: clear slow growth [Re: Apples in Mono]
    #26698103 - 05/26/20 10:00 PM (3 years, 7 months ago)

That culture is far from healthy vigorous growth in any event... if he was getting vigorous rhizomorphs and now is getting nothing but slow tomentose growth, I'd suspect contamination, or some poor luck with genetic isolation... the 3rd from last plate looks most promising imo


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OfflineMad Hatter
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Re: clear slow growth [Re: Caps McGee]
    #26698145 - 05/26/20 10:21 PM (3 years, 7 months ago)

I see bacteria in the second pic as well and defenitly agree thats not very healthy growth. The amount of transfer really depends on the amount of spores used and size of transfers. But i defenitly agree 12 isnt a ridiculous amount but typically when you transfer for isolation you dont let the culture grow out so much. Growing out over 12 full agar dishes over the same media can cause slow weaker growth not necessarily senescence expecially if its on a nutritionally weak agar to achieve faster growth. transfering to a different media may reinvigorate it.

Shouldnt of used the word senescence.


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InvisibleApples in Mono
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Re: clear slow growth [Re: Mad Hatter]
    #26698303 - 05/27/20 12:12 AM (3 years, 7 months ago)

There's definitely a "halo" of condensation there, as well as a reflection of a dude in a baseball cap with a smartphone in front of his face. Also looks to me like a reflection of something else a bit higher up and to the right.

It certainly doesn't look like super vigorous growth and it looks a bit unorganized(though only right around the takeoff point). This could be a result of bacteria, but I really can't make out any VISIBLE bacteria. And as far as growth slowing down after a transfer, that isn't necessarily a sign of an unhealthy or bacterial culture, certainly not without knowing whether or not there were any shifts in things like agar recipe and temperature


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OfflinetubbyToLoose
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Re: clear slow growth [Re: Apples in Mono]
    #26701552 - 05/28/20 11:02 AM (3 years, 7 months ago)

Hey, thanks for the responses. A little more info for ya. This culture was started two years ago via spore syringe to grain jar. As soon as the grains had some mycelium i transfered to agar and did a transfer of clean growth. The subsequent plates were used to inoculate grain jars and several of the nicer plates were saved and refrigerated. The plates were then removed from the fridge and had a transfer done. most of those plates were used for innoculating grain and again several plates were saved in the fridge for the next grow. This has been the cycle so far over and over. Ive been using mea agar for the entire time. Been doing 15g agar, 15g lme to 750ml water pcd for 45 mins at 15psi.the plates i posted were actually dropped to 11g of malt.


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OfflineSockadin
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Re: clear slow growth [Re: tubbyToLoose]
    #26701578 - 05/28/20 11:14 AM (3 years, 7 months ago)

Looks like the Parafilm in the back of the plate.

But yeah the growth doesn't look healthy. Try a low nutrient plate and see if it jumps back up interns of plate colonization.
Like 5-7lme and 8-9 agar. 500ml


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InvisibleLadysKnight
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Re: clear slow growth [Re: Caps McGee]
    #26701658 - 05/28/20 11:49 AM (3 years, 7 months ago)

Quote:

Caps McGee said:
12 transfers is FAR from an isolate; even if you started from a clone (yes, single fruit will still contain hundreds, even THOUSANDS of individual strains)







Quote:

cronicr said:
Quote:

LadysKnight said:
Isn't a clone still multiple strains, thereby MS?



nobody can prove it, as far as published studies go, one mushroom one strain.
people have put out theories about there being more but that's about it




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InvisibleCaps McGee
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Re: clear slow growth [Re: LadysKnight]
    #26706767 - 05/30/20 09:34 AM (3 years, 7 months ago)

I've seen sectors develop immediately, and perform differently, even had albino pop up from a clone culture several transfers from tissue... I don't have a scope or genetic testing equipment, but seems fairly concrete to me


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