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OfflineEclipse3130
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Agar To Grain Slow/Stalled Growth?
    #26495134 - 02/20/20 12:52 PM (3 years, 11 months ago)

Been having continuous trouble with my grain jars stalling out after a week or so of growth, I am open to it being bacterial or contamination related, but wouldn't make the most of sense as all my other practices don't yield any contamination, and I always inoculate from clean material - I just recently made 30 agar plates and only 1 of them received a small amount of contamination.

I understand agar to grain is a slower process than LC or G2G most of the time.

I have had over 50 jars not finish, to varying degrees and can't seem to pin down the problem.

Here are some pictures of what usually happens.

(7 days post agar inoculation, 4 days post shake)
This is where my jars usually stall out, and stop growing.


This jar is around 25 days old after a shake a good few weeks ago, it just never recovered. Most of my jars will stall out at various levels of growth, and 0 ever reaching full colonization. Always inoculating from a clean plate.




Visible contamination never develops in any of the jars, even after 2 months. I am guessing it is bacteria? What would be the cause of this, jars are pressure cooked for 120 minutes.

Update:
I have been cooling my scalpel with alcohol post flame sterilization, I am guessing this is what is causing the jars to get bacteria? But this also wouldn't make much sense and it is the same method I use to make the plates and they turn out fine(I will stop cooling with isopropyl anyway)


--------------------
"In The Material World One seeks retirement and grows Old
In The Magical World One seeks Enlightenment and grows Wiser
In The Miraculous World One seeks nothing and grows Lighter
As we all tread the Homeward Path we will explore many Realms
And one day... we will all Realize that all experiences are Simply
Different ways in which The
All-That Is
Perceives Itself"


Edited by Eclipse3130 (02/20/20 01:37 PM)


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InvisiblePiptheGreAtest
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Re: Agar To Grain Slow/Stalled Growth? [Re: Eclipse3130]
    #26495171 - 02/20/20 01:10 PM (3 years, 11 months ago)

I think this method is much quicker in achieving results full colonization 7-10 days hope it helps out.
https://www.shroomery.org/forums/showflat.php/Number/22833314/fpart/1/vc/1


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OfflineSynKyd
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Re: Agar To Grain Slow/Stalled Growth? [Re: PiptheGreAtest]
    #26495183 - 02/20/20 01:14 PM (3 years, 11 months ago)

Posting the plates pics immediately after you take your wedge to grain will get you a better answer here.

If you rule out the plates and grains contam then it’s probably a low hydration issue.

:hippie:


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OfflineEclipse3130
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Re: Agar To Grain Slow/Stalled Growth? [Re: PiptheGreAtest]
    #26495191 - 02/20/20 01:17 PM (3 years, 11 months ago)

Quote:

PiptheGreAtest said:
I think this method is much quicker in achieving results full colonization 7-10 days hope it helps out.
https://www.shroomery.org/forums/showflat.php/Number/22833314/fpart/1/vc/1




Interesting, i'll consider it, as well as do some test runs without alcohol wiping my flame sterilized needle :facepalm:


--------------------
"In The Material World One seeks retirement and grows Old
In The Magical World One seeks Enlightenment and grows Wiser
In The Miraculous World One seeks nothing and grows Lighter
As we all tread the Homeward Path we will explore many Realms
And one day... we will all Realize that all experiences are Simply
Different ways in which The
All-That Is
Perceives Itself"


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OfflineEclipse3130
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Re: Agar To Grain Slow/Stalled Growth? [Re: SynKyd]
    #26495199 - 02/20/20 01:22 PM (3 years, 11 months ago)

Quote:

SinysterKyd said:
Posting the plates pics immediately after you take your wedge to grain will get you a better answer here.

If you rule out the plates and grains contam then it’s probably a low hydration issue.

:hippie:




I usually just throw a whole plate in. But here's what my plates look like, 95% of my plates are contamination free, and I only use the ones that are good to transfer into grain, I'm assuming it's because I alcohol wipe my scalpel after flame sterilizing that is introducing all the bacteria into the jars as that is the only thing I can think of that I am doing wrong, but this also wouldn't really make much sense as it is the same technique I use to make my plates and they turn out fine. I know this is the wrong way to sterilize your blade



--------------------
"In The Material World One seeks retirement and grows Old
In The Magical World One seeks Enlightenment and grows Wiser
In The Miraculous World One seeks nothing and grows Lighter
As we all tread the Homeward Path we will explore many Realms
And one day... we will all Realize that all experiences are Simply
Different ways in which The
All-That Is
Perceives Itself"


Edited by Eclipse3130 (02/20/20 01:36 PM)


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OfflineEclipse3130
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Re: Agar To Grain Slow/Stalled Growth? [Re: Eclipse3130]
    #26495254 - 02/20/20 01:55 PM (3 years, 11 months ago)

I'm going to try inoculating inside of a SAB rather than using my flowhood to see if anything changes.


--------------------
"In The Material World One seeks retirement and grows Old
In The Magical World One seeks Enlightenment and grows Wiser
In The Miraculous World One seeks nothing and grows Lighter
As we all tread the Homeward Path we will explore many Realms
And one day... we will all Realize that all experiences are Simply
Different ways in which The
All-That Is
Perceives Itself"


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Offlineshevanel
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Re: Agar To Grain Slow/Stalled Growth? [Re: Eclipse3130]
    #26495349 - 02/20/20 02:42 PM (3 years, 11 months ago)

Whats your lid tek?


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OfflineEclipse3130
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Re: Agar To Grain Slow/Stalled Growth? [Re: shevanel]
    #26495359 - 02/20/20 02:46 PM (3 years, 11 months ago)

Quote:

shevanel said:
Whats your lid tek?




Micropore tape

Making some unmodded lid jars now to see if it makes any difference. Really confusing me what's going on, but obviously it must be bacteria halting the growth, 0% success rate, just doesn't make much sense how bacteria would enter my jars if the same technique is used to make my plates and they are fine.

Going back to the box.. i'm sure things will be just fine in there :lol:


--------------------
"In The Material World One seeks retirement and grows Old
In The Magical World One seeks Enlightenment and grows Wiser
In The Miraculous World One seeks nothing and grows Lighter
As we all tread the Homeward Path we will explore many Realms
And one day... we will all Realize that all experiences are Simply
Different ways in which The
All-That Is
Perceives Itself"


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Offlineshevanel
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Re: Agar To Grain Slow/Stalled Growth? [Re: Eclipse3130]
    #26495439 - 02/20/20 03:20 PM (3 years, 11 months ago)

Micropore tape sucks imo. It may or may not be your issue but man polyfill lids are easy, cheap and work great.


Edited by shevanel (02/20/20 03:20 PM)


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OfflineEclipse3130
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Re: Agar To Grain Slow/Stalled Growth? [Re: shevanel]
    #26495454 - 02/20/20 03:25 PM (3 years, 11 months ago)

Quote:

shevanel said:
Micropore tape sucks imo. It may or may not be your issue but man polyfill lids are easy, cheap and work great.






I've never had an issue with micropore tape in the past, when working out of a box, but I will change it up as something is not working right. I have been using a flowhood for a few months, but haven't gone back to box yet to see if I can get a jar of clean spawn


--------------------
"In The Material World One seeks retirement and grows Old
In The Magical World One seeks Enlightenment and grows Wiser
In The Miraculous World One seeks nothing and grows Lighter
As we all tread the Homeward Path we will explore many Realms
And one day... we will all Realize that all experiences are Simply
Different ways in which The
All-That Is
Perceives Itself"


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Invisiblefilthyknees
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Re: Agar To Grain Slow/Stalled Growth? [Re: Eclipse3130]
    #26495810 - 02/20/20 06:31 PM (3 years, 11 months ago)

For your consideration, undoubtedly sfds are a better option. How many layers?

https://www.shroomery.org/forums/showflat.php/Number/13643478#13643478

Quote:

stonesun said:
Well I didn't measure the the pore sizes,
instead took a piece of tape and mounted it on a slide.
Hydrated some P. cubensis spores and with an inoculation loop
mounted them on the tape.
The gaps are huge comparing to cubensis spores, which are ~12-17µm.
I circled the fibers with individual spores sticked to them.
Sorry for the kinda crappy images, didn't put a whole lotta effort in it...

100X


400X


400X





--------------------
But if you're in a hurry, and really got to go
If you're in a hurry, might have to find out slow
That it's one thing to try and another to fly
You get there quicker just a step at a time
It's one thing to bark, another to bite
The show ain't over till you pack up at night


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OfflineEclipse3130
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Re: Agar To Grain Slow/Stalled Growth? [Re: filthyknees]
    #26495938 - 02/20/20 07:33 PM (3 years, 11 months ago)

It seems to be a gas exchange issue for sure, my holes are puny small. 1/16th... With 2 layers of micropore. :lol: need 1/4 inch minimum, all jars are extremely slow, and none with really any visible contamination, some develop a bacterial look after a long time, even after 30-60 days, but far and few between - This would explain a lot. Some reach near full colonization, but none ever completely finish, and very very few develop a visible contamination.

Thank you for the feedback


--------------------
"In The Material World One seeks retirement and grows Old
In The Magical World One seeks Enlightenment and grows Wiser
In The Miraculous World One seeks nothing and grows Lighter
As we all tread the Homeward Path we will explore many Realms
And one day... we will all Realize that all experiences are Simply
Different ways in which The
All-That Is
Perceives Itself"


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InvisibleInfiniteDreams
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Re: Agar To Grain Slow/Stalled Growth? [Re: Eclipse3130]
    #26495946 - 02/20/20 07:36 PM (3 years, 11 months ago)

If you want SFD check our vendors, some made you buy in bulk but others were piecemeal and they are like less than $1 a piece


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OfflineSynKyd
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Re: Agar To Grain Slow/Stalled Growth? [Re: Eclipse3130]
    #26496024 - 02/20/20 08:26 PM (3 years, 11 months ago)

Quote:

Eclipse3130 said:
I'm assuming it's because I alcohol wipe my scalpel after flame sterilizing that is introducing all the bacteria.




This was a problem for me, went back to flaming blade for every cut and my bacteria plates are down for sure.


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InvisibleHans Wermhat
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Re: Agar To Grain Slow/Stalled Growth? [Re: SynKyd]
    #26496128 - 02/20/20 09:24 PM (3 years, 11 months ago)

Well there's the issue of the scalpel like you said, which is good to recognize and correct, but tbh I doubt that's resulting in bacterial contams in every single one of your a2g jars. You said they all seem to stall around the same time, that suggests that it's a gas exchange issue (which in itself could exacerbate what might otherwise have been a minor and unnoticeable bacterial problem, although jars don't look bacterial to me anyway).

A 1/4" hole is 16 times larger than a 1/16" hole. Does a 1/16" hole with 2 layers of micropore tape supply enough GE? I don't know. Maybe it's enough, maybe not, but in addition to the scalpel thing I would definitely also increase the hole size.


--------------------
Thank you for taking the time to read this message. Have a wonderful day.


Edited by Hans Wermhat (02/20/20 09:25 PM)


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