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Offlinejohnny six-guns
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Registered: 08/06/15
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Newbie First Post - G2G stall from lack of moisture?
    #22071172 - 08/10/15 10:29 AM (8 years, 5 months ago)

Hello everyone. This is my first post here. I've been brought to the shroomery dozens of times over the years when searching for information pertaining to my various hobbies. Now I have decided to join and ask a question.

I've moved on from PF tek to starting my first monotubs. I've went through 3 tubs that contaminated that I traced back to a bad jar of LC (I have since moved on to g2g and agar, but that's only been in the last two weeks).

This is where I've run into a problem - I did a g2g transfer from 1 half pint jar to 5 pint jars as a test. My half pint jar colonized fast and smelled like fresh mushrooms, so I knew it was good. The jars I transferred to smelled like Wbs, so I am assuming it was fine too (sterile procedure involves PCing in an autoclave at 18-30 psi for 75mins). This was 7 days ago.

After 5 days, 2 of the jars are almost done colonizing, while 3 have pretty much stalled. They look no different from before the transfer, and the sniff tests just smells like Wbs still. So I'm not leaning towards a contamination. Nor do I see any signs of bacteria.

Some things to narrow down differences between jars are that these ones were much more full than the ones that colonized. Also, while the other jars "fluffed up" their mycelium as they grew, the stalled jars look exactly the same. It doesnt look at though they "woke up". Also, as the other jars colonized, I could see condensation build in them, whereas this never happened at any point with the stalled jars. They literally look almost exactly as the did when I did the transfer. No better, no worse (which keeps me hopeful).

Some other things to keep in mind are that I have some colonized other jars from the same culuture, and they have taken off fine. Fluffed up and munching along.

So I'm wondering if it's a moisture issue? Last night I injected 12cc of sterile water into each jar to see if that helps, but I haven't been home to check on them since.

To save some questions - I know the "fluffiness" is not contam, because I see the rhizo growth all around it as well, and thanks to my LC I got a nice crash course in trich and cobweb.

I open-air transfer in a (IMO) very sterile and clean room. No carpet. Meticulously dusted. Meticulously bleached and disinfected even when no work is done; with a HEPA running constantly and lots of FAE.

My sterile tech for substrate is for wbs. Clean. Soak for 24hrs. Clean. Bring to a 10 min boil, drain, rinse and throw it in the jar asap to get sterilized.

Sterilization is done in an autoclave. 250-270 degrees at 18-28 psi for 75 mins.

The only thing I can think is maybe some jars are gething too dried out for some reason during the PCing, and that's the problem. Some jars definitely have the Wbs clinking and tinking around.

So any thoughts or experience with this problem? Solutions? I'm at work right now but will answer as I can. Thanks in advance for any help you can give.

-j6g


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Offlinetetherface
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Re: Newbie First Post - G2G stall from lack of moisture? [Re: johnny six-guns]
    #22071205 - 08/10/15 10:43 AM (8 years, 5 months ago)

do you let your grains dry completely in a colander before loading into jars? i'm sure its not from lack of moisture if you indeed soaked your wbs for 24 hrs. do you let you pc naturally return to 0psi before opening it? and are you sure your grains were cool enough when you did your G2G? also G2G in open air is asking for failure rig up a SAB and watch your contam rates drop significantly (especially with Lc's)


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Offlinespacechildo
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Re: Newbie First Post - G2G stall from lack of moisture? [Re: tetherface]
    #22071216 - 08/10/15 10:45 AM (8 years, 5 months ago)

its a very long post, but it seems your master jar colonized fine. do you have any pics?

how do you know what the jars smell like? only open them in a SAB very quickly.

if you have a hole in the lid, and the hole is covered with a filter, your jars are good.

its most likely a contamination thats stalling the growth, now to figure out how it got inside your jar!


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OfflinetaGyo
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Re: Newbie First Post - G2G stall from lack of moisture? [Re: spacechildo]
    #22071258 - 08/10/15 10:58 AM (8 years, 5 months ago)

TLDR.

Condense it for me please.


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Offlinespacechildo
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Re: Newbie First Post - G2G stall from lack of moisture? [Re: taGyo]
    #22071276 - 08/10/15 11:04 AM (8 years, 5 months ago)

OK, this happened

I open-air transfer in a (IMO) very sterile and clean room. No carpet. Meticulously dusted. Meticulously bleached and disinfected even when no work is done; with a HEPA running constantly and lots of FAE.

build a still air box.


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Invisiblewowimflabbergasted
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Re: Newbie First Post - G2G stall from lack of moisture? [Re: spacechildo]
    #22071292 - 08/10/15 11:10 AM (8 years, 5 months ago)

PC at 15psi for 90 minutes, not 18-30psi for 75 minutes. How are you even getting that thing up to 30psi???

Your "sterile" room is far from sterile. Make a $10 SAB. Or get a leftover cardboard box and convert that. You can literally do it for free.


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Offlinejohnny six-guns
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Re: Newbie First Post - G2G stall from lack of moisture? [Re: wowimflabbergasted]
    #22071627 - 08/10/15 12:58 PM (8 years, 5 months ago)

I'm still at work but will send a picture as soon as I get home.

As far as tl;dr, I write long posts that's contain pertinent info, to avoid a lot of the same questions. I refrain from condensing as I like give as much info as possible. Sorry it was too long for you.

I have a SAB and I willingly don't use it. Its cumbersome and awkward and so far I'm having about a 10% fail rate by obvious contam/bacteria. Thats fine with me given that im not using the SAB. This is my first flat stall out, and that's what I'm looking for a reason for, because there is no obvious reason. I know sterility is a factor, but I'd says it minimal in this case. 3 of 25 jars done g2g stalled. I also did all my PF work open air, and as did all 3 tubs. Everything was going beautiful until trich from an LC popped up (yes I know I could have used an SAB, but I learned the greater value of testing LCS before mass innoculating).

To answer other questions.. I'm not using a PC. I'm using a medical grade autoclave. it's quite different in form and function from a PC.

Jars are innocu later anywhere from 24-72 hours after they are pulled, so heat shouldn't be a problem. I smelled them through the hole yesterday and before innocing. They've smelled the same both times, and again there is no growth.

One thing that may be a factor is that no, it is not returning to 0 psi naturally. I have to vent the autoclave, so they lose pressure but maintain temperature very quickly. I think that may be where I'm losing moisture.

And no guys, I'm not being a difficult dick. Sorry if its construed that way. My methods have just been compiled from the million ways to skin a cat I've read across OMC boards, trying and testing different methods. So if I'm using it, it's because it worked somewhere (this is in reference to the SAB and prep temps).

I'll send pics tonight.

Knowing about my prep method, is it maybe the combo of heat and venting that is making me lose moisture and stalling jars?


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OfflinetaGyo
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Re: Newbie First Post - G2G stall from lack of moisture? [Re: johnny six-guns]
    #22071634 - 08/10/15 12:59 PM (8 years, 5 months ago)

The problem is I've read and written, along with a lot of members here, thousands of posts just like that and they all contain the same info and end up with the same problem.

The other guys can help :thumbup:


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Offlinespacechildo
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Re: Newbie First Post - G2G stall from lack of moisture? [Re: johnny six-guns]
    #22071648 - 08/10/15 01:03 PM (8 years, 5 months ago)

Quote:

johnny six-guns said:
My methods have just been compiled from the million ways to skin a cat I've read across OMC boards




just stick to the tried and true teks of shroomery man, lots of bogus BS out there!

I suggest you read through frankhorrigans list of shit! :thumbup:


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Offlinejohnny six-guns
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Re: Newbie First Post - G2G stall from lack of moisture? [Re: johnny six-guns]
    #22071677 - 08/10/15 01:09 PM (8 years, 5 months ago)

Also.. my PCing time is 18-28 psi because that is the nature of the machine as it builds to pressure. I start my time at 15 psi/250 degrees. It will fluctuate between 18 to 28 and 250 to 270. I have no control of that. I can stop the venting process by killing the power to it though.

And again, I'm not trying to be a rude noob. I am just the type of person who never takes the attitude of "my way is the only way, or failure." And I know there are many methods that can be brought to good use simply by troubleshooting the methods.


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Invisiblewowimflabbergasted
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Re: Newbie First Post - G2G stall from lack of moisture? [Re: spacechildo]
    #22071694 - 08/10/15 01:12 PM (8 years, 5 months ago)

Quote:

RogerRabbit said:
Both sterilize with steam pressure.  Some autoclaves automatically open a solenoid at the end of the cycle allowing pressure to escape all at once.  This can cause agar to boil over or jars to dry out.  If you have an automatic autoclave, disable that function so that pressure slowly decreases after the cycle.

For grain jars, you want at least 60 minutes after reaching 15 psi.  For brf jars, get at least 45 minutes after reaching 15psi.  As said above, don't let pressure suddenly release after the cycle, which is fine for medical instruments, but not fine for grain jars.
RR




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Offlinejohnny six-guns
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Re: Newbie First Post - G2G stall from lack of moisture? [Re: wowimflabbergasted]
    #22071744 - 08/10/15 01:22 PM (8 years, 5 months ago)

Quote:

wowimflabbergasted said:
Quote:

RogerRabbit said:
Both sterilize with steam pressure.  Some autoclaves automatically open a solenoid at the end of the cycle allowing pressure to escape all at once.  This can cause agar to boil over or jars to dry out.  If you have an automatic autoclave, disable that function so that pressure slowly decreases after the cycle.

For grain jars, you want at least 60 minutes after reaching 15 psi.  For brf jars, get at least 45 minutes after reaching 15psi.  As said above, don't let pressure suddenly release after the cycle, which is fine for medical instruments, but not fine for grain jars.
RR







Thank you, flab. And from RR himself even.

So I'm looking at the moisture as being the source of my problems, I think.

I'm guessing noone on here has actively experimented with anything like this? Such as what I did with adding sterile water to the grain to add moisture?

As far as correcting the issue long term, I'll just start killing the autoclave when time is done, and let it cool down on its own rather than venting.


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Invisibleblindingleaf
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Re: Newbie First Post - G2G stall from lack of moisture? [Re: johnny six-guns]
    #22071970 - 08/10/15 02:16 PM (8 years, 5 months ago)

do the grains in the jars that stalled look any different?  if u prepped them similarly, why would a few have one moisture content, and a few have a different moisture?

usually, if a grain jar stalls, and u see no color difference (still just white) its bacteria.  mold is usually colorful and very fast, so it wouldn't appear like its stalling.


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Offlinejohnny six-guns
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Re: Newbie First Post - G2G stall from lack of moisture? [Re: blindingleaf]
    #22072006 - 08/10/15 02:27 PM (8 years, 5 months ago)

Quote:

blindingleaf said:
do the grains in the jars that stalled look any different?  if u prepped them similarly, why would a few have one moisture content, and a few have a different moisture?

usually, if a grain jar stalls, and u see no color difference (still just white) its bacteria.  mold is usually colorful and very fast, so it wouldn't appear like its stalling.





No, BL, they don't look any different. They look exactly as they did before the transfer, and the same as the sterile jars sitting on the shelf that were done in the same batch.

There are no signs of excess moisture, or anything like that, nor discoloration or strange growth. It is literally as though the colonized grains that I transferred have just never done anything. Like I did the transfer and time stood still. :confused:

I just got home. I'll be adding some pics very shortly.


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Offlinejohnny six-guns
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Re: Newbie First Post - G2G stall from lack of moisture? [Re: johnny six-guns]
    #22072096 - 08/10/15 02:50 PM (8 years, 5 months ago)

This pic is all 5 jars. They were colonized all on the same day.


This is a picture of a stalled jar.  It looks no different since the transfer.


This is a picture of a sterile jar, of the same substrate, done in the same batch and conditions.


This is one of the colonized jars.  It was colonized with the rest 6 days ago.


For comparison, these next two jars in the grow room that I am letting go and keeping under observation, so I can test out different ideas on them.  The first is jar from the same batches, done with a MS syringe to grain.  I have what looks to be a small spot of trich.  It hasn't grown much, and Im just wanting to personally see for myself how things go once the mycelium gets to it.


This is a jar in my room that was losing a battle to what I believe to be some bacterial contamination due to too much innoculant.  But it also looks like the jar has dried out some, and it is allowing the myc to make a comeback, as its gotten progressively better looking over the last few days.



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Offlinejohnny six-guns
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Re: Newbie First Post - G2G stall from lack of moisture? [Re: johnny six-guns]
    #22072187 - 08/10/15 03:16 PM (8 years, 5 months ago)

Also, BL, I can only speculate that the difference in moisture content is possibly due to different amounts of steam/moisture escaping the jars went I vent the autoclave, possibly even having to do with where they're positioned in the autoclave. I do prep my jars differently than other tech I have read.


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Invisibleblindingleaf
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Re: Newbie First Post - G2G stall from lack of moisture? [Re: johnny six-guns]
    #22072260 - 08/10/15 03:34 PM (8 years, 5 months ago)

what do the top of the jars look like?  whats the filter?


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Re: Newbie First Post - G2G stall from lack of moisture? [Re: blindingleaf]
    #22072264 - 08/10/15 03:34 PM (8 years, 5 months ago)

Yeah why is their foil under the lid??


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Offlinejohnny six-guns
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Re: Newbie First Post - G2G stall from lack of moisture? [Re: wowimflabbergasted]
    #22072337 - 08/10/15 03:53 PM (8 years, 5 months ago)

Top of the jars are just a single self-healing port for inoculation when using an LC or Ms syringe.

Foil is under the lid. I screw the band and lid on around it. It comes off either during transfer, or birthing. For GE I crack the lid about halfway through colonization for a period of time.

Actually, just in typing that I realize that the foil is another vector for moisture to escape during venting of the autoclave.

This stalling without signs of contamination has only shown up with use of the autoclave. Before that I did use a PC.

Some of my methods are unorthodox, but they work as efficiently as anything else. I've never been a believer that there is only one right way to do something, and that it's more important to understand why something doesn't work than to understand why it does, if that makes sense.


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Re: Newbie First Post - G2G stall from lack of moisture? [Re: johnny six-guns]
    #22072364 - 08/10/15 04:00 PM (8 years, 5 months ago)

yes, it makes sense.  do what works for you. 

maybe try cracking the lids on those more.  might be a GE issue, because i can't see any bacteria, and i don't think the grains look dry, u have some burst kernels, which means on the whole, they had enough water before autoclave.  if i prep rye too dry by accident, it will still leap off and grow, but take a bit longer to get to 100%.
the fact that they aren't even fuzzing up makes me think they are not getting enough o2
filters for GE are a pretty standard thing though dude.  i would really trust that more than cracked lid.  poly, tyvek, etc.  when ur jars start getting larger, might be hard to get them to 100%, and because the only entrance for gas is an unfiltered crack, the chance of mold is much higher.  if ur getting away with it now, then thats cool, but just know that it is risky and easily avoidable


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Offlinejohnny six-guns
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Re: Newbie First Post - G2G stall from lack of moisture? [Re: johnny six-guns]
    #22072372 - 08/10/15 04:02 PM (8 years, 5 months ago)

And sorry ahead of time to anyone who I come across as a dick to. I am not like that at all, I've just browsed these boards and others alot, and I realize that as a noob what I say or why I do things just makes me seem misinformed or wrong. I just want to say that's not the case.

I know and have done some of the standard ways of doing things, I just ultimately end up mixing them altogether to find my own rhythm of doing things. They're not always as efficient or make sense, but I try to have some reasoning behind them :grin:.

If you have any questions about why I am doing something, shoot. Because I'm also always open to suggestions on easier ways. I'm just much more of a troubleshoot and modify type of person than I am a follow the directions type. :grin:


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Offlinejohnny six-guns
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Re: Newbie First Post - G2G stall from lack of moisture? [Re: blindingleaf]
    #22072405 - 08/10/15 04:08 PM (8 years, 5 months ago)

Quote:

blindingleaf said:
yes, it makes sense.  do what works for you. 

maybe try cracking the lids on those more.  might be a GE issue, because i can't see any bacteria, and i don't think the grains look dry, u have some burst kernels, which means on the whole, they had enough water before autoclave.  if i prep rye too dry by accident, it will still leap off and grow, but take a bit longer to get to 100%.
the fact that they aren't even fuzzing up makes me think they are not getting enough o2
filters for GE are a pretty standard thing though dude.  i would really trust that more than cracked lid.  poly, tyvek, etc.  when ur jars start getting larger, might be hard to get them to 100%, and because the only entrance for gas is an unfiltered crack, the chance of mold is much higher.  if ur getting away with it now, then thats cool, but just know that it is risky and easily avoidable




I'll give that a try right now.

Those 3 jars really perplexed me because they have literally done nothing.  I transferred 12 jars lastnight and they're already acting lively and getting to work.

I'm floating around the realm of dry (even though I noticed the burst kernels too), the o2 suggestions you had, or maybe I was very rough on them? After the initial transfer, they were very bruised and blue around the edges, but as you can see that faded. So maybe they're still recovering.

For now I suppose I will check the moisture as well as I can, crack the lids some more, and give it time. :thumbup:


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Offlinejohnny six-guns
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Re: Newbie First Post - G2G stall from lack of moisture? [Re: johnny six-guns]
    #22072834 - 08/10/15 05:55 PM (8 years, 5 months ago)

Thank you spacechildo and blindingleaf.

The culprit turned out to be bacteria, like you guys had said. I was looking for signs of it by wet spots or clumping, or any settled discoloration. While there were no visual signs of any of that in the jars, the nose test let me know that things had gone south.

Now a question of where it came from? I can only assume it was something left over from my substrate. But I thought the long soak and PCing would have taken care of that.

Thanks guy.


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Invisibleblindingleaf
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Re: Newbie First Post - G2G stall from lack of moisture? [Re: johnny six-guns]
    #22072867 - 08/10/15 06:03 PM (8 years, 5 months ago)

if I'm working with agar and clean plates, and i get bacteria, IME, its either PC time was fucked up and short, or the temps were a little high in the room.
if ur using MS syringe, can be that too.

any grain prep will work.  but if u start getting bacteria a lot even with clean plates, its good to look at how ur prepping it or how long ur PCing it

if bacteria shows in many places in the jar, up ur PC time.
if its around the g2g grains, check ur OG plate, or possibly ur master jar was not PC'd long enough, and did not recover from the shake/g2g


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Offlinejohnny six-guns
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Re: Newbie First Post - G2G stall from lack of moisture? [Re: blindingleaf]
    #22074855 - 08/11/15 04:53 AM (8 years, 5 months ago)

After looking at some of my notes, I think I have never the problem down.

I believe those 3 jars were the last of the last batch I had done in my PC before switching to the autoclave. They were done for 75mins at 15 psi. They also look much more full compared to the rest IMO.

There was also a short heat wave through my area right around the time that I transferred those which set the temperature in my house and room inot the high 80s for a couple days.

I think the shorter PC time, coupled with the heat wave and possibly too much innoculating (3 cc of LC) set those jars up for failure.


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