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InvisibleThomsen
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Registered: 09/27/19
Posts: 364
Bacterial spawn - or not yet?
    #26940752 - 09/17/20 01:48 PM (3 years, 4 months ago)

Hey guys.

I've been struggeling with bacterial spawn lately and i'm having kind of a hard time to pinpoint where it goes wrong.
Although i'm quite sure that is not when doing agar thing goes wrong.
I've been doing NTC plates everytime i've made a agar batch and they always turn out blank. But, just to be sure i stepped it up a notch to see how'd go.


NTC after pouring (coagulating)
I poured my agar plates and waited for them to coagulate, and whilest doing that i left one plate with the lid half off (just over the edge and resting on the table).
When doing so, i actually left the lab where my flowhood is placed, and i entered it again about 2 hours later. Had to be sure, right?

The plate looks like this:


Just a single colony of what could look like Staphylococcus aureus
Not bad for an open plate which was places about 40 cm from the door i opened and closed twice. This picture is 5 days later.


NTC whilest cleaning edibles
But of course i needed to test it some more.
So left another plate open right on the edge of my flowhood whilest i cleaned up about 15 plates with edibles.
Also, my torch was standing 5 cm away from it, which i used to sterilize my scapel between every transfer from old plates to new.

The plate looks like this:


Not even a single colony to spot, 5 days later.
So the conclusion here is that i'm not introducing bacteria when doing agar work. The impurities you see on the plate is from the agar, so it's inside the puck.


Clean cultures
I clean my cultures about 3-4 times before inculating bags and after about 5 days they look like this:

PESA


Stropharia - note the rings?


The plates have been in the fridge for a couple of days and i just took them out today. The only thing i see here is the rings. But i read that they can form by temp. swings i.e. the fridge.
Almost all of my cultures have tomentose morphology, but i have been using a  pretty high concentration the past time, which was 12g MEA for 350-400mL.
What do you say?


Last but not least; the bags.
Theres a tendency that my bags looks like the following pictures, and after the secound shake (i make the secound shake about 4 days before this state) they just show a lot of bacteria in form of uniform growth and what not.
The they even smell very slightly sweet (some more than others)

To my eye these bags look pretty darn good. Am i blind?



These bags a B+ and LGT but the technique is the same all the way though. Has also been cleaned may times. And they haven't had the secound shake just yet.
The bags are inoculated with agar because G2G is not possible due to the freakin bacteria...

Hope you can help guys!


Edited by Thomsen (09/17/20 01:53 PM)


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InvisibleJosex
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Re: Bacterial spawn - or not yet? [Re: Thomsen]
    #26940772 - 09/17/20 02:00 PM (3 years, 4 months ago)

Can't read it all right now but those bags look spawn-able to me too. If you were to crumble them up right now it could tell you a lot by the way they recover, a last shake can make more apparent what wasn't obvious before.


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OfflineForresterM
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Re: Bacterial spawn - or not yet? [Re: Josex]
    #26940808 - 09/17/20 02:24 PM (3 years, 4 months ago)

I see signs of bacteria in the bags.  Spawn-able, maybe bacteria that cubes can beat, but I think there's bacteria in there.  It's that slimy-wet look up against the sides of the bag.

Have you left out a test grain bag to rule out your sterilization process?  How long are you sterilizing the bags for?


--------------------
Repugnant is a creature who would squander the ability to lift an eye to heaven, conscious of his fleeting time here.
-------------------

Have some medicinal mushrooms and want to get the most out of them?  Try this double extraction method.


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InvisibleJosex
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Re: Bacterial spawn - or not yet? [Re: Forrester]
    #26940835 - 09/17/20 02:44 PM (3 years, 4 months ago)

Quote:

Forrester said:
I see signs of bacteria in the bags.  Spawn-able, maybe bacteria that cubes can beat, but I think there's bacteria in there.  It's that slimy-wet look up against the sides of the bag.

Have you left out a test grain bag to rule out your sterilization process?  How long are you sterilizing the bags for?




:whathesaid:
Also, myc looks stressed and growth is a bit too dense and thick. Spawn-able is one thing and clean spawn is another.

I'm also inclined to believe it may have something to do with the prep and an inssuficient cycle.


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InvisibleThomsen
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Re: Bacterial spawn - or not yet? [Re: Josex]
    #26940872 - 09/17/20 03:00 PM (3 years, 4 months ago)

Quote:

Forrester said:
I see signs of bacteria in the bags.  Spawn-able, maybe bacteria that cubes can beat, but I think there's bacteria in there.  It's that slimy-wet look up against the sides of the bag.

Have you left out a test grain bag to rule out your sterilization process?  How long are you sterilizing the bags for?





In what picture do you see this?
I PC for 2,5 hours. 4x1kg bags each time. :shrug:


Quote:

Josex said:
Quote:

Forrester said:
I see signs of bacteria in the bags.  Spawn-able, maybe bacteria that cubes can beat, but I think there's bacteria in there.  It's that slimy-wet look up against the sides of the bag.

Have you left out a test grain bag to rule out your sterilization process?  How long are you sterilizing the bags for?




:whathesaid:
Also, myc looks stressed and growth is a bit too dense and thick. Spawn-able is one thing and clean spawn is another.

I'm also inclined to believe it may have something to do with the prep and an inssuficient cycle.




Is it stressed in the bags or on the plates?


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InvisibleJosex
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Re: Bacterial spawn - or not yet? [Re: Thomsen]
    #26940887 - 09/17/20 03:07 PM (3 years, 4 months ago)

Bags


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OfflineForresterM
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Re: Bacterial spawn - or not yet? [Re: Thomsen]
    #26941050 - 09/17/20 05:04 PM (3 years, 4 months ago)

Quote:

Thomsen said:
In what picture do you see this?




All of them. 

If you're certain about your sterilization, it's gotta be getting in there when you inoculate with that wedge.


--------------------
Repugnant is a creature who would squander the ability to lift an eye to heaven, conscious of his fleeting time here.
-------------------

Have some medicinal mushrooms and want to get the most out of them?  Try this double extraction method.


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InvisibleThomsen
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Re: Bacterial spawn - or not yet? [Re: Josex]
    #26941624 - 09/18/20 01:52 AM (3 years, 4 months ago)

Quote:

Josex said:
Bags




Actually I'm suspecting the bags to be the problem.

Weather it's when inoculating or shitty bags idk. I place the PC on the floor in the lab, opens the lid, wearing clean gloves and what not. Exactly the same procedure as when moving agar from the pc.
And then place the bags in front of the hood, and leaves them there for about 20-30 mins often.
I do not wipe the bags down with ethanol before inoculation though.


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OfflineForresterM
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Re: Bacterial spawn - or not yet? [Re: Thomsen]
    #26941648 - 09/18/20 02:54 AM (3 years, 4 months ago)

If you want to keep using bags, and your sterile technique with agar/jars is good, you could always make a LC and inoculate the bags with a syringe.  Then your not having to open them and worry about stuff getting in.  But then again if you're having issues with bacteria I'm not sure if LC is the best idea. :shrug:


--------------------
Repugnant is a creature who would squander the ability to lift an eye to heaven, conscious of his fleeting time here.
-------------------

Have some medicinal mushrooms and want to get the most out of them?  Try this double extraction method.


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InvisibleThomsen
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Registered: 09/27/19
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Re: Bacterial spawn - or not yet? [Re: Forrester]
    #26941688 - 09/18/20 04:39 AM (3 years, 4 months ago)

I don't have any bags with injection ports.
Could I just poke it through the bag and micropore tape it?
Although im not really a fan of one-use bags.

Idk where it goes wrong. Could it be the filter in the bags? They should be 0,2 microns though...


Edited by Thomsen (09/18/20 10:37 AM)


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OfflineForresterM
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Re: Bacterial spawn - or not yet? [Re: Thomsen]
    #26942926 - 09/18/20 06:10 PM (3 years, 4 months ago)

Quote:

Thomsen said:
Could it be the filter in the bags? They should be 0,2 microns though...




I haven't heard of this happening, but it's always possible there's a bad batch of something like that. 

You say you don't like one-use bags - are these bags you've run through a PC cycle before?


--------------------
Repugnant is a creature who would squander the ability to lift an eye to heaven, conscious of his fleeting time here.
-------------------

Have some medicinal mushrooms and want to get the most out of them?  Try this double extraction method.


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InvisibleThomsen
I'm a teapot


Registered: 09/27/19
Posts: 364
Re: Bacterial spawn - or not yet? [Re: Forrester]
    #26943784 - 09/19/20 09:43 AM (3 years, 4 months ago)

Okay, I guess it must be when inoculating then.

No these bags haven't been used before.
Other bags I've used have had holes in the bottom where it's closed after a run though.
I fund it after spawning.


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Offlineverum subsequentis
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Re: Bacterial spawn - or not yet? [Re: Thomsen]
    #26944091 - 09/19/20 12:48 PM (3 years, 4 months ago)

What kind of PC are you using?
Are you maintaining a pretty even psi or blasting steam?
Microscopic pinholes can be deadly to a grow and can be caused by dramatic pressure and temperature fluctuations while the plastic bags are hot.

I'm not saying that this is your issue here but it is one possibility.


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InvisibleThomsen
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Re: Bacterial spawn - or not yet? [Re: verum subsequentis]
    #26944153 - 09/19/20 01:15 PM (3 years, 4 months ago)

I'm using a AA921 and i set the stove on max under the whole cycle.
I raises to a bit over 16,5 psi and then i turn it a bit down so it sets at 15 psi for the rest of the cook. So i'd say it's even throughout the cycle.


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Offlineverum subsequentis
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Re: Bacterial spawn - or not yet? [Re: Thomsen]
    #26944306 - 09/19/20 02:39 PM (3 years, 4 months ago)

sounds good. You may want to rub some Vaseline on on the over pressure plug while under pressure and see if it bubbles. Be careful as it's rather hot. If it bubbles than either the plug needs replaced or the sealed came or has become scratched. I once lost a whole bunch of bags in a new machine and finally found out it was because the over pressure plug was leaking due to faulty fabrication of the cover by AA. They sent me a new one but it still sucked.

you can also tell if something is up based on whether or not the unit holds a vacuum after you let it cool. If not it could be the same issue or the stop cock (or the cover seal itself)


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InvisibleThomsen
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Re: Bacterial spawn - or not yet? [Re: verum subsequentis]
    #26944381 - 09/19/20 03:15 PM (3 years, 4 months ago)

Okay will try that one the next cycle. You mean vaseline on there the plug is screwed in the lid, right?


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Offlineverum subsequentis
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Re: Bacterial spawn - or not yet? [Re: Thomsen]
    #26944386 - 09/19/20 03:17 PM (3 years, 4 months ago)

no. the red (or black) rubber over pressure plug


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InvisibleThomsen
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Re: Bacterial spawn - or not yet? [Re: verum subsequentis]
    #26944408 - 09/19/20 03:28 PM (3 years, 4 months ago)

The AA921 uses a wiggle weight


Edited by Thomsen (09/19/20 03:29 PM)


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Offlineverum subsequentis
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Re: Bacterial spawn - or not yet? [Re: Thomsen]
    #26945262 - 09/20/20 12:49 AM (3 years, 4 months ago)

oh, shit. really? Never had one. Didn't know they made one with a wiggle


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InvisibleThomsen
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Registered: 09/27/19
Posts: 364
Re: Bacterial spawn - or not yet? [Re: verum subsequentis]
    #26945303 - 09/20/20 01:07 AM (3 years, 4 months ago)

Yeah. Be glad you haven't, they are a pain in the ass to listen to when cooking. I'm actually thinking about buying an autoclave instead, just because of the noise :lol:


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