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Offlineverum subsequentis
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Re: LC Testing + Grow Log [Re: Forrester]
    #26990157 - 10/17/20 02:18 PM (3 years, 3 months ago)

:goodday:


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InvisibleMateja
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Re: LC Testing + Grow Log [Re: eLeSDenes]
    #26991847 - 10/18/20 06:43 PM (3 years, 3 months ago)

Quote:

eLeSDenes said:
I think clearness of LC is not an indication of being clean ( there is no bacteria in it.



To estimate bacterial growth in a liquid media, microbiologists measure the turbidity of the broth using a spectrophotometer. So basically how turbid a broth is, is directly related to the amount of bacterial cell mass present. But keep in mind that cell mass can mean both microbial activity and it can mean the presence of dead/inactive bacteria.


Quote:

eLeSDenes said:
I had lots of super clean looking LC which somehow seemed to stall on grains after shake



Your definition of 'super clean looking' was probably not established by observing an identical but sterile broth side by side, thus making it very hard to determine if there was an increase in turbidity or not during incubation. And I really hope I don't sound like a Hitler or anything lol I only recently started to dive into reading research papers and lurking the microbiology forums. I started doing this as a way to better understand the science behind the concept we in MushCult call 'contamination'. I'm learning as I go, the way I phrase the scientific data in my posts may sound like I know alot more about it than I actually do so don't take my word blindly for anything, I encourage everyone to question everything and see for themselves as well. I'm not always right or always have the correct assessment of the sceicne so this thread should have all sorts of inquiries and healthy debates I hope.
:havesomescience:


Edited by Mateja (10/18/20 07:15 PM)


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OfflineForresterM
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Re: LC Testing + Grow Log [Re: Mateja] * 1
    #26992221 - 10/19/20 02:02 AM (3 years, 3 months ago)

So it seems like you are saying that more turbidity/cloudiness indicates how much bacteria is present.

This could quite well be true, and likely is.

But let's just remember anytime anyone says "clearness of an LC does not prove it's clean", they are not refuting the above. 

They are simply stating that just because an LC looks nice and clear to the human eye it is not proof that there isn't some small amount of bacteria in there.  And we haven't begun discussing mold spores yet.

I think this is where we got into disagreement before, so I just wanted to clarify - to say that clearness of an LC does not prove it's 100% clean does not at all contradict what you're trying to show here, that turbidity is an indicator of bacterial presence.  It only says that the human eye might not be able to detect a very small amount of turbidity.  Say there's 100 bacterial cells that survived a PC cycle - you're not gonna see 100 cells.  But when you transfer them onto grain, they will replicate.

Just wanted to clear that up.


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InvisibleMateja
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Re: LC Testing + Grow Log [Re: Forrester] * 1
    #26992570 - 10/19/20 09:26 AM (3 years, 3 months ago)

Quote:

Forrester said:
just because an LC looks nice and clear to the human eye it is not proof that there isn't some small amount of bacteria in there.



I am totally with you in what you're trying to convey and theoretically your conclusion is very reasonable and I really wouldn't object to this statement at all per se. But from a couple of perspectives I also totally disagree with this. The first one being that if you are to introduce bacteria to a sterile broth it will occur upon inoculation due to insufficient sterile Tek. Or it could also be due to for example some thermophilic microbes surviving the sterilization process. But in the end no matter which type of bacterial contamination occurs it will nevertheless be present from the start and will thus have a sufficient incubation time to result in prolific growth causing obvious turbidity. I can't imagine a scenario where you would have no bacterial growth at all for the majority of the incubation period and only have healthy myc growth only to in the last day or last hours of finishing the successful LC to somehow suddenly introduce a bacterial colony to the broth that starts to grow but doesn't become visible because of the very short incubation time. I'm of course open to hearing and discussion such scenarios if you are thinking of some specific ones that are realistic.



Quote:

Forrester said:
Say there's 100 bacterial cells that survived a PC cycle - you're not gonna see 100 cells.  But when you transfer them onto grain, they will replicate.



This statement doesn't make sense to me either according to my current understanding of how bacterial colonies grow on a solid vs a liquid media. Considering the relatively high requirements bacteria has to sustain growth compared to molds, strickly speaking of the minimum levels of water activity needed, to assume that a bacterial activity would be able to hide or remain dormant in a liquid broth (optimal environment) and somehow still be able to grow prolifically on a solid media such as grain just isn't likely at all when considering the science behind how bacterial colonies spread. Even the most motile or osmophilic kinds of bacteria would still have an immensely bigger advantage in a liquid broth compared to for example overly wet grain. In fact a bacterial colony in a liquid media will almost certainly be able to reach logarithmic growth even if other growth conditions aren't optimal such as temperature. But on solid media (with high enough water activity) very motile bacteria (and some non motile) will definitely be able to grow to some extent until the dealing with the osmotic pressure eventually puts a 'limit' on the growth rate. As always I'm just stating my current understanding of the science behind this and much of everything is still not known to me on any kind of deeper level of understanding, and that is also the reason for this grow log to explore all of these possibilities and to discuss them thoroughly and to finally also be able to have some hard data and documents from this experiment to help us conclude what is actually what at the end. :takingnotes:


Quote:

and we haven't begun to discuss mold spores yet



I have many different thoughts considering mold in this discussion but I don't know at this moment what you're trying to say about mold spores so I'm all ears! :mushroom2:


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OfflineForresterM
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Re: LC Testing + Grow Log [Re: Mateja]
    #26993100 - 10/19/20 02:24 PM (3 years, 3 months ago)

Quote:

Mateah said:
But in the end no matter which type of bacterial contamination occurs it will nevertheless be present from the start and will thus have a sufficient incubation time to result in prolific growth causing obvious turbidity.




This could very well be true.  I just think it would take a lot more than a few jars and some agar tests to prove.  There's a lot of different kinds of bacteria.


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InvisibleMateja
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Re: LC Testing + Grow Log [Re: Forrester]
    #26993493 - 10/19/20 07:06 PM (3 years, 3 months ago)

I just hope my predictions at least turn out to be in the general direction of truth, then it could mean that LC's will become a lot more practical/reliable to work with.


Also just finished sterilizing 8 new broths from rye boil water. 4 of the broths are rye water diluted with 9 parts water and the other 4 are diluted with 19 parts water. I also made 3 types of agar that I'll pour tonight. No rest for the wicked :rockon:


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InvisiblemushboyMDiscord
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Re: LC Testing + Grow Log [Re: Mateja]
    #26993503 - 10/19/20 07:11 PM (3 years, 3 months ago)

:popcorn:


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InvisibleMateja
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Re: LC Testing + Grow Log [Re: mushboy] * 1
    #27009586 - 10/29/20 02:51 AM (3 years, 3 months ago)

Some updates.

The rye jars inoculated with the master LC on the 12th are finishing up and they still look good



The oatmeal cakes inoculated with the master on 14th are also finishing



Here are some cakes inoculated on 24th with LC#2 looking good as well



Here are a couple of qt jars with the overcooked burst rye inoculated on 24th as well with LC#2 one shook and one not.



If we compare the overcooked rye with the 'normally prepped' one it's obvious that elevated water activity plays a huge roll.
Both these pics were taken 5 days after inoculation.


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InvisibleMateja
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Re: LC Testing + Grow Log [Re: Mateja]
    #27013052 - 10/30/20 10:56 PM (3 years, 3 months ago)

How it looked when I tested LC#1 and LC#2 on agar

LC#1



LC#2



Also I noticed this really broad outer ring when LC cultures grow on agar


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Offlineverum subsequentis
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Re: LC Testing + Grow Log [Re: Mateja]
    #27013075 - 10/30/20 11:09 PM (3 years, 3 months ago)

I've seen that too. Not only from LC though.


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Re: LC Testing + Grow Log [Re: verum subsequentis]
    #27013105 - 10/30/20 11:53 PM (3 years, 3 months ago)

a assumed by now that ring around culture is micro-mycellium.. at one time i thought it could be bacteria riding under the mycellium but likely just paranoid.


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InvisibleMateja
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Re: LC Testing + Grow Log [Re: trippleblack]
    #27013135 - 10/31/20 12:28 AM (3 years, 3 months ago)

Well since bacteria doesn't grow filaments I'd be hard to assume that the outer edges would be bacteria, myc on the other hand is filamentous in nature so it's only logical that it's myc especially when those thin filaments are coming out of something that's undeniably cube myc :super:


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Offlineverum subsequentis
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Re: LC Testing + Grow Log [Re: Mateja]
    #27013137 - 10/31/20 12:31 AM (3 years, 3 months ago)

First i saw it I was rather convinced that it was myc trying to make it's way through some nasty something. You know that super insecure noob feeling where you're just tripping ever living balls looking at cultures on agar. I did a lot of that.


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OfflineSingularFusion
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Re: LC Testing + Grow Log [Re: verum subsequentis]
    #27013162 - 10/31/20 12:59 AM (3 years, 3 months ago)

:takingnotes:


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Re: LC Testing + Grow Log [Re: SingularFusion]
    #27019447 - 11/03/20 01:24 PM (3 years, 3 months ago)

Thank you for your work! :dancer:


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Invisiblemushhead
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Re: LC Testing + Grow Log [Re: CapMeh]
    #27019780 - 11/03/20 04:04 PM (3 years, 3 months ago)

:takingnotes:


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OfflineTight Lunchbox
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Re: LC Testing + Grow Log [Re: Forrester]
    #27019843 - 11/03/20 04:47 PM (3 years, 3 months ago)

Quote:

Forrester said:


It only says that the human eye might not be able to detect a very small amount of turbidity.





I've read somewhere that the human eye can't detect turbidity lower than 5 NTU.


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InvisibleMateja
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Re: LC Testing + Grow Log [Re: Tight Lunchbox]
    #27025558 - 11/06/20 06:48 PM (3 years, 3 months ago)

Updates


Originally 9 rye jars were inoculated with the Master LC. 3 jars were lost early to different types of mold, honestly I cant really make sense of the contamination I've been noticing, I just have to assume that the inoculate was clean and that my sterile tek was to blame. One jar sprouted something cobweb-like on top of the grain, looked like this:



And then there was this jar which looked fine after inoculation, it also recovered beautifully after the first shake and then after the other shake I notice green sporulation from every single piece of grain. :shrug:


Then there was one jar which sporulated green from one single spot at the bottom of the jar. This jar wasn't shook upon inoculation making this scenario all the more weird.


Anyhow 6 rye jars made it to spawning the other day. At this stage all of the jars had been shaken 2-3 times each and this last recovery (a few days before spawning) looked as healthy as all the previous ones did. (to my eyes at least, this looks like healthy recovery after multiple shakes. But if anyone sees something I don't then please chime in)




A few days after spawning I noticed that one of the subs wasn't recovering all that well, and when I went to check on it I was met with a smell that would make your eyes water, like the worst smelling cheese in the world gone sour + death. And also there's this other sub that has a single green sporulating grain on the surface, I'm leaving this tub for observation.

This is the look of the 4 subs that seem to be "healthy" day 5 after spawn.


And the sour cheese and green mold




If anyone has an idea on why I'm seeing such huge variety in the contamination characteristics from both colonizing jars and bulk subs please share :shrug:


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Re: LC Testing + Grow Log [Re: Mateja]
    #27025575 - 11/06/20 06:56 PM (3 years, 3 months ago)

These two Rye/coir jars were also shaken two times each and one of them is obviously not recovering well post second shake while the other one seems to be doing good. This is how they looked on day 5 after inoculation (one shook one not)


And this is how they look right now, shook last time on Oct 31st.


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Offlineverum subsequentis
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Re: LC Testing + Grow Log [Re: Mateja]
    #27025717 - 11/06/20 08:40 PM (3 years, 3 months ago)

How do you feel about the last two pics?


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