WARNING!!!
This thread was made with the intention of trying to showcase a persistent problem I've had over the years of dealing with terrible local grain.
I had all signs in my jars of what's commonly agreed upon to be bacteria and yet, measures were always taken to ensure there was no user error, hence why at some point, I became convinced it must be surviving endospores.
Many tests were done in this thread, but the final and most important can be found in the section "The bad prep: Addendum", where it was made clear that whatever was causing the spawn to look so bad it was not bacteria.
With that out of the way, hope you can learn as much from the following documented tests as I did!
I think we can all agree that grain prep is one of the most important aspects of cultivation.
In my own experience, I've seen time and time again that a grain prep has the potential to make or break a grow, so I think it's safe to say that there's a bit more to clean spawn than good technique, axenic inoculant and a properly executed PC/autoclave cycle.
In this thread, I'm going to be doing three different preps, using the same grain for each (local grain that has proved to be rather problematic), so that we can observe first hand the impact that each prep will make on the final result, if any.
This thread was also made with the intention of trying to shed light on whether or not some endospores in the grains can survive a PC cycle.
I'm aware only a microscope and someone with the knowledge to use it could tell us for sure, but on the other hand, I'm hoping the tests we're going to run here with the humble means I have will at least help us draw what I hope will be some valuable conclusions.
I've always believed that a small percentage of endospores in the grain can survive a standard PC cycle, and the degree in which this happens will hinge upon different factors.
Prep can be one of them, as well as the more obvious such as a properly/improperly executed PC cyle. Although IMO, one very important factor is the endospore count the grains start with. This is why I think it's so important to source clean quality grain if we can, in order to succeed.
I also believe that the activity of a live mycelial colony actually colonizing the spawn and moving moisture creates the perfect environment for these surviving endospores to recover and activate.
IME, they won't recover by just letting the jar sit on a shelf uninoculated (unless there was an insufficient PC cycle).
In this thread, I will try to show/prove all this and let you guys take a peek at what I've been experiencing over years of dealing with a terrible source for grain with unacceptably high endospore counts.
The following is a list of the conditions and rules that will be followed:
- Three preps were done and will be documented:
A "bad" prep (in my opinion), a standard prep and a "hard" prep.
- The grain used for the three preps is the usual bad quality barley that I can source locally.
- All jars from the three preps (18 in total) were sterilized in the same PC cycle for 140 minutes at 15 psi.
- For each prep, a total of 6 jars was made. 4 of those were inoculated with LC. 1 jar was left out uninoculated as a control and the last jar was inoculated with 20ml of sterile broth (LME water at 3% strength).
- The same LC was used to inoculate all jars, ~5ml per jar.
- The LC will be tested on agar (results in a couple of days).
- Each prep will have its own section and will be updated periodically with pictures, until its conclusion.
RESULTS OF THE AGAR TESTS (7/13)

I squirted a tiny bit of LC on 5 separate plates the same day I inoculated the jars and this is the result today after exactly 72 hours.
It doesn't matter how little you squirt on agar, it is always a bit messy compared to using an inoculation loop. I do it because it's easier/more convenient for me and it gets the job done too.
When testing an LC on agar, what grows or doesn't grow until the colony recovers tells you everything about its cleanliness.
If there was bacteria, it would have taken off fast and would have made itself visible in a matter of hours, same with mold.
So I think it's safe to say the LC tested clean.
THE BAD PREPBarley was soaked for 72 hours in cold tap water. The stink was as good as the picture suggests, yummy:

Then it was strained and loaded wet into jars when it wasn't dripping anymore.
The barley had still some hydrating to do, so they absorbed the extra water during the PC cycle.
From left to right, jar inoculated with LC, control jar "inoculated" with 20ml of nutrient broth and uninoculated jar.
7/13
Absolutely no signs of life yet on these jars 72 hours after inoculation. They look the same way as the day they were inoculated, no growth of any kind.
This wasn't expected, I think it's safe to say there's something wrong going on here.
7/16
These are the fermented grains six days after inoculation. Still, no signs of life.
If I don't see changes within 3 days I will need to re-do this prep but this time, soaking for 48hours instead of 72.
7/24Two weeks have past since the jars were inoculated, so today I took a peek and to my surprise I found some growth in one single jar. The others remain without changes.

Here's a pic of each individual jar, showing the bottom too because the LC inevitably pools there a little after inoculation:




I don't know when the colony started to grow in that single jar but growth is very thick and I think the colony is finding it hard to colonize the grains for whatever reason.
From the other jars that are showing no life yet we can at least draw some conclusions so far after 2 weeks:
- The PC cycle was successful, otherwise we would have seen a bacterial bloom by now.
- The filters and the lids are working properly.
- There are no signs of improper technique.
- The LC couldn't recover on these fermented grains (for reasons unknown). However, there are no signs of bacteria or any other organism either, which in my view means that the LC proved clean once again.
All these 4 jars got shook today.
Also the control jars after 2 weeks. On the left, the jar where I squirted 20ml of sterile LME broth (3% strength) and on the right, the uninoculated jar:



No changes, as expected. From this we can draw some conclusions too after 2 weeks:
- The PC cycle was successful.
- The filters and the lids are working properly.
- There are no signs of improper technique even though a much larger amount of liquid was squirted in the control jar.
The control jars got shook today, as well.
8/6


These 3 pics belong to the same jar from the bad prep, the only jar out of four that could grow any mycelium.
I let it go 2 weeks after the initial shake but growth has been very slow and the colony still seems to be having trouble colonizing the grains. Growth is very thick and it caked so much that it was impossible to break it up by shaking:

And here we have the 3 remaining jars from the bad prep:

Still, no changes at all after the initial shake 2 weeks ago, no signs of bacteria or mycelium, just nothing. These are the last updates from these 3 jars, unless I see changes.
All the jars from the bad prep were inoculated with 5ml of the same LC, so I don't understand why the LC could only recover and grow in that one jar. However, the LC took many days recover in that jar, so there must be something in these jars inhibiting normal, healthy growth.
No news on the 2 control jars. Won't post pics because I don't want to clutter up the thread more than already is. There will be no more updates on the control jars, unless I see changes.
8/11


These are 3 pictures from the same jar 5 days after the second shake, the only jar in which the LC could recover and grow.
It's been 5 days since the second shake and a month since inoculation.
The spawn is starting to look bad at this point. There are a lot of wet kernels pressed against the glass and an a concerning excess of moisture that wasn't there the day the jar got inoculated.
This jar got a final shake today and it's after this shake when I expect things to really go south.
The other 3 jars that were also inoculated with LC remain without changes. They look exactly the same as when I took them out of the PC.
The Bad Prep: Addendum.With the recent arrival of my humble flowhood and considering the unexpected situation we've witnessed with these four jars from the "bad prep" where the colony could only recover and grow in one single jar, I've decided to add a final twist to the plot that was not in the original plan.
I'm going to run a final test that I think is going to be interesting.
Some people have asked me why I didn't put any kernels directly on agar (uncolonized and colonized) in order to test for cleanliness. I know it would make apparent sense to do this in a thread about bacteria in spawn but the answer as to why I didn't bother is simple, I don't think it would work... Source: experience.

Dropping an uncolonized kernel won't ever activate the endospores in case there are any, only an insufficient cycle would give us bacteria in this case. Similarly, the bacterial jars we've seen in this thread where thorougly colonized by mycelium even if the colony was showing signs of stress from bacteria.
Therefore, if I had put a colonized kernel on agar, myc would have leapt off just fine, very likely leaving any existing bacteria behind.
Maybe we could have seen bacteria on agar if I had managed to snatch a single wet kernel from those that were pressed against the glass, but how am I to do that without shaking the jars?
So, an awesome member gave me an awesome idea that can hopefully allow me to test the cleanliness of the spawn: Grain Liquid Culture (GLC).
For those of you who may not know, GLC is an old technique that consists of squirting sterile water in a fully colonized jar (usually with the help of a syringe through a SHIP) and then aspirating the now myc-laden water back into the syringe, to be used immediately.
Since my lids weren't made for GLC, I had to improvise and make use of the jars I have for LC, which are designed for aspirating.
I was debating with myself if I should use plain sterile water for the GLC's or nutritios broth instead, but in the end I opted for broth, just to make it a bit easier for any possible "dormant" bacteria from the 3 jars that showed no growth of any kind.
8/16

Both pics belong to the same jar from the bad prep, the only jar that got any growth. The spawn looks bad but I was expecting even worse.

Shook the jar and transferred a small amount of spawn to an empty (and sterile) LC jar.

I sterilized some LME water (jar on the right) and aspirated some broth from it with a 60ml syringe that I then emptied in jar on the left:

Then I used a 10ml syringe to aspirate the GLC and immediately squirted a bit of this liquid into two separe agar plates.

The idea behind this test is that in the event that there's any vegetative bacteria in the spawn (and it looks like it), the water should be laden not only with mycelium but also with bacteria, so hopefully it will show quickly on agar with the help of the extra liquid. But the truth is that I don't know what is going to happen nor am I sure this is even a proper approach to test for cleanlines. However, I am dead curious to see the results from this.
The same exact procedure was done with the 3 jars from the bad prep that showed no growth.
In addition and just for bro-science, the four 10ml syringes containing GLC from the 4 grain jars (technically only one is GLC) were put in a previously sterilized gallon jar and will be kept there for 7 days to allow any possible nasties to grow some more. Then they will be tested on agar again.
8/20We've got some unexpected results from the "GLC to agar" tests. Even though the spawn looked horrible, the GLC tested clean on agar.
Here's the 2 plates that were inoculated with the GLC from that iffy jar we saw earlier:


I didn't realize the first pic was a bit blurry when I took it, but the second is a bit better.
I've been monitoring the plates closely from day one and I could see absolutely no signs of life until the ~72 hour mark when the mycelium started to recover.
Since I squirted a generous amount of GLC, we can see in the picture that the liquid congealed around the edges of growth and this is caused by the colony itself when it recovers, something that to the untrained eye may look a bit suspect.
The following pics are the 2 plates from jar 2, which are perfectly clean and clear (let's remember there was no mycelial growth in this jar because the LC could not recover for unknown reasons):

Pics of the plates from jar 3 which are perfectly clean too:

Pics of the plates from jar 4. Clean:

I'm not going to lie that a huge "what if" crossed my mind. What if what we're seeing in that jar is not actually bacteria?
We still have to test the 4 GLC syringes in 3 days from now (remember they're being left to grow for 7 days) so that we can have further proof, but on the other hand these first agar tests are rather conclusive IMO, because measures were taken to purposely give ample oportunity to any existing vegetative bacteria to grow. Like using LME broth instead of plain water and also squirting a liberal amount of liquid on the plates to facilitate bacterial growth.
Nonetheless, I thought of doing yet another test.

This is the recovery we have in the jar from the bad prep after that last shake 4 days ago when I made the GLC.
It looks terrible and the colony could not even recover at all on a great number of kernels.
So I shook the jar one more time and put an individual kernel in 3 separate jars containing sterile LME broth at 0.2% strength. Let's see what grows!
8/226 days in, the GLC syringes from the four jars look like this today.
From left to right, syringe from jar 1 (the only spawn jar that got to 100% colonization), syringe from jar 2, syringe 3 and syringe 4:




The GLC from jar 1 got some obvious myc growth. The others look like the day they were aspirated. The turbidity in those is due to a good amount of sediment that was collected when the liquid was aspirated.
One agar plate was inoculated with each syringe.
8/25It's been five days since I inoculated some agar plates with the 4 GLC syringes that were left to grow for 6 days and we've got the results today.
This is the plate inoculated with the GLC from the jar that was colonized. It's a bit overgrown and growth is all over the place and quite messy from having used a generous amount of GLC. Been monitoring the plate everyday from the beginning and no bacteria could be seen at any given time.

Jar 2, clean:

Jar 3, clean:

Jar 4, clean:

I want to bring attention to the fact that these GLC's were done with LME broth at 0.2% strength and that the syringes were kept in a jar for 6 days to give ample time for any living organisms to grow.
Since no signs of bacteria could be observed on the agar, I have to admit that to me this is sufficient proof that the nasty looking jar was actually free of bacteria and that something else was causing the erratic, unhealthy looking growth.
These are the LC's that were inoculated with a grain kernel from said jar, five days in:


It wasn't easy to take good pics. The uncolonized broth is as clear as the day it was inoculated, no turbidity or visual sings of bacteria whatsoever.
I could have tested the LC's today because in 5 days bacteria would have had ample time to thoroughly contaminate the LC, but I'm going to wait until the LC's are actually ready.
All seems to indicate that no vegetative bacteria was responsible for the bad looking spawn we've seen and all seems to indicate that I may have to recant and admit I have been wrong in saying endospores could survive a proper cycle.
As to what could be causing these issues, I have no answer to give you besides pure conjecture.
8/31I put the 3 LC's to agar today to test them:


Although they look perfectly fine to me, no turbidity whatsoever.
As a curious note, this is the first time I inoculate broth with anything other than a biopsy and I was surprised to see how long they took to colonize, considering the myc sample is orders of magnitude bigger than a myc biopsy. These took 11 days and still don't even look 100%, whereas currently I'm getting my agar LC's ready in 5-7 days from biopsy.
The thread will conclude after we have the results from the agar tests.
9/4Happy to be giving the final update to conclude this thread.
It's been quite a journey from which I've personally been able to learn some interesting new things but it's also left me with some important unanswered questions.
Let's recapitulate a bit:
The LC could only recover on one single jar from the 4 that were done for the "bad" prep. This was recovery from said jar after a shake at 100%:

I don't need to tell anybody that that jar looks absolutely horrible and I think I'm not wrong in assuming everybody on this forum (including myself) would have agreed that this jar was contaminated with bacteria.
Several tests were done to see if that jar was contaminated. First, some GLC's, but all of them tested clean. And lastly, just to make sure, 3 LC broths were inoculated with a whole kernel from that jar.
The LC's looked clean but were also tested on agar for good measure. These are the 3 agar plates from the 3 LC's today, 3 days later:


Absolutely no signs of bacteria. If there was a test that would determine the presence of bacteria in that spawn jar, I think this is the one.
I think we can confidently say that despite the looks of it, that spawn jar was free from bacteria.
The question still remains what is causing the spawn to look like this when all the tests seem to indicate that there's no vegetative bacteria present.
To a lesser or greater degree, I've experienced iterations of this exact same issue many times over the years. I don't know if it's the bad quality of the grain I can source, or the way they dry and store the grain, but it's certainly been a huge problem for me.
This thread was made in order to prove that endospores could survive a cycle but I may have to recant and admit that I have been wrong all along after seeing the results from these tests.
It's been years experiencing the same... poor recovery on grains, spawn that looked terrible and with signs of what it's commonly agreed upon by the OMC to be bacteria.
There comes a point after seeing a lot of this where it was easy for me to realize I was not the one fucking up. Since I assumed it was bacteria all along, the more plausible explanation in my eyes was survivinig endospore but... live and learn.
This thread will not be edited, but instead a warning will be issued at the beginning where I explain the huge misunderstanding.
THE GOOD PREPLet's go with something more standard for this one.
The barley was rinsed and then boiled in a pot until I was satisfied with the level of hydration (a good 50 minutes). Then it was left to dry on a screen for some hours with the help of a box fan.
From left to right, jar inoculated with LC, control jar "inoculated" with 20ml of nutrient broth and uninoculated jar.
7/11Here's the jars from the standard prep 24 hours later. Recovery was not bad at all.

7/1248 hours later. Jars got shook!

7/1324 hours after shaking.
7/1448 hours after the initial shake, looking healthy in my opinion. Jars got shook!

7/1648 hours after the second shake.




Seen this time and time again. It is usually when I shake seemingly healthy jars when they're past ~60% colonization (like I did this time for the second shake) that I start seeing things I don't like once the colony recovers and starts re-colonizing.
For instance:
- Speed of colonization slows down almost as if all of a sudden the colony was struggling.
- Areas of very thick and patchy growth can be seen. In my opinion, this is a clear sign of stress.
- The spawn does that thing where it never finishes colonizing or struggles to do so. The colony has trouble colonizing some individual kernels.
- Slimy, wet kernels pressed against the glass. (Not really seen this time).
I have to say this is a "mild" case of stressed spawn that we're seeing here, have seen way worse from inoculant that proved clean.
I have no way to prove what's causing the colony to grow stressed in such an advanced stage of colonization.
Could it be bacteria? If so, why hasn't it shown signs earlier? The LC proved clean and the colony started colonizing the grains in a healthy, vigorous fashion.
Could it be that the grains were prepped a bit wet? Well, the colony was growing just fine up until this point. It looked really happy and for what it's worth, I've seen this happening too with grains prepped on the drier side.
Could it be that at this stage of colonization the conditions the colony creates in the jar are ideal for the (allegedly) surviving endospores to activate? Again, no clue. But in all honesty and having witnessed the same thing countless times, I'd say that currently I do see this as a plausible explanation.
Take of this what you will, think what you must.
Worth mentioning again that the PC was purged for more than 15 minutes, jars were sterilized for 140 minutes (not 120), the LC proved clean and my method of inoculation is one that carries a high success rate, which is barely cracking a lid for a second to quickly squirt a bit of LC through a flamed needle.
My source for grain also happens to be terrible, I really think it's important to highlight this fact
Jars got shook, let's see how they recover.
7/1848 hours after the recovery shake.





The jars are looking way roughed up to bother spawning them.
7/24Made a tub with the four sketchy looking jars, cuz science...
Also, here we have the control jars for the standard prep. On the left, the jar where I squirted 20ml of sterile LME broth (3% strength) and on the right, the uninoculated jar:

No signs of bacteria in the uninoculated jar or the jar inoculated with sterile broth, as expected:

Conclusions so far:
- The PC cycle was successful.
- The filters and the lids are working properly.
- There are no signs of improper technique even though a much larger amount of liquid was squirted in the control jar.
The control jars got shook today.
7/28Tub from the standard prep 4 days in:

And a side picture of both tubs to see the difference (from the standard and hard preps):

This tub continues to give off a rather strong sweet smell and it's rather far behind the tub from the hard prep. Let's see if it makes it.
8/2Tub from the standard prep 9 days in:



Bacteria is slowing things down a lot and the colony is exuding metabolites, hence the yellowish tinge that can be observed.
The tub from the hard prep took only 4 days to fully colonize and let's not forget it's the exact same culture in both tubs, so there's no question something is wrong with the tub from the standard prep.
8/6
The tub from the standard prep triched out before first flush, was wondering when it would happen.
I did the good 'ol trashcan-tek™.
No news on the 2 control jars. Won't post pics because I don't want to clutter up the thread more than already is. There will be no more updates on the control jars, unless I see changes.
THE HARDY PREPLet's go overboard with this one.
Put some dry barley in a bucket (polypropylene) and covered with more than double the volume in hot tap water.
Then put the bucket inside my AA 941, vented for 15 minutes and run the cycle for 90 minutes at ~28 psi, yup.


Let the PC drop pressure naturally and then rinsed the hell out of the barley to break up all clumps and get all the starchy water out.
Then put the grain on a screen to dry for several hours. The problem is that the only place I could put the screen was in the backyard (the other screen was being occupied for the other prep), I got careless and the sun got the barley a bit too dry, so I'm expecting slow-ish growth.
From left to right, jar inoculated with LC, control jar "inoculated" with 20ml of nutrient broth and uninoculated jar.
7/11Here's the jars from the hard prep 24 hours later. Really happy with the recovery.

7/1248 hours later. Jars got shook!

7/1324 hours after shaking.
7/1448 hours after the initial shake, looking healthy in my opinion although a little behind of the boiled spawn. Jars got shook!

7/1648 hours after the second shake.




The spawn for this prep is looking better than the jars from the standard prep, although rather roughed up too.
Jars got shook, let's see how they recover.
7/1848 hours after the recovery shake.





Looking slightly better than the jars from the standard prep but to be honest I was expecting a bigger difference between both preps.
7/24Made a tub with the four
slightly less sketchy looking jars, cuz science...
Also, here we have the control jars for the hard prep. On the left, the jar where I squirted 20ml of sterile LME broth (3% strength) and on the right, the uninoculated jar:

No signs of bacteria in the uninoculated jar or the jar inoculated with sterile broth, as expected:

Conclusions so far:
- The PC cycle was successful.
- The filters and the lids are working properly.
- There are no signs of improper technique even though a much larger amount of liquid was squirted in the control jar.
The control jars got shook today.
7/28Tub from the hard prep 4 days in:

And a side picture of both tubs to see the difference (from the standard and hard preps):

This tub gave off a slight sweet smell the first two days but it seems to have subsided and it's now colonizing at a nice speed. Let's see if this tub pulls out some mushies.
8/2Tub from the hard prep 9 days in:


This tub is starting to knot up. So far there are no observable or at least obvious indications that something is wrong but it could also look better, IMO.
8/6
First pins popping up in the tub from the hard prep. They're not easy to see but they are there.
No news on the 2 control jars. Won't post pics because I don't want to clutter up the thread more than already is. There will be no more updates on the control jars, unless I see changes.
8/11
At least we have some mushrooms growing, they still have a couple days to go.
8/13We've got a harvest.

Enough for an ol bouquet 'o dicks!
