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So I got these endospores...

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#1 jkdeth

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Posted 18 April 2017 - 07:45 PM

Ok trying to wrap my head around endospores. Got the basics. I know what an endospore is. Kind of doing a crash course in advanced endospore mechanics. One for instance, why would endospores be more of a concern in a whole grain than in a whole grain flour?

Edited by jkdeth, 18 April 2017 - 07:45 PM.


#2 jkdeth

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Posted 18 April 2017 - 08:11 PM

Just I'd share, oldest endospore germinated and grown in a lab, estimated 25 Million years old. Tough little forkers.
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#3 wharfrat

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Posted 18 April 2017 - 10:56 PM

yeah, to say we can kill endospores is kinda not true.. i think we just try and avoid unwanted contaminates the best we can.. I and others do a pre soak to hopefully germinate any endospore before PC'n, but this might be unnecessary, i'm really not sure. Some say it don't matter, some say it does. I personally do a 24 hour pre soak, not just in case it helps germinate the endospore, but also it helps hydrating the grain so u do not need to cook so long. and as far as grain or flour, i would say they are both pretty much equal so would need the same. Bacteria is a fun one to diagnose when you are dealing with contamination issues.


Edited by wharfrat, 18 April 2017 - 10:56 PM.

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#4 CatsAndBats

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Posted 19 April 2017 - 09:59 AM

Bacterial Endospores

Microorganisms sense and adapt to changes in their environment. When favored nutrients are exhausted, some bacteria may become motile to seek out nutrients, or they may produce enzymes to exploit alternative resources. One example of an extreme survival strategy employed by certain low G+C Gram-positive bacteria is the formation of endospores. This complex developmental process is often initiated in response to nutrient deprivation. It allows the bacterium to produce a dormant and highly resistant cell to preserve the cell's genetic material in times of extreme stress. 

Endospores can survive environmental assaults that would normally kill the bacterium. These stresses include high temperature, high UV irradiation, desiccation, chemical damage and enzymatic destruction. The extraordinary resistance properties of endospores make them of particular importance because they are not readily killed by many antimicrobial treatments. A variety of different microorganisms form "spores" or "cysts", but the endospores of low G+C Gram-positive bacteria are by far the most resistant to harsh conditions. 

endospore.jpg Endospore Structure

The resilience of an endospore can be explained in part by its unique cellular structure. The outer proteinaceous coat surrounding the spore provides much of the chemical and enzymatic resistance. Beneath the coat resides a very thick layer of specialized peptidoglycan called the cortex. Proper cortex formation is needed for dehydration of the spore core, which aids in resistance to high temperature. A germ cell wall resides under the cortex. This layer of peptidoglycan will become the cell wall of the bacterium after the endospore germinates. The inner membrane, under the germ cell wall, is a major permeability barrier against several potentially damaging chemicals. The center of the endospore, the core, exists in a very dehydrated state and houses the cell's DNA, ribosomes and large amounts of dipicolinic acid. This endospore-specific chemical can comprise up to 10% of the spore's dry weight and appears to play a role in maintaining spore dormancy. Small acid-soluble proteins (SASPs) are also only found in endospores. These proteins tightly bind and condense the DNA, and are in part responsible for resistance to UV light and DNA-damaging chemicals. Other species-specific structures and chemicals associated with endospores include stalks, toxin crystals, or an additional outer glycoprotein layer called the exosporium.

stages-410x597.png Endospore Development

The process of forming an endospore is complex. The model organism used to study endospore formation is Bacillus subtilis. Endospore development requires several hours to complete. Key morphological changes in the process have been used as markers to define stages of development. As a cell begins the process of forming an endospore, it divides asymmetrically (Stage II). This results in the creation of two compartments, the larger mother cell and the smaller forespore. These two cells have different developmental fates. Intercellular communication systems coordinate cell-specific gene expression through the sequential activation of specialized sigma factors in each of the cells. Next (Stage III), the peptidoglycan in the septum is degraded and the forespore is engulfed by the mother cell, forming a cell within a cell. The activities of the mother cell and forespore lead to the synthesis of the endospore-specific compounds, formation of the cortex and deposition of the coat (Stages IV+V). This is followed by the final dehydration and maturation of the endospore (Stages VI+VII). Finally, the mother cell is destroyed in a programmed cell death, and the endospore is released into the environment. The endospore will remain dormant until it senses the return of more favorable conditions. [A sigma factor is a small protein that directs RNA polymerase to specific cites on DNA to initiate gene expression.]

  Endospores and Epulopiscium

Some Epulopiscium-like surgeonfish symbionts form mature endospores at night. These spores possess all of the characteristic protective layers seen in B. subtilis endospores and also contain large amounts of dipicolinic acid. These are the largest endospores described thus far, with the largest being over 4000 times larger than a Bacillus subtilis endospore. 

epulospore.jpg

The formation of endospores may help maintain the symbiotic association between these Epulopiscium-like symbionts and their surgeonfish hosts. Since endospore formation coincides with periods in which the host surgeonfish is not actively feeding, the cells do not need to compete for the limited nutrients present in the gut at night. The protective properties of the endospores also allow them to survive passage to new surgeonfish hosts. The fish may also benefit from this relationship because it is able to maintain stable microbial populations that assist in digestion and may receive a nutritional gain from microbial products released during mother cell death and spore germination.

lifecycle.jpgDaily life cycle of endospore-forming Epulopiscium-like symbionts.

Endospore formation in some Epulopiscium-like symbionts follows a daily cycle:
A) Polar septa are formed at the poles of the cell.
B) Forespores become engulfed.
C) Forespores gradually increase in size within the mother cell through the day.
D) In late afternoon, final preparations for endospore dormancy.
E) Endospores mature and remain dormant throughout most of the night.
F) Just before sunrise, the endospores germinate and are released from mother cell to repeat the cycle.

 

 

 

 

Found here:

https://micro.cornel...rial-endospores

 

Here's some "fun" reading:

https://mycotopia.ne...-sterilization/

 

This is a great resource:

https://www.khanacad...acteria-archaea

 

For more fun:

http://movies.nation...e-unseen-world/


Edited by CatsAndBats, 19 April 2017 - 10:05 AM.

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#5 sandman

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Posted 19 April 2017 - 11:52 AM

Ever since switching to RO water for my projects I do no soak, even no rinse, for my milo grain with 100% success. Just pour it into a pot, add water, bring to simmer, drain through screen. The true enemy of grain is too much moisture in the jars, gotta strain in a super shallow pile or it will be too wet. Even a pile filling up a normal strainer will not drip enough after many hours IME. Spread it out so it can air dry and not look wet!


Edited by sandman, 19 April 2017 - 11:52 AM.

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#6 jkdeth

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Posted 19 April 2017 - 12:19 PM

Forgive my lack of knowledge, I don't know RO water, what is that?

#7 CatsAndBats

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Posted 19 April 2017 - 12:22 PM

reverse osmosis prolly



#8 sandman

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Posted 19 April 2017 - 12:31 PM

Yes, reverse osmosis. My local water is really bad.


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#9 jkdeth

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Posted 19 April 2017 - 01:14 PM

Thank you, water here is pretty bad too. There is a local spring a lot of people use, I may try some of that, other than that I most likely would buy water.

#10 CatsAndBats

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Posted 19 April 2017 - 01:21 PM

Thank you, water here is pretty bad too. There is a local spring a lot of people use, I may try some of that, other than that I most likely would buy water.

 

 

What's your major concern/question? True destruction of endospores or moisture/hydration/tyndallization?



#11 jkdeth

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Posted 19 April 2017 - 01:39 PM

Researching non PC sterilization. Endospores I see are the biggest problem. Research of course causes my mind to tangent off in multiple directions.

I've read a lot about pftek working without a PC, that brings the question of flour vs grain, would there not be endospores in any whole grain flour?

That brings the question of how do these endospores enter the media, are grains particular susceptible to the bacteria involved? Or is it just environmental. In other words are endospores just eveywhere, point of contamination being unknown.
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#12 jkdeth

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Posted 19 April 2017 - 01:46 PM

Additionally when I asked about mechanics, I'm wondering about triggering germination of endospores, in order to kill the bacteria. I've also learned that new endospores can be formed in as little as 6 hours, so now I'm wondering if FS as I've read, 3 sessions each a day apart might even increase the endospore load.

#13 CatsAndBats

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Posted 19 April 2017 - 08:21 PM

https://mycotopia.ne...tion/?p=1315948

 

more bioicidal info there.. including effectiveness of killing endospores..


Edited by CatsAndBats, 19 April 2017 - 08:21 PM.

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#14 Needles

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Posted 20 April 2017 - 12:02 AM

I used to think that the grains we used for spawn needed to be soaked in order to germinate bacterial endospores so that they could be killed in the PC.
After testing five different grains by growing out and isolating bacteria from each grain I was unable to stain and identify any endospore producing Gram negative bacillus. Not to say that they don't exist in the grain, but just not as common as I thought they would be.
There was definitely a collection of bacteria and mold to be found.
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#15 CatsAndBats

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Posted 20 April 2017 - 06:24 AM

There was definitely a collection of bacteria and mold to be found.

 

After annihilation in the PC?



#16 Microbe

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Posted 20 April 2017 - 01:20 PM

The PC will destroy endospores. No life form will survive a proper cycle at 250° at 15 psi. Many believe that even after pc'ing that contaminating molds, yeasts, and or bacteria may be present at a very limited quantity setting the mycelium up for success. Sure this is true if one didnt run a proper cycle ie time,temp, and pressure. I have grain sit for 6 weeks before inoculation, trust me when i say all life is destroyed. The secondary measure for maintaining sterility is proper sealing and filter design.

If endospores, or any life form is still alive then it was not a proper cycle. There is no need to germinate endospores.

To your question why isnt endospores a issue with brf as it is with grains? They are a issue if they are in there because some endospores can survive over 24 hours after being exposes to a constant boil.

The reality is very few bacteria (relatively speaking) can go the endosporulation process. Most of the time when we get a bacteria contamination its not the result of endospores. Its from improper sterilization processes or poor filter design.

Theoretically you place jars of grains in a constant boil for 48 to 72 hours and destroy all life but this is not even feasible and if one would attempt this, well after a few projects it will end up costing you more in energy consumption then it would buying a pc.

My point is brf jars are just as prone to endospores as grains are and even more so because im certain noone here is boiling brf jars for a few days.

So as i always do, i have to leave a contradiction clause. If there is a threat of endospores being present, and assuming you are sterilizing via water bath, one SHOULD allow endospores to germinate before boiling.

Finally we shouldnt be worried about endospores. Again there are few endosporulating bacterium. Proper preparation, sterilization cycle and effective filter design is what one should focus on.

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#17 CatsAndBats

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Posted 20 April 2017 - 01:39 PM

The secondary measure for maintaining sterility is proper sealing and filter design.

 

 

Letting your PC/jars cool in a clean area (relatively), without too many mold spores (like a garage or outside), is key as well and speaks to microbe's secondary measure. Proper jar lids too.


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#18 Cuboid

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Posted 20 April 2017 - 01:50 PM


The secondary measure for maintaining sterility is proper sealing and filter design.


Letting your PC/jars cool in a clean area (relatively), without too many mold spores (like a garage or outside), is key as well and speaks to microbe's secondary measure. Proper jar lids too.
Is that to say Garage/outdoors has higher mold spore count than other indoors areas?

#19 CatsAndBats

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Posted 20 April 2017 - 02:01 PM

 

 

The secondary measure for maintaining sterility is proper sealing and filter design.
 


Letting your PC/jars cool in a clean area (relatively), without too many mold spores (like a garage or outside), is key as well and speaks to microbe's secondary measure. Proper jar lids too.
Is that to say Garage/outdoors has higher mold spore count than other indoors areas?

 

 

 

mine does

 

https://mycotopia.ne...ease/?p=1290109



#20 Microbe

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Posted 20 April 2017 - 02:20 PM

The secondary measure for maintaining sterility is proper sealing and filter design.

Letting your PC/jars cool in a clean area (relatively), without too many mold spores (like a garage or outside), is key as well and speaks to microbe's secondary measure. Proper jar lids too.
Is that to say Garage/outdoors has higher mold spore count than other indoors areas?
Hard to determine. Regoin in which you live in along with indoor conditions such as old carpet, how many people whom live there, pets, and etc. Some homes can have a greater particulate count then outdoors.

I dont worry about where i cool my pc and typically leave mine in the garage. Although cats advice is accurate and best practice, it doesnt mattwr how dirty the air is where you cool the pc as long as you have a efficient filter and the container is sealed properly such as impulse sealing bags before pc'ing or using silicone gaskets with plastic storage caps.

Im confident with my filter design for jar lids and even the filters that are integrated with spawn bags. However im not confident in the tyvek tek where one leaves the bag open and folded. I also cram 7 2lb bags of oats in a 24 qt presto so i need to stand them up vertically and submerge the bottom 1/3 in water.

Anyway i used to sit my spawn bags after pc'ing on a table in my garage to inflate and without any issues. Again it comes all comes back to proper pc cycle, lid and filter design. I have since stopped waiting for the bags to inflate and just cut them open in my flow hood then twist them and apply a cable tie to close them back up.

Are you having issues with mold contamination?

Edited by Microbe, 20 April 2017 - 02:21 PM.

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