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InvisibleStipe-n CapMDiscord
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Autoclave/Pressure Cooker effectiveness against Bacterial Endospore * 15
    #27376048 - 07/06/21 09:13 AM (2 years, 6 months ago)

Abstract


Bacterial endospores are a hot topic of debate within the community, anecdotal experiences vary between Trusted and accomplished cultivators. This post will seek to hold the feet of our fast held beliefs to the fire of science.

Autoclaves are utilized by many industries  for the sterilization of products ranging from foods to pharmaceutical articles. Products to be sterilized must first undergo bioburden   testing to determine microbial levels, these microbes are counted and then represented by units referred to as colony forming units  (CFU) .

Autoclaves are verified by a number of tests, but the benchmark test for verification is by biological indicator with a known bioburden.
Biological indicator testing uses ampules filled with thermophilic bacterial endospores from the species geobacillus stearothermophilus (formerly Bacillus stearothermophilus) containing 106 or more spores/vial.

Validation requires the testing of dense materials like folded surgical garments and towels with biological indicator packets placed at their centers. These materials effectively mimic the density of our grain jars in terms of the potential denial of steam penetration.

Autoclave Validation

For sterilization, it is required  to achieve a sterility assurance level (SAL) of 10-6. One log 6 reduction to reduce bioburden to 1 CFU, and an additional log 6 reduction to achieve a SAL of 10-6which is a 1 in 1,000,000 chance of a single non-sterile unit survival.

A total 12 log reduction is required for pharmaceutical practices. A 6 log SAL where 12 log reduction is required, the sterilization time should be a min of 121°C for 30 mins.

Our sterilization cycle runs 121°C for 2 hours.



SAL and log reduction


Quote:

"Bacillus stearothermophilus was, until the discovery of archaebacteria in hot springs, the most heat-resistant organism known. Spores withstand 121°C for up to 12 min, and this has made the organism ideal for testing autoclaves that run on a time–temperature cycle designed to ensure the destruction of spores."




Quote:

Because the destruction of endospores has been the benchmark of sterilization since the autoclave was invented, the only test of sterilization effectiveness accepted by regulatory agencies such as O.S.H.A. and state boards is endospore testing.




Sterilization Monitoring Service

Steam sterilization is achieved by exposing the items to be sterilized with saturated steam under pressure. Steam enhances the ability of heat to kill microorganisms by reducing the time and temperature required to denature or coagulate proteins in the microorganisms.



Quote:

Adam Savage said:
"The difference between screwing around and science is writing it down"





I've worded the Hypothesis in such a way that it will lend itself to falsification:

Hypothesis

Bacterial endospores are destroyed completely by the autoclaves and pressure cookers employed by growers within the community. A time–temperature cycle designed to ensure the destruction of endospores has been set for a minimum of 121°C for up to 2 hours which ultimately results in completely sterile media.


Equipment and materials

For this experiment I will be using the following equipment:

1.  One All American 1915x autoclave;
2.  One seedling heat pad;
3.  One electric Cadco hot plate;
4.  Laminar flow cabinet 99.999% efficient to .1 micron;
5.  Seven quart jars;
6.  Seven no leak ball lids with SFD;
7.  Wheat;
8.  Stainless steel prep pot;
9.  One Plastic bucket;
10. Petri dishes;
11. LME agar;
12. 70% ISO;
13. Infrared thermometer.

Procedure

 
1. Wheat will be prepped by soaking for 72
  hours to encourage endosporulation(1);
2. Once hydrated the grain will then be
  loaded into quart jars at three cups
  per jar;
3. Once loaded, jars will be foil capped
  and then sterilized at a min of 15 psi
  for two hours;
4. The autoclave will then be allowed to
  cool overnight;
5. The following day: jars will be removed
  from the autoclave and placed on a
  shelf  atop a seedling mat to
  incubate for 10 days at ~30 deg C(2)
  (Temperature will be tracked and recorded with an infrared thermometer)

The intention is to allow for the possibility of any surviving endospore to reactivate and enter the vegetative state when incubated in a nutrient rich environment, any surving endopores will take approximately two days to reactivate and then quickly divide and multiply thereafter.

After the ten day incubation period, jars will be thoroughly sanitised using 70% ISO and then placed in front of the flow-hood, clusters of grains will be poured onto LME agar to be cultured for bacterial growth.
Petri dishes will be placed on a shelf for observation at room temp (24°C) 

The remaining grain in the donor jars will then be inoculated with  axenic agar culture and placed back on the shelf to colonize at room temperature (24°C)




Notes


(1) A sustained soak should provide a suitable environment to trigger endosporulation:

Endospores in Cerial Grains Discussion 

"Under conditions of starvation, especially the lack of carbon and nitrogen sources, a single endospore
forms within some of the bacteria through a process called sporulation. When a bacterium detects environmental conditions are becoming unfavourable it may start the process of endosporulation, which takes about eight hours." Wiki

"In rich medium, B. subtilis cells divide by binary fission approximately every 30 minutes. By contrast, deterioration of environmental conditions triggers sporulation, a developmental process that takes about 8 to 10 hours. Thus, endospore formation represents a formidable investment of time and energy and is considered to be a survival pathway of last resort, as B. subtilis cells only commit to sporulation after they failed to deal with starvation in other way"
Making a spore: morphological stages of sporulation and formation of protective structures


(2)  Of the ten temperatures tested, ranging from 4°C through 50°C, the optimum reduced temperature for spore activation was 30°C.

Activation and germination characteristics observed in endospores

(3) On Cry toxins:

Quote:

Both the Bt bacterium and its toxin can be inactivated by usual physical (for example, heating at 60 to 90°C) and chemical (formaldehyde, chlorine, chlorine dioxide, hypochlorite, or strong acid solution) sterilization methods




https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/delta-endotoxin


:havesomescience:


Results:

Mycelium progressed normally without indication of bacterial infection. All jars maintained healthy growth: leap-off, spawn run, post shake recovery (3 shakes) were observed to be normal and healthy, grains broke apart readily both while growing and just before spawning.

full colonization was achieved without stalling, producing wet spots, excessive condensation, condensation below the grain line, stressed/erradic growth, or metabolites.

 More on the identification of bacterial spawn

If bacteria was present I would expect all of these symptoms of bacterial infection to occur simultaneously, however none of the symptoms were observed throughout the entire vegetative cycle.

Grains were then transferred to a shoebox where colonization of the substrate continued to be swift and healthy, lacking all indication of bacterial infection.

The shoeboxes were not fruited.


conclusions:


Autoclaves are designed to destroy bacterial endospores, if reproductively viable bacterial endospores were present they would reproduce. The argument that grains under these conditions don't grow bacteria is clearly incorrect, if that was a correct statement then we need not worry about bacteria to begin with.


Bacterial endospores prefer the incubation temperature provided for them (30°C):

https://link.springer.com/article/10.1007/BF00407754


Bacillus bacteria are motile on dry surfaces due to the secretion of surfactants:

https://www.nature.com/articles/nrmicro2405

I've provided information regarding autoclave verification: Verification procedures and equipment that are designed to produce barriers to conductivity and convection which I believe reasonably matches or exceeds the barriers presented by the loads that we are working with;

I have also excluded the possibility of Cry toxins affecting performance:

Quote:

Both the Bt bacterium and its toxin can be inactivated by usual physical (for example, heating at 60 to 90°C) and chemical (formaldehyde, chlorine, chlorine dioxide, hypochlorite, or strong acid solution) sterilization methods




https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/delta-endotoxin

I've reproduced all of the primary starting conditions that are agreed upon by the community to trigger bacterial blooms;

I have also supplied the information and requisite sources to confirm the operation and expected statistical efficacy of autoclaves.

When reviewing all of this information alongside the real time application of these principles, under the correct conditions: I conclude that this test has produced bacteria free spawn due to the efficiency/efficacy of the equipment we employ for that purpose.

The destruction of bacterial endospores is the primary reason that we employ these devices, it stands to reason that they actually work.

My conclusion is that a proper sterilization cycle, including purging, will sterilize your grains, wet or not.

Sterile wet grains are sterile wet grains. Bacteria doesn't require excessive moisture to survive or multiply. Temperature swings, moisture, extended shelf time, etc: none of these are sufficient conditions to contaminate your spawn either on their own or in conjunction so long as the sterilization cycle has been completed properly.

The only caveat is to begin with materials that can be sterilized. There are barriers that can prevent autoclave efficiency.

Time, Temperature, and pressure will destroy the microorganisms it was designed to destroy providing that you do not over-burden the unit.

Any of the above conditions will expose contamination by providing a hospitable environment for bacteria to flourish.

Finding bacteria in your media will be an indication that you have made one of 3 mistakes.

1. You have chosen media that has far too much overburden to insure that your equipment runs efficiently;

2. You have improperly operated your equipment;

3. You have introduced a contaminant.

These statements seem reasonable.

This is subject matter continues here:

Autoclave Validation and Load/Cycle Verification

 






Edited by Stipe-n Cap (10/28/22 08:11 PM)


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Invisiblecoversall
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Re: Autoclave/Pressure Cooker effectiveness against Bacterial Endospore [Re: Stipe-n Cap]
    #27376058 - 07/06/21 09:16 AM (2 years, 6 months ago)

:retrocool:


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Offlinegabbk
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Re: Autoclave/Pressure Cooker effectiveness against Bacterial Endospore [Re: Stipe-n Cap]
    #27376059 - 07/06/21 09:16 AM (2 years, 6 months ago)

subbed :awesomenod:


--------------------


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InvisibleJosex
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Re: Autoclave/Pressure Cooker effectiveness against Bacterial Endospore [Re: gabbk]
    #27376080 - 07/06/21 09:30 AM (2 years, 6 months ago)

:pope:


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OfflineEclipse3130
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Re: Autoclave/Pressure Cooker effectiveness against Bacterial Endospore [Re: gabbk]
    #27376091 - 07/06/21 09:36 AM (2 years, 6 months ago)

:thumbsup:

Just comments/questions soaking the wheat in water for 3 days will turn it into mush and completely over-hydrate it, I'm sure you know this though, and is probably a point of the experiment - when you are to repeat the process with the mushy wet grain it will be interesting to see.

What is the desired incubation temperature? May I suggest do 2 separate tests some at room temperature(70-75f) as well as some in the 85-90f range

My hypothesis on the experiment: I expect the wet jars to be bacteria laden incubated at above average temperature when you take some grain to agar. I don't expect the first jars properly hydrated to be contaminated or show any signs of growth on agar. The wet grain environment will encourage surviving bacteria to replicate at a much greater speed


--------------------
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OfflineLtLurker
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Re: Autoclave/Pressure Cooker effectiveness against Bacterial Endospore [Re: Eclipse3130]
    #27376095 - 07/06/21 09:37 AM (2 years, 6 months ago)

:popcorn:


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Offlinedeadmandave
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Re: Autoclave/Pressure Cooker effectiveness against Bacterial Endospore [Re: Josex]
    #27376097 - 07/06/21 09:38 AM (2 years, 6 months ago)

:popcorn:

Can you see endospores with a microscope?


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InvisibleJosex
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Re: Autoclave/Pressure Cooker effectiveness against Bacterial Endospore [Re: deadmandave]
    #27376109 - 07/06/21 09:43 AM (2 years, 6 months ago)

Only if you "stain" them first, afaik.


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Offlinegone-pear-shaped
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Re: Autoclave/Pressure Cooker effectiveness against Bacterial Endospore [Re: Josex]
    #27376145 - 07/06/21 10:06 AM (2 years, 6 months ago)

I'm happy you'll be using a flow hood for your tests, since my own pet theory is that nothing is as clean as people think it is. (Every time a jar is opened in a SAB, it gets filled with spores which by luck may not germinate, and the jar may continue looking clean.)


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InvisibleBenson
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Re: Autoclave/Pressure Cooker effectiveness against Bacterial Endospore [Re: LtLurker]
    #27376182 - 07/06/21 10:27 AM (2 years, 6 months ago)

:popcorn::takingnotes:


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InvisibleJosex
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Re: Autoclave/Pressure Cooker effectiveness against Bacterial Endospore [Re: gone-pear-shaped]
    #27376183 - 07/06/21 10:28 AM (2 years, 6 months ago)

Besides my own experiences, one thing that reinforces my convintion that endospores can recover after a cycle is having seen over the years countless instances of good cultivators that own a FH consistently produce bacterial spawn even from plates that looked good.

Some of them understand what's happening, some of them would rather think there's some sneaky ninja bacteria hitching a ride on their plates even if they looked perfectly fine.


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InvisibleModularMind
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Re: Autoclave/Pressure Cooker effectiveness against Bacterial Endospore [Re: Stipe-n Cap]
    #27376195 - 07/06/21 10:41 AM (2 years, 6 months ago)

Maybe shake several midway.


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InvisibleStipe-n CapMDiscord
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Re: Autoclave/Pressure Cooker effectiveness against Bacterial Endospore [Re: Eclipse3130]
    #27376196 - 07/06/21 10:43 AM (2 years, 6 months ago)

Quote:

Eclipse3130 said:
:thumbsup:
What is the desired incubation temperature? May I suggest do 2 separate tests some at room temperature(70-75f) as well as some in the 85-90f range




Yeah, thanks for reminding me, I'm running 7 jars because I will have some sitting at room temp. I failed to mention in the OP.


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InvisibleStipe-n CapMDiscord
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Re: Autoclave/Pressure Cooker effectiveness against Bacterial Endospore [Re: deadmandave]
    #27376199 - 07/06/21 10:45 AM (2 years, 6 months ago)

Quote:

deadmandave said:
:popcorn:

Can you see endospores with a microscope?




https://en.wikipedia.org/wiki/Endospore_staining


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OfflineDERRAYLD
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Re: Autoclave/Pressure Cooker effectiveness against Bacterial Endospore [Re: Stipe-n Cap]
    #27376216 - 07/06/21 10:54 AM (2 years, 6 months ago)

Is the point to prove or disprove your hypothesis?
If you prove the hypothesis to be false, are we all fcked? Or is there a solution in mind?

Just trying to understand if this is more for science to have a case study of sorts or to prove a hypothesis?


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OfflineBrownBear
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Re: Autoclave/Pressure Cooker effectiveness against Bacterial Endospore [Re: Stipe-n Cap]
    #27376219 - 07/06/21 10:55 AM (2 years, 6 months ago)

:takingnotes:


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Offlinegone-pear-shaped
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Re: Autoclave/Pressure Cooker effectiveness against Bacterial Endospore [Re: DERRAYLD]
    #27376236 - 07/06/21 11:07 AM (2 years, 6 months ago)

He's trying to disprove it. If he does, not much changes. We continue trying to use clean grain and pressure cooking sufficiently for the container size and quality. (Paul Stamets actually wrote that autoclave time had to be adjusted based on how dirty the grain was, but he's not considered a serious authority on this anymore.)

Actually, if endospores can survive a full PC cycle, one possible solution is to do one round of fractional sterilization: boil, wait eight hours (not 24), then pressure cook. But that might not work since the grain center ideally won't be fully hydrated by the boil, so spores in the center won't germinate. Maybe higher temperature would be a solution. But using relatively clean grain will always be the easiest solution (if p9 disproves the hypothesis).


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InvisibleJosex
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Re: Autoclave/Pressure Cooker effectiveness against Bacterial Endospore [Re: gone-pear-shaped]
    #27376242 - 07/06/21 11:12 AM (2 years, 6 months ago)

Quote:

But using relatively clean grain will always be the easiest solution




This is key, quality matters.


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InvisiblemushboyMDiscord
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Re: Autoclave/Pressure Cooker effectiveness against Bacterial Endospore [Re: Josex]
    #27376251 - 07/06/21 11:18 AM (2 years, 6 months ago)

:god2:
i dedicate this post to meteah.


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InvisibleJosex
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Re: Autoclave/Pressure Cooker effectiveness against Bacterial Endospore [Re: mushboy]
    #27376260 - 07/06/21 11:25 AM (2 years, 6 months ago)

Quote:

mushboy said:
:god2:
i dedicate this post to meteah.



:manofapproval:

Ma main man Mateha is always very busy reading stuff about microbes so he can cult much much better, I doubt he'll pop up.


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