Home | Community | Message Board


This site includes paid links. Please support our sponsors.


Welcome to the Shroomery Message Board! You are experiencing a small sample of what the site has to offer. Please login or register to post messages and view our exclusive members-only content. You'll gain access to additional forums, file attachments, board customizations, encrypted private messages, and much more!

Shop: Kraken Kratom Red Vein Kratom   PhytoExtractum Buy Bali Kratom Powder   Unfolding Nature Unfolding Nature: Being in the Implicate Order   Bridgetown Botanicals CBD Concentrates   Left Coast Kratom Buy Kratom Extract   Original Sensible Seeds Autoflowering Cannabis Seeds   North Spore Bulk Substrate   Mushroom-Hut Substrate Bags

Jump to first unread post Pages: 1 | 2 | Next >  [ show all ]
InvisibleStipe-n CapMDiscord
Male User Gallery

Registered: 08/04/12
Posts: 7,623
Loc: Canada Flag
Trusted Cultivator
Autoclave Validation and Load/Cycle Verification (Gourmet edition) * 3
    #28061273 - 11/20/22 08:07 AM (1 year, 2 months ago)

Autoclave Validation and Load/Cycle Verification




 


First, what is an autoclave?:

Autoclaves are also known as steam sterilizers, and are typically used for healthcare or industrial applications. An autoclave is a machine that uses steam under pressure to kill harmful bacteria, viruses, fungi, and spores on items that are placed inside a pressure vessel. The items are heated to an appropriate sterilization temperature for a given amount of time. The moisture in the steam efficiently transfers heat to the items to destroy the protein structure of the bacteria and spores.

In healthcare, the term "autoclave" is typically used as the nomenclature to describe a Steam Sterilizer.

Steam sterilization can be accomplished either under pressure or at standard atmospheric pressure (1 atmosphere = 0.101325 MPa), water boils at approximately 100 degrees Celsius. That is simply another way of saying that the vapor pressure of water at that temperature is 1 atmosphere.

Some of us possess the All American benchtop autoclaves, fewer have larger volume digital laboratory autoclaves, large atmospheric drum sterilizers, etc, but  most of us use the standard 23 quart Presto pressure canner; all of these pressurized vessels perform essentially the same function utilizing the exact same mechanisms, atmospheric sterilization simply takes longer exposure to achieve the same goal.

Autoclaves require validation in the laboratory setting on a weekly basis, we do not require this level of quality control for obvious reasons, however, understanding the process is beneficial for the hobby level enthusiast and professional cultivator alike.

The intention of this post is to test the penetration capabilities of the 23 quart pressure canner with different loads and will be an extension of my Bacterial Endospore Experiment



Autoclave Cycle Verification Procedure



Initial Process

1. Retrieve a self-contained biological indicator (SCBI) ampoule (Geobacillus stearothermophilus).

2. Identify the indicator (spore ampoules) by labeling it with the proper information (date,
autoclave number, control, test, etc.).

3. Place the spore ampoule in a horizontal position with representative materials to be sterilized.
The ampoule should be located in the part of the load that would be considered most difficult to
sterilize (center of grain jar).

Remember to attach a piece of autoclave tape onto the load.

4. Select appropriate cycle to process the load. (90+ mins for grain spawn)

5. Once the cycle has come to an end, allow the autoclave to depressurize and cool down.

6. Remove the load from autoclave and allow it to further cool down (10 - 15min).

7. Retrieve the spore test ampoule from the load.

8. It is common to see a color change from the spore ampoules from purple to black; usually this
distinguishes indicators that have been exposed to steam to those that have not.



Incubation Process


1. Remove the SCBI from the load and use the Verify activator to gently crush the interior glass
ampoule in order to have the spores come into contact with the growth media. Place the
indicator in the preheated incubator.

2. Take a second spore ampoule (label control ampoule) that was not run in the load and proceed
to break the glass ampoule as above and also incubate in the same incubator.

3. Incubate both spore ampoules for 24 hours.

Interpretation Process

1. Examine indicator ampoules for any color change after 24 hours. The presence of yellow
indicates a failed test and positive bacterial growth. Cloudy media within the ampoule also
indicates growth. No color change and clear liquid indicate a passing test and proper
sterilization of sample. The control ampoule should have a yellow color change.

I will be following this process using the following instruments:



Bionova Biological Indicator Incubator:

FEATURES:
For use with traditional 24 Hour Biological Indicators
26 Positions (0.9 cm for biological indicators)
10 Positions  (1.3 cm for culture medium and spore ampoules)
On-board ampule crusher
Dual Temperature Option:  37°C or 60°C




McKesson Sterilization Biological Indicator Vial Steam:

McKesson Self-Contained Steam BI
For use in monitoring steam sterilization processes.
Product features include final read-out in only 24 hours and fits existing incubators.

Steam self-contained biological indicators are inoculated with viable Geobacillus stearothermophilus bacterial spores and are intended for monitoring the efficacy of saturated steam sterilization processes operating at 121°C, self contained culture media is tryptic soy broth validated for growth
promotion.

Growth of surviving spores has been documented in as little as 2 1/2 hours.





McKesson Steam Indicator Strip:
For monitoring Steam sterilizers operating at 250-285 Degrees F (121-140 Degrees C).
The indicator strip provides distinct color change when exposed to the steam sterilization process.



Maximum Registering Autoclavable Thermometer:

Autoclave Mercury Thermometers are for validating autoclaves by monitoring the highest temperatures attained during a sterilization cycle.



Load Penetration and Cycle Verification



Phase 1:

I will be subjecting the biological indicators/strips/thermometer to common loads to ascertain effective cycle duration with accuracy, exposures will begin with: 90 minute cycle at 15psi with a 10 minute purge/vent for 1L/Q grain jars;


Wheat

Corn

Oats

Millet




Phase 1 Results:

All four grains have passed verification for the 90 minute exposure at 15psi (121C).
There were minor variations of ~1 degree Celsius between some of the cycles which falls within the margin of error for the max registering thermometer and the accuracy tolerances of the Presto pressure gauge. The positive sterile result for each biological indicator validates the 23 quart Presto pressure canner as an autoclave while simultaneously verifying that these loads can be penetrated and that the time temperature cycle is sufficient. 

Despite having passed at 90 mins, less is not more when dealing with sterility.

Thermophilic microorganisms though thermophilic have limitations to their resistance. Longer venting ensures trapped gasses are purged, replaced with steam and surfaces have ample time to evenly heat, uneven heating leads to "cold" spots, cold relative to working temperature.

Longer cycles ensure that microorganisms have had sufficient exposure to heat and serve as redundancy failsafe's for inadvertent mistakes made somewhere in the process.

Running temperature/pressure in excess of 15psi or 121°C adds another layer of redundancy. Redundancy measures are integral to successful planning, failsafe measures are cheap insurance policies; this is why many of us recommend more than the standard 90 minute cycle at 15psi with 10 minute purge.

More is more, less is not more (Within reason).


Phase 2:

I will reduce the 90 minute cycle by 30 mins; I will choose only one grain (millet) for this as the cycle appears to have the same outcome despite grain selection. My intention here is to determine the lower threshold for cycle efficiency.

A time-temperature cycle of 1 hour at 15psi with a 10 mins vent will be attempted.

Phase 2 Results:

I did not make a YouTube video for this as it is pretty straight forward, the 60 min cycle passed verification by biological indicator:



I will not be reducing the 90 min cycle for the Presto or IP as I believe that it's unnecessary due to the existing sterility assurance standard set by the pharmaceutical industry which is 6 log sterility assurance level (SAL) where 12 log reduction is required (10-6), the sterilization time is set to a min of 121°C for 30 mins.

So reducing to 30 seems pointless when we know that 60 has passed. 30-60 mins should be considered the minimum threshold for assured sterility.


Phase 3:

I will now move onto 6 x 3 quart spawn bags following the same protocol. The bags will be stacked in 3 tiers of two bags each. The BI ampoule will be loaded into a bag on the center tier, central in the load.




The max registering thermometer will be wedged between the tiers at a 45° angle:




The time temperature cycle will be set for 3.5 hours at 15psi with a 15 minute purge/vent.




Phase 3 Results:








I kept a close eye on the gauge, it didn't climb outside of the long dash for 15 psi so I'm not sure what to make of the core temp reading; either way the 3.5 hour cycle at 15 psi has been verified.








Autoclave Validation



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 ampoules filled with thermophilic bacterial endospores from the species geobacillus stearothermophilus (formerly Bacillus stearothermophilus) containing 106 or more spores/vial.

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.






Common electric kitchen pressure cookers including the Instant Pot have already been tested in a laboratory setting and have been confirmed to be capable of sterilizing liquids:

Quote:

It was determined that a maximum of 1.5 L of liquid (split between one 1.5 L beaker holding 1 L of liquid and two 0.5 L beakers holding 0.25 L of liquid each) could fit comfortably in the metal insert within the pressure cookers without bubbling over and losing some of the contents during sterilization. To determine whether pressure cookers could sterilize microbial culture media, the minimum amount of time necessary to reliably sterilize 1.5 L of nutrient broth (NB) was determined (Table 3). The nutrient broth was prepared en masse on the laboratory bench, portioned out into the three beakers for a total of 1.5 L in each pressure cooker, and sterilized in five-minute intervals from 5–30 mins using the manual setting at the highest setting. It was found that 15 mins was the minimum time required to consistently sterilize 1.5 L of NB in all of the pressure cookers and as evidenced by a lack of microbial growth when incubated at 30°C for one week. Similarly, when agar was added to the medium to pour plates, 15 mins was the minimum time required to consistently sterilize 1.5 L of NA plates.

Less time in the pressure cookers or no sterilization resulted in turbid growth in NB and colonies on NA plates within 1–3 days of incubation at 30°C. These results indicate that pressure cookers provide sufficient heat and pressure to reliably sterilize 1.5 L of microbiological growth medium for laboratory use.




Source:

Research article: Assessment and verification of commercially available pressure cookers for laboratory sterilization


Quote:

To verify that an autoclave is consistently reaching the desired temperature and pressure for sterilization, biological tests are regularly conducted to demonstrate that the most heat-resistant organisms are inactivated, which implies that all other more sensitive contaminants would be similarly sterilized. In one of the more common tests, a suspension of especially heat-resistant Geobacillus stearothermophilus (originally deposited as Bacillus stearothermophilus Donk) endospores are autoclaved and growth is assessed either by plate-based growth or by the appearance of turbidity and color change of an indicator dye. Failure of the endospores to germinate and grow indicates that the autoclave is functioning properly.






Instant Pot (Duo Plus):




Phase 1:

I will be subjecting the Instant Pot to autoclave verification by biological indicator ampoule and chemical indicator strip. The initial cycle will be on max pressure using the pressure cook function. The unit will be allowed to vent for 10 minutes prior to the cycle which will be run for 3 hours on high pressure which fluctuates between 9.4-12.3 psi.

There wasn't sufficient space to accommodate the max registering thermometer so it was not included in this test.

A brand new 6 quart Instant Pot Duo Plus was used for this validation sequence.


Phase 1 results:



The 1L grain jar was unable to fit standing vertically so I placed the jar on its side supported by the wire trivet, the jar was not submerged in water. I filled the insert with 4 cups (1 quart) of water to run this cycle.




Post cycle the steam indicator on the exterior of the biological indicator had changed colour which indicates sterilization temps had been achieved, the same with the stand alone chemical indicator strip:









The 3 hour cycle produced a positive sterile result with the BI ampoule which validates this unit as a functional autoclave.

Phase 2:

I will be reducing the cycle duration to 90 mins, should the ampoule fail to sterilize I will increase the cycle duration by 30 mins.

Phase 2 Results:

The 90 min cycle has been verified. 4 pint jars are all you need to inoculate 4 x 5lb supplemented hwfp blocks, so potentially a great tool for low volume gourmet.




Phase 3:

I will attempt to load the IP with a 4lb 3 quart spawn bag; bag will be the standard 3t unicorn myco-bag. The  cycle will be set for 3.5 hours on max pressure setting using the pressure cook mode with a 15 minute vent.



The spawn bag fit comfortably within the stainless insert supported by the IP' wire trivet, 1 quart of water was used for this cycle.

Ampoule is currently incubating, standby.

Phase 3 Results:








Conclusions:



On the left you'll notice that the integral chemical indicator strip is black, this is the cycled ampoule, the one on the right with the blue strip is the control. I have been keeping a close eye on the control amps to determine how long it takes to observe colour change from purple to yellow which indicates the presence of bacteria. This picture was taken at the 5 hour mark. I know that the incubator is functioning, this is why a control is required every cycle to ensure that the incubator is actually capable of reactivating the endospores. The product literature states that:

Quote:

Growth of surviving spores has been documented in as little as 2 1/2 hours.





Each cycle and every load so far has passed the verification process. What does this tell me?

If we can be assured that endospores are destroyed regardless of grain selection, size, etc, we then know that there are other factors to consider when problems arise post cycle. It appears as though our pressure cookers easily penetrate the common loads and thoroughly defeat thermophilic endospores which means that if we have any remaining issues with grains post cycle (so long as the  cycle was performed correctly) those issues will not be due to reactivated endospores. The chance of a single CFU survival is 10-6.

I believe that the OMC has made a myth and a legend out of bacterial endospores, gave them an air of invincibility and mystique. It appears as though the pharma 12 log SAL achieved with a 30 minute exposure at 15psi is applicable to our loads. Folks have argued endlessly on the boards that endospores are somehow exceptional materials which are not governed by the regular laws of physics and chemistry; they are somehow a different class of stubborn, exceptionally septic, unsterilizable material which can only be super pasteurized at best.


However, these validation/verification protocols serve only to verify the units/cycles ability to effectively sterilize endospores only, and by extension any other living organism. What these tests cannot touch on are the subject of bacteriocin-like inhibitory substances (BLIS), cry toxins, fungicides, Chitinases, etc.
Cereal grains are mass produced agricultural products meant for mass consumption which implies the use of chemical fungicides/pesticides, etc. Vegetative bacteria produce chitinases, dying bacteria produce BLIS, not to mention the unknown unknowns; molds, blights, rots, mycotoxins....I'm starting to think that endospores are the least of our worries.

There is much more to spawn production than meets the eye, I'm willing to wager that user error plays a huge role in bacterial infections however when competent cultivators have exhausted all other possibilities I believe we're dealing with chemical compounds either produced by agro or are organic compounds produced by the very organisms we're destroying, the result of a very ancient and competitive arms race between microorganisms.

To what extent heat exposure effects these compounds is yet to be determined. This is why I recommend long purge cycles with extended exposures at greater pressure and temperatures just to be safe.

Clearly with the case of Josex' stubborn grain there are instances where heat and pressure seem to have little effect. Thankfully those instances are far and few between. Should a cultivator run into some grain that causes exceptional stress to their productivity, cut your loss and find a new grain to work with.

Should you practice proper technique and cycle your loads correctly then bacteria, though omnipresent should be manageable.


So yeah, there's likely to be other variables that haven't been factored into our sterilization process that are not so easily solved by simple heat exposure. Perhaps it's the case that relatively short exposures are effective against endospores while longer exposures serve to breakdown  chemicals, toxins, or other inhibitory compounds.

Our grain jars/bags contain a microscopic universe of complex ineractions and organisms completely alien to our preconceptions.



The tl;dr/didn't understand/ please just fucking tell me in plain english version:



The focus here is that thermophilic bacterial endospores can be sterilized by both the 23 qt Presto and by the 6 qt IP duo.

Furthermore each load type despite variation in grain size and species can be penetrated by the cycles we commonly subject them to:

The minimum being 90 min @ 15psi with 10 min purge; although the units both seem to be effective at shorter exposures which serves only to validate the pharma standard for sterility assurance which is a 12 log reduction from a 30 min cycle at 15 psi.


Short exposures at minimum pressure/temperature can be effective against endospores but there is more to the story than just bacterial endospores. 

For 1L/Q jars:

Cycle jars for 2 hours @ 15-18 psi with a 10-15 min vent with preference for the higher end.

For spawn bags:

3.5-4 hours at 15-18 psi with a 15-20 min vent with preference for the higher end.

Less is not more with sterility assurance.


On purging/venting:

Every vessel is filled with air, whether it be your pressure cooker, grain jar, or spawn bag all of these vessels are filled both with the material intended for sterilization and air. When you place the lid on any pc you are now encapsulating that air (gas).

Venting is required to purge this trapped air from the unit/vessels while simultaneously replacing it with steam, if you fail to purge the pc/autoclave you're pressurizing the mixed trapped gases which will result in a lower temperature than if you allowed the steam to occupy the vessel alone, via the vent.

Daltons law covers this phenomenon:

Quote:

According to Dalton's law of partial pressures, the total pressure by a mixture of gases is equal to the sum of the partial pressures of each of the constituent gases. The partial pressure is defined as the pressure each gas would exert if it alone occupied the volume of the mixture at the same temperature.




Edited by Stipe-n Cap (11/27/22 12:57 PM)


Extras: Filter Print Post Top
InvisibleStipe-n CapMDiscord
Male User Gallery

Registered: 08/04/12
Posts: 7,623
Loc: Canada Flag
Trusted Cultivator
Re: Autoclave Validation and Load/Cycle Verification (Gourmet edition) [Re: Stipe-n Cap]
    #28061275 - 11/20/22 08:07 AM (1 year, 2 months ago)

Wheat:

A standard 1L grain jar with modified SFD lid containing 24 ounces of hydrated wheat and 1 self-contained biological indicator ampoule;

The grain was measured out with this 8oz spoodle:



The 2nd jar: swing top jar containing 32 ounces of grain and the max registering thermometer along with a chemical indicator strip buried alongside, foil capped.



Both Were cycled in the Presto 23qt for 90 minutes @ 15psi with a 10 minute vent/purge.

Post cycle the 2 jars were removed from the Presto and allowed to cool, once sufficiently cooled the max registering thermometer was removed and left to cool further so as not to attempt a reading while the mercury was still warm; once sufficiently cooled the thermometer was read vertically:



Max core temperature reached was 119C.

The chemical indicator strip has changed from blue to black which indicates sterility:



The BI ampoule was removed and placed into the BI incubator along with a control ampoule, both ampoules were crushed to allow the spores to come into contact with the self contained growth medium and then incubated for 24 hours at 60°C.





The ampoule on the left is the test ampoule, the amp on the right is the control; the colour indicates that the test amp has been successfully sterilized while the control amp has turned yellow indicating bacterial growth, which is to be expected.




Results:

The load has been successfully sterilized by the 23qt Presto utilizing the standard 90 cycle @ 15psi, however the core temperature failed to reach 121°C. More time or a longer purge may be required to raise the core temperature, this didn't prevent the load from achieving sterilization, however this may become more of an issue with larger/denser loads like spawn bags.

I would recommend that 1L/1Q grain jars still be run for 2 hours, as always with sterility more is more.










Edited by Stipe-n Cap (11/20/22 08:55 AM)


Extras: Filter Print Post Top
InvisibleStipe-n CapMDiscord
Male User Gallery

Registered: 08/04/12
Posts: 7,623
Loc: Canada Flag
Trusted Cultivator
Re: Autoclave Validation and Load/Cycle Verification (Gourmet edition) [Re: Stipe-n Cap]
    #28061278 - 11/20/22 08:08 AM (1 year, 2 months ago)

Corn:

Framework/protocol will be the same for each cycle.


Results: 








Cycle has been successfully verified for corn, max internal temperature was 121.5°C; I had to run the thermometer twice due to a lack of concentration :lol:




Edited by Stipe-n Cap (11/20/22 08:58 AM)


Extras: Filter Print Post Top
InvisibleStipe-n CapMDiscord
Male User Gallery

Registered: 08/04/12
Posts: 7,623
Loc: Canada Flag
Trusted Cultivator
Re: Autoclave Validation and Load/Cycle Verification (Gourmet edition) [Re: Stipe-n Cap]
    #28061280 - 11/20/22 08:08 AM (1 year, 2 months ago)

Oats:





Results:


Max temperature registered was 120°C







Edited by Stipe-n Cap (11/20/22 08:58 AM)


Extras: Filter Print Post Top
InvisibleStipe-n CapMDiscord
Male User Gallery

Registered: 08/04/12
Posts: 7,623
Loc: Canada Flag
Trusted Cultivator
Re: Autoclave Validation and Load/Cycle Verification (Gourmet edition) [Re: Stipe-n Cap]
    #28061282 - 11/20/22 08:08 AM (1 year, 2 months ago)

Millet:







Cycle was successfully verified with millet; max internal temp was 121°C.


Edited by Stipe-n Cap (11/20/22 08:59 AM)


Extras: Filter Print Post Top
InvisibleStipe-n CapMDiscord
Male User Gallery

Registered: 08/04/12
Posts: 7,623
Loc: Canada Flag
Trusted Cultivator
Re: Autoclave Validation and Load/Cycle Verification (Gourmet edition) [Re: Stipe-n Cap]
    #28061285 - 11/20/22 08:08 AM (1 year, 2 months ago)

Reserved for future indexing.


Extras: Filter Print Post Top
InvisibleStipe-n CapMDiscord
Male User Gallery

Registered: 08/04/12
Posts: 7,623
Loc: Canada Flag
Trusted Cultivator
Re: Autoclave Validation and Load/Cycle Verification (Gourmet edition) [Re: Stipe-n Cap]
    #28061293 - 11/20/22 08:15 AM (1 year, 2 months ago)

I thought this would be just as useful in gourmet considering that spawn production remains the same for most cultivated species.


Extras: Filter Print Post Top
Offlinedeadmandave
Slime
Male User Gallery

Registered: 02/16/10
Posts: 3,355
Loc: Flag
Last seen: 3 hours, 58 minutes
Trusted Cultivator
Re: Autoclave Validation and Load/Cycle Verification (Gourmet edition) [Re: Stipe-n Cap]
    #28061338 - 11/20/22 09:09 AM (1 year, 2 months ago)

Interesting. I would like to see indicator strips or bi ampoule testing in the center of some 5-10lb substrate bags that get steamed for 24hrs. Steamers don't get to 121, they stay under 100c so it would be curious to have results from them.


Extras: Filter Print Post Top
InvisibleStipe-n CapMDiscord
Male User Gallery

Registered: 08/04/12
Posts: 7,623
Loc: Canada Flag
Trusted Cultivator
Re: Autoclave Validation and Load/Cycle Verification (Gourmet edition) [Re: deadmandave]
    #28061341 - 11/20/22 09:11 AM (1 year, 2 months ago)

I agree, that would be a great addition but unfortunately I am not set up to run that atm....very unfortunate. Maybe in the future.


Extras: Filter Print Post Top
OnlineStromriderM
This must be the place
Male User Gallery


Registered: 06/02/13
Posts: 7,326
Loc: Dept of know what I'm say... Flag
Last seen: 2 seconds
Trusted Cultivator
Re: Autoclave Validation and Load/Cycle Verification (Gourmet edition) [Re: deadmandave]
    #28061500 - 11/20/22 10:57 AM (1 year, 2 months ago)

Quote:

deadmandave said:
Interesting. I would like to see indicator strips or bi ampoule testing in the center of some 5-10lb substrate bags that get steamed for 24hrs. Steamers don't get to 121, they stay under 100c so it would be curious to have results from them.





I've ran indicator strips in the middle of 5lb sub blocks during a 20 hour atmospheric steam cycle (ran at 205f)

They all indicated sterilized. I have a theory that sterilization of grain may not be the whole story in a long steam cycle like that though. I think it over cooks the sugars in the grain making it hard for the mycelium to digest the grain allowing bacteria to flourish. Maybe the combination of not quite killing all the endospores and over cooked grains. Idk just a thought


Extras: Filter Print Post Top
Offlinedeadmandave
Slime
Male User Gallery

Registered: 02/16/10
Posts: 3,355
Loc: Flag
Last seen: 3 hours, 58 minutes
Trusted Cultivator
Re: Autoclave Validation and Load/Cycle Verification (Gourmet edition) [Re: Stromrider]
    #28061775 - 11/20/22 01:58 PM (1 year, 2 months ago)

I mean based on these test strips/bi ampoules the efforts needed to sterilize grains/sub seem straightforward but in practice it seems less clear.

For instance based on strom's test it should be possible to cook grains in a low temp steamer for 20 hours and generate sterility. However any grains i have cooked this way end up with a high chance of growing bacteria.

From P9's vantage it seems like a simple pressure cook run of even only 60 minutes at 15psi for a quart size load is sufficient to nuke the grains and leave a sterile environment. But if you look at the subjective or anecdotal evidence on this forum you'll see loads of people who struggle to get clean spawn even in long cycles at or above 15 psi. im mostly referencing josex's thread

so where are the contaminants introduced? are we missing bacteria in our agar work? are these test strips not accurate for mycology? is working with bags too cumbersome? do some species carry symbiotic bacteria within that can get out of balance if the conditions are right?

there's always the possibility that what we percieve as bacterial contam is something altogether different in certain situations. Im mostly thinking about steamed grains.


Edited by deadmandave (11/20/22 02:04 PM)


Extras: Filter Print Post Top
InvisibleStipe-n CapMDiscord
Male User Gallery

Registered: 08/04/12
Posts: 7,623
Loc: Canada Flag
Trusted Cultivator
Re: Autoclave Validation and Load/Cycle Verification (Gourmet edition) [Re: deadmandave]
    #28061848 - 11/20/22 02:54 PM (1 year, 2 months ago)

I think the OMC has made a myth and a legend out of bacterial endospores, gave them an air of invisibility and mystique.

It appears as though the pharma 12 log SAL achieved with a 30 minute exposure at 15psi is applicable to our loads as well. Folks have argued endlessly on the boards that endospores are somehow exceptional materials which are not governed by the regular laws of physics and chemistry; they are somehow a different class of stubborn, exceptionally septic, unsterilizable material which can only be super pasteurized at best.


As I mentioned at the end of the OP these validation/verification protocols serve only to verify the units/cycles ability to effectively sterilize endospores only, and by extension any other living organism. What these tests cannot touch on are the subject of bacteriocin-like inhibitory substances (BLIS), cry toxins, fungicides, Chitinases, etc.

Cereal grains are mass produced agricultural products meant for mass consumption which implies the use of chemical fungicides/pesticides, etc.

Vegetative bacteria produce chitinases, dying bacteria produce BLIS, not to mention the unknown unknowns; molds, blights, rots, mycotoxins....I'm starting to think that endospores are the least of our worries.

There is much more to spawn production than meets the eye, I'm willing to wager that user error plays a huge role in bacterial infections, however when competent cultivators have exhausted all other possibilities I believe were dealing with chemical compounds either produced by agro or are biological compounds produced by the very competitive arms race between microorganisms.

To what extent heat exposure effects these  compounds is yet to be determined. This is why I recommend long purge cycles with extended exposures at greater preasure and temperatures just to be safe.

Clearly with the case of Josex' stubborn grain there are instances where heat and pressure seem to have little effect. Thankfully those instances are far and few between.

Should a cultivator run into some grain that causes exceptional stress to their productivity, cut your loss and fimd a new source or different grain to work with. Should you practice proper technique and cycle your loads correctly then bacteria, though omnipresent should be manageable.


Edited by Stipe-n Cap (11/20/22 03:26 PM)


Extras: Filter Print Post Top
OnlineStromriderM
This must be the place
Male User Gallery


Registered: 06/02/13
Posts: 7,326
Loc: Dept of know what I'm say... Flag
Last seen: 2 seconds
Trusted Cultivator
Re: Autoclave Validation and Load/Cycle Verification (Gourmet edition) [Re: Stipe-n Cap] * 1
    #28061966 - 11/20/22 04:19 PM (1 year, 2 months ago)

I've been making spawn at a relatively large scale for many years now. The more I learn the more I realize I still have a lot to learn.

I will say I seem to have the least amount of issues with rye as opposed to anything else. How much of that is in my head I don't know. I just know when I'm running certified seed grade rye I rarely have issues. If I buy feed grade wheat I see problems pop up. Oats are even worse. When buying grain by the pallet you tend to see patterns

I say these things only for small scale growers to scream that popcorn or oats or whatever works great for them. I don't doubt that it does. Just keep in mind it's a very different thing at a large scale

Thanks for posting this info here p9


Extras: Filter Print Post Top
InvisibleStipe-n CapMDiscord
Male User Gallery

Registered: 08/04/12
Posts: 7,623
Loc: Canada Flag
Trusted Cultivator
Re: Autoclave Validation and Load/Cycle Verification (Gourmet edition) [Re: Stromrider]
    #28061995 - 11/20/22 04:32 PM (1 year, 2 months ago)

Yeah man I've noticed the same thing, I don't run as much spawn volume especially not these day's but feed grains are absolute garbage, especially oats.

I've had my best luck with either food grade or bird seed like millet. Sometimes I can source some very clean wheat even from the feed shop, but if it's covered in filth and bran it can be hit or miss.

This is why I like bird seed, the harvesting and processing of feed grains for livestock produces contamination from the processing machinery; this is monitored by the ministry of agriculture in Canada; not sure who would track that sort of thing in the US.

Anyways, I've learned from this endeavor so I hope it has the same effect on others.


Extras: Filter Print Post Top
OnlineStromriderM
This must be the place
Male User Gallery


Registered: 06/02/13
Posts: 7,326
Loc: Dept of know what I'm say... Flag
Last seen: 2 seconds
Trusted Cultivator
Re: Autoclave Validation and Load/Cycle Verification (Gourmet edition) [Re: Stipe-n Cap] * 1
    #28062086 - 11/20/22 05:16 PM (1 year, 2 months ago)

I'm not a hundred percent how it works I just know i can go to my feed store and buy "feed grade" grains or I can pay a little more and get "certified seed grade" grains. It's a huge difference. The certified seed grade is nearly spotless with no busted grains or anything floating. Feed grade is tons of straw, trash, and undeveloped floating grains. Cost a few more dollars a bag but definitely worth it


Extras: Filter Print Post Top
InvisibleStipe-n CapMDiscord
Male User Gallery

Registered: 08/04/12
Posts: 7,623
Loc: Canada Flag
Trusted Cultivator
Re: Autoclave Validation and Load/Cycle Verification (Gourmet edition) [Re: Stromrider]
    #28062178 - 11/20/22 05:59 PM (1 year, 2 months ago)

Cheap insurance for sure.

OP is now complete.


Extras: Filter Print Post Top
InvisibleStipe-n CapMDiscord
Male User Gallery

Registered: 08/04/12
Posts: 7,623
Loc: Canada
Trusted Cultivator
Re: Autoclave Validation and Load/Cycle Verification (Gourmet edition) [Re: Stipe-n Cap]
    #28100495 - 12/16/22 12:54 PM (1 year, 1 month ago)

Whoops, wromg thread, lol.


Edited by Stipe-n Cap (12/16/22 06:17 PM)


Extras: Filter Print Post Top
Offlinetinyflush
 User Gallery


Registered: 08/09/23
Posts: 26
Loc: US
Last seen: 4 months, 23 days
Re: Autoclave Validation and Load/Cycle Verification (Gourmet edition) [Re: Stromrider]
    #28446534 - 08/25/23 02:57 PM (5 months, 1 day ago)

This thread is sorta old, but I just wanted to say that chemical indicators are not reliable compared to biological ones. Steam sterilization chemical indicators are not adequate for monitoring real steam sterilization cycles
Also, this is a great thread Stipe, nice work!


--------------------
Tiger got to hunt, bird got to fly;
Man got to sit and wonder "why, why, why?"
Tiger got to sleep, bird got to land;
Man got to tell himself he understand.


Extras: Filter Print Post Top
InvisibleStipe-n CapMDiscord
Male User Gallery

Registered: 08/04/12
Posts: 7,623
Loc: Canada
Trusted Cultivator
Re: Autoclave Validation and Load/Cycle Verification (Gourmet edition) [Re: tinyflush]
    #28446560 - 08/25/23 03:22 PM (5 months, 1 day ago)

I followed the protocol utilized by laboratories to verify autoclaves, employing the same equipment, including self contained biological indicator ampoules coupled with the appropriate incubators. The equipment is listed in the OP and is mentioned often throughout.

I went a step further by using a max registering autoclave thermometer in order to verify whether R-value played a role in grain selection. The chemical indicator strips were secondary but are included in the protocol literature, which is posted in the OP.

All loads, every cycle was validated by biological indicator. The BI ampoules have integral chemical indicator strips.


Extras: Filter Print Post Top
Offlinetinyflush
 User Gallery


Registered: 08/09/23
Posts: 26
Loc: US
Last seen: 4 months, 23 days
Re: Autoclave Validation and Load/Cycle Verification (Gourmet edition) [Re: Stipe-n Cap]
    #28446690 - 08/25/23 05:52 PM (5 months, 23 hours ago)

Ah sorry I’m not doubting your methodology at all! Clearly you were extremely thorough.

My comment was just in reply to strom and deadman in regards to their chemical indicatory test giving unreliable results.

Sorry for the confusion, not a frequent forum user, I should have quoted.


--------------------
Tiger got to hunt, bird got to fly;
Man got to sit and wonder "why, why, why?"
Tiger got to sleep, bird got to land;
Man got to tell himself he understand.


Edited by tinyflush (08/25/23 06:00 PM)


Extras: Filter Print Post Top
Jump to top Pages: 1 | 2 | Next >  [ show all ]

Shop: Kraken Kratom Red Vein Kratom   PhytoExtractum Buy Bali Kratom Powder   Unfolding Nature Unfolding Nature: Being in the Implicate Order   Bridgetown Botanicals CBD Concentrates   Left Coast Kratom Buy Kratom Extract   Original Sensible Seeds Autoflowering Cannabis Seeds   North Spore Bulk Substrate   Mushroom-Hut Substrate Bags


Similar ThreadsPosterViewsRepliesLast post
* Autoclave pics
( 1 2 3 all )
crazyredhead 9,817 47 01/13/03 11:27 AM
by Anno
* anyone seen good prices on small autoclave or retorts? Imorph 1,837 3 01/16/04 11:22 PM
by Imorph
* gourmets easier for the newbz????
( 1 2 all )
sBUD 8,811 26 06/21/17 07:46 AM
by Kevs0923
* The Most Delicious Gourmet Mushroom
( 1 2 all )
bluepie 4,297 20 07/08/20 03:02 PM
by BiGF0007
* Gourmet and Medicinal Mushrooms DVDs thallus 1,985 9 12/18/06 12:07 PM
by thallus
* Gourmet Mushrooms Business? f8L 6,448 8 07/23/16 11:48 PM
by micelio
* What about opening a subsection ONLY 4 MYCELIUM CULTURES PICS into gourmet section?Say your opinion FreeSporePrints 1,893 6 09/03/06 08:07 AM
by falcon
* Opinions on the best gourmet mushrooms azcrash 2,024 4 06/30/04 08:04 AM
by Aneglakya

Extra information
You cannot start new topics / You cannot reply to topics
HTML is disabled / BBCode is enabled
Moderator: RogerRabbit, Pastywhyte, Forrester, Stromrider, SHROOMSISAY01
885 topic views. 2 members, 8 guests and 2 web crawlers are browsing this forum.
[ Show Images Only | Sort by Score | Print Topic ]
Search this thread:

Copyright 1997-2024 Mind Media. Some rights reserved.

Generated in 0.043 seconds spending 0.005 seconds on 14 queries.