Autoclave Validation and Load/Cycle VerificationFirst, 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 Process1. 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 Process1. 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 Process1. 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 VerificationPhase 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;
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:The 60 min cycle passed verification by biological indicator:

I will not be reducing the 60 min cycle for the Presto or IP as I believe 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.
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 was set for 3.5 hours at 15 psi 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 ValidationAutoclaves 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 10
6 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.
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.
Phase 3:
I will attempt to load the IP with a 4lb, 3 quart spawn bag; bag will be a 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:
Pass: The bags seal on cool down, it failed to vacuum seal completely before opening the lid, perhaps more time was required to seal.
Conclusions:

On the left you'll notice 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 there are other factors to consider when problems arise post cycle. It appears as though our pressure cookers easily penetrate the common loads to thoroughly defeat thermophilic endospores which means if we have 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 the OMC has made a myth and a legend out of bacterial endospores, we've given 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 do not address are 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's much more to spawn production than what 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 organic compounds produced by the very organisms we're destroying during the cycle, 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 with proper autoclaves or pressure cookers that are capable of reaching and maintaining the minimum threshold of 121°C 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, bacteria, though omnipresent, should be manageable.
So yeah, there's likely to be other variables that haven't been factored into our sterilization process which 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,and/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 gas. 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 gases. When you place the lid on any pc you are now encapsulating that gas.
Venting is required to purge this trapped gas 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.