The biggest mistake in sterilization isn’t temperature. It’s assuming steam went where you think it did.
Weeks after sterilization, a batch starts showing bacterial contamination. Immediately the investigation begins.
Was the spawn contaminated? Did somebody make a mistake during inoculation? Did the bags leak?
But sometimes none of those things happened.
Sometimes the contamination survived the sterilization itself. Not because the autoclave failed. But because steam never reached the place where the microorganisms were hiding.
That sounds impossible. After all, the autoclave displayed 121°C. So how could anything survive?
Because sterilization is not simply about reaching a temperature. It is about delivering heat to every part of the substrate. And those are not the same thing.
Temperature doesn't kill microorganisms
This surprises many people.
The display on your autoclave tells you the temperature in the vessel. It does not automatically tell you what happened inside every substrate bag.
Microorganisms die because they receive enough lethal energy.
That energy reaches them through condensing steam, not through hot air alone. Steam releases most of its heat when it condenses onto cooler surfaces. If condensation cannot occur where it should, heat transfer becomes inefficient even if the chamber temperature appears correct.
Think of steam as a delivery system.
The temperature is only the address.
Steam still has to arrive.
The bag is changing while you sterilize it
Most people imagine substrate as something passive. It isn’t. Even before sterilization starts, microorganisms inside the substrate are still alive. They consume oxygen. They produce carbon dioxide.
As temperature rises, chemical reactions release even more CO₂. Meanwhile pressure inside the bag changes dramatically. Cold air becomes compressed. Steam enters through the filter. CO₂ is being released. Everything is moving simultaneously.
Venting therefore isn’t simply “letting steam out.”
It is allowing all those gases to leave so steam can replace them. The Substrate & Wood Fungi Production Course describes how microbial respiration produces CO₂ before sterilization and why heating above about 90°C releases additional CO₂, making proper venting considerably more complex.
Bigger production makes everything harder
This becomes even more important as production grows.
A larger load means:
- more substrate to heat
- longer heat penetration
- more trapped air
- greater differences between bags
- increased risk of cold spots
Two autoclaves can both reach 121°C. Only one may deliver an effective sterilization throughout the load.
Load arrangement, substrate density and accurate temperature measurement inside the substrate all influence whether sterilization is truly successful. This is one of the reasons why recipes that work beautifully on a small scale often become unreliable after expansion.
Not because biology changed.
Because the physics changed.
Sterilization continues after the heat is off.
Keeping substrates sterile after sterilization.
Another misconception:
People often think sterilization ends when heating stops. In reality, cooling introduces a new biological risk.
As steam condenses, pressure inside the autoclave drops. Without the replacement of steam by sterile air, non-sterile air can be drawn back into the bags.
A perfectly sterilized substrate can therefore become contaminated during cooling if the process isn’t properly managed. This is one of the reasons why, in our Substrate & Wood Fungi Production Course, we don’t focus only on reaching sterilization temperature. We spend considerable time understanding the complete sterilization cycle – including cooling – and the biological principles behind each step.
The sterilization cycle isn’t only about heating: It is about heating, venting, holding and cooling.
Validation instead of assumptions
Many producers still evaluate sterilization with a simple question: “Did we reach 121°C?”
Experienced producers ask different questions.
- Where was the coldest point?
- Was venting complete?
- How was the load arranged? How were the substrates positioned?
- Did steam reach every bag?
- Was the process validated inside the substrate, not only inside the autoclave vessel?
That is one of the fundamental differences between reacting to contamination and preventing it.
The experienced producer asks these questions before production starts – not after contamination appears.
What we explore
Autoclaving receives an entire theory session and practical workshop during the Substrate & Wood Fungi Production Course, where participants learn what actually happens inside substrate bags during sterilization. Beyond operating an autoclave, youexamine steam production, heat transfer, venting, the F-value concept, validation, common mistakes and the biological consequences of poor sterilization, followed by practical work on autoclaves and steam generators.
