Contamination is the most common source of failure in any mushroom lab. Almost invariably, that contamination comes from improper preparation of bulk substrate prior to inoculating it with mushroom culture. So, whether it’s for research or cultivation, properly preparing your fungus substrate is a vital first step.
But there are so many complicating factors: Different substrates (and even different batches of the same substrate) have different compositions, densities, microbial loads, nutrient concentrations, chemical profiles, etc. That said, every bulk substrate will arrive with its own natural load of competing organisms and dormant spores that you need to reliably reduce or eliminate, lest they crowd out your mushrooms.
In order to meet these challenges, it’s extremely important to establish a validated, consistently reproducible mushroom substrate preparation procedure. Having a self-contained, programmable, research-grade laboratory autoclave makes it significantly easier to develop such a procedure.
Options for Mushroom Substrate Preparation: Pasteurization vs. Sterilization
You have many options when preparing your mushroom substrate. These tend to break into two broad categories:
- true sterilization (e.g., steam autoclaving)
- pasteurization (e.g., “traditional” heat-based pasteurization, or “cold pasteurization” methods like lime bath, peroxide bath, or cold-water fermentation)
An important distinction here is that pasteurization seeks only to weaken microorganisms and kill pathogenic bacteria present in the substrate. Pasteurization does not actually remove/kill/deactivate all forms of life present in the substrate (e.g., fungi, bacteria, viruses, spores, unicellular eukaryotic organisms, and so on). Sterilization, on the other hand, aims at complete destruction of all microorganisms and their spores.
Pasteurization is favored by many edible mushroom growers, because it scales easily and demands no specialized equipment. Provided you’re working non-supplemented straw substrate, it’s both effective and forgiving. But supplemented substrates (and especially denser ones, like hardwood sawdust) cannot be prepared using pasteurization. They harbor mold spores that easily survive pasteurization, and will take over the substrate long before most mushrooms have a chance to gain a foothold.
Supplemented substrates—and even plain straw substrate, in some applications—need to be fully sterilized.
Heat-based Substrate Sterilization for Mushroom Cultivation
But not all sterilization methods are created equal. There is an array of laboratory sterilization options, each with its strengths and weaknesses. Mushroom labs almost invariably opt for heat-based sterilization. With respect to microorganisms and macromolecules sheltered in soils and cultivation substrates, heat is much more destructive than other strategies, including gaseous/liquid chemical sterilization (e.g., ethylene oxide or hydrogen peroxide) and gamma radiation.
Steam sterilization is, by far, the most common approach used in mushroom labs. (Some fungus cultivators do use dry heat for their non-straw mushroom substrate, but that’s only in special cases; in general sterilizing substrate with dry heat is counter-productive, as you will then need to rehydrate the substrate to cultivate fungus). While some mushroom cultivators rely on something as simple as a kitchen pressure cooker or bulk atmospheric sterilizer unit, for most lab applications, autoclave-based steam sterilization will prove to be the most cost-effective, consistent, and quick strategy (relative to other solutions). It’s also the simplest to validate (which is important, as undesirable resting spores can be so resilient in mushroom substrate).
Steam Sterilization in the Mushroom Lab
Purpose-built laboratory autoclaves flood a sealed chamber with high-pressure steam under constant monitoring. These programmable units make most lab sterilization tasks extremely easy. For common tasks (like sterilizing glassware or preparing agar), it’s as simple as loading a dishwasher and hitting “START.”
Most research-grade lab autoclaves will sterilize equipment and supplies (scalpels, beakers, etc.) in just 15 to 20 minutes. Growth media preparation is another common task, especially if your autoclave has special liquid processing or “media prep” cycles. With a programmable lab autoclave, preparing agar, bulk nutrient/growth media, or Petri dishes takes between 15 and 45 minutes (depending on volume).
That said, substrate sterilization is trickier, because of the variety of substrates, and because the sterilization time varies wildly (and not necessarily linearly) by substrate volume. For example, hardwood sawdust in small volumes can be sterilized in under 20 minutes, while the same sawdust in blocks or large bags could take upwards of 2.5 hours. Jars of grain spawn can likewise take from 45 minutes to two hours.
Regardless of your sterilization task, every lab needs to develop several substrate sterilization protocols (for the various substrates, sizes, and preparations). Each of these will need to be validated carefully with a quality biological indicator (such as Self-Contained Biological Indicators (SCBI), Mini Self-Contained Biological Indicators (MSCBI), or self-contained spore ampoules).
Proper Mushroom Lab Autoclave Installation
We occasionally hear about cultivators “recycling” the steam from their autoclave and injecting it back into the fruiting room as a cost effective way to maintain the high humidity mushrooms like. While this might be acceptable in smaller mushroom farms and cultivation operations where they strictly handle edible fungus and do no other lab operations, we advise against the practice in any mycological laboratory setting.
First and foremost, as a rule, we advise that labs vent their autoclaves outside of the building, well above ground level, using a heat-resistant pipe not less than 30mm (~1 in) in diameter. There is always the potential that viable spores can be carried a short distance by the water vapor during some portions of the autoclave cycle. Additionally, if you’re regularly using your autoclaves to process potentially hazardous/pathogenic/contaminated waste, it’s wise to both vent outside your building and to plumb a direct connection to the building sanitary sewer drain.
As a practical matter, if you are going to run cycles that include freesteaming or vacuum options, you’ll also want to vent outside the building and directly to the sanitary sewer: these operations can potentially produce large volumes of waste water and steam.
Additionally, “recycling” steam tends to create positive pressure in the fruiting room, which runs the risk of dispersing mushroom spores and contaminants from the fruiting room elsewhere in your lab (including the inoculation and incubation spaces).
Developing Reliable Substrate Sterilization Procedures
Here’s a good starting point to establish your autoclave-based substrate sterilization process:
- Prepare several identical metal trays of substrate, layering 2–4 inches evenly in the tray
- Embed biological indicators (BIs) in the sample, being sure to have one at the center of mass, and several along the length of the tray at various depths
- Process the tray for 30min at 121ºC or 20min at 135ºC
- Incubate your BIs
- If the BIs show no activity (i.e., don’t change color/turbidity after 48 hours of incubation), your procedure is validated
- If the initial sterilization procedure did not inactivate the BIs, repeat with an identical tray of substrate (same style tray, same soil type, same soil depth, same number and placement of spore ampoules), but process it for twice as long as the first. Incubate those BIs. If biological activity is still evident, repeat again processing for three times the original cycle time. (NOTE: Many labs find a single 30 minute cycle sufficient, although 90 minute cycles aren’t unusual.)
There is one way to be sure that your autoclave will perform to your specification: Have it built to your specifications. Every Priorclave is built to your specification, optimized for low-energy/low-water/low-maintenance operation, and is backed by our free lifetime technical support. Contact us now to discuss the challenges your lab needs to meet.