Pasteurization & Sterilization A Complete Guide for Mushroom Cultivators

Sterilization Time & Pressure Reference

Use this table to determine cook times based on your pressure cooker’s operating pressure and the type of substrate you are sterilizing.

Pressure (PSI)	Temp (°F)	Temp (°C)	Liquids	Grain / Substrate
10 PSI	240°F	116°C	30 min	120+ min
15 PSI	250°F	121°C	15-20 min	90-120 min
20 PSI	259°F	126°C	10-15 min	60-90 min

Note: Times start when the cooker reaches the target pressure. Denser loads and larger vessels may need additional time. When in doubt, add time rather than cut it short.

Agar Preparation

Agar plates are used for isolating cultures, storing genetics, and testing for contamination. Because agar is a high-nutrition medium, it must be sterilized in a pressure cooker.

Traditional Pour Method

1. Choose a heat-tolerant vessel — a glass flask, Pyrex pitcher, or liquor bottle works well.
2. Mix agar powder with water and nutrients per your recipe (e.g., malt extract agar, potato dextrose agar).
3. Cover the vessel with foil to prevent contamination after sterilization.
4. Add 1-2 inches of water to the pressure cooker and place the vessel inside on a trivet or rack.
5. Cook at 15 PSI for 20-30 minutes.
6. Turn off heat and allow pressure to equalize naturally — do not force-release.
7. Cool the liquid agar in front of a flow hood or inside a SAB until it reaches 120-140°F (still liquid but cool enough to handle).
8. Pour into sterile petri dishes, filling each about halfway. Work quickly and keep lids partially open only long enough to pour.

No-Pour Technique

The no-pour technique eliminates the riskiest step — pouring hot agar in open air. Instead, you pre-fill containers before sterilization so the agar solidifies in place.

1. Mix your agar medium according to the recipe.
2. Pour the liquid agar into small containers with tight-fitting lids (glass jars, plastic cups, or ketchup cups). Fill to about 1/4 inch depth.
3. Seal containers with lids or foil. If using glass jars, loosen lids slightly to allow pressure equalization during sterilization.
4. Sterilize in the pressure cooker at 15 PSI for 20-30 minutes.
5. Allow the pressure cooker to cool and depressurize naturally.
6. Remove containers carefully and let them cool to room temperature. The agar solidifies as it cools.
7. Inoculate using sterile technique — transfer spores or mycelium onto the agar surface with a sterilized needle, scalpel, or inoculation loop.

Liquid Culture

Liquid culture (LC) is a nutrient broth that supports rapid mycelium growth. A single jar of colonized LC can inoculate dozens of grain bags, making it one of the most efficient ways to expand a culture.

1. Prepare the broth. Common recipes: 4% honey water, light malt extract (10g per liter), or Karo corn syrup (3-4%). Mix with distilled or deionized water in mason jars.
2. Prepare the lids. Each jar lid needs a self-healing injection port (for inoculation with a syringe) and a gas exchange filter (synthetic filter disc or polyfill) for air exchange during colonization.
3. Load the pressure cooker. Place a trivet or steamer basket in the bottom. Add 1-1.5 inches of water. Arrange jars upright — avoid stacking directly on top of each other. Use a secondary rack if you need to stack.
4. Sterilize. Bring to 15 PSI and maintain for 15-20 minutes. Minimize steam release — just enough to hold pressure.
5. Cool naturally. Turn off the heat and let the pressure drop on its own. This can take 30-60 minutes depending on your cooker. Do not force-release.
6. Remove and cool. Use heat-resistant gloves to remove jars. Let them cool to room temperature before inoculating.
7. Inoculate. Using a sterilized syringe, inject spores or mycelium through the self-healing injection port. Work in front of a flow hood or in a SAB.
8. Incubate. Store jars at room temperature (70-75°F). Swirl or shake gently every few days to break up mycelium clumps and encourage even colonization. LC is typically ready in 1-3 weeks.

Fruiting Substrate Pasteurization

Pasteurization is the standard treatment for bulk fruiting substrates — straw, wood chips, coco coir, and unsupplemented sawdust. Because these substrates are low in nutrition, pasteurization kills enough competitors for your mushroom culture to colonize successfully.

1. Hot Water Bath (160-180°F for 1-2 hours)

The most common pasteurization method. Hydrate your substrate first to activate dormant spores (activated spores are less heat-resistant), then hold the temperature in the pasteurization range.

• Submerge substrate in water heated to 160-180°F (71-82°C).
• Maintain temperature for 1-2 hours. Use a thermometer — overheating kills beneficial microbes.
• Drain thoroughly, cool to below 100°F, and inoculate.

2. Steam Pasteurization

Uses steam instead of submersion. Effective for straw and wood chips packed into bags or containers.

• Place substrate in heat-tolerant bags or containers with some ventilation.
• Arrange above a water reservoir in a large pot, barrel, or purpose-built steam chamber.
• Generate steam to bring substrate temperature to 160-180°F and hold for 1-2 hours.
• Cool, drain any condensation, and inoculate.

3. Cold Water Lime Bath (12-24 hours)

A no-heat method that uses high pH to kill contaminants. Effective for straw and wood chips.

• Mix hydrated lime (calcium hydroxide) with cold water at a ratio of 6-20g per gallon. Higher concentrations are more aggressive.
• Submerge substrate in the lime water for 12-24 hours. Weigh it down to keep it fully submerged.
• Drain the lime solution, rinse the substrate well, then drain again to optimal hydration.
• Inoculate once drained.

4. Cold Water Fermentation (5-7 days)

The simplest method — submerge substrate in cold water and let anaerobic fermentation kill aerobic contaminants. Best for straw.

• Pack substrate into a large container or barrel.
• Fill with cold water and weigh the substrate down to keep it fully submerged.
• Let it soak for 5-7 days. The water will become foul-smelling — this is normal.
• Drain, rinse well, and drain again to the appropriate hydration level before inoculating.

5. Boiling Water Incubation (12-24 hours)

A quick, low-effort method that works well for coco coir, straw, and wood chips in smaller batches.

• Add dry substrate to a heat-safe container (a 5-gallon bucket with a lid works well).
• Pour enough boiling water over the substrate to hydrate it to the correct moisture level, or plan to drain excess later.
• Seal the container and wrap it in towels or blankets to retain heat.
• Let it rest for 12-24 hours. The extended heat exposure pasteurizes the substrate as it slowly cools.
• Once cooled below 100°F, drain any excess water and inoculate.

Fruiting Substrate Sterilization

Substrates supplemented with wheat bran, soy hulls, or other nitrogen-rich additives are too nutritious for pasteurization alone — contaminants like Trichoderma will outcompete your mushroom culture on the extra nutrients. These substrates must be sterilized.

Standard Process

1. Prepare your substrate according to the recipe — typically hardwood sawdust with 10-20% wheat bran or soy hulls, hydrated to proper moisture content.
2. Pack into containers. Use autoclavable grow bags (with filter patches), mason jars, or other heat-tolerant vessels. Leave some space — do not pack too tightly.
3. Load the pressure cooker. Arrange bags or jars with gaps between them so steam can circulate freely. Tight packing leads to cold spots and incomplete sterilization.
4. Sterilize at 15 PSI for 90+ minutes. Larger or denser loads may need 2-2.5 hours. Start timing when the cooker reaches full pressure.
5. Cool and depressurize naturally. Do not force-release. Let the cooker cool completely before opening.
6. Inoculate under sterile conditions. Work in front of a flow hood or inside a SAB. Sterilized substrates have zero microbial protection — any contamination introduced during inoculation will grow unchecked.