The incubation of substrates for mushroom cultivation

What is incubation?

The incubation of mushroom substrates corresponds to the vegetative phase during which the mycelium explores and digests the substrate, before fruiting and mushroom formation. Specifically, after inoculation, the white filaments invade the particles of straw, grain spawn, or sawdust to create a dense and homogeneous network.

This step takes place in a controlled environment: moderate temperature (22 – 26 °C), internal bag humidity, low light, and limited gas exchange. The goal is simple: achieve complete colonization, leaving no chance for contaminations (Trichoderma, bacteria). Poorly controlled incubation lengthens the cultivation cycle and reduces yield.

• Average duration: 10 days (oyster mushroom) to 60 days (shiitake on wood).
• Success indicator: uniform white, bag fully tight around the substrate.
• Key parameter: internal temperature < 28 °C to avoid bacterial “cooking.”
• Risks: overheating, contaminations, substrate under- or over-hydrated.

Practically, 90% of a culture's success is decided here: a well-colonized substrate ensures speed, health, and quality of harvests — and thus a better final yield. Understanding the basics (mycelial respiration, CO₂ management, thermal thresholds) gives you an edge, whether you cultivate fast oyster mushrooms or slower shiitakes.

⚗️ Grower's tip: Check and record the internal temperature of the bags daily; a simple spike of +3 °C often signals intense mycelial activity… or the start of contamination. Meat probe thermometers are inexpensive and can perfectly suit this purpose.

Frequently asked question 1: why is my grain spawn not colonizing?

Often, the incubation temperature is too low or the substrate lacks oxygen. Check that the room stays between 22 °C and 26 °C, gently shake the bag to restart aeration, then wait 48 hours before taking action.

 

Frequently asked question 2: should incubation be done in the dark?

Not necessarily; diffuse light (≤ 200 lux) does not affect colonization, but darkness lowers temperature and limits some contaminations.

Now, let's look in detail at the optimal incubation conditions essential for your success.

 

2. The 4 optimal incubation conditions

A successful substrate incubation for mushroom cultivation relies on four key parameters: temperature, internal humidity, darkness/diffuse light, and gas exchange. Well managed, they speed up colonization, reduce contamination risks like Trichoderma, and ensure better yield.

Make sure to measure the incubation temperature at the core of the bags, not just in the room; a 2 °C difference is enough to slow down the grain spawn. Maintain a "closed" humidity (non-perforated bag) and allow just enough fresh air to avoid CO₂ buildup.

2.1 Ideal temperature

• Oyster mushroom: 22 – 24 °C (fastest colonization).
• Shiitake: 24 °C then "rest" at 20 °C after 60% colonization.
• It is better to have temperatures a bit too cool than too high. Avoid exceeding 25°C.
• Absolute max: 28 °C — beyond this, bacterial flora explodes and growth is significantly impaired.

Note that substrates rich in sugars, and heavy grain spawn colonization will encourage heat production by the mycelium. This heat encourages the metabolism of surrounding mycelia, which create even more heat. There is thus a risk of a spiral that can quickly raise your room temperature to levels too high for your mycelia. 

 

2.2 Humidity

The closed bag creates its own microclimate. If you see internal droplets, there is a thermal gradient: move the bags closer to a warmer area or add a gentle fan. 

Condensation inside the bags can indicate the presence of bacteria in the substrate, which also generate a lot of heat. It can also be due to too large temperature variations in the room. That said, it is very common to have small condensations in the bags, it is normal and not necessarily a cause for concern. Over time, you will be able to detect what is normal and what indicates a problem.

 

2.3 Darkness & light

Most species tolerate up to 200 lux. According to various studies, low and dim light during incubation would be beneficial for shiitakes, allowing them to properly develop their "pop-corning" and "browning" phase.

For oyster mushrooms, light triggers the appearance of primordia, so complete darkness is recommended.

 

2.4 Gas exchange

The mycelium breathes: provide a 0.5 µm filter or hydrophilic cotton. Clogged filters cause a CO₂ high, stopping colonization. Depending on the amount of substrate in your room, it may be necessary to add air renewal. If possible, filter the incoming air and put your room under positive pressure.

Frequently asked question: can I stack my incubation bags?

No, it is not recommended. Heat and CO₂ trapped in the center slow down colonization and promote molds.

Now focus on contamination prevention to secure this sensitive phase.

 

3. Hygiene & contamination prevention: 

The incubation phase rarely takes place in a cleanroom, but a clean and stable environment remains essential. Dust, competing spores, and bacteria love the warmth and moisture of the substrate; without minimal hygiene, they appear even before complete colonization.

A good protocol limits air turbulence, reduces handling, and enforces regular visual inspection (1-2 times/week). Maintain a “clean zone”: surface washed with isopropyl alcohol, gloved hands, and bags sealed immediately after inoculation. Thus, incubation remains dominated by the mycelium, not by Trichoderma or filamentous bacteria.

Color	Most common name	Probable cause
Bright green	Trichoderma	Excess moisture, poorly sterilized grain
Pink / orange	Bacteria (Bacillus)	Too high temperature, clogged filter
Black / blue	Competing molds	Airborne dust, poorly sealed bag

• Cleaning : work surface + walls disinfected with 70% IPA.
• Gentle ventilation : laminar flow or calm room, without sudden drafts.
• Observation : note color, smell, pattern of each anomaly.
• Quick reaction : isolate or discard any suspicious bag to protect the batch.

Photo showing a rather aesthetic trichoderma contamination.

 

4. Monitoring of incubation

During incubation, visual vigilance remains your best tool: healthy mycelium forms a uniform white fuzz, without gray halos or green spots. Check each bag every two days; note the speed at which the white "eats" the substrate — it is a direct indicator of vitality and future yield.

Get a permanent marker: draw a progress line on the bag wall to assess advancement. Coupled with a quick weighing (1 to 3% water loss accepted), this objective method reveals any stagnation before it becomes critical.

Day	Visual state	Action
D0	Sterile substrate	Inoculation
D3	Whiteness < 25 %	Optional: shake to distribute the grain spawn
D6	Whiteness 50 %	None – OK
D9	Whiteness 75 %	None – OK
J12	Whiteness 100 %	Ready for “rest” or fruiting

• Stagnation index: no visible progress ≥ 48 h.
• Reflexes: check internal temperature, substrate moisture, CO₂ filtering.
• Logbook: date, degree of colonization, odor/color observations.

Frequently asked question: my mycelium stops at 80% – what to do?

Increase air renewal (briefly open the bag), lower ambient temperature by 1–2 °C, and gently massage the substrate to redistribute moisture.

 

5. Specifics according to the substrate

Incubation varies greatly depending on the nature of the substrate. Each matrix has its own pace of colonization, its risk level, and its impact on future yield. Adapting your parameters (incubation temperature, humidity, filling density) to each substrate avoids many surprises.

Substrate	Colonization speed	Contamination risk	Key advantage
Rye grains	⚡ Very fast (3–7 d)	High — nutrient rich	Excellent grain spawn expansion; ideal for duplicating
Enriched sawdust	🐢 Slower (15–30 d)	Medium	Optimal nutrition for abundant fruiting
Pasteurized straw	Fast (7–12 days)	Low if well pasteurized	Low cost, perfect for oyster mushrooms
Logs / raw wood	Very slow (3–12 months)	Low — outdoor environment	Multi-year production, authentic aroma

 

• Grains : monitor moisture; too wet ⇒ bacteria.
• Sawdust : compress slightly to limit air pockets.
• Straw : inoculate as soon as cooled after pasteurization.
• Logs : seal inoculation holes with wax to prevent drying out.

Frequently asked question: which substrate to choose to start?

Pasteurized straw is ideal for oyster mushroom cultivation: inexpensive, quick to colonize, and tolerant to temperature variations. Mycosphere also offers certified organic straw pellets, which are very easy to use and less prone to contamination.

 

6. Common mistakes to avoid

Even experienced growers sometimes make mistakes that sabotage substrate incubation. Knowing these pitfalls protects your colonization and your future yield.

• Stacking bags excessively : metabolic heat → +4 °C in the center → contamination.
• Non-uniform incubation temperature: gradients > 2 °C = slow grain spawn, fast bacteria.
• Clogged filter: CO₂ stagnates, grain spawn exhausts and yellows. An overly wet substrate can clog a filter, which then becomes a source of bacterial contamination.
• Initial under-hydration: substrate too dry → incomplete colonization.
• Starting fruiting too early: uncolonized areas attract Trichoderma.

Keep in mind that every mistake slows down incubation by at least 24 h, or even dooms the entire batch in case of massive contamination.

Frequently asked question: can I scrape off the green contaminant to save my bag?

No: Trichoderma spores quickly. Remove the entire bag to avoid spreading, disinfect the area, then check the temperature and humidity of neighboring bags.

Now that the traps are known, let's move on to the transition to fruiting to harvest your first mushrooms with complete peace of mind.

 

7. Transition to fruiting

When incubation reaches 100% uniform whiteness — and only then — the grain spawn is ready to change phase. A bag still spotted or showing inert areas stops future production cold: wait for complete colonization to preserve your yield.

• Visual cue: surface entirely white, substrate has become compact and solid.
• Smell: fresh mushroom note, no sour or ammonia scent.
• Sometimes: appearance of primordia (oyster mushrooms) — a sign that the grain spawn demands fresh air.

Depending on the species, triggering fruiting requires a "shock":

Species	Thermal shock	Mechanical shock	"Rest" duration
Oyster mushroom	Thermal shock: 15 °C / 12 h (depending on the strain)	Open the bag + fresh air	0 d
Shiitake	4 °C (24 h) ➔ 18 °C	Hammer or tap the block	7 d
Reishi	-	Cut the top of the bag	0 d

Frequently asked question: should you always do a “shock”?

No: some species (pink oyster, enoki) fruit as soon as exposed to air. Check the strain's datasheet before any change.

Ready? Open your bags, adjust light and humidity, and get ready to see your first mushrooms emerge!

Conclusion

The incubation phase is the silent stage that determines 90% of a culture's success. Maintaining temperature, humidity, and gas exchange at the right level allows for a rapid, healthy, and homogeneous colonization; everything else – yield, quality, harvest duration – naturally follows.

• Monitor each bag; the white must cover the entire substrate.
• Record your parameters (°C, % RH, time) to reproduce them.
• Act quickly at the first sign of contamination – better to discard one bag than sacrifice the entire batch.

Ready to take action?

Save time with our ready-to-grow substrates: already incubated under optimal conditions, they start fruiting in a few days. If you prefer to prepare your own supports, our highly vigorous grain spawn will ensure rapid colonization.