Your Guide To TTVBI

🍄 How to Prepare Malt Extract Agar (MEA) for Mushroom Cultivation

✅ What You’ll Need:

Ingredients (Standard 1 Liter Batch – Makes ~35–40 Petri dishes):

• Malt Extract: 20 grams
• Agar-Agar Powder: 15 grams
• Distilled Water: 1000 mL
• (Optional: Peptone 1–2g or yeast extract for additional nutrients)

🥣 Step 1: Mixing the Ingredients

1. Measure out 20g of malt extract and 15g of agar powder.
2. Add both to a clean 1-liter Erlenmeyer flask or heat-safe container.
3. Pour in 1 liter of distilled water.
4. Stir well until all solids are dissolved. Use a magnetic stirrer or manual stirring. The solution may be cloudy.

🔍 Tip: If you're using powdered malt extract, mix slowly to avoid clumping.

🔥 Step 2: Sterilization

1. Cover the flask with aluminum foil or a loose cap — not airtight.
2. Place it in a pressure cooker or autoclave.
3. Sterilize at 15 PSI (121°C / 250°F) for 20–30 minutes.

⚠️ Do not exceed 30 minutes to avoid caramelizing the sugars in the malt, which may inhibit fungal growth.

❄️ Step 3: Cooling

1. Allow the sterilized MEA to cool to around 50–55°C (122–131°F). It should still be liquid but not too hot to kill spores if pouring into inoculated media.
2. If using a thermometer, monitor closely. This is the ideal pouring temperature.

🧊 Do not wait too long — it will start to solidify around 40°C (104°F).

🧪 Step 4: Pouring Plates

1. Sanitize your work area thoroughly or use a laminar flow hood/still air box.
2. Pour ~20 mL of agar into each sterile Petri dish.
3. Slightly crack the lids and let them sit for 15–30 minutes until solid.
4. Once solidified, close the lids and allow the plates to fully dry for 24 hours before sealing.
5. Optional: Store plates upside down to prevent condensation from dripping onto the surface.

🧼 Label your plates with date and medium type.

📦 Storage Instructions

• Store sealed plates in ziplock bags or parafilm-wrapped stacks
• Keep refrigerated at 2–8°C (35–46°F)
• Plates are viable for up to 4–6 weeks if properly stored

🌱 Optional Additions & Notes

• Antibiotics (e.g., gentamicin) can be added after cooling (below 50°C) to prevent bacterial contamination
• Activated charcoal (0.5g/L) can be added to promote some fungal species
• Color indicators can be added for pH sensitivity, though not usually necessary for mushroom work

🍄 How to Prepare Liquid Culture (LC) Jars for Mushroom Mycelium

Liquid culture is a sterile sugar-water solution used to grow mycelium for easy inoculation of grain, agar, or other substrates. It enables fast, clean, and efficient propagation of fungal cultures.

✅ What You’ll Need

Ingredients (Standard 500 mL Batch):

• Light Malt Extract (LME): 10 grams
  (Alternative: Karo Light Corn Syrup – 4% concentration or 20mL per 500mL water)
• Distilled Water: 500 mL

🥣 Step 1: Prepare the Liquid Culture Mix

1. Add 10 grams of light malt extract (LME) to 500 mL of distilled water in your jar.
2. • If using Karo syrup, add 20 mL per 500 mL water.
2. Mix thoroughly to dissolve all sugars. Use a stir rod or magnetic stirrer for even mixing.
3. Add a magnetic stir bar to the jar if you plan to use a stir plate later (optional but recommended).

🔍 Tip: Use clear jars to visually monitor mycelium growth and contamination.

🧴 Step 2: Jar Setup & Lid Prep

1. Use a lid fitted with:
2. • Self-healing injection port (SHIP) for inoculation
   • Micropore filter or synthetic filter disc for gas exchange
2. Tighten the lid lightly to allow some venting during sterilization.
3. Cover the top of the jar with aluminum foil to prevent condensation or water from the pressure cooker entering the lid.

🔥 Step 3: Sterilization

1. Place your LC jar into a pressure cooker or autoclave.
2. Sterilize at 15 PSI (121°C / 250°F) for 20–25 minutes.
3. • Longer sterilization can caramelize sugars and darken the solution.

⚠️ Always keep jars upright and avoid overfilling the pressure cooker with water. Use a rack or trivet inside.

❄️ Step 4: Cooling

1. Let the jar cool fully to room temperature before use (may take several hours).
2. Do not inoculate while warm — it will kill your spores or culture.

💉 Step 5: Inoculation (In Sterile Conditions)

1. Use a still air box or laminar flow hood.
2. Sterilize a syringe needle with flame or alcohol.
3. Inject 1–2 mL of spore solution or agar wedge into the LC through the SHIP.
4. Optionally swirl gently or place on a magnetic stir plate for 30 mins daily.

🧬 Step 6: Incubation & Growth

• Incubate at 21–25°C (70–77°F).
• Shake or stir once daily to prevent clumping and increase oxygenation.
• Mycelium should begin showing in 3–7 days, fully colonized in 10–21 days depending on strain and environment.

🧪 Monitoring & Use

• Look for cloudy, web-like mycelium suspended in the liquid.
• Discard if you see:
• • Sediment or grainy debris
  • Smell of rot or sourness
  • Color other than white (green, black, pink = contamination)

Once colonized, the LC can be used to:

• Inoculate grain spawn
• Transfer to agar
• Create additional liquid culture jars

📦 Storage Instructions

• Store at room temperature if in use
• For long-term: refrigerate for up to 2–3 months
• Always flame sterilize the needle before every use

🍄 Grain Spawn Prep & Inoculation Guide

For 8 x 2-Quart Jars (16 Quarts Total)

✅ Ingredients & Materials

Grain Batch (for 16 quarts):

• Whole grains (e.g. rye berries, millet, wheat): 4 kg (~500g per 2-quart jar)
• Distilled or filtered water: ~6–7 liters (for soaking & simmering)
• Gypsum (optional): ~3–4 tablespoons (1 tsp per jar)

🥣 Step 1: Rinse & Soak the Grains

1. Weigh 4 kg of dry grain (500g per 2-quart jar).
2. Rinse grains thoroughly to remove dirt and excess starch.
3. Soak in clean water (6–7 liters) for 12–24 hours.
4. • Add gypsum (3–4 tbsp) if desired.
   • Soaking helps hydrate grains and begin germination (weakens bacterial spores).

🔥 Step 2: Simmer the Grains

1. After soaking, bring grains to a gentle simmer in clean water for 15–20 minutes.
2. Check readiness: grains should be swollen, soft but not split or mushy.
3. Stir occasionally to prevent sticking.

❄️ Step 3: Strain and Dry

1. Pour into a colander or strainer.
2. Let steam dry for 30–45 minutes.
3. • Surface moisture should evaporate.
   • Grains should feel dry on the outside but remain hydrated inside.
   • No pooling or clumping.

🧴 Step 4: Load Jars

1. Load each 2-quart jar 2/3 to 3/4 full with hydrated grains.
2. • Leave room to shake for redistribution.
2. Add glass marbles or stir bar (optional) to aid future shaking.
3. Close with modified lids:
4. • Injection port for LC inoculation
   • Filter for gas exchange
4. Cover lids with foil to protect from condensation during sterilization.

🔥 Step 5: Sterilize the Jars

1. Place jars upright in your pressure cooker.
2. Sterilize at 15 PSI (121°C / 250°F) for 2.5–3 hours.
3. • Use a trivet inside and ensure water level is correct.
   • You may need to run in multiple batches depending on PC size.

⚠️ Let jars cool completely (ideally overnight) before inoculation.

💉 Step 6: Inoculate with Culture

Option A – Liquid Culture (LC):

1. Flame-sterilize your syringe needle.
2. Inject 2–4 mL of LC per jar through the injection port.
3. Gently swirl or shake.

Option B – Agar Wedge:

1. In a sterile box or flow hood, use a flame-sterilized scalpel.
2. Transfer a clean agar wedge to each jar.
3. Close and shake lightly if needed.

🌱 Step 7: Incubation

• Incubate jars at 70–77°F (21–25°C).
• First signs of growth: 3–7 days
• Full colonization: 2–3 weeks

Shake jars once 25–50% colonized to redistribute and accelerate growth.

🧪 Signs of Healthy vs Contaminated Grain

Healthy:

• Even, white mycelial growth
• Earthy mushroom smell

Contaminated:

• Sour, musty, or wet smell
• Colored patches (green, black, orange, pink)
• Slimy or soggy grain

🗑️ Discard contaminated jars immediately to prevent spore spread.

📦 Storage or Use

• Fully colonized jars can be used to inoculate bulk substrate (coir, straw, manure).
• Store colonized jars in the fridge for up to 2 months if sealed tightly.

🔥 Step-by-Step CVG Pasteurization (Bucket Tek):

1. Boil Water:
   Boil ~4 quarts (1 gallon) of water to 190–200°F (88–93°C).
2. Layer Dry Ingredients:
   In a clean 5-gallon bucket, place:
3. • 1 coco coir brick
   • 2 quarts vermiculite
   • 1 cup gypsum (optional)
3. Add Boiling Water:
   Carefully pour the boiling water evenly over the dry ingredients.
4. Stir & Seal:
   Stir thoroughly until the coir starts to break up. Seal the bucket tightly and let it sit for 4–6 hours to pasteurize.
5. Mix & Cool:
   After the soak, open the lid and mix thoroughly again, breaking up clumps. Let it cool to room temperature (important before inoculation).
6. Field Capacity Test:
   Squeeze a handful of substrate:
7. • Should release a few drops of water, not a stream
   • Too wet? Add dry vermiculite
   • Too dry? Add a bit more water

🍄 Inoculation: Mixing Colonized Grain Spawn into CVG

⚙️ What You Need:

• Fully colonized grain spawn (e.g., rye, wheat, millet – shaken once at ~30%)
• Prepared CVG substrate (cooled)
• Clean monotub with liner (optional)
• Rubbing alcohol and gloves
• Face mask or still air box (for cleanliness)

🧼 Clean Workspace:

• Disinfect monotub and tools with 70% isopropyl alcohol
• Wear gloves and mask
• Work in a low-draft, dust-free room

📦 Mixing Procedure:

1. Line the Tub (optional):
   Use a trash bag or liner to reduce side pinning.
2. Break Up the Grain Spawn:
   Gently break up the colonized spawn inside the jar or bag to separate kernels.
3. Layering (optional) or Mix Thoroughly:
4. • Option 1: Mix CVG and spawn thoroughly (most common)
   • Option 2: Use a bottom layer of substrate, mix in spawn, top with a thin layer of plain CVG
4. Ideal Ratios:
5. • 1:1 to 1:3 spawn to substrate ratio (more spawn = faster colonization)
5. Level & Compress Lightly:
   Spread evenly and gently press down the surface to compact slightly.
6. Apply Microclimate Layer (optional):
   Add a ½" casing-like top layer of unspawned CVG for better humidity control.
7. Cover & Incubate:
   Close the tub with the lid or plastic wrap. Incubate at 68–75°F (20–24°C) in a dark or indirect light environment. Wait for full surface colonization (7–14 days typically).

🌱 Next Step: Fruiting

Once the surface is fully colonized and starts showing signs of rhizomorphic mycelium, introduce fresh air exchange and light (i.e., fruiting conditions).

✅ Primordia Triggers – What Conditions to Introduce

To initiate pinning, adjust the following environmental conditions:

🌱 Step-by-Step: Initiating Fruiting (Pinning)

1. Wait for Full Surface Colonization
2. • The top layer of substrate should be entirely white with mycelium.
   • No uncolonized patches should remain.
   • Look for condensation under the lid and hyphal knots (tiny white dots or threads).
2. Introduce Fresh Air Exchange (F.A.E.)
3. • Remove lid and fan the tub lightly 2–3 times a day.
   • OR flip the lid upside down to create a small air gap.
   • Alternatively, open polyfill holes (if using) or loosen micropore tape.
3. Maintain High Humidity
4. • You want small water beads on the substrate surface — this is the “glistening” look.
   • Mist the walls or substrate lightly ONLY if it appears too dry (no mist or beads).
   • Avoid heavy misting or pooling water.
4. Provide Light (12/12 Cycle)
5. • Place under indirect natural light or use a 6500K daylight bulb.
   • Light acts as a directional signal for pin formation, not as a heat source.
5. Keep Surface Conditions Optimal
6. • Surface should stay moist with no pooling, no dry crusts, and visible condensation.
   • Ideal surface = glistening, evenly moist, and colonized.

📆 Timeline: What to Expect

For CVG-Based Monotubs (Post-Setup)

After your spawn and CVG substrate are fully colonized, it's time to manage the most critical stage: fruiting. This guide focuses on how to observe your tub, maintain ideal conditions, identify contamination, and harvest at the perfect time.

🔍 1. Monitor for Full Colonization

Fruiting should only begin after:

• The entire substrate surface is evenly covered in white mycelium
• No bare spots or exposed substrate remain
• No visible contamination is present

🕒 Colonization typically takes 10–14 days. Patience ensures better yields and fewer issues.

💨 2. Initiate Fruiting Conditions

Once colonized:

1. Crack or flip the tub lid to introduce Fresh Air Exchange (FAE)
2. Loosen polyfill or micropore tape to allow passive airflow
3. Introduce 12 hours of 6500K daylight-spectrum light per day
4. Keep room temp between 68–74°F (20–23°C)

💧 3. Maintain Proper Humidity

Fruiting success depends on moisture balance. The substrate surface should have tiny beads of water — not puddles.

✅ Healthy surface signs:

• Micro-droplets on the mycelium ("glistening")
• Slight condensation on tub walls

💦 Adjusting conditions:

• Too dry? Mist lightly from ~18" away. Avoid drenching.
• Too wet? Increase FAE and remove pooling water.

⚠️ Avoid misting directly on forming pins. Mist the walls or above the surface gently.

🧪 4. Watch for Contamination

Even in the fruiting phase, vigilance is essential. Catching contamination early can save your flush.

🚫 Action: If contamination appears, isolate and discard the tub if severe. Minor mold can be salted and contained, but is not recommended for new growers.

🍄 5. Pinning to Maturity: Observation & Timing

Within 5–10 days of fruiting, you’ll see primordia (pins) forming — tiny mushroom heads.

Monitor development:

• Pins grow rapidly over 3–6 days
• Ideal harvest point is just before or as the veil breaks

✂️ 6. Harvesting Mushrooms

✅ When to Harvest:

• Veil is just beginning to tear from the stem (look under the cap)
• Cap edges are still slightly curled downward
• Avoid letting them fully flatten or release spores

🚫 Avoid:

• Overgrown mushrooms (flattened caps, dropped spores)
• Wet, soggy mushrooms from over-humidity

✂️ How to Harvest:

1. Wash hands or wear sterile gloves
2. Twist and pull gently at the base
   OR
   Snip cleanly with scissors or a blade close to the surface
3. Avoid damaging surrounding pins

✅ Harvest the entire flush in one session if possible for uniform second flush development.

🔁 7. Second & Third Flush Management

1. Remove leftover stubs or aborts after harvest
2. Lightly mist the surface if needed
3. Maintain same fruiting conditions
4. Pins should reappear in 3–7 days

Most monotubs produce 2–4 flushes, with diminishing returns each time.

🧼 8. Post-Flush Cleanup

After your final flush:

• Dispose of spent substrate in a sealed bag (compostable if uncontaminated)
• Scrub tub with hot water and dish soap
• Wipe down with 70% isopropyl alcohol or hydrogen peroxide
• Air-dry thoroughly before reuse