Objective

To document, maintain, and develop the facility's genetic library through systematic isolation, hybridization, and selection of mycelial cultures; to prepare and verify the viability and sterility of liquid and agar culture media for mycelial expansion; and to continuously source, isolate, and improve novel and existing mushroom genetics.

Part A: Strain Portfolio & Sourcing

1. Primary Families

The facility cultivates strains from three primary families:

• Cubensis: Includes standard albino strains and all Enigma variations. Enigma is a mutated expression of a Cubensis strain.
• Natalensis: A distinct species, referred to as "Peanut."
• Cyanescens: A distinct family, referred to as "Pancyan."

2. Strain Sourcing and Collaboration

• Source new genetics from a global community of cultivators and via online platforms (e.g., Instagram).
• Focus on developing stable genetics from historically difficult-to-grow strains, such as Enigma.
• Participate in a collaborative community by exchanging shippable cultures to accelerate development.

3. Strain Log Maintenance

Maintain a log of all active strains with their corresponding genetic lineage and known characteristics. Documented strains include:

• Calypso: A hybridization of Mooncakes and the "smurf" variety of TAT (True Albino Teacher).
• Iceberg: An isolation of Albino Thai Lipi Yai.
• Maria Sabina: An Albino Huautla isolation from creator Yoshi Armano.
• Mooncakes: An in-house developed culture.
• Nebula: A stabilized blob mutation of the Maria Sabina strain.
• Omni Blue Wave: A blob mutation of an Omni x Penis Envy.
• Snozzberries: An in-house hybridization of Shakti x Phobos.
• Tattoo: A mutated blob isolation from the strain Gandalf.
• White Lotus: A hybrid of TAT Smurf x Albino Rollercoaster.
• Psilocybe natalensis Low Spore: A sporeless variety from Yoshi Armano.
• Meringue: An in-house enigma strain, isolated from True Albino Teacher: Yeti.
• Chocolate Krinkle Brain: An enigma variation of Chocolate Krinkle.
• White Dwarf: An in-house hybrid of Jack Frost x TAT Yeti.
• Hobbits Feet: An in-house hybrid of TAT Ghost x Jack Frost.
• Panaeolus Cyanescens (BVI Tamarind Tree): Originating from the British Virgin Islands.

Part B: Culture Initiation, Isolation, and Transfer

1. Initiation (T0/T1)

• From Spores: Transfer the 3 best (furthest growing, most rhizomorphic) and cleanest swabs to one large agar plate.
• From Clones: Transfer 2-3 separate clones to one plate, using tiny pieces of tissue. Cloning should be done from the part of the mushroom closest to the desired phenotype.

2. Isolation Transfers (T2-T6)

• From the T1 plate, select the best sections (clean, strong, rhizomorphic).
• Perform a 3-to-1 transfer (3 sections to 1 new plate) onto mini plates.
• Continue this process until transfer T5 or T6, with the goal of achieving a monoculture.

3. Master Plate Creation (T5 & T6)

• T5: Create 2 large master plates from the best T4 plate.
• T6: From the best T5 plate, create 2 new large master plates and 1 slant.

Part C: Liquid Culture (LC) Formulation & Quality Control

1. Formulation

• Standard LC (Per 1-Quart Jar): 600 ml boiling distilled water with 12 g honey or 15 g liquid malt extract.
• Semi-Solid LC (SSLC): To 600 ml boiling hot distilled water, add 12 g honey or 15 g liquid malt extract, 0.6 g peptone, and 1.2-1.3 g agar powder.

2. Quality Control (QC)

• Visual Inspection: Mycelium should form a single "ball," and the liquid should be clear.
• Streak Testing: Before large-scale use, dispense 3-8 drops onto a QC agar plate and incubate for 5-10 days to monitor for contamination.

Part D: Agar Plate Media Preparation

1. Recipes (Per 1 L water)

• Cam's Agar (PHA+): Dry: 25 g agar, 25 g potato starch, 1 g yeast, 1 g peptone. Wet: 14 g honey. Final: 0.15-0.2 g activated charcoal.
• Standard MEA: 25 g agar, 20 g malt extract powder, 2 g nutritional yeast powder.
• Standard PDA: 25 g agar, 25 g potato starch, 14 g dextrose, 2 g nutritional yeast powder.
• Distilled Water Agar: 1 L distilled water, 25 g agar powder.

Objective

To stabilize desirable genetic traits, maintain viability, and prevent genetic deterioration over successive generations; to create pure, isolated monocultures; and to combine genetics from spores and clones to maximize genetic variation.

Part A: Genetic Stabilization (Backcrossing)

After several generations, a culture may deteriorate. To revitalize the strain, cross the current generation back with an archived first-generation clone or spores from a desirable fruit.

Method: Take a spore sample (swab) and a tissue clone from the same desirable fruit. After isolating the clone on agar, introduce spores from the swab onto the same plate. Allow the two to grow and fuse, then select the strongest resulting growth.

Part B: Genetic Diversification (V-Series)

• V1 Plate: Prepare separate plates for spores (2 swabs/plate) and clones (2 clones/plate).
• V2 Plate: Transfer the 2 strongest sections from the swab plate and the best section from the clone plate onto a single new plate.
• V3 (Inoculation): Transfer the V2 plate culture to a grain jar to create a G1 bag.
• V4 (Grain Isolation): Once V3 is colonized, place 3 colonized grains back onto a new agar plate to create a grain isolation master ("gi").
• V5 (Master Creation): Transfer 3 sections from the V4 plate to one new master plate and one new grain jar.
• Test Fruiting: Use the V5 grain jar for test fruiting. If successful, make a new V6 master plate.

Part C: Distilled Water Agar (DWA) Preparation

To prepare DWA for culture purification.

Objective

To safely store master cultures for long-term use and disaster recovery; to formally document and secure rights to internally developed hybrid strains.

Part A: Genetic Verification and Intellectual Property

1. For new, proprietary hybrid strains, submit samples for PCR and CRISPR DNA sequencing.
2. The purpose of sequencing is to prove the strain is genetically distinct from parent strains and to establish a unique DNA code.
3. Compare the new sequence against a database to verify its genetic uniqueness. This data is used to secure legal rights to the strain's DNA.

Part B: Culture Archiving and Storage

1. Master Plates: For every finalized phenotype, prepare two master plates. These are working copies and should not be used for production if older than three months.
2. Slants (Medium-Term Storage): From the best master plate, prepare one agar slant. Slants are stored in a refrigerated environment.
   [CONFLICTING DATA]: Viability is stated as 1-2 years [1729] and approximately 5 years [105].
3. Long-Term Archival: For long-term archival, create a backup via cryogenic storage or in distilled water.
   [CONFLICTING DATA]: Viability for long-term methods is stated as 20+ years [1732] and 20-40+ years [106].

Objective

To create redundant, long-term backups of all finalized master cultures.

Procedure

1. Once a culture is finalized and proven commercially viable, create two forms of backup: agar slants (medium-term) and cryogenic storage (long-term).
2. Redundancy: Store a primary set of backups on-site. Store a secondary, duplicate set of all backups in a secure safe at an off-site location.

Objective

To accurately mix, hydrate, and prepare bulk substrate media prior to sterilization, efficiently producing hydrated substrate using a two-person team.

Part A: Standard Coco Coir Substrate (83% Moisture)

1. Materials (9-Brick Batch):

• Coco Coir: 9 bricks (46.962 kg)
• Vermiculite: 11.25 kg
• Gypsum (Diamond K): 4.95 kg
• Azomite: 0.64 kg
• Water: 326.2 L total

2. Procedure:

1. Coco Pre-Hydration: Divide 9 coco coir bricks between large bins. Add 195.7 L of hot water. Allow to soak for 1-2 hours.
2. Mixing (Next Day): Load pre-hydrated coco into the ribbon mixer. Add dry ingredients (vermiculite, gypsum, Azomite). Incrementally add the remaining 130.5 L of water while mixing.

Part C: General Substrate Mixing (Twin Caps Method)

1. Formulations:

• Batch 1 (240 x 3 lb bags): 11 bags Coco Coir, 1 bag Vermiculite (20% of mix), 3 kg Gypsum, 105-111 L Water. Target Bag Weight: 1050-1150 g.
• Batch 2 (300 x 3 lb bags): 14 bags Coco Coir, 1 bag Vermiculite (15% of mix), 3 kg Gypsum, 131-140 L Water. Target Bag Weight: 1600-1700 g.

Part E: Quality Control and Adjustments

Field Capacity Verification: The substrate is at "field capacity" when fully saturated but does not drip when compressed. A quick, light squeeze should yield 1-2 drops. A long, firm squeeze should produce a steady drip.