Agar work
2 to 4 weeks per isolation round
Growing mycelium on nutrient agar in petri dishes, to isolate a clean vigorous culture from a contaminated or mixed one, and to store genetics indefinitely. It is the step that turns cultivation from repeatedly buying syringes into keeping a strain.
What you need
- Light malt extract, agar agar powder
- Petri dishes, 90 mm, sterile or reusable glass
- Pressure cooker
- Scalpel or inoculation loop
- Still air box or laminar flow hood
- Parafilm or micropore tape
Procedure
- 01
Make the medium
Light malt extract agar is the standard general-purpose medium. Mix, then pressure cook in a loosely capped bottle rather than in the dishes.
- Light malt extract
- 20 g per litre
- Agar agar
- 20 g per litre
- Water
- 1 litre
- Sterilise
- 15 psi, 30 minutes
Watch out: Agar settles hard at the bottom of the bottle. Swirl before pouring or the last dishes will not set.
- 02
Pour plates
Pour at around 60 °C inside a still air box. Hotter and condensation floods the lid; cooler and it sets in the neck of the bottle. Let plates set undisturbed, then leave inverted for a day so any condensation clears.
- Pour temperature
- ~60 °C, bottle warm to hold, not hot
- Volume per dish
- 15–20 ml, about 5 mm deep
- Set
- 1 hour, then invert 24 hours
- 03
Inoculate
Transfer spores, a spore-syringe droplet, or a tissue fragment from a healthy mushroom's interior. Tissue cloning preserves an exact genetic individual; spores produce a new genetic mix each time.
- Incubate
- 24–26 °C, dark
- Germination visible
- 2–5 days
- 04
Isolate a sector
A germinated spore plate contains many genetically distinct mycelia competing. Cut a wedge from the leading edge of the fastest, cleanest, most evenly rhizomorphic sector and move it to a fresh plate. Repeat two or three times until growth across the plate is uniform.
- Rounds
- 2–3 transfers
- Cut from
- The leading edge, never the centre
- Wedge size
- 5 mm
Watch out: Cutting from the middle of a colony transfers older, slower mycelium. The edge is the growing front.
- 05
Store
Seal a clean isolated plate with parafilm and refrigerate. Alternatively transfer a wedge to sterile water in a vial for long-term storage, years rather than months.
- Refrigerated plate
- 4 °C, 6–12 months
- Agar in sterile water
- 4 °C, several years
Troubleshooting
| What you see | What it is | What to do |
|---|---|---|
| Plates set cloudy or never set | Agar not dissolved before sterilising, or the ratio was wrong. | Swirl thoroughly and verify by weight rather than by eye. |
| Condensation floods the plate surface | Poured too hot, or dishes stacked while warm. | Invert the plates. Pour cooler next time. |
| Bacterial colonies, shiny wet dots, sometimes yellow | Airborne or from the transfer tool. | Flame the scalpel between every cut and let it cool. Move the still air box away from foot traffic. |
| Mycelium grows as a thin flat sheet with no texture | Tomentose rather than rhizomorphic growth. Often a weaker or older isolate. | Select a different sector. Rhizomorphic, ropey growth generally spawns and fruits better. |
Sources
- [1]Shroomery cultivation archives
- [2]Stamets & Chilton, The Mushroom Cultivator (1983)