Agar work

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

1. 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.

2. 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

3. 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. For spores, use a clean print kept covered and dry, flame the tool and let it cool, touch it to the print so it picks up only a few spores, and streak lightly. Open the plate only as far as the transfer needs, then close, label with date and source, and incubate upside down. Watch daily for the first week: small white points or fuzzy patches are germination, while fast fluffy growth that changes colour or glossy wet spots are contamination, not your culture.

   Incubate

   24–26 °C, dark

   Germination visible

   2–5 days

   Spores per transfer

   A few, not a smear

   Watch out: Too many spores crowd the plate and the sectors cannot be told apart. A hot tool kills the spores, so touch it to clean agar first if unsure.

4. 04

   Isolate a sector

   A germinated spore plate contains many genetically distinct mycelia competing. A basidiospore germinates into a monokaryon, which does not fruit on its own; compatible monokaryons must fuse into a dikaryon before the culture can make mushrooms. So the plate is a mixture that varies in speed, texture and vigour, and some sectors will grow poorly or fail to fruit. 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.

5. 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