What is inoculation?

Clean inoculation matters because the mushroom's mycelium has to get established before anything else can take hold, and that's hardest on rich substrates. Cornell's Small Farms Program says straw, wood chips and logs are fine to inoculate in open air with a clean table and clean hands. "Sterility becomes much more critical" with agar, grain and supplemented sawdust, which are easier for ambient microorganisms to grow on and need a lab-like setting (Cornell). At home, that means a still air box or a flow hood.

Each stage of a grow uses a different inoculant and method.

You inoculate	With	Method
Sterilized grain	Liquid culture	Once the grain has cooled to room temperature, wipe the lid with alcohol (GroCycle) and inject 1–2 mL per quart jar (GroCycle)
Sterilized grain	Agar	Cut actively growing culture into small cubes; UF/IFAS uses a 60 mm plate the culture has "just filled" for 400–450 g of grain (UF/IFAS)
Straw or supplemented sawdust	Grain spawn	Break it up and mix it through; see spawn to bulk
Logs	Sawdust or plug spawn	At least 30–40 holes per log, 7/16 inch wide and 1.25 inches deep for sawdust spawn, staggered in a diamond pattern and sealed with hot food-grade wax (Cornell)

After inoculating grain, UF/IFAS shakes it so growth starts "from multiple points", then incubates it at 73°F (23°C) in darkness (UF/IFAS). Logs should be inoculated between one day and three weeks after the tree is felled; the fewer days, the greater the production per log (Cornell).

Contamination that starts at the injection point usually traces back to the inoculation. Rootlab, which sells grain bags, says contamination that seems to start at the site of inoculation likely means the syringe was contaminated or sterile technique slipped (Rootlab). Our grow log guide covers telling a substrate problem from a technique problem, and our contamination ID guide shows what each mold looks like.

• Hot grain. GroCycle lets sterilized jars cool in the cooker for 8 hours or overnight before inoculating (GroCycle).
• A hot needle or too much liquid. Flame a needle outside any still air box and away from wet alcohol, as our liquid culture syringes guide explains, then let it cool before it goes in. Don't empty a syringe into one bag: Rootlab, which sells grain bags, warns that extra liquid sits at the bottom, where bacteria thrive (Rootlab).
• An untested culture. Put a drop on agar first; our liquid culture syringes guide covers the test.
• Old logs. Cornell says not to wait longer than three weeks after felling, or the log is more likely to dry out or pick up competing fungi; trees felled in winter are the exception (Cornell).

In Hypha, create each jar as a transfer from its culture, and compare each strain's contamination rate to see whether failures cluster in one strain.