Clean work, agar and liquid culture

Mould spores and bacteria are on every surface, in the air and on your skin. You cannot get rid of them, only keep them out of the moments when sterile food is open. That means still air, clean surfaces and hands, quick work, and never opening anything you do not need to. Nothing short of a laminar flow hood is truly sterile, so make a few extra jars to cover losses (Bible, p. 64).

• A large clear tub with two arm holes, used upside down or on its side. Bigger is better than you think (Bible, p. 57, 62).
• Wipe every surface, including the gloves, with a 1:9 bleach to water solution, then wait five to ten minutes for the air to settle (Bible, p. 62–63).
• Do not spray alcohol inside a closed box and then light a flame. That is a fire risk; dilute bleach is safe near a flame (Bible, p. 62).
• Move slowly inside the box. Fast movements stir up air.

• Shower or wash arms to the elbow, wear clean clothes, and use gloves wiped with alcohol.
• Flame needles until they glow, then let them cool for a moment before they touch culture.
• Wipe injection ports and jar tops before and after.
• Let sterilized jars cool fully before opening the cooker, and open it only when ready to use them (Bible, p. 91).
• Label everything with what it is and the date.
• Keep jars and plates at least 2–3 feet off the floor, ideally chest height; floor air carries the most spores (Bible, p. 247).
• Sterilize one jar you do not inoculate. If it stays clean while the others fail, the problem is your technique or your culture, not the cooker (Bible, p. 260).

Liquid cultureLCLiquid culture: a sterilized sugar-water broth with living mycelium suspended in it, injected like a spore syringe but faster to take. is a sterilized sugar broth with living mycelium growing in it. It colonizes grain much faster than spores because the mycelium is already alive; injected spores take several days just to germinate (Bible, p. 125).

Broth

About 4% sugar by weight: 12 g honey in 288 g water for a 16 oz jar (Bible, p. 133–134)

Sterilizing

About 15 minutes at 15 psi; longer caramelizes the sugar, turning it amber and slowing growth (Bible, p. 133)

Inoculating

1–2 ml of spore solution per culture jar; 1–2 ml of culture per grain or PF jar (Bible, p. 136)

Needle

Wide enough not to clog with mycelium, roughly 14–21 gauge (Bible, p. 136)

Ready

White cloudy clumps that thicken over days; fully grown in about 2 weeks to a month (Bible, p. 136–138)

A healthy culture is clear broth with white clouds in it; stirring once a day breaks the clouds into more growth points. Dark spots that grow, a murky or odd-colored broth, or a sour smell mean throw it out (Bible, p. 141). Because early green mould looks white too, test a new culture on one PF jar or a small bag of grain before trusting it with a whole batch (Bible, p. 140).

AgarAgarA firm gel in a petri dish that mycelium grows across, used to check a culture is clean and to pick strong, fast sectors before they go to grain. plates let you see contamination before it reaches grain. Spores, a drop of culture or a piece of tissue go onto a plate; clean, fast-growing mycelium is cut from the leading edge and moved to fresh plates until only clean growth is left. That clean wedge then starts liquid culture or grain (Bible, p. 234–241).

• A simple batch: about 12 g of potato dextrose agar powder in 200 ml water, enough for up to 30 thin plates (Bible, p. 219).
• Seal plates and keep them a few days before use; any that grow something on their own were not clean (Bible, p. 221, 224).
• Pick stringy, fast, branching rhizomorphicRhizomorphicMycelium that grows in branching, rope-like strands, as opposed to even, cottony growth. growth over fluffy tomentoseTomentoseEven, cottony mycelium without strands; normal for many cultures and not a sign of contamination. growth when transferring (Bible, p. 239).
• When cloning a mushroom, use a fresh one and tear it open rather than cutting, so the blade does not drag surface spores inside (Bible, p. 226–228).
• Bacteria show as a small round, often yellowish spot beside the mycelium; transfer from the edge farthest from it (Bible, p. 234).