Tutorials/Agar/Three-Transfers

Overview

When working with agar plates, you'll often see them labeled with a T number (T1, T2, T3) and hear the advice to "transfer three times." This page explains what that means, why it works, and how to do it.

The short version: every time you move the best-looking piece of a culture to a fresh plate, you leave behind contamination and weaker genetics. After about three rounds, what's left is usually a single clean, fast-growing culture.

Plates at different transfer stages, each labeled with the strain, its transfer number, and the date.

Why transfer at all

Cleaning up a culture

Spore prints and swabs are never sterile. Spores, bacteria, and stray mold spores all come along for the ride. On agar they all grow, but they grow in different places on the plate. Mycelium outgrows most contamination at its leading edge, so a wedge from that edge is usually cleaner than the plate it came from. Each round leaves more of the passengers behind.

Genetic selection

A plate started from spores holds many genetically different individuals, each from a different spore. They grow side by side and often look visibly different: one sector fast and ropey, another fluffy and slow. These distinct regions are called sectors.

Fast colonization is one of the most valuable traits in any cultivated mushroom. A faster culture:

• Leaves contaminants less time to establish
• Shortens the whole cycle from inoculation to harvest
• Tends to produce stronger, more aggressive mycelium

By always picking from the fastest, healthiest sector, you're selecting for those traits.

Rhizomorphic vs. tomentose

• Rhizomorphic growth is ropey, with thick strands that radiate outward like roots. Growers generally favor it as a sign of vigorous genetics.
• Tomentose growth is fluffy and cottony, without clear strands. It isn't contamination, but it usually grows slower, and many growers avoid selecting it.

Healthy mycelium is normally white. Fluff that turns gray, green, or another color is probably not yours.

The method

T1: the starting plate

Inoculate a plate from your spore syringe, swab, print, or tissue sample. Give it time to grow out. Expect it to look messy: several sectors, possibly some contamination at the inoculation point.

T2: the first transfer

1. Find the healthiest-looking sector: fast, white, ideally rhizomorphic, and as far from any contamination as possible.
2. Working in a still air box or flow hood, flame your scalpel and let it cool.
3. Cut a small wedge, about the size of a grain of rice, from the leading edge of that sector.
4. Place it in the center of a fresh plate, mycelium side down.
5. Label the new plate with the strain, T2, and the date.

T3: the second transfer

Repeat the process from the T2 plate. By now growth should look much more even. Again take the leading edge of the best sector.

Checking the result

A good T3 plate grows out evenly in every direction with one consistent look and no sectoring. If it still shows distinct sectors, keep going to T4 and beyond. "Three" is a rule of thumb, not a magic number. Some cultures are clean by T2; others need five or more rounds.

Tips

• Transfer from the edge, not the middle. The center of the plate is the oldest growth, where the spores and contamination started.
• Label everything. Strain, transfer number, and date at minimum, as in the photo. Plates look identical in a stack.
• Run two or three plates per transfer. If one contaminates, you still have the others.
• Don't transfer too soon. Wait until the sector has clearly established itself and is growing away from the rest of the plate.
• Keep your winners. Once you have a clean, fast culture, store a few plates or slants in the fridge so you can start from it again later.

What's next

A clean plate can go straight to grain, or into liquid culture to expand it into many jars at once.