To estimate the initial number of colony-forming units in a mycangium, use the technique of serial dilution. See Database protocols for Isolations databse for how to accurately input this information into the database.

Isolation

1. Prepare two tubes per each sample, label them “0.1” and “0.01”, fill each with 500 ul of water or PBS.
2. Suspend mycangium in the tube “0.1” and vortex.
3. Plate 50 ul of the suspension on media. Record the plate as “0.1 dilution” in [PLATES].[note] in the Isolations database.
4. Transfer 50 ul of the initial suspension to the second tube (“0.01”) and vortex.
5. Plate 50 ul of the second suspension on second media plate, and record that plate as “0.01 dilution”.
6. Plate 5 ul of the second suspension on a third plate, and record it as “0.001 dilution”.

Calculation of CFU count

Multiply the number of colonies on the plate by the inverse of the initial dilution factor.