1. Initial inoculum:

- From solid media, touch an isolated colonies with a sterile applicator or toothpick.

- From liquid media, touch the culture with the end of a sterile applicator.

- From a frozen culture, scrape the surface with a sterile applicator; you need not take visible clumps.

- From a stab tube, insert a sterile applicator into the agar and withdraw; do not take agar chunks.

2. Draw the inoculated end of the applicator across an agar media plate, keeping toward the edge of the plate.3. Select a fresh applicator, and make a second single streak across the first one.4. Unskilled - Selected a fresh applicator, Skilled - Rotate the applicator 180 degrees and use the sterile edge.5. Streak across the second streak, avoiding the first streak, and continue to make dense streaks until the designated area has been filled. 6. Single colonies will form where a single bacterium was deposited on the medium. It is easy to streak four (4) strains for single colonies on a single 100 mm plate; twelve or more strains can be done by skilled workers. 7. Invert plate so that the media face points down; this prevents water droplets from dripping onto the plate surface. Incubate at appropriate temperature.