A student is provided with a pure culture and asked to make a three-phase streak plate. The student takes a sample of the culture and streaks the first sector of the plate. What is the next step?

The following step is to sterilize the wire loop, turn the plate 90 degrees, collect some organisms from the first area and continue to streak on the second area. Repeat for the third area.

Explanation:

The goal of three-phase streak is to obtain isolated colonies in the plate. To do this, the culture is diluted by spreading it through the entire plate.

In the first phase, one third of the plate is used to streak the initial inoculum in a zig-zag motion. As the wire spreads, the densitiy of organisms diminishes and colonies start to be further apart. Doing this only one time, is usually not enough to obtain isolated or separated colonies. So two more phases are used.

Before the second phase, the wire is sterilized, and colonies are collected by passing through the first area two or three times, so the inoculum now is much smaller than at the beginning. The organisms are spread in the second third of the plate, also in a zig-zag motion.

The same process is repeated in the third phase, by filling the remaining area of the plate.

By doing this, it is very probable that the initial culture is diluted enough to obtain single, isolated colonies in the plate which can then be grown separately in order to identify them.