15 Streaking for Isolation
Emilie Miller, Ph.D
If a single bacterial cell is placed on the surface of a TSA agar plate and allowed to multiply for 24 to 48 hours, it would grow into a mass of cells visible to the human eye called a colony. Colonies formed by the same microbial species growing on the same medium will all look alike. This is because the cell shape, pigmentation, division plane, rate of cell division and other characteristics of the organism result in the progeny cells stacking on one another in a pattern resulting in a characteristic colony form.
If you swab a door handle, where bacteria are likely to be present, and then pass the swab across the surface of a TSA plate, cells would be deposited onto the surface from the swab. Initially, the swab may have a fairly high concentration of cells and the area touched by it will have lots of different cell types placed close together. After these grow up, the cells’ progeny will crowd together and overlap with other cells’ progeny forming areas called confluent growth. This is the type of growth you observed on the surface of the TSA slants in the Basic Aseptic Transfers exercise.
As the swab moves across the agar leaving cells on the agar in a zig zag pattern, regions touched later in the process will have fewer and fewer cells. Individual cells are far enough apart that each one would grow into a discrete isolated colony. The result may look something like the figure to the right, Because the door handle likely has a variety of microbes on it, there are numerous colony forms. This technique is called a zig zag streak and is one type of isolation streak method.
Now consider streaking a sample from a pure TSA broth culture prepared for you. If there is visible turbidity, there will be a high density of cells. If you used the same zig zag streaking pattern, the cells would never be reduced in concentration such that you could get isolated colonies. In order to reduce the cell density on the surface of the plate, we can use a T-Streak or a quadrant streak technique.
The T-streak and quadrant streak are forms of dilutions on a solid surface (Franklund, 2018). In these two techniques the plate is divided into sections. Bacteria are deposited in the first section at full strength from the source. Then the inoculating loop is sterilized. From this point on, no additional cells are added to the agar surface. The sterile loop is used to spread out cells that have already been placed on the plate. After spreading cells from the first area to the second, the loop is sterilized again. This eliminates extra cells from the loop. The sterile loop is used to spread some cells from the second area into the third area diluting them further. (In a quadrant streak, cells are spread into a forth area as described.) After all the regions have been inoculated, the hope is that in the last section cells are far enough apart so that they grow up into isolated colonies.
This technique allows one to observe isolat