After feeding your cells, you should wait three to four days and then go into the lab to observe your cells. If they are growing well, you can plan on subculturing them. If your cells aren’t growing well, work with one of the lab faculty to troubleshoot.

Your cells are ready to subculture if they are confluent or stretch across the whole plate. The reason we subculture cells growing in culture is to decrease the total number of cells so that the cells are not overgrown. Adherent eukaryotic cells grow best when there is space to expand. If they grow to confluency in the T-flask the cells actually send out biochemical signals to restrict growth. Subculturing decreases the cell population so that they have room for robust growth.

There are a few skills you’ll need to have in hand before you’re ready to complete the subculturing protocol. In the next couple of pages, you’ll learn what a hemocytometer is, how to use it, and how to complete the equations to count your cells.

### Learning Objectives:

- 1.4 - Count cells using a hemocytometer
- 1.5 - Subculture cells.