sorry for that i'm not a native English speaker, so my expression may confuse you sometimes.

Generation of a growth curve can be useful in evaluating the growth characteristics of a cell line.From a growth curve, the lag time, population doubling time, and saturation

density can be determined.

Procedure:

1. Trypsinize and centrifuge the cells.

2. Resuspend the pellet in 5 mL of medium and count the cells(see Hemocytometer Counting).

3. Dilute the cell suspension in order to have an appropriate amount of medium and cells to achieve a seeding density of 2 x 103 cells per cm2 of surface area. Cell seeding

densities vary by cell line (you should choose an appropriate seeding density according to your cell, if you cell grow quickly, the density would be relatively low).

4. Mix well and seed the dishes/flasks with the appropriate amount of diluted cell suspension.

5. Count some of the leftover cell suspension in order to determine the actual seeding density

6. Put the plates in an incubator.

7. Count the duplicate plates every 24 hours (also up to your cells, if they grow too fast, this time should be shorten).

8. Plot the results on a log-linear scale. The population-doubling time can be determined by identifying a cell number along the exponential phase of the curve, tracing the

curve until that number has doubled, and calculating the time between the two.

u mean I should inoculate some amount from previous medium in to fresh medium? and keep doing that,

yes, I mean you should keep doing the cell passage, let the cell keep growing, and you may understand the "growing habit" of your cell and find a optimized time lag to do cell passage.