cell culture confusion - (Sep/16/2011 )

HI

I need to establish a growth curve for my cultures and exploring the best way to do so. Some help would be appreciated with the following points:

- I am considering using a 24 well plate to culture, however if i counted three wells per day would that be a good way of determining the growth curve?
- I have to read an absorbance from my cultures. What confuses me is:
if I do a subculture and take a volume of that to determine the absorbance, would that not theoretically effect the cell numbers?
-eg: if I had a 10ml suspension and used 2ml to do a cell count on the hemocytometer and an absrobance reading would that effect the growth curve if only 8ml is re-seeded?

- I am new to this so any help would be great!

The easiest way to do growth curves is to avoid the cytometer and plate count methods, and to use absorbance (or color change, if you have acid fermenting bacteria). Set up a plate (300 ul wells) with 200 ul of your culture medium in the first column, and 100 ul in adjacent columns. Inoculate your culture into the 200 ul column. Do 2x serial dilutions of the culture across the plate. Grow the plate in a plate reader to measure absorbance over time. You will be able to measure doubling time directly by measuring the length of time required for adjacent columns to reach the same OD reading.

This avoids having to carefully quantify the inoculated amount, avoids issues of measurement linearity, and provides a built-in replicate measurement from the multiple serial dilutions.