Question

How do I calculate the number of cells recovered in a suspension? What information do I need? Also, calculate the percent increase in cell density from the time of IPTG addition to the time when the cells were harvested. The OD of the E.coli culture is around OD 2-2.5. 10ml of culture was added to each flask.

Components	IPTG (+) OD600	IPTG(-) OD600
E.coli+Glucose+Ampicillin	0.6263	0.5764
E.coli+Glucose+Ampicillin after IPTG and growth	0.2870	0.2041

Answer 1

Counting Cells in a Hemacytometer

The procedure below provides some general directions on how to use the hemacytometer. 1. Clean the chamber and cover slip with alcohol. Dry and fix the coverslip in position. 2. Harvest the cells. Add 10 μL of the cells to the hemacytometer. Do not overfill. 3. Place the chamber in the inverted microscope under a 10X objective. Use phase contrast to distinguish the cells. 4. Count the cells in the large, central gridded square (1 mm2 ). The gridded square is circled in the graphic below. Multiply by 104 to estimate the number of cells per mL. Prepare duplicate samples and average the count.