36 Growth Rate
349
Conventional Method for Substrate-Adherent Cells
Materials
- Inverted microscope
- Ocular micrometer (grid type)
- Objective micrometer
- Culture flasks
- Pasteur pipettes
Equipment
- The solutions are the same as those given for the Replicate Tissue Cul- Solutions
ture Method
Procedure
1. Harvest cells from the culture to be examined by suspending the cells Cell culture
in the culture medium, and by centrifuging the cells at 150 g for 5 min.
2. Suspend the cells in the experimental medium at a density of between
1 x 10 5 and 5 x 10 5 cells/ml.
3. Dispense the cell suspension to 4-5 vessels.
1. On cell counting time point, select at least five microscopic fields at
random by placing a culture flask on the stage of the inverted microscope and by moving the stage.
2. Count the cells in the selected fields using the projected grid micrometer.
3. Perform the above cell counting with 3 cultures leaving 1-2 cultures
as extra cultures for accidental contamination.
Results
Using data for the mean number of cells per unit area, the area of the
bottom of the culture flask, and the volume of the culture medium, calculate the number of cells/ml and the standard deviation. Plot the calculated values on a graph.
Comments
This method is suitable for counting substrate-dependent cells. When
this method is applied to suspended cells, first shake the culture vessel to
make a homogeneous cell suspension, and then leave the suspension for
several minutes to allow the cells to settle. Select fields for counting after
the cells have settled.
Enumeration
of cells
349
Conventional Method for Substrate-Adherent Cells
Materials
- Inverted microscope
- Ocular micrometer (grid type)
- Objective micrometer
- Culture flasks
- Pasteur pipettes
Equipment
- The solutions are the same as those given for the Replicate Tissue Cul- Solutions
ture Method
Procedure
1. Harvest cells from the culture to be examined by suspending the cells Cell culture
in the culture medium, and by centrifuging the cells at 150 g for 5 min.
2. Suspend the cells in the experimental medium at a density of between
1 x 10 5 and 5 x 10 5 cells/ml.
3. Dispense the cell suspension to 4-5 vessels.
1. On cell counting time point, select at least five microscopic fields at
random by placing a culture flask on the stage of the inverted microscope and by moving the stage.
2. Count the cells in the selected fields using the projected grid micrometer.
3. Perform the above cell counting with 3 cultures leaving 1-2 cultures
as extra cultures for accidental contamination.
Results
Using data for the mean number of cells per unit area, the area of the
bottom of the culture flask, and the volume of the culture medium, calculate the number of cells/ml and the standard deviation. Plot the calculated values on a graph.
Comments
This method is suitable for counting substrate-dependent cells. When
this method is applied to suspended cells, first shake the culture vessel to
make a homogeneous cell suspension, and then leave the suspension for
several minutes to allow the cells to settle. Select fields for counting after
the cells have settled.
Enumeration
of cells