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Related Techniques
Counting 1. Culture the cells for 7-10 days.
Equipment
and
solutions
2. Count the cells either every day or every 2nd day by harvesting three
cultures selected at random.
Results
Using counting data, the cell number per ml can be calculated. Calculate
the mean number of cells per ml and the standard deviation. By plotting
the calculated values on a graph, a growth curve for the culture can be
constructed.
Repeating Sampling from the Same Culture
If the volume of the culture is large enough (culture volume is more than
15 ml), a small quantity (0.3-0.5 ml) of the cell suspension may be
sampled repeatedly from one culture every cell enumeration. Otherwise,
a decrease in the volume of the culture medium as a result of sampling
may affect cell density in the culture. This method of repeated sampling
should not be applied to substrate-adherent cells, because detachment
at each enumeration time may cause considerable injury to the cells, and
also disturb cell growth.
Materials
The equipment and solutions are the same as those given for the Replicate Tissue Culture Method. However, the culture should be prepared in
larger-volume vessels such as 250 ml flask.
Procedure
Cell culture 1. Follow steps 1-3 of the seeding protocol given for the Replicate Tissue
Culture Method.
2. Prepare three cultures in large vessels. It is preferable to make one or
two extra cultures in case of accidental contamination.
3. Culture the cells for 7-10 days.
Sampling 1. At each counting time point, make a single cell suspension by shaking
the vessels or by pipetting the cells in the culture medium under a
laminar flow cabinet, and remove several hundred fll (just enough to
enable cell counting).
2. Count the number of cells in the sample with a hemocytometer.
3. Return the culture to the incubator and continue the cell culture.
Results
The results obtained with this method are the same as those for the Replicate Tissue Culture Method.
Related Techniques
Counting 1. Culture the cells for 7-10 days.
Equipment
and
solutions
2. Count the cells either every day or every 2nd day by harvesting three
cultures selected at random.
Results
Using counting data, the cell number per ml can be calculated. Calculate
the mean number of cells per ml and the standard deviation. By plotting
the calculated values on a graph, a growth curve for the culture can be
constructed.
Repeating Sampling from the Same Culture
If the volume of the culture is large enough (culture volume is more than
15 ml), a small quantity (0.3-0.5 ml) of the cell suspension may be
sampled repeatedly from one culture every cell enumeration. Otherwise,
a decrease in the volume of the culture medium as a result of sampling
may affect cell density in the culture. This method of repeated sampling
should not be applied to substrate-adherent cells, because detachment
at each enumeration time may cause considerable injury to the cells, and
also disturb cell growth.
Materials
The equipment and solutions are the same as those given for the Replicate Tissue Culture Method. However, the culture should be prepared in
larger-volume vessels such as 250 ml flask.
Procedure
Cell culture 1. Follow steps 1-3 of the seeding protocol given for the Replicate Tissue
Culture Method.
2. Prepare three cultures in large vessels. It is preferable to make one or
two extra cultures in case of accidental contamination.
3. Culture the cells for 7-10 days.
Sampling 1. At each counting time point, make a single cell suspension by shaking
the vessels or by pipetting the cells in the culture medium under a
laminar flow cabinet, and remove several hundred fll (just enough to
enable cell counting).
2. Count the number of cells in the sample with a hemocytometer.
3. Return the culture to the incubator and continue the cell culture.
Results
The results obtained with this method are the same as those for the Replicate Tissue Culture Method.