general_dicty_techniques.doc.docVIP

Cells grown in axenic medium have slower growth rates: they have a doubling time of roughly 8 to 12 hours depending on temperature, medium, the presence of selective drugs, and most likely several unknown factors. When growing cells, it is important that they remain in exponential phase rather than allowing them to reach stationary phase. Therefore it is important to establish and maintain a reasonable schedule for splitting the cultures to insure an adequate supply of cells that develop well. Cells should never be allowed to grow beyond 3x106 cells/ml if development is going to be analyzed. Cultures of higher densities begin to secrete factors that trigger entry into development, therefore changing the physiology of the culture. On the other hand, it is generally not a good idea to split the cells so that the cell density is less than 1x104 cells/ml. Cultures of lower cell densities lag for a period that can be difficult to estimate. Optimal growth of Dictyostelium is observed at a temperature of 22°C. For maintaining the culture do not allow the cells to grow beyond 5x106 cells/ml. Do not maintain the same strain for more than a month growing since they might accumulate mutations. Thaw the strains regularly. Determination of Cell Density Cell density is easily determined by taking an aliquot of the culture and counting it in a hemocytometer. The standard hemocytometer carries a grid that is 3 mm on a side (see image below). Count 5 squares 1 mm on a side. Divide that number by 5 to obtain the average number of cells per sq. mm. Since the coverslip sits 0.1 mm above the surface of the hemocytometer, the volume of the counting chamber is actually 0.1 cubic mm. Therefore, to obtain the cell density in cells per ml, the number of cells counted must be multiplied by 1x104. When the cells approach densities of greater than 1-5x106 it is usually a good idea to do a 1:10 dilution of the culture sample prior to putting it in the hemocytometer. Estimation of Growth Rat