Hypotonic allows cells to take up water (H2O) through osmosis. This swells the cells to provide better dispersal of the chromosomes at time of spreading. The osmosis is accomplished by setting up a concentration gradient between the cytoplasm of the cell and the hypotonic solution surrounding the cells.
The rate of osmosis is dependent on the composition of hypotonic reagent, its concentration in the hypotonic solution, the time of exposure and the solution temperature. Higher temperature or increased time in hypotonic can lead to excessive swelling leading to cell breakage and overspreading of the chromosomes. Decreased temperature and/or decreased time can lead to under-spread chromosomes (too “tight” chromosomes).
From dilute balanced salt solutions or mixtures, a ratio of potassium chloride (KCl) and sodium citrate, to dilute media, and distilled water, there are several types of hypotonic solution that are utilized by laboratories.
The most widely used hypotonic is 0.075M KCL for peripheral blood and bone marrow cultures. This inorganic salt has long-term stability and stays readily dissolved for a broad range of concentrations and temperatures.
For more sensitive cell culture types like in-situ prenatal amniocytes, chorionic villus samples (CVS) and Products-Of-Conception (POC) samples, 0.8% sodium citrate is preferred. As an organic salt it is found to be gentler on the cells, resulting in more-optimal chromosome morphology.
Overall, the type of hypotonic used by laboratories is an individual preference due to a balancing of many factors.