An improved method to investigate staining kinetics in single cells

A new method to analyze staining processes in single cells of histochemical and cytochemical specimens in situ is described. The combination of a microscope photometer with a perfusion cuvette developed in our laboratory allows the continuous observation of a cell during the staining process. The flow rate dependence of the staining process has been examined demonstrating the strong suppression of the diffusional boundary layer adjacent to the cell surface by sufficiently high flow rates. Experiments to find optimal conditions for the kinetic analysis of the staining reaction of nuclei in lymphocytes, neutrophile granulocytes and monkey kidney cells with thionin are described. Half-staining times of the binding of monomer dye molecules and aggregates to nuclei have been calculated; they depend on the pretreatment of the cells. The addition of electrolytes decreases the rate of staining. The formation of aggregates obeys approximately a first-order reaction law and the binding of monomers provides an order of reaction of n = 0.5.