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To examine the relationship between the growth and pH gradients of Debaryomyces hansenii at a single-cell level.Using bioimaging techniques, the cell areas and early pH gradients (ΔpH10), i.e. the pH gradients determined 10 min after initiation of experiments, were determined for single cells of two D. hansenii strains in fluid and on solid (agar) substrate with and without 8% (w/v) NaCl. The combination of NaCl and solid substrate prolonged the growth initiation of both D. hansenii strains additively. In all our experiments, primarily two groups of cells existed; a vital group consisting of growing single cells with intact early pH gradients, and a group of dead cells without early pH gradients.Our results show that growth initiation of the D. hansenii cells is severely affected by NaCl and to a lesser extent by the type of substrate in an additive and strain dependent way. Moreover, the early pH gradient of a vital D. hansenii cell cannot be correlated with the rate of its subsequent growth.Our study reveals new knowledge on the growth and pH gradients of D. hansenii on solid surfaces in the presence of NaCl.