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It has been reported that the intracellular pH of the budding yeast Saccharomyces cerevisiae is asymmetric between mother and daughter cells, and this asymmetry in pH underlies replicative aging and rejuvenation. S. cerevisiae growth morphology changes between the yeast form and pseudohyphal form, according to nutrient availability. A previous study reported that the replicative life span of pseudohyphal form cells is longer than that of yeast form cells in S. cerevisiae. However, the intracellular pH of pseudohyphal cells is unknown. To examine the intracellular pH of S. cerevisiae cells during pseudohyphal growth, vital staining was performed with neutral red, which is a pH indicator, of cells growing on nitrogen starvation (SLAD) medium. The results showed that the vacuoles of S. cerevisiae cells during pseudohyphal growth induced by nitrogen starvation formed polar pH gradients. The relationship between cell size and shape and the neutral red staining patterns suggested that the pH of cell vacuoles during pseudohyphal growth changed from uniformly near pH 6.8 to steep gradients of pH from vacuole ends along the long axis of the cell. The results of time-lapse imaging to examine vacuolar dynamics and neutral red staining suggested that the pH gradients were not formed simply by inheritance of vacuolar contents accompanying vacuolar movements.