Staining Nerve Fibers with Methylene Blue. An Evaluation of Variables Used in An Immersion Technic

A study of the effects of osmotic pressure, pH, the presence of dextrose, acetate, pyruvate or lactate, and agents affecting cell permeability during supravital staining by methylene blue was made by means of an immersion technic. Mesentery and intestine of dogs and cats were used. Penetration of the dye was limited to the mesentery and more superficial layers of intestine. Conditions which facilitated the characteristic differentiation of of nerve fibers were: continuous oxygenation of the staining solution, pH about 5.6 stabilized by phosphate buffer, and the presence of small amounts of acetate and lactate. Young animals' tissue stained better than old. Methylene blue was a much more effective staining agent than less completely methylated thionins.