Absorbances of hematoxylin solutions at varying pH levels

An alum hematoxylin was prepared (0.5 g hematoxylin, 8 g potassium alum, distilled water 477.5 ml and Na10(3) solution, 1 mg/ml, 22.5 ml) and divided into 50 ml aliquots. The pH of these aliquots was adjusted with 2 1/2% NaOH or 10% H2SO4. As soon as the pH of a sample was adjusted the absorbance from 400 to 700 nm was recorded with a Cary 15 spectrophotometer using specially designed cuvettes and undiluted samples. The following day the absorbances at peak were measured on a Coleman Jr. spectrophotometer and three days later a variety of tissues was stained with each solution. The pH and peak of absorbance of the aliquots were pH = 2.0 450 NM, 2.5 505, 2.6 507, 2.7 515, 2.8 520, 2.9 530, 3.0 540, 3.1 550, 3.3 560, 3.5 560. In the stained material in the intensity of nuclear staining was about the same at all pH levels but non-specific staining was greatest in slides stained at pH = .5 and virtually absent in slides stained at pH = 2.0. At intermediate pH levels the amount of non-specific staining was intermediate.