Reduction of Colour Fading in the PNPH Test for DNA in Bovine Spermatozoa

Abstract

COLORIMETRIC tests are employed routinely for estimating the deoxyribonucleie acid (DNA) content of tissues. The diphenylamine test, originally described by Dische¹, or subsequent modifications of it, are the most frequently used. Other colorimetric tests include Ceriotti's indole² and the p-nitrophenylhydrazine (PNPH) proposed by Webb and Levy³. The latter is the most sensitive, but possesses the disadvantage of rapid colour fading. In 1963 Brown et al.⁴ proposed a modification of the PNPH test for plant leaf tissue that is more sensitive and stabilizes the colour. This modification, applied to animal tissue, does not increase the sensitivity of the original method, but it does reduce the colour fading. Essentially this test differs from the Webb–Levy technique in the extraction procedure since iso-amyl acetate replaces the butyl acetate. This is then followed by an extraction of ethyl ether to remove residual amyl acetate. Colour is developed in n-butyl alcohol made strongly alkaline by the addition of an equal volume of 50percent sodium hydroxide.