The assessing of acidophilia with Biebrich scarlet, ponceau de xylidine and woodstain scarlet.

Horobin and Bennion stated in 1973 that the bonding of Biebrich scarlet at an alkaline pH is hydrophobic as the isomers of Biebrich scarlet (ponceau de xylidine and woodstain scarlet) do not stain at that pH. If correct this would negate the use of Biebrich scarlet as proposed by Spicer and Lillie in 1961 as a measure of acidophilia. We have found all three isomers to stain similarly at an alkaline pH. Goldstein stated in 1963 that Biebrich scarlet saturated with urea stained elastic fibers only faintly or not at all. This we confirmed; we also found that the staining of other acidophilic structures (Paneth cell granules, sperm heads, etc.) was blocked. If only hydrogen bonding and not ionic bonding is blocked in an aqueous dye solution saturated with urea then the exact meaning of alkaline Biebrich scarlet staining is unknown.