Staining properties of alizarin red S for growing bone in vitro.

The pH-metric titration curve of alizarin red S (ARS) showed that ARS ionizes in three stages: firstly the -SO3Na group, secondly the beta-hydroxyl group, and finally on the alkaline side the alpha-hydroxyl group. The titration of ARS solution with calcium ions indicates that complex and chelate formation between ARS and calcium are distinct. The in vitro staining experiments were carried out at different pH levels. Completely ionized ARS had the best affinity for growing bone surfaces. The next most effective staining occurred just prior to neutralization. The least effective staining by ARS was found after the neutralization point had been reached. The presence of calcium made the dye solution inactive for staining the growing bone in vitro.