Evaluation of alternative pretreatment solutions for the Masson’s trichrome stain: replacements for Bouin’s solution

Masson's trichrome stain is a standard stain used to differentiate extracellular matrix components, primarily collagen, in histologic tissue samples. The classic pretreatment solution, Bouin's solution, contains formaldehyde, picric acid, and acetic acid. The chemical components introduce safety concerns encompassing toxicity, respiratory, explosive hazards, and environmental hazards. Three pretreatment solutions (citric acid solutions pH 4 and pH 6, and Gram's iodine) as well as a no pretreatment condition, were assayed alongside Bouin's solution on various human and animal tissue samples by two laboratories using manual methods. The investigators determined that tissue sections heated at 60-70°C for 30 min in either Gram's iodine or citric acid solution pH 4 produced similar staining results to those using Bouin's solution and therefore may be safe and effective replacement solutions for the trichrome stain.