Evaluation of alternative pretreatment solutions for the Masson's trichrome stain: replacements for Bouin's solution
In brief
Gram's iodine and pH 4 citric acid matched Bouin's staining in tissue tests
In two laboratories, human and animal tissue sections pretreated with Gram's iodine or pH 4 citric acid and heated at 60 to 70 degrees C for 30 minutes stained similarly to sections treated with Bouin's solution. These alternatives avoid hazardous components in the traditional solution, but broader testing is needed to confirm their performance across tissues and workflows.
- Journal
- Journal of histotechnology (Q2)
- Published
- 19 October 2026
- Study design
- Unclassified
- Evidence level
- Level 5, Expert Opinion (CEBM 5)
- Authors
- Amber Moser, Hannah Benton, Elizabeth Chlipala, Sheila Criswell
- PMID
- 42895190
- DOI
- 10.1080/01478885.2026.2748813
Why clinicians should know about it
- Picked for Histology (top studies of the week, 25 October 2026): Direct comparison of safer pretreatments for a standard histological stain
Abstract
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.
Abstract as published, via PubMed.
For healthcare professionals. The summary is generated by AI from the published abstract, and the evidence level is assigned automatically from the study design on the Oxford CEBM hierarchy. Neither is medical advice. Read the full paper before changing practice.