Colourimetric Detection of Formalin Concentration in Fixing Solutions using Schiff’s Reagent: An In-vitro Study

Introduction: Formalin is a widely used tissue fixative in histopathology. Maintaining a precise formalin concentration in fixing solutions is essential, as both over-fixation and underfixation can compromise tissue quality and pose health risks to laboratory personnel and Pathologists. Existing techniques such as gas chromatography and spectrophotometry are accurate but expensive and impractical for routine use. Hence, a simple, affordable, and reliable method for monitoring formalin concentrations in routine laboratory procedures is required. Aim: The present study aimed to develop a method to detect and quantify formalin concentration using Schiff’s reagent and to further validate it using a colour palette. Materials and Methods: The present in-vitro study was conducted in the Department of Oral Pathology, Saveetha Dental College and Hospitals, Chennai, Tamil Nadu, India, from July 2024 to November 2024. Formalin solutions with concentrations of 2%, 5%, 10%, 20%, 25%, 50%, 75%, and 100% were prepared. Schiff’s reagent was added to each solution, and the resulting colour changes were recorded. A colour palette was generated based on this gradient, covering the full spectrum of formalin concentrations from 2 to 100%. This colour palette was patented and published in the Official Journal of The Patent Office (202541045779). Fifty filter paper strips (10 for each concentration) soaked in Schiff’s reagent were immersed in the different formalin solutions. Two Oral Pathologists independently assessed all 50 strips and compared them with the colour palette to confirm concentration-dependent colour changes. Kappa statistics were calculated using Statistical Package for Social Sciences (SPSS) software version 23 to assess interrater reliability. Results: A gradient of purple colour intensity corresponding to formalin concentration was observed, confirming the specificity of Schiff’s reagent for formalin detection. Higher concentrations of formalin produced a more intense dark violet, while lower concentrations resulted in lighter shades. Kappa statistics demonstrated excellent inter-rater agreement, with a κ value of 0.92. Conclusion: According to the present study findings, Schiff’s reagent reliably detects formalin concentrations, providing an accessible, cost-effective colourimetric method for the safe and effective use of formalin in routine laboratory practice.