Impact of Reagent Age and Storage Temperature on the Diagnostic Accuracy of the Gram Stain: A Quality Control Study in a Microbiology Laboratory at Tertiary care setting in North Gujarat, India

Background: Gram staining is still one of the most commonly applied diagnostic assays in the clinical microbiology laboratory. Technical skill is not alone responsible for the quality of Gram stain results; the stability and correct storage of staining reagents are also crucial. Staining quality may be affected by reagent degradation resulting from exposure to inappropriate storage temperatures and/or the passage of time, which can cause errors in diagnosis. The study was a cross-sectional test that involved a total of ninety-two isolates of various bacterial species. This study revealed that the diagnostic accuracy of Gram staining is affected by the age of the reagent and by the storage temperatures. Quality control recommendations for routine Microbiology laboratory for the use of Gram staining were made. Material and Methods: A quality control study was performed in the laboratory using the standard strains of bacteria such as staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis. Reagents used were crystal violet, Gram's iodine, decolorizer and safranin stored at 0, 25, 37°C for 0, 1, 3 and 6 months respectively. The reagents were used to stain standardized bacterial smears. The quality of staining was done by evaluating the preservation of the morphology, staining intensity, clarity of background, and correct Gram reaction. The accuracy of diagnosis was estimated by comparing with expected staining characteristics. Results: Fresh reagents showed the greatest accuracy in diagnosis (99.2%). They could be used for six months without noticeable loss of performance and were stored at the low temperature of 4°C. Samples of reagents held at 37ºC showed a significant deterioration, with diagnostic accuracy down to 86.5% after six months. The biggest degradation was found with the crystal violet and the iodine reagents. Aged reagents showed reduction in staining intensity, irregular Gram reaction and background artifacts. Conclusion: The performance of gram stain is greatly affected by the age of reagents and the temperature of the storage. Refrigerated storage helps to maintain the stability of the reagents while maintaining the diagnostic value. To maintain reliable microbiological diagnosis both routine quality control procedures and scheduled replacement of reagents are necessary.

Keywords: Gram stain, quality control, stability of reagents, storage temperature, laboratory diagnosis, microbiology laboratory.