Modified string test to improve and confirm by molecular characterization for bacterial identification

Rapid and reliable identification of bacteria is essential in clinical and environmental microbiology. Gram staining remains a widely used method for preliminary classification; however, it may require additional steps and can be difficult to interpret under certain conditions. To address these limitations, we evaluated a modified potassium hydroxide (KOH) string test incorporating methylene blue to enhance visualization of DNA release and facilitate differentiation between Gram-positive and Gram-negative bacteria. A total of 185 samples from clinical and environmental sources (including hospitals, soil, salt mines and honeycombs) were analysed. Samples were first assessed using Gram staining and subsequently tested using the modified KOH method. In this procedure, a 3% KOH solution was applied to bacterial colonies, followed by the addition of methylene blue to improve visualization of filament formation. All Gram-negative isolates demonstrated visible string formation, while Gram-positive isolates showed no string formation, indicating complete concordance with Gram staining results within the tested dataset. Selected isolates were further examined using 16S rRNA gene analysis to support taxonomic identification. The modified KOH string test provides a rapid, simple and low-cost approach for preliminary bacterial differentiation. While it does not replace conventional or molecular identification methods, it may serve as a useful complementary tool, particularly in resource-limited or high-throughput laboratory settings.