Bacteriophages are bacterial viruses that have shown increasing use in recent years across medicine, food industry, and as research tools. For these purposes, accurate and rapid determination of their concentration and monitoring of their stability over time are of crucial importance. Our work attempts to evaluate the use of the fluorescent dye fluorescein isothiocyanate (FITC) for tracking the infectivity of morphologically distinct bacteriophages T4, T7, and ϕ6. Following the covalent labeling of capsid proteins, we measured the fluorescence intensity. We investigated the effect of chemical modification on the preservation of phage infectivity and found that the staining procedure reduced the infectivity of bacteriophage T7, whereas did not reduce the infectivity of phages T4 and ϕ6. The correlation between fluorescence intensity and PFU titer in fresh samples was good, reaching its highest value in bacteriophage T7. In the second part of the study, we monitored the stability of the bacteriophages over time for 624 hours. The results showed that both the fluorescence signal and infectivity, measured by PFU, were stable and well-correlated in a refrigerator at 4 °C; however, a critical discrepancy emerged at room temperature (25 °C) under constant illumination. Infectivity dropped quickly to zero in all viruses, albeit at different rates, while the fluorescent signal remained detectable. This indicates that FITC fluorescence measures the presence of viral biomass rather than biological functionality, causing misleading results under non-optimal conditions. The method is useful as a rapid complementary tool for fresh samples, whereas the PFU method remains an indispensable standard for the precise determination of active particle titers.
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