Photoinduced Fluorescence Changes in Selected Bacterial Strains Under 405 nm Laser Excitation: An In Vitro Hue-Based...

Bacterial fluorescence generated by endogenous fluorophores may provide an optical approach for monitoring microbial fluorescence responses. The aim of this study was to analyse temporal changes in the Hue parameter, defined as an image-derived component of the HSB colour space representing the dominant colour tone, and to characterize strain-associated fluorescence response profiles under 405 nm violet-light excitation. A total of 28 reference bacterial strains were examined. Each strain was inoculated at eight distinct locations on a separate BHI agar plate, yielding eight colonies per strain. At 24, 48, 72, 96, and 168 h of incubation, the same colonies were repeatedly exposed for 45 s to a 405 nm continuous-wave diode laser operating at 200 mW and imaged in a completely darkened room using an iPhone 14, a yellow emission filter, and fixed acquisition geometry. The JPEG images were converted to the HSB colour space in ImageJ software (version 1.54), and background-corrected Hue values were calculated for each colony as ΔHue = Hmean - Hmean_BG using the corresponding colony and agar-background regions of interest. A complementary qualitative visual assessment was performed, and aligned rank transform ANOVA was used to evaluate the effects of bacterial strain, incubation time, and their interaction. ΔHue values differed significantly between strains and across incubation time points, with significant strain × time interactions observed in both rod-shaped and coccal bacteria (all p < 0.001). Exploratory planned contrasts also showed differences between the predefined Gram-staining groups (p = 0.0002) and between rod-shaped and coccal bacteria (p < 0.0001); however, these comparisons do not establish independent effects of Gram status or cellular morphology. The recorded fluorescence signal was generally weak, although visible temporal changes occurred in selected strains, including Staphylococcus aureus ATCC 6538P and Proteus mirabilis PCM 543. Overlap between ΔHue distributions limited separation of some strains. Background-corrected Hue analysis enabled exploratory within-dataset monitoring of temporal fluorescence colour changes under the applied in vitro imaging protocol. However, the overlap between ΔHue distributions and the technical limitations of the imaging system preclude the use of ΔHue as a standalone bacterial identification or diagnostic parameter.