Abstract
This paper addresses the problem of multi-class classification of bacterial microscopic images using a rigorous experimental protocol designed to prevent information leakage and improve performance. The dataset consists of 2034 images representing 33 taxa, organized by class. Data integrity checks confirmed the absence of corrupted or unreadable files. To formalize image characteristics and ensure quality control, indirect geometric and textural features were calculated, including minimum frame size, brightness statistics (mean and standard deviation), Shannon entropy, Laplace variance, and Sobel gradient energy. Quality checks revealed a small proportion of images with extreme brightness (2.5074%), while no samples with critically low sharpness according to the selected criteria were detected. Statistical analysis of interclass differences using the Kruskal–Wallis test with multiple comparison correction demonstrated the high discriminatory power of texture features, specifically gradient energy (ε2 = 0.819987) and Laplace variance (ε2 = 0.709904). Feature correlations were consistent with their physical interpretation, revealing a strong positive relationship between sharpness and gradient energy. Principal component analysis confirmed a strong structural pattern, with the first two components explaining 75.5766% of the total variance. For a unified comparison, classical machine learning, transfer learning, and modern deep architectures were evaluated within a single protocol.