A novel method for evaluation of bacteria colonies concentration based on optical spectra examination, is proposed. The influence of bacteria colonies number on Fourier spectrum properties is considered in term of scalar diffraction theory and corresponding theoretical model is presented. Computational simulations are performed to confirm the theoretical predictions. Additionally, optical Mellin transform is used to omit the dependence of Fourier spectrum pattern on bacteria colonies size fluctuations and to provide a scale-invariant analysis. Presented results have shown high potential of the proposed approach for comparative study of bacteria colonies grown on solid medium in vitro.© (2009) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.