Recent worldwide foodborne outbreaks emphasize the need for the development of rapid and accurate method for pathogen detection. To address such issues, a new colony based label-free detection method working on the principles of elastic light scattering was introduced. In order to build libraries of scattering images for bacterial pathogens, it is pertinent to determine the effect of preparation and storage of the agar media on the scatter patterns. Scatter patterns of three Escherichia coli serovars (O26, O111 and O157) were studied and used in a model system, after growth on Sorbitol-MacConkey agar plates that were prepared and stored at different conditions in the laboratory. Quantitative image processing software was used to analyze variation in scatter patterns of the same serovar on media prepared under various standard laboratory conditions and to generate a cross-validation matrix for comparison. Based on the results, it was determined that attention should be given during preparation of media so that the agar plates are not air-dried more than 10 - 20 min after solidification at room temperature. The plates could be stored in sealed bags in cold room (4^oC - 10^oC) for up to a month before use. The findings of this study should provide guidelines in preparation, storage, and handling of media for generation of reproducible scatter patterns of bacterial colonies with the light scattering sensor for pathogen detection.