The priority operation on process of analyzing bacteria automatically on the microscopic images is realization of optimal focusing. Reliable imaging can be achieved by examining multiple images in sequential images formed with Hand-eye coordination of the laboratory on Z axis of microscope. In this study, instead of selection of a single frame with maximum auto-focus function value on sequential images, automatic generation of optimal color image based on the processing more frames around frame with maximum auto-focus value is proposed. Microscopic imaging with optimal focusing is generated by processing images with different focusing on the Z axis of microscope. Firstly, Probability density function (pdf) of image focus based on auto-focus function is obtained. The image with highest value is assigned to the reference image. Optimal focusing in all areas of color image is achieved by applying image fusion approach in a multi-dimensional color space of reference image and images with pdf values near it to ensure optimum focus in all areas(in pixels) of the image. Proposed method is compared with other methods in the literature by testing in image sequences taken from a different slides containing sputum smear painted with ARE for the diagnosis of TB.