The objective of this study was to compare and determine the optimal sample preparation techniques to observe the adherence of probiotic bacteria, Lactobacillaceae sp. to human colorectal adenocarcinoma cell line HT-29. Different fixation time and coating thickness were compared to evaluate the integrity and quality of image observed using field emission scanning electron microscope (FESEM). Based on the qualitative assessments, the one-hour fixation was proposed as a promising fixation period as it is better than overnight fixation in terms of showing clearer parts and the adhesive features on the surface of both bacteria and HT-29 cells. Platinum sputter-coating step is recommended at shorter time to increase conductivity of sample and reduce electron beam damage. Thicker platinum coat could obscure fine structural visualization although it helps in eliminating charging during the imaging process. This finding provides an important sample preparation optimization to enable better structural integrity and quality of image captured using FESEM.