We have developed a three-channel microfluidic device with embedded chambers useful for the study of bacterial chemotaxis and sample processing. The device is capable of generating linear chemical gradients in the embedded chambers that are free of flow disturbances. These linear chemical gradients are established by diffusing a chemical through widely available nitrocellulose material that serves as a permeable membrane between the adjacent supply channels and embedded chambers. The generated gradients were confirmed by measuring the fluorescent molecules diffused through the source channel into the chamber on a fluorescence microscope. As the gradients were generated without through-flow, cell movement in the chambers is caused solely by the cells' random motility or chemotactic response up or down a chemical gradient and not interfered by fluid flow. A precise laser cutting technique was utilized to create the microfluidic design in the membrane layer. The advantages of this microfluidic design are (i) a rapid implementation of a static chemical gradient can be established; (ii) two chemoeffectors can be simultaneously studied and compared; (iii) multiple chambers can be used for a reproducible study; (iv) the design can be fabricated at a low cost. Using a Nikon Ti microscope, food borne pathogen E Coli O157 was observed in the microfluidic device to swim toward the attractant, L-aspartic acid (500 uM) and away from the repellent, NiCl2 (500 uM). The chemotactic responses to the chemicals were quantified using the cell population ratios in the chambers close to the source channel and the buffer channel respectively. (C) 2013 The Electrochemical Society. All rights reserved.