Identification of unknown bacteria is a critical part of microbiology. The purpose of the experiment is for students to learn different characteristics of bacteria, construct a proper dichotomous key, carry out tests to differentiate and narrow down bacteria, and use the results to identify two unknown bacteria. By carrying out this experiment, students learn how to properly identify bacteria, and this knowledge can be useful later in life, whether it is for research purposes or applications in clinical settings. Two strains of bacteria, one Gram positive and one Gram negative, were provided in a broth tube. Using a sterilized inoculating loop, these bacteria were cultured on four plates: two Tryptic Soy Agar plates, one Mannitol Salt Agar plate, and one Eosin Methylene Blue plate. Following this, the two bacteria were isolated onto separate plates using the isolation streak technique in order to determine which one was Gram positive and which one was Gram negative. Then, using the dichotomous key that was created, various tests were carried out on each of the bacteria to narrow down possibilities and ultimately identify the two different bacteria. Materials and Methods To begin the experiment, broth tube Unknown #2A containing two unknown bacteria, one Gram positive and one Gram negative, was used as the master stock. Additional subcultures would be created from this master stock. First, a Gram stain was performed on the mixed culture to observe the size, shape and Gram reaction of the organisms. Using a sterilized inoculating loop, the mixed bacterial culture was then streaked onto four plates: three plates, Tryptic Soy Agar (TSA) plate, Mannitol Salt Agar (MSA) plate, and Eosin Methylene Blue (EMB) plat>