the size and cost of isolators, making gnotobiotic research more universally accessible. After numerous advances in gnotobiotic research and technologies, the main challenges facing gnotobiotic research today are cost, space, efficiency, and operational procedure requirements. In 2015, the costs of maintaining gnotobiotic mice cages was greater than 4 times the cost of maintaining those of non-gnotobiotic mice, creating a challenge for establishing and maintaining facilities using typical funding sources, such as federal grants from institutions like the NIH. Applications The early focus of the field of gnotobiology was on proving that an organism could live in the absence of microorganisms, which ultimately resulted in the development of gnotobiotic organisms as a tool for research. Between the 1950s and 1970s, germ-free models were used to study the effects of the absence of bacteria on host organism metabolism and physiology, which later evolved into intentionally infecting germ-free organisms with specific microorganisms to investigate their functions and other questions relating to the biomedical field. In the early 1970s, gnotobiotes were used to study the role of microorganisms in host nutrition acquisition and immune response; however, this was limited because animals reared in a gnotobiotic colony often have poorly developed immune systems, lower cardiac output, and thin intestinal walls, which make them highly susceptible to infectious pathogens. After the early 1970s, gnotobiotic research decreased until the mid-1980s. Within the 21st century, gnotobiotic model systems have become an important tool for investigating interactions between host organisms and their commensal microbiota, as they allow for researchers to investigate specific microbes in a highly controlled host system. Historically, mouse models have been used to investigate the impacts of the microbiota composition (which microorganisms are present) on host immune system, nervous system, metabolism, and physiology; however, an increasing interest in this field has led to the incorporation of other model organisms to address a larger variety of questions relating to these topics. Animals A gnotobiotic animal (gnotobiote) is an animal in which all microorganisms interacting with it are known and controlled. Gnotobiotic animals are typically born under aseptic conditions, which may include removal from the mother by Caesarean section followed by immediate transfer of the newborn to an isolator where all incoming air, food and water is sterilized. Gnotobiotes are usually raised in a sterile laboratory environment, and are only intentionally exposed to microorganisms of interest to researchers. Mice and rats are common gnotobiotic animals used in research,