Gnotobiosis (from Greek roots gnostos "known" and bios "life") refers to an engineered state of an organism in which all forms of life (i.e., microorganisms) in or on it, including its microbiota, have been identified. The term gnotobiotic organism, or gnotobiote, can refer to a model organism that is colonized with a specific community of known microorganisms (isobiotic or defined flora animal) or that contains no microorganisms (germ-free) often for experimental purposes. The study of gnotobiosis and the generation of various types of gnotobiotic model organisms as tools for studying interactions between host organisms and microorganisms is referred to as gnotobiology. History The concept and field of gnotobiology was born of a debate between Louis Pasteur and Marceli Nencki in the late 19th century, in which Pasteur argued that animal life needed bacteria to succeed while Nencki argued that animals would be healthier without bacteria, but it wasn't until 1960 that the Association for Gnotobiotics was formed. Early attempts in gnotobiology were limited by inadequate equipment and nutritional knowledge, however, advancements in nutritional sciences, animal anatomy and physiology, and immunology have allowed for the improvement of gnotobiotic technologies. Methods Guinea pigs were the first germ-free animal model described in 1896 by George Nuttall and Hans Thierfelder, establishing techniques still used today in gnotobiology. Early methods for maintaining sterile environments involved sterile glass jars and gloveboxes, which developed into a conversation surrounding uniformity of the methods in the field at the 1939 symposium on Micrurgical and Germ-free Methods at the University of Notre Dame. Many early (1930-1950s) accomplishments in gnotobiology came from Notre Dame University, The University of Lund, and Nagoya University. The Laboratories of Bacteriology at the University of Notre Dame (known as LOBUND) was founded by John J. Cavanaugh and is cited for making some of the most notable achievements in the field of gnotobiotic research. Under the direction of James A. Reyniers, early work at LOBUND focused on obtaining gnotobiotes by sterilizing animals and maintaining the animals using high-pressure steam sterilized steel isolators; however, later work at the institute shifted the focus of the field towards establishing colonies of animals born germ-free. The first germ-free rat colony was generated and maintained using a steam sterilized isolator in 1946 by Swedish scientist Bengt Gustafsson. Flexible film isolators using peracetic acid vapor for sterilization began being developed in the 1950s. Refined sterilization techniques and manufacturing changes from LOBUND significantly reduced