Gnotobiotics: A New Discipline in Biological and Medical Research

GNOTOBIOTICS: A NEW DISCIPLINE IN BIOLOGICAL AND MEDICAL RESEARCH THOMAS G. WARD, M.D., Dr. P.H., and P. C. TREXLER, M.S.* The development of pure culture methods revealed to the early bacteriologists the complexity of the microbic world and the possibility of rearing thehigher organisms in a similar state ofpurity. Theutility of pure living specimens in the laboratory was recognized, as well as the probable difficulty in isolating higher animals from the microbes with which they have been intimately associated throughout evolutionary history. A few seed plants were grown in a sterile environment, and guinea pigswere reared in a sterile environment by Nuttal and Thierfelder (1895). These studies were continued by a few investigators and extended to chickens, goats, and frogs. Only limited success was had by the early workers because of difficulties in maintaining a sterile environment and in devising an adequate sterile diet. In recent years these problems seem to have been solved for the laboratory rat and mouse (1). Reproducing colonies and practical means for the distribution and use of these animals are available. The germfree animal must be considered a new type of biological material rather than just another laboratory animal, since it opens up new areas to investigation. Thus a new discipline developed: "gnotobiotics" (2, 3). The word is derived from Greek (gnotos, "known"; and bios, "life"). Gnotobiotics is defined as the study of a species in the absence of all other species or in the presence of only known species. The absence of all species other than the ones under investigation cannot be demon- * Virus Research Laboratory and the Technology Division, Lobund Institute, University ofNotre Dame, NotreDame, Indiana. Readinpart beforethethirty-fourth annual meeting, AmericanEpidemiological Society, Albany, New York, April 6, 1957. Supported in part by an Office ofNaval Research Contract NR:i3i-i67 and in part by grants from the United States Public Health Service, National Institutes of Health, E 1223 and DAEG-63r. 447 strated unequivocally, but the absence of certain groups of organisms can be attested by specific examinations. Provided that the subject organism reproduces in a sterile environment, these examinations, of both samples and investigators, can be very extensive. Thus the presence ofknown species and the absence of all others can be determined with a high degree ofcertitude. This cannot be done by even the most elaborate examination ofvertebrates in their ordinary environment, for the teeming associated microbiota of most species can be recognized only by studies ofpure cultures. In contrast, one may synthetically assemble microbiota so that all varieties involved are known. The term "gnotobiota" is appropriate to designate biota ofknown composition assembled from pure isolates. An individual organism is termed a "gnotobiote," and their derivation and maintenance alone or in known association is termed "gnotobiotics." I. The Methods ofRearing and Handling Gnotobiotes Isolators constructed of rigid material for rearing "germfree" animals in closed systems are of three types: (a) a cylindrical "steam sterilizer," 28-34 inches in diameter, bearing sight ports and shoulder-length rubber gloves attached to the walls; (b) a light-walled, rectangular vessel ofabout the same capacity as above but equipped with an entire glass top and sterilizedwithin alarge steam sterilizer; (c) a large cylinder, 8 feet in diameter and 15 feet long, sterilized by steam under pressure and serviced by an attendant in a plastic suit. Isolators with transparent flexible plastic walls are now under test. Sterile materials are introduced or removed without disturbing the sterility ofthe isolator by means ofa liquid germicidal trap or a double door-pass autoclave attached to the wall. The 28-inch steel units have been used successfully to rear several species of gnotobiotes. Germfree rats have been reared to the ninth generation, Swiss mice to the ninth, and C3H mice to the seventh. Although no great effort has been expended to breed chickens through several generations, a germfree chicken was hatched from one offour eggs laid by a germfree hen mated with a germfree rooster. The bird survived eleven days and died of unknown causes. Despite considerable effort for several years, no success has been obtained in breeding gnotobiotic guinea pigs. Results have been similar with rabbits , though the effort has not been so great. The germfree...