Divergent views exist concerning the morphology of Bacterium tularense. McCoy and Chapin's (1912) original description depicted an apparently immotile, small, questionably encapsulated, pleomorphic organism occasionally presenting enlarged, irregular, and apparent involution fonns; sometimes with predominant large globular forms. Ohara, Kobayashi, and Kudo (1935) claimed to have demonstrated flagella on both Japanese and American strains, stated that motility was observed repeatedly, and described clubbed, comma-shaped, dumbbell, and triangular forms. They further stated, "The more virulent is the bacterium, the greater is the pleomorphism"; also, "Virulence, pleomorphism, and motion are closely related and vary together." Galli-Valerio (1938), after working with cultures isolated by Drbohlav in Czechoslovakia, stated that both coccoid and bacillary forms were absolutely immotile and that no flagella were demonstrable by the Casares-Gil stain. He also failed to find any enlarged, elongated, filamentous, or involution forms. Most European language reports and textbooks omit all reference to flagella, state that the bacterium is nonmotile, present inadequate descriptions of the extraordinary pleomorphism, give scant mention to encapsulation except as an occasional finding in tissues or in tissue smears, and classify the organism among either Pasteurella or Brucella. Since the most extensive studies on morphology were conducted by Ohara and his associates, we reviewed thoroughly the Japanese literature. Ota (1936) states that Kudo and Kobayashi, working in Ohara's laboratory, first demonstrated a single polar flagellum in 1934, using the silver deposition method of Nishigawa and Sugahara (apparently the same as the Saisawa-Sugawara method mentioned by Ohara). They also observed capsules. Ota confirmed this work. He demonstrated flagella also with Victoria blue (4R), Burri's India ink method, and by his own modification of Benian's Congo red method. In his experience the methods of Loeffler, Benian, Zettnow, Inouye, Yokota, and Uyeno either failed entirely or showed few poorly stained flagella. Under dark-field illumination, "Refractile flagella were demonstrated." With regard to motility, "I found some actively motile, definitely changing their position." Capsules were well demonstrated by Ota's modification of Benian's method, mercurochrome negative staining, and by Gin's India-ink carbol-thionine method. The methods of Johne, Wadsworth, Hiss, Welch, and Friedlander were said to stain them poorly or not at all. Ota stained Bacterium tutarense and Yato-byo bacteria, also their flagella, in tissues with the Levaditi method. Successful preparations were made from human lymph node and skin, guinea pig spleen, and rabbit liver. 1 In partial fulfillment of the thesis requirements for the degree of Doctor of Philosophy.

Throughout our morphologic study of Bacterium tularense in liquid medium cultures we noted constantly the presence of very minute forms (Hesselbrock and Foshay, 1945). These appeared to grade downward in size from the most prevalent coccoid or ring forms of about 0.5 ,u in diameter to bodies so small that neither size nor shape was discernible. Such particles were never seen in uninoculated media, either before incubation or for 96 hours thereafter. Some particles were discrete and freely suspended; others were attached by delicate short filaments to larger morphologic units. Many additional ones were seen lying in, and apparently arising within, delicate filaments of variable length. The number of discrete particles was never large at any time in any of the many cultures that were examined. Single particles in suspension, as well as those filamentously attached.or interconnected, stained a brick-red color when supravirtally stained with 2 per cent aqueous malachite green, whereas all large morphologic units stained green. None of six other common pathogenic bacteria, cultivable in the same medium, produced particles of this size. Prolonged intermittent observations of cultures, although inadequate to establish the fact, suggested strongly that these particles were capable of reproducing all other formns of this pleomorphic organism and, apparently by means of larger units, of reproducing themselves. Our provisional interpretation was that they represented true morphologic units of Bacterium tularense, possibly to some degree analogous in size and function to the minute elementary bodies formed by organisms of the pleuropneumonia group, appropriately called minimal reproductive units, M.R.U. (Sabin, 1941).