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L6hnis and Smith (1923) pointed out that not less than seven different morphological types can be developed and stabilized from Azotobacter cultures. This extreme transmutation of cells has been questioned by subsequent investigators (Lewis, 1937, 1941). Nevertheless, peculiar morphological phases do occur in Azotobacter cultures and no satisfactory explanation has been given as to their function. Several new techniques, as well as some new concepts, have been developed since these earlier workers debated the significance of the morphological cell types in Azotobacter species. The purpose of the investigation now being reported was to see whether phase microscopy, electron microscopy, and nuclear staining might add information concerning the cytology of Azotobacter. Also, recent discussions of sexuality and peculiar reproductive processes in bacteria (Dienes, 1946; Lederberg, 1948) encouraged the authors of this paper to search for nuclear arrangements among large bacteria, such as in the genus Azotobacter. MATERIALS AND METHODS About 20 strains of organisms representing all species of the genus Azotobacter were observed. One culture, however, was chosen for this report so that results would be comparable. The organism selected for this study was Azotobacter agile (University of Illinois strain ZN 350). This species was grown on several different media, but the results in this report are from cells grown on Ashby's nitrogen-free medium. Cultures ranging in age from 3 hours to 60 days were examined. Each culture was observed with phase microscopy after light nigrosin background staining, according to a method previously described (Eisenstark and McMahon, 1949), to determine morphology and to view internal structures at different ages. Electron microscopy was done with the R.C.A. type EMC instrument. Cultures were prepared by suspension of cells in distilled water followed by centrifugation and resuspension. With full recognition that this method may produce slightly distorted cells,

MINOPTERIN may be used to select readily thymineless ( t h y ) mutants in A Escherichia coli ( OKADA, HOMMA, and SONOHARA 1962) ; however, this selection is more difficult in Salmonella typhimuriun strain LT2. Since this may be due to the rarity of thy mutation in this organism, the mutagen N-methy1-N'nitro-N-nitrosoguanidine (NG) was combined with aminopterin to successfully mutate and select t h y mutants (NISHIOKA and EISENSTARK 1964).Approximately 300 t h y mutants of S. typhimurium were collected (Table 1 ) and many of these were analyzed, seeking answers to these questions: ( 1 ) What is the precise position of t h y on the chromosome map of S. typhimurium (see SANDERSON and DEMEREC 1965 for detailed map)? (2) What loci are included in the same phage P22 transducing fragment with thy? (3) What is the clockwise map orientation of the thy locus and its neighbors? (4) Do all thy fall into a single locus? (5) Are all thy physiologically identical? (6) By reversion tests with a series of mutagens, which thy appear to be single-site and which multisite mutants? In the course of analyses, a number of observations led to additional probing of the degree of heterogeneity of transducing particles, sensitivity of cells to deoxyribosides, and the mapping of neighbors of thy.While this investigation was in progress, a report appeared (ALIKHANIAN et al. 1966) of similar experiments with E. coli. MATERIALS A N D METHODSConjugation methods for S. typhimurium were presented by SANDERSON and DEMEREC (1965). Most other materials and methods were described by DEMEREC et al. (1956), and special materials and techniques are presznted in RESULTS.Induction and selection of thy mutants: A series of liquid cultures was inoculated with a small number (ca. 103-104) of S. typhimurium-LT2 cells. The medium contained thymine (200 pg/ml), aminopterin (200 pg/ml), NG ( 3 @g/ml), K,HPO, ( I O pg/ml), KH,PO, (4.5 pg/ml), (NH,), SO, (1 pg/ml), sodium citrate (0.5 pg/ml), MgSO, (0.05 Pg/ml) and glucose (4 mg/ml). The salts were prepared in one batch, and each of the other components was prepared separately; all ingredients were mixed prior to use. NG and aminopterin w x e not sterilized.T w o days of incubation were required before turbidity appeared in the broth cultures. A loopful of grown culture was taken from each of the tubes and passed through single colony isolation on minimal f thymine (0.5 to 5 pg/ml) agar plates. Aminopterin and NG were omitted from the agar. Plates were incubated overnight. About 1 to 10% of the colonies were t h y and ' Slipported by National Science Foundation Grant GB4702. Contribution No. 976,