420 
Journal of Agricultural Research 
Vol. XXIII, No. 6 
mannite-nitrate solution, especially with Ph 7.5, the tendency to reproduce large 
nonsporulating Azotobacter cells was sometimes very marked, and transformations 
were effected accordingly. 
Staining after Gram was always positive. 
Motility was always absent. 
Gonidia are often liberated in great numbers especially from the slimy sheaths 
(fig. 40 on PI. 4) and w r ere frequently seen to grow up to rods or to pass over to the 
yellow, dwarfed growth. 
Gonidangia. —The large, slimy threads containing numerous gonidia may be 
accepted as such. In addition the large, globular cells, produced in soil and in 
mannite-nitrate solution, which later grew as regular Azotobacter, are typical goni¬ 
dangia. Such globules, when developed from the symplastic stage on potato agar, 
quickly dissolved into heaps of small coccoid cells (regenerative bodies). 
Regenerative bodies were produced regularly in lateral or in terminal position. 
The former were seen to reproduce small spore-free rods, while the latter were inclined 
to become endospores. 
Arthrospores and microcysts were frequent on all substrates, but most abundant 
in water. 
Endospores were developed from the terminal regenerative bodies in five cases. 
When the microscopic picture was similar to that shown in figure 45 on Plate 4, the 
material was heated in beef broth or in mannite-nitrate solution first to 70° C., later 
to higher temperatures up to 90° C., and from successful tests cultures were made on 
potato and on beef agar. Heated material gave at first development intermediate 
between normal fungoid growth and small sporulating rods. Plating and repeated 
heating permitted final separation. 
Symplasm produced very manifold growth (Figs. 38, 47, and 48 on PI. 4, Figs. 100, 
102, and 107 on PI. 9), although the typical pale, irregular sheaths w T ith dark granules 
appeared most frequent. The large spore-free cells originated always from the sym¬ 
plasm. Irregular sclerotia, like those reproduced in figure 108 on Plate 9, were also 
found with this type of growth. 
Colonies on mannite-nitrate agar and on beef agar were in the beginning smaller, 
but otherwise very similar to regular Azotobacter colonies. When the fungoid growth 
was more firmly established,, the colonies appeared macroscopically whitish, fiat, with 
raised center and irregular edge, microscopically brown in the center, of hairy struc¬ 
ture, with thin, transparent, fringed edge, very similar to Figure X of Table 69 in 
Lehmann’s and Neumann’s atlas (1#). When the tendency prevailed to assume a 
more regular rod-shaped growth, the colonies became circular with sharp edge and fine 
granulation, whereas the inclination to change to sporulating rods caused the appear¬ 
ance of smaller colonies with hairy structure and irregular moss-like edge, similar to 
small colonies of Bacillus mesentericus. 
Agar slants. —A flat, dry, grayish or whitish growth with thin, irregular edge is 
typical on beef agar as well as on mannite-nitrate agar. Often white spots are scattered 
over the surface. If the small spore-free rods are developing a thicker slimy white or 
slightly pink growth appears, while the change to small sporulating rods is accompanied 
by a slightly yellowish color of the layer, which may show a few wrinkles. Potato agar 
produced a thick yellowish pinkish growth with yellow secondary colonies, from which 
a pure growth of yellow coccoids was branched off. 
Beef-gelatine. —Irregular, thin, grayish or pinkish surface growth, little develop¬ 
ment in stab; no liquefaction. 
Beef broth. —Uniform turbidity, loose grayish film, whitish ring, heavy slimy 
white sediment. 
Milk unchanged during the first days, then slowly peptonized, turning brown, 
becoming alkaline and very slimy; on the surface often a brittle film and a whitish 
ring are visible. After repeated transfers from milk to milk the peptonization and 
brown discoloration ceased and the milk remained neutral and became very ropy; the 
cells had changed from the fungoid type to small nonsporulating rods. 
Potato.—T ypical raised, dry, pink growth with irregular surface and edge. Rarely 
a yellowish grayish brownish, more or less slimy growth was produced; in these cases 
the irregular cells assumed rod shape, 
Mannite-nitrate solution became slightly acid, remained practically clear; on 
the surface a thin grayish film developed, which often ascended on the walls of the 
tubes and frequently showed the white dots characteristic of the growth of Azotobacter 
Beijerinckii; on the paper above the solution a thick slime developed, which later 
turned brownish, and a heavy, white, slimy sediment was formed in the solution. 
Nitrogen fixation by pure cultures in mannite solution was not noticeable, but 
there was a pronounced tendency to produce the wrinkled dotted film, characteristic 
of crude Azotobacter cultures, especially when the fungoid cells grew in symbiosis 
with the dwarfed cells. 
