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ROOT-NODULES OF LEGUMINOSAE. 137 
grew readily, these distant sub-cultures being used so as to avoid carrying over any of the numerous 
bacteroids. 
For planting, pots of washed and dried soil wrapped in paper were sterilized 1 hour in the 
autoclave at 134°C. Seeds of Vicia faba were soaked for half an hour in alcohol at 60° C., repeatedly 
washed in sterile distilled water and then dipped into one of the inoculating preparations. Inocu- 
lations were also made by adding 1 cc. of this inoculating material to the soil after sowing. Control 
pots for each experiment were prepared. No attempt was made to protect the soil from aerial con- 
taminations, but as the control plants produced no nodules the author thinks such a precaution 
unnecessary. After 2.5 months growth, no trace of nodules appeared on the roots of the controls or 
of plants inoculated with subcultures from the colonies which grew readily on his plates, while those 
inoculated with the bacteroids had formed many nodules. 
To meet the objection that failure to obtain nodules from his subcultures was due to loss of 
virulence, de Rossi states that loss of virulence commonly occurs in pathogenic bacteria only after 
a considerable time and many transfers, never after so short a period and so few transfers as in case 
of his organisms. Here loss of virulence is really loss of the invisible and hitherto unrecognized 
organism, viz., the non-germinating bacteroids. What has commonly been considered to be the right 
organism is only an intruder. 
In the course of further studies de Rossi discovered and isolated a very slow-growing organism 
which did not lose its virulence, and which he states to be quite unlike Bacillus radicicola in its 
morphology and cultural characters, which are described as follows: 
Young (non-vacuolated) bacteroids remain unchanged upon the plates (gelatin, agar, etc.) even 
when observed for 20 or 30 days or longer. As soon, however, as they become vacuolated they grow 
and form colonies. In this phase intruding colonies are fewer and often, for a time, the plates appear 
to be sterile. When, however, the plates are observed for a longer time, small formations are seen 
to appear (between the sixth and ninth days) which must be regarded as real colonies. 
These colonies develop from the vacuolated bacteroids, a process which de Rossi states he 
watched under the microscope (compare with statements by Hiltner). The bacteroid lost its typical 
form, changing to an amorphous heap of more or less spherical, tiny, bodies. Multiplication of these 
went on, enlarging the colony which, however, remained microscopic. ‘The colony was round and 
granular and composed of spherical, elongated, or bluntly branched bodies, held together by an 
apparently gelatinous substance. Photomicrographs of these are given. 
From this stage on, the various nutrient media had different effects. Upon simple nutrient gelatin 
nofurther development took place; on gelatin with Vicia faba extract the colonies enlarged so as to be 
visible to the naked eye as minute points. This requires 12 to 3c days. ‘These colonies were some- 
what raised and non-liquefying. In three cases the spherical and branched forms occurring in the 
colonies were all transformed into rods. In eight other strains from as many plants this change did 
not occur or remained incomplete, roundish and irregularly branched forms being mixed in with 
rods, up to the close of the observations. Further observations convinced him that all of these were 
pure cultures. ; 
Streak and stab cultures, in gelatin with Vicia faba extract, were made from colonies on these 
8 plates. The development was extraordinarily slow; within 8 to 10 days a very minute growth was 
observed, which after 25 to 30 days had taken on a characteristic appearance. At the point of inocu- 
lation a tiny drop of colorless gelatin-like substance rose strongly above the surface of the gelatin, 
containing and surrounding a white stearine-like bacterial growth. This substance colored intensively 
in microscopic preparations, and showed that it surrounded the usual peculiar forms. 
Four or five transfers in series were made and each time the growth was more luxuriant, the 
drops were whiter and the enveloping substance less abundant. At the same time the organism began 
to take on the form of rods. These rods stained with aniline dyes, but not by Gram’s method. In 
fresh cultures diluted with water and watched in hanging drops many motile individuals were seen. 
De Rossi was unable to demonstrate the flagella. After 6 or 8 days the rods in these little colonies 
became vacuolated and, when placed on peptonized nutrient gelatin, developed just as the vacuolated 
bacteroids from the nodules had done, 7. e., they became swollen and granular and then changed 
into tiny heaps of more or less rounded bodies. On this medium no further development took place. 
When, however, these swollen granular bodies were placed on gelatin with Vicia faba extract, they 
multiplied by division, reproducing the bacterial form. 
On other media, as bouillon, agar, potatoes, or carrots there was no development. In Moore’s 
agar (containing little nitrogen), and in silicate jelly (no nitrogen) growth was good, forming a thin, 
white, wide-spreading layer. 
Inoculations on Vicia faba, made as before, but with this newly isolated organism, gave most 
positive results. 
