126 BACTERIA IN RELATION TO PLANT DISEASES. 
i. e., sproutings which stained strongly on nearly every rod and which developed sidewise often 
the whole length of the rod. 
Asparagin in 1 per cent grape-sugar gave results similar to those of saltpeter and grape-sugar, 
- that is the differentiation of nuclear plasma from cell-plasma took place only when the greater part 
of the asparagin had been used up by the bacteria. 
From these results and similar ones obtained with peptone and grape-sugar solutions many of 
which are figured, Hiltner concludes that the differentiation of the plasma and theAussprossungen 
connected therewith takes place only when the source of nitrogen is largely exhausted by the bacteria.* 
In his experiments with phosphoric acid, Hiltner failed to verify Hartleb’s statement that only 
by the addition of alkaline salts of phosphoric acid is bacteroid formation induced in liquid media 
containing carbon and nitrogen. In Hiltner’s carbon-free cultures the potassium phosphates were 
unable to bring about bacteroid formation. In cultures containing, in addition to the potassium 
phosphate, 0.1 per cent to 5 per cent grape-sugar, growth took place, but bacteroid formation was 
less marked than in the corresponding pure grape-sugar solutions. He concludes, therefore, that in 
such fluids the bacteroid formation is due exclusively to the presence of the carbon compounds and 
that the addition of phosphates, while it favors multiplication, retards bacteroid formation and the 
differentiation of the plasma. The réle of the phosphate is solely that of nutrition. Saltpeter alone, 
of all the non-carbon substances tried, was able to cause bacteroid formation by itself. 
The effect of ten other carbohydrates was tested in comparison with that of grape-sugar. One 
per cent sugar solutions were used (0.5 per cent with the pentoses) with and without o.1 per cent 
asparagin. Laevulose wasespecially active in its influence on soy-bean bacteria. In 5 days the plasma 
had gathered into a spore-like body. ‘Two weeks after the bacteroids were very large with outgrowths 
and a month later were immense bodies staining poorly with carbol fuchsin but well with tincture of 
iodine. All possessed very large, granular, honey-combed outgrowths which stained strongly. 
Raffinose, cane-sugar, mannit, and galactose had the greatest effect on pea bacteria, while 
laevulose had here almost no effect. 
Robinia bacteria grew well in cane-sugar and mannit, fairly well in laevulose and lactose but 
poorly in all the other solutions. Cane-sugar had the greatest effect and grape-sugar the least effect 
on bacteroid formation and plasma differentiation. 
Tests with organic acids showed that these have a variable effect on bacteroid formations 
expressed by branching, plasma differentiation, and outgrowth. Hartleb’s objections to Stutzer’s 
statements are, therefore, untenable. Succinic acid was most active in its effect while citric acid was 
least so. The simultaneous addition of grape-sugar hastened the breaking up of the peculiar forms 
(outgrowths) developed from the bacteroids. 
The most striking fact in bacteroid formation is the outpushing of the nuclear plasma, a process 
which occurs in the nodules as well as in artificial media. This process does not leave behind an 
empty membrane, but rather consists of a differentiation of the plasma into an out-pushing nuclear 
plasma and a nutrient plasma that remains behind. As shown by the power of a bacteroid to resume 
growth after this outgrowth has occurred, some of the nuclear plasma must remain within the rodlet, 
just as the outgrowth must contain some nutrient plasma. 
The red-brown stain is not a reaction of the nuclear plasma as such, but rather of an unorganized 
substance arising from its activity. This may be easily separated from the plasma by different 
solutions and also may be worked over and used by it so that the red-brown color does not always 
appear when iodine is applied. For example, one finds very often in the same culture bacteroids 
with outgrowths which take this red-brown color along with those which stain pure yellow. 
Prazmowski pointed out this plasma differentiation and considered the refractive red-brown sub- 
stance as a peculiar form of albumen, formed under the influence of the plant. Frank’s results also 
agree with these so far as the differentiation of the plasma is concerned. Frank further distinguished 
in the pea two sorts of nodules, one containing albumen, the other amylodextrin. Mdller has shown. 
however, that this latter substance stains with iodine like glykogen and not like amylodextrin. 
Further evidence against the starchy nature of these granules lies in the fact that they do not swell 
in concentrated calcium nitrate and are not changed by boiling water. They are insoluble in cold 
dilute potash lye, cold or boiling concentrated ammomia, in hot ethyl alcohol and amyl alcohol, in 
ether, benzine or carbon bisulphide. This substance is not vaporized or otherwise changed by careful 
heating over the flame. It is easily dissolved by chloroform, aceton, glacial acetic acid, clove oil, 
and less easily by benzol. ‘These reactions indicate that the ground substance is neither a carbo- 
hydrate nor albumen, but a fatty or waxy substance corresponding most nearly to cholesterin, but 
not identical. 
*Similar extrusions from the cells have been observed by Kuntze and others in the lactic acid group of bacteria 
grown in whey, these bodies being Gram negative (see White and Avery, Centralb. f. Bakt., 1 Abt.,Bd. 25, 1909, p.165). 
