ROOT-NODULES OF LEGUMINOSAE. 129 
studied this question carefully would, for example, take for granted that a colony of true nodule 
bacteria taken from a sound pea nodule would be composed of pea bacteria. Hiltner, however, from 
many observations and much reflection has learned to be very cautious on this point. He asks, for 
instance, whether he has any guarantee that he is obtaining true pea bacteria when he uses for obtain- 
ing his pure cultures pea nodules from Dahlem soil. In the Dahlem soil peas, vetches, clovers, lupins 
and serradella produce root-nodules without inoculations. ‘Therefore, might not clover or lupin 
bacteria have wandered in after the way had been prepared by the true pea bacteria, and in conse- 
quence, might not the colonies chosen for further cultivation consist of clover bacteria instead of 
pea bacteria. To avoid this possibility, cultures for inoculation should not be set from single colonies. 
If the nodule bacteria comprise only one widely adapted species, as Nobbe and Hiltner formerly 
believed, it might be assumed that even these stray forms would by growth in the pea attain a greater 
or less degree of adaptation so that they could produce noduleson thisplant. This conclusion is correct 
so long as the hypothesis holds true, but this is the case only to a limited degree. More recent experi- 
ments have led Hiltner to discard the single species theory and to distinguish two sharply defined 
groups which have the character of distinct species: One group contains Pisum, Vicia, Lathyrus, 
Phaseolus, Trifolium, Medicago, Anthyllis, Onobrychis, and Robinia; the other contains Lupinus, 
Ornithopus, Soja, Genista(?) and Sarothamnus(?). Buhlert in reaching his conclusion that but one 
species exists used only bacteria from the first of these groups, 7. e., from Vicia faba and Pisum 
sativum. ; 
Hiltner states that at no time has he considered a thoroughly adapted form as an unalterable one. 
Mazé rejected the adaptation theory and distinguished two groups based on the acidity or alka- 
linity of the soil in which theylive. Hiltner’s experiments on this subject makesuch aview untenable, 
e. g., the Robinia bacteria do not cause tubercles on pea roots or vice versa, and yet the Robinia is 
not injured by lime. In the experimental garden at Hohenheim, of 13 kinds of lupins growing close 
together, 11 bore root-nodules and 2 were free. The soy-bean bacteria certainly belong with the 
lupin bacteria inmorphology and biological peculiarities, yet the soy is a kind of bean and not hostile 
to lime. 
Mazé’s view that nitrogen assimilation takes place whenever an organism is able to cause nodules 
on a plant is also incorrect; for example, pea bacteria may penetrate into bean roots forming hundreds 
of nodules, yet often no nitrogen assimilation takes place. 
His assumption that inoculation is of no use in soil containing no nodule bacteria since this fact 
of itself shows that they cannot grow there is unjustified because Hiltner’s first work sufficed to show 
the contrary. Moreover, a careful study of literature would have shown both Mazé and Stoklasa 
that pure cultures of the pea bacteria were obtained direct from the soil at Tharandt as long ago as 
1890. 
In general, Mazé’s theories are of this character that in their expression he disregards the results 
accumulated in abundance by different investigators during 10 years. Concerning the strange 
pleomorphisms reported by Mazé, Hiltner says comment is unnecessary. 
As a nutrient medium, Hiltner has used Beyerinck’s medium, viz., gelatin with extract of legu- 
minous leaves, 0.5 per cent cane-sugar, 0.25 per cent asparagin and a little malic acid, but has found 
it advantageous to substitute root-extract for leaf-extract. After evaporating anddrying the extract 
at 102° C. so that definite amounts might be taken each time, a 0.2 per cent solution was made to 
which was added 1 per cent grape-sugar and o.1 per cent to 0.2 per cent asparagin. Only the best 
gelatin, e. g., Griibler’s, must be used. Very acid gelatin should be avoided since its neutralization 
with sodium or potassium hydroxide introduces too much chloride. After the first cooking, the 
gelatin is neutralized with soda or potash and made moderately acid (deutlich) to litmus paper with 
malic acid. A second good medium for nodule bacteria is agar containing 2 per cent legume extract, 
I per cent grape-sugar, and 0.1 to 0.2 per cent asparagin. ‘The root-extract gives enough acidity. 
No alkalies should be added, nor malic acid, since excess of acid in agar interferes with growth, which 
is not the case in the gelatin. Both the gelatin and agar should be heated as little as possible. 
All the forms prosper on the agar but not all on the gelatin as already mentioned, 7. e., lupin, 
serradella, and soja do not. The gelatin was not rendered suitable to them by omission of the aspara- 
gin, or by the addition of 10, 20, or 30 cc. of normal soda solution, or by the same amount of normal 
malic acid solution, or by addition of CaCO3 after acidifying with malic acid. ‘he addition of 
asparagin had no good effect. For these bacteria agar was found the most suitable medium when 
neither acid nor alkaline, but made either neutral or nearly neutral to phenolphtalein, with CaCO3. 
The following composition gave the best results: 1.5 per cent agar, 2 per cent root-extract, 1 per cent 
grape-sugar, heated in the autoclave 20 minutes at 120°. To 0.5 liter of this solution is addeda knife 
point full of carbonate of lime. The mixture is then heated 10 minutes at 120° and filtered. 
Tests with the addition of different amounts of pepton gave varying results. Soja bacteria were 
not injured even by 10 per cent pepton, while when 1 per cent pepton was present in the agar, pea 
