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 nodules on this plant. 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 denned 

 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(l) and Sarothamnus(?). Buhlert in reaching his conclusion that but one 

 species exists used only bacteria from the first of these groups, i. e., from Vicia j aba and Pisum 

 sativum. 



Hiltner states that at no time has he considered a thoroughly adapted form as an unalterable one. 



Maze 1 rejected the adaptation theory and distinguished two groups based on the acidity or alka- 

 linity of the soil in which they live. Hiltner's experiments on this subject makesuch a view 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, 1 1 bore root-nodules and 2 were free. The soy-bean bacteria certainly belong with the 

 lupin bacteria in morphology and biological peculiarities, yet the soy is a kind of bean and not hostile 

 to lime. 



Mazd'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 Maz and Stoklasa 

 that pure cultures of the pea bacteria were obtained direct from the soil at Tharandt as long ago as 

 1890. 



In general, Maze'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 Maz6, 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 and drying 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 i per cent grape-sugar and o.i 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 o.i 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, i. 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. The 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 : i .5 per cent agar, 2 per cent root-extract, i per cent 

 grape-sugar, heated in the autoclave 20 minutes at 120. To 0.5 liter of this solution is added a 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 i per cent pepton was present in the agar, pea 



