It was observed that in a series of pots of peas to which no nitrogen was added most of the plants were apparently limited in their growth by the amount of nitrogen locked up in the seed. Here and there, however, a plant, under apparently the same circumstances, grew luxuriantly and possessed on its rootlets abundant nodules. The experiments of Sir John Lawes and Sir Henry Gilbert at Rothamsted[46] demonstrated further that under the influence of suitable microbe-seeding of the soil in which _Leguminosæ_ were planted there is nodule formation on the roots, and coincidentally increased growth and gain of nitrogen beyond that supplied either in the soil or in the seed as combined nitrogen. Presumably this is due to the fixation, in some way, of free nitrogen. Nobbe proved the gain of nitrogen by non-leguminous plants (Elœagnus, etc.) when these grow root nodules containing bacteria, but to all appearances, bacteria differing morphologically from the _Bacillus radicicola_ of the leguminous plants. [Illustration: NITROGEN FIXING BACTERIA _IN SITU_ IN NODULE ON ROOTLET OF PEA × 400 --Cellular sheath of Rootlet forming capsule of nodule. --Colonies of bacteria _in situ_.] [Illustration: NITROGEN FIXING BACTERIA _IN SITU_ IN ROOT-NODULE OF PEA (Section of Nodule) × 500] [Illustration: NITROGEN FIXING BACTERIA _IN SITU_ IN ROOT-NODULE OF PEA (Section of Nodule) × 600] These facts being established, the question naturally arises, How is the fixation of nitrogen to be explained, and by what species of bacteria is it performed? In the first place, these matters are simplified by the fact that there is very little fixation indeed by bacteria in the soil apart from symbiosis with higher plants. Hence we have to deal mainly with the work of bacteria in the higher plant. Sir Henry Gilbert concludes[47] that the alternative explanations of the fixation of free nitrogen in the growth of _Leguminosæ_ seem to be: "1. That under the conditions of symbiosis the plant is enabled to fix the free nitrogen of the atmosphere by its leaves; "2. That the nodule organisms become distributed within the soil and there fix free nitrogen, the resulting nitrogenous compounds becoming available as a source of nitrogen to the roots of the higher plant; "3. That free nitrogen is fixed in the course of the development of the organisms within the nodules, and that the resulting nitrogenous compounds are absorbed and utilized by the host." "Certainly," he adds, "the balance of evidence at present at command is much in favour of the third mode of explanation." If this is finally proved to be the case, it will furnish another excellent example of the power existing in bacteria of assimilating an elementary substance.