But the condition which more than all others controls the quantity and quality of the contained bacteria is the degree and quality of the organic matter in the soil. The quantity of organic matter present in soil having a direct effect upon bacteria will be materially increased by placing in soil the bodies of men and animals after death. Dr. Buchanan Young two or three years ago performed some experiments to discover to what degree the soil bacteria were affected by these means. "The number of micro-organisms present in soil which has been used for burial purposes," he concludes, "exceeds that present in undisturbed soil at similar level, and this excess, though apparent at all depths, is most marked in the lower reaches of the soil."[35] The numbers were as follows:—
Virgin soil, 4 ft. 6 in. = 53,436 m.o. per gram of soil.
Burial soil (8 years), 4 ft. 6 in. = 363,411 m.o. per gram of soil.
Burial"lsoil(3 " ), 6 ft. 6 in. = 722,751m.o. pe"per gra"
Methods of Examination of Soil. Two simple methods are generally adopted. The first is to obtain a qualitative estimation of the organisms contained in the soil. It consists simply in adding to test-tubes of liquefied gelatine or broth a small quantity of the sample, finely broken up with a sterile rod. The test-tubes are now incubated at 37° C. and 22° C., and the growth of the contained bacteria observed in the test-tube, or after a plate culture has been made. The second plan is adopted in order to secure more accurate quantitative results. One gram or half-gram of the sample is weighed on the balance, and then added to 1000 cc. of distilled sterilised water in a sterilised flask, in which it is thoroughly mixed and washed. From either of these two different sources it is now possible to make sub-cultures and plate cultures. The procedure is, of course, that described under the examination of water (p. 41 et seq.), and Petri's dishes, Koch's plates, or Esmarch's roll cultures are used. Many of the commoner bacteria in soil will thus be detected and cultivated. But it is obvious that this by no means covers the required ground. It will be necessary for us here to consider the methods generally adopted for growing anaërobic bacteria, that is to say those species which will not grow in the presence of oxygen. This anaërobic difficulty may be overcome in a variety of ways.
1. The air contained in the culture tube may be removed by ebullition and rapid cooling. And whilst this may accurately produce a vacuum, it is far from easy to introduce the virus without also reintroducing oxygen.
2. The oxygen may be displaced by some other gas, and though coal-gas, nitrogen, and carbon dioxide may all be used for this purpose, it has become the almost universal practice to grow anaërobes in hydrogen. The production of the hydrogen is readily obtained by Kipp's or some other suitable apparatus for the generation of hydrogen from zinc and sulphuric acid. The free gas is passed through various wash-bottles to purify it of any contaminations. Lead acetate (1–10 per cent. water) removes any traces of sulphuretted hydrogen, silver nitrate (1–10) doing the same for arseniated hydrogen; whilst a flask of pyrogallic acid will remove any oxygen. It is not always necessary to have these three purifiers if the zinc used in the Kipp's apparatus is pure. Occasionally a fourth flask is added of distilled water, and this or a dry cotton wool pledget in the exit tube will ensure germ-free gas. From the further end of the exit tube of the Kipp's apparatus an india-rubber tube will carry the hydrogen to its desired destination. With some it is the custom to place anaërobic cultures in test-tubes, and the test-tubes in a large flask having a two-way tube for entrance and exit of the hydrogen; others prefer to pass the hydrogen immediately into a large test-tube containing the culture (Fränkel's method). Either method ends practically the same, and the growth of the culture in hydrogen is readily observed. Yet another plan is to use a yeast flask, and after having passed the hydrogen through for about half an hour, the lateral exit tube is dipped into a small flask containing mercury. The entrance tube is now sealed, and the whole apparatus placed in the incubator. The interior containing the culture is filled with an atmosphere of hydrogen. No oxygen can obtain entrance through the sealed entrance tube, or through the exit tube immersed in mercury. Yet through this latter channel any gases produced by the culture could escape if able to produce sufficient pressure.
Kipp's Apparatus
For the Production of Hydrogen
3. The Absorption Method. Instead of adding hydrogen to the tube or flask containing the anaërobic culture, it is
Fränkel's Tube
For Cultivation of Anaërobes feasible to add to the medium some substances, like glucose