118 



BACTERIA IN THE SOIL 



t 



7 



from zinc and dilute sulphuric acid, or it may be provided in a cylinder. The free 

 gas is passed through various washbottles to purify it of any contaminations ; e.t?. 

 lead acetate (1-10 of water) removes any traces of sulphuretted hydrogen, silver 

 nitrate (1-10) doing the same for arsenlaled hydrogen ; vi-hilst a flask of pyrogallate 

 of potash will remove any oxygen. It is not 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 stopper in the exit tube, will ensure 

 germ-free gas. From the further end of the exit tube of the Kipp's apparatus an 

 indiarubber tube will carry the hydrogen to its desired destination. With some it is 

 the custom to place anaerobic cultures in test-tubes, and the test-tubes in a large 

 flask, tube, or desiccator, having a two-way tube for entrance and exit of the 

 hydrogen, or Petri dishes may be used and placed in well-sealed jar or desiccator ; 

 others prefer to pass the hydrogen immediately into a large test-tube containing the 

 culture (Frankel's method). Either method, if properly carried out, will be found 

 effectual, and the growth of the culture in hydrogen is readily observed. 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 capsule containing mercury 

 (as in Plate 9). The entrance tube is now sealed, and 

 the whole apparatus placed in the incubator. The 

 interior of the flask 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 may 

 escape. 



2. The Absorption Jisi^od— Instead of adding hydro- 

 gen to the tube or flask containing the anaerobic culture, 

 it is feasible to add to the medium substances, such as 

 glucose or pyrogallic acid, which will absorb the oxygen 

 which is present, and thus enable the anaerobic require- 

 ment to be fulfilled. To various media — gelatine, agar, 

 or broth (the latter used for obtaining the toxins of 

 anaerobes) — 2 per cent, of glucose may be added. 

 Pyrogallic acid, or pyrogallic acid one part and 20 per 

 cent, caustic potash one part, is also readily used for 

 absorptive purposes. A large glass tube of 25 c.c. height, 

 termed a Buchner's cylinder, having a constriction near 

 the bottom, is taken ; and about two drachms of the 

 pyrogallic solution are placed in the bulb. A test-tube 

 containing the culture is now lodged in the upper part 

 above the constriction, and the mouth of the Buchner 

 tube is carefully sealed. The apparatus is then placed in the incubator at the desired 

 temperature, and the contained culture grows under anaerobic conditions. As the 

 pyrogallic solution absorbs the oxygen it assumes a darker tint. 



3. Mechanical Methods. — These include various ingenious methods for preventing 

 an admittance of oxygen to the culture. An old-fashioned one was to plate out the 

 culture and protect it from the air by covering it with a plate of mica. A more 

 serviceable mode is to inoculate, say, a tube of agar with the anaerobic organism, 

 and then pour over the culture a small quantity of melted agar, which will readily 

 set, and so protect the culture itself from the air. Oil or vaseline may be used 

 instead of melted agar. Another mechanical method is to make a deep inoculation, 

 and then melt the top of the medium over a Bunsen burner, and thus clo§e the 

 entrance puncture and seal it from the air. 



4. Absorption of Oxygen by an Aerobic Culture. — This method takes advantage 

 of the power of absorption of certain aerobic bacteria, which are planted over the 

 culture of the anaerobic species. It is not practically satisfactory, though occasionally 

 good results have been obtained. 



5. Lastly, there is the Vacuum Method. — By this means it is obviously intended 

 to extract air from the culture and seal it in vacuo. The culture tubes are connected 



X^ 



Fig. is.— Frankel's Tubk. 

 For Cultivation of Anaerobes. 



