and Laboratory Methods. 1741 



A Review of the Existing Methods of Cultivating Anaerobic 



Bacteria. 

 II. 



REPLACEMENT OF AIR BY INERT GASES. 



This principle has been employed by Pasteur in the study of his Vibrion 

 butyricus as early as 1861. He cultivated this organism in an atmosphere of 

 hydrogen and carbonic acid. Since then many methods of this type have been 

 introduced and put in practice. Of the various gases that have found applica- 

 tion as a means of replacing the air, hydrogen proved to be, everything consid- 

 ered, the most satisfactory. 



Hydrogen is produced most conveniently by means of the Kipp Generator, 

 dilute hydrochloric acid and metallic zinc being used. The hydrogen thus pro- 

 duced is freed from traces of AsHg, SHg and PHg that may be present, by 

 passing the gas through concentrated KMn04, from acids that may have been 

 carried over from the generator by passing it through concentrated KOH, from 

 traces of oxygen by passing it through an alkaline solution of pyrogallol, and 

 from water by passing it through dry CaCl2 or concentrated H2SO4. If 

 chemically pure zinc is used, washing is not necessar}-. 



When a union is to be made between two glass tubes by means of rubber 

 tubing the ends of the glass tubes should meet. This avoids the direct exposure 

 of large surfaces of rubber tubing to the action of hydrogen and prevents the 

 diffusion of hydrogen through the porous walls of the rubber tubing. In any 

 case it is advisable to vaselinate the rubber tubing. 



If the apparatus is sealed by means of glass turn-cocks, only cocks with 

 diagonal openings should be used. They afford a perfect seal. 



High pressure in the apparatus tends to seriously affect the cultures and 

 should therefore be avoided. Slight over-pressure which will not materially dis- 

 turb growth is desirable as a means of preventing any possible diffusion of 

 gases. Where convenient, it is well, instead of filling the apparatus with hydro- 

 gen and sealing it hermetically, to pass the hydrogen through the apparatus con- 

 tinually during the whole period of cultivation. In this case the hydrogen, upon 

 leaving the culture apparatus, is conducted through a doubly perforated rubber 

 stopper to the bottom of a wash bottle containing distilled water. The water 

 forms a hermetical seal, preventing the air from entering the apparatus in case 

 the gas pressure should diminish. 



The purity of the gas in the culture apparatus is tested by filling a test tube 

 with the escaping hydrogen and applying a match to it. If the gas burns with 

 explosion, the apparatus still contains some atmospheric oxygen, a quiet flame 

 indicates pure or nearly pure hydrogen. 



A. CULTURES IN TUBES AND FLASKS. 



Hauser and Liborius (1886) introduced the apparatus shown in Fig. 8. A 

 short distance above the surface of the medium the test tube carries a lateral 



