ABSTRACTION OF AIR FROM CULTURE MEDIA 89 



in commerce guaranteed to contain 99'6 per cent, of hydrogen, the remaining 

 O4 per cent, being almost if not entirely composed of air, which represents 

 O08 per cent, of oxygen. When used for the cultivation of anaerobic 

 organisms in a Bulloch's apparatus (pp. 96 and 100) which is the method 

 usually adopted in England the gas requires no preliminary washing, 

 but is passed direct from the cylinder into the bell jar containing the 

 cultures.] 



B. Carbonic anhydride. Carbonic anhydride is harmful to a large number 

 of organisms, and its use for that reason is not to be recommended in the 

 present connexion. The apparatus described above for the preparation of 

 hydrogen can be utilized for the preparation of the gas, if pieces of white 

 marble be substituted for the zinc, and hydrochloric acid for the sulphuric 

 acid. The gas should be washed by passing it through a solution of sodium 

 hydrosulphite contained in the wash-bottle F (fig. 75). 



C. Nitrogen. The preparation of this gas is so difficult that its use should 

 be abandoned in practical bacteriology. [Nitrogen can however now be 

 obtained as a commercial product in the form of cylinders of the compressed 

 gas, which on analysis is found to contain very little oxygen. In our experi- 

 ence the results obtained with this compressed gas in the growth of anaerobic 

 organisms have been quite satisfactory.] 



D. Coal gas. The use of coal gas is not to be recommended in anaerobic 

 methods, because many of the component gases comprising the mixture 

 are inimical to micro-organisms. 



Note. Before passing any gas into a culture medium it must be sterilized by 

 filtration through a sterile cotton- wool plug. The technique of this operation will 

 be referred to later. 



3. By absorbing the oxygen. 



A. Advantage may be taken of the affinity possessed by some substances 

 for combining with oxygen to remove the latter from culture media. In 

 practice oxygen is generally absorbed by resting the culture-tube on a glass, 

 or metal, support inside a much larger tube (about 20 to 25 cm. in length), 

 and then pouring the following solution into the latter : 



Pyrogallol, - 1 gram. 



Alcoholic potash, - 1 



Water, - - 10 c.c. 



Plug the outer tube with a tightly-fitting india-rubber bung. Under 

 these conditions oxygen diffuses through the wool plug of the inner culture- 

 tube and, being absorbed by the pyrogallol, turns the solution brown. 



Sellards, using a similar apparatus, substitutes fragments of phosphorus for the 

 potassium pyrogallate solution. 



B. In some cases it will be found convenient to add to the medium some 

 easily oxidizable substance, which does not interfere with the growth of the 

 organism ; e.g. glucose (2 per cent.), formate of soda (0'5 per cent.), sodium 

 sulphindigotate (0*1 per cent.), fragments of tissue, etc. This method is 

 generally adopted in the case of deep stab cultures in agar (vide infra). 



C. By sowing the surface of an anaerobic culture in a solid medium with 

 some aerobic organism which absorbs a good deal of oxygen, air can be 

 prevented from reaching the anaerobic culture, the growth of the latter 

 taking place beneath the growth of the aerobic organism (Roux). This 

 method will be described in detail when dealing with stab cultures. 



