CULTTJRING ANAEROBES 



79 



without interfering with the anaerobic condition producing properties 

 of the fresh tissues. This method is practically the same as that recom- 

 mended by Smith (see Tetanus). This is also a feature of Noguchi's 

 method of culturing Treponema pallidum. 



The Method of Liborius 



In this it is necessary to have a test-tube containing about 4 inches of a i% 

 glucose agar. Glucose acts as a reducing agent and furnishes energy. It is con- 

 venient to add about i/io of i% of sulphindigotate of soda; the loss of the blue color 

 at the site of the colony enabling us to pick them 

 out. The tube of agar should be boiled just be- 

 fore using' to expel remaining oxygen from the 

 tube. Now rapidly bring down the temperature 

 to about 42C., by placing the tube in cold water, 

 and inoculate the material to be examined. A 

 second or third tube may be inoculated from the 

 first, just as in ordinary diluting methods for plate 

 cultures. Having inoculated the tubes, solidify 

 them as quickly as possible, using tap water or 

 ice-water. The anaerobic growth develops in the 

 depths of the medium. Some pour a little sterile 

 vaseline or paraffin or additional agar on the top 

 of the medium in the tube as a seal from the air. 

 Others have recommended the inoculation of some 

 aerobe, as B. prodigiosus, on the surface. This 

 latter method is not advisable. A deep stab 

 culture is often sufficient. 



The same technic can be applied to gelatin 

 cultures for anaerobes, pouring in at the comple- 

 tion of the inoculation an inch or so of melted 

 glucose agar to act as a stopper for the gelatin 

 layer below. 



FIG. 21. Arrangement of 

 tubes for cultivation of anae- 

 robes by Buchner's method. 

 (Mac NeaL) 



The Method of Buchner 



In this method i gram each of pyrogallic acid 

 and caustic potash or soda for every 100 c.c. of 

 space in the vessel containing the culture is used 

 to absorb the oxygen. It is convenient to drop 

 in the pyrogallic acid; then put in place the in- 

 oculated tubes or plates; then quickly pouring in the amount of caustic soda, in a 

 10% aqueous solution, to immediately close the containing vessel. A large test- 

 tube in which a smaller one containing the inoculated medium is placed, and which 

 may be closed by a rubber stopper, is very convenient. A good rubber-band fruit 

 jar is satisfactory. A desiccator may be used for plates. An excellent method for 



