METHODS USED IN CULTIVATION OF BACTERIA 183 



For anaerobic cultivation upon solid media, the inoculated tubes or plates 

 are placed in an apparatus such as the Novy jar. This is connected with 

 the hydrogen apparatus and hydrogen allowed to flow through it for five 

 or ten minutes, and the stop-cocks then closed. 



In applying the hydrogen method to fluid media, flasks containing the 

 broth are fitted with sterile, tightly fitting rubber stoppers perforated by 

 two holes, through which glass tubes are passed. One of these tubes, the 

 inlet, passes below the surface of the liquid. The other one, the outlet, 

 extends only a short distance below the stopper and is always kept above 

 the surface of the medium. The flasks are inoculated and hydrogen is 

 passed through the medium so that it enters the long tube, bubbles up through 

 the fluid, and leaves by the short tube. The broth may be covered with a 

 thin layer of liquid paraffin or albolin. 



The Use of Pyrogallic Acid Dissolved in Alkaline Solutions for Oxygen 

 Absorption. Buchner 8 has applied the principle of chemical absorption for 

 the removal of oxygen to the cultivation of anaerobic bacteria. This has 

 been made use of in a number of different ways. The method is based upon 

 the fact that alkaline solutions of pyrogallol possess the power of absorbing 

 large quantities of free oxygen. At first such solutions are of a light 

 straw-color, which becomes dark brown as oxygen is absorbed. The absorp- 

 tion of all the oxygen in the environment may be assumed when there is no 

 further deepening of the brown color. 



Buchner first utilized this principle by placing a small wire or glass holder 

 within a large test tube, dropping dry pyrogallol (pyrogallic acid) into the 

 bottom of this tube, then running thirty per cent sodium hydrate solution 

 into it, and inserting within this large tube a smaller test tube containing 

 the inoculated culture medium. The large tube was then tightly closed 

 with a rubber stopper. In this way, the air space surrounding the smaller 

 tube was rendered oxygen free. An excellent little trick with which to 

 employ the Buchner tube method is to pack lightly over the dry pyrogallic 

 acid in the bottle a small piece of absorbent cotton. This prevents the 

 immediate solution of the pyrogallic acid, and allows one time to pour in 

 the KOH solution, insert the smaller tube inside the Buchner tube, and 

 tightly insert the rubber stopper in place before solution and oxygen absorp- 

 tion has begun. 



A simple modification of the preceding method of Buchner has been 

 devised by Wright. 9 Stab-cultures of gelatin or agar in test tubes are made 

 in the usual way. The cotton stopper closing the tube is then thrust into 

 the tube to such a depth that its upper end lies at least 1 cm. below the 

 mouth of the tube. A small quantity of sodium or potassium hydrate solution 



* Buchner, Cent. f. Bakt., I, iv, 1888. 



9 Wright, Jour, of the Boston Soc. of Med. Sci., Dec., 1900. 



