186 BIOLOGY AND TECHNIQUE 



one into the other as do the halves of a Petri dish, and similar to these 

 in every respect except that they are higher, and that a slightly greater 

 space is left between their sides when they are placed together. The dishes 

 should be about three-fourths to one inch in height, they need be of no 

 particular diameter, although those of about the same size as the usual 

 Petri dish are most convenient. An important requirement necessary for 

 the success of the method is that the trough left between the two plates, 

 when put together, shall not be too broad, a quarter of an inch being the 

 most favorable. 



Into the smaller of these plates the inoculated agar is poured exactly 

 as into a Petri dish in the ordinary aerobic work. Prolonged boiling 

 of the agar before plating is not essential. When the agar film has 

 become sufficiently hard on the bottom of the smaller dish, the entire ap- 

 paratus is inverted. The smaller dish is now lifted out of the larger, and 

 placed, still inverted, over a moist surface a towel or the wet surface of 

 the table to prevent contamination. Into the bottom of the larger dish, 

 which now stands open, there is placed a quantity (about 3 grams) of dry 

 pyrogallic acid. Into this, over the pyrogallic acid, the smaller dish, still 

 inverted, is then placed. A five per cent solution of sodium hydrate is 

 poured into the space left between the sides of the two dishes, in quantity 

 sufficient to fill the receiving dish one-half full. While this is gradually 

 dissolving the pyrogallic acid, albolin, or any other oil (and this is the only 

 step that requires speed), is dropped from a pipette, previously filled and 

 placed in readiness, into the same space, thus completely sealing the chamber 

 formed by the two dishes. 



If these steps have been performed successfully, the pyrogallic solution 

 will at this time appear of a light brown color, and the smaller plate, with 

 its agar film, will float unsteadily above the other. Very rapidly, as the 

 pyrogallic acid absorbs the free oxygen in the chamber, this plate is drawn 

 down close to the other, and the acid assumes a darker hue, which remains 

 without further deepening even after three or four days' incubation. 



We have described a considerable number of methods of anae- 

 robic cultivation which have been in use. Following our general 

 purpose, however, of emphasizing the methods that we ourselves 

 have found most useful, we will describe in the following paragraphs 

 the methods which we are using as routine for anaerobic work in 

 our own laboratory, and which we believe are the most practical. 



For anaerobic cultivation on agar slants, we use the Buchner 

 tube method as described above. 



For spirochaete cultivation, etc., we use the Noguchi method 

 of narrow deep agar or broth tubes with tissue at the bottom, sterile 



