CHEMICAL CRITERIA OF ANAEROBIOSIS 37 



reduces this discussion, in so far as it relates to chemical reduction 

 by means of an agent in the air chamber, to the most valuable 

 agent with which we have to deal, i.e., alkaline-pyrogallol. 



Inasmuch as this reagent finds a legitimate use only in con- 

 nection with attempts to secure surface colonies on solid media, 

 it is to this that we have limited our attention. Buchner's 

 (1888)' original technic involving the use of a small tube contain- 

 ing slanted culture medium (2 per cent agar, methylene blue 

 1:100,000, n/100 NaOH) in a longer one containing the usual 

 alkaline pyrogallol mixture gave satisfactory results when the 

 cotton stopper was left out of the inner tube, but otherwise did 

 not, unless the tube were inverted. Decolorization begins, as 

 does growth of obligate anaerobes on suitable media, in the 

 thinnest portion of the slant. In Wright's (1901) modification, 

 which involves the saturation of the plug with the mixture, 

 sealing by rubber stopper, and inversion, even better results 

 were obtained. In either case partial decolorization appeared 

 at the surface in less than twenty-four hours at 37°C. and con- 

 tinued to completion within two to three days. Both these 

 methods, but notably the last, have given quite satisfactory 

 results in the surface cultivation of B. tetani, B. welchii, B. 

 sporogenes, B. chauveauii, B. botulinus, and many unidentified 

 anaerobes. Wright's method has been relied upon largely for 

 repeated surface colony isolation of the writer's cultures. 



Two plating methods for surface culture have been studied 

 in connection with the methylene blue test, namely Lentz's 

 (1910) pyrogallic acid saturated cardboard, and McLeod's (1913) 

 divided plate. Both showed the dye-agar decolorized at 37°C. 

 within eighteen hours. The latter method has refused consist- 

 ently however to yield us surface colonies of well known anaerobes 

 on either blood or glucose agar identical with that used in con- 

 trols by Wright's method. Dr. Oskar Klotz at the University 

 of Pittsburg has stated his belief that aromatic substances in 

 the clay are sufficiently germicidal to explain the failure of 

 bacterial growth and has overcome the difficulty through the 

 use of a special cement containing paraffine and some other 

 substances. At any rate, McCleod's device seems constructed 



