MALIGNANT (EDEMA 69 



An excellent method for anaerobic plates, either in a desiccator with the pyrogallic 

 acid and caustic soda, or less satisfactorily in the open air, is to sterilize the parts 

 of the Petri dish inverted; that is, the smaller part is put bottom downward in 

 the inverted cover (as one would set one tumbler in another). Then, in using, 

 unwrap the Petri dish, lift up the inner part, pour in the inoculated medium 

 into the upturned cover. Then immediately press down the inner dish, spreading 

 out a thin film of the medium between the two bottoms. 



J. H. Wright's Method. 



Make a deep stab culture in glucose agar or gelatin, preferably boiling the media 

 before inoculating. Then flame the cotton plug and press it down into the tube so 

 that the top lies about three-fourths of an inch below the mouth of the test-tube. 

 Next fill in about one-fourth of an inch with pyrogallic acid; then add 2 or 3 c.c. of 

 a 10% solution of caustic soda, and quickly insert a rubber stopper. This method 

 is one of the most convenient and practical, and is to be strongly recommended. 



Method of Vignal. 



In this a section of glass tubing (1/4 in.) is drawn out at either end, as in making a 

 bacteriological pipette, with a mouth-piece containing a cotton plug. The liquid 

 agar or gelatin is then inoculated and the medium drawn up into the tube. In a 

 very small flame the capillary narrowings are sealed off, and we have inside the tube 

 very satisfactory anaerobic conditions. To get at the colonies, file a place on the 

 tube and break at this point. 



To obtain material for examination and isolation in pure culture from the deep 

 agar stab-tube, it is best to loosen the medium at the sides of the tube with a heated 

 platinum spud or a flattened copper wire. Then shake the mass out into a sterile 

 Petri dish. It is dangerous to break the tubes with a hammer as some do. 



A Combination Method. 



Recently as shown in the illustration in Fig. 7, I have been combining various 

 methods so that very satisfactory anaerobic conditions are obtained. First, a 

 deep agar stab of freshly sterilized glucose agar is made. The surface of this is then 

 covered with sterile paraffin oil. The proper amount of pyrogallic acid is then 

 deposited in a salt mouth bottle. The rubber stopper with the glass and rubber 

 tubing is then firmly pushed in and connection made with a filter pump. 



In five to ten minutes almost all the air will be exhausted when the Hofmann 

 clamp is screwed up tight and the bottle disconnected from the vacuum pump. 

 The glass tubing end is then inserted into a graduate holding 10% caustic soda 

 solution, the Hofmann clamp unscrewed, and the necessary amount of caustic soda 

 having been run in, as noted under Buchner method, we again close the screw clamp 

 and incubate. 



B. oedematis maligni (Pasteur, 1877). This is the vibrion septique 

 of Pasteur. It is found in garden soil and in street sweepings. It is 



