FLAGELLATION OF BACTERIA OF LEGUMINOSAE 241 



for a few minutes after washing. With a pair of forceps a nodule 

 was then placed in a disinfecting solution prepared by adding 

 2.5 cc. of concentrated hydrochloric acid to 500 cc. of a 1:500 

 corrosive sublimate solution and allowed to remain in this solution 

 for one and a half to two minutes. It was then removed with a 

 pair of flamed forceps, rinsed in sterile tap water, and placed in 

 a drop or two of sterile tap water in the center of a sterile Petri 

 dish. The nodule was crushed by using a flamed and cooled 

 glass rod, after which a tube of sucrose agar which had been 

 melted and properly cooled was added, and thoroughly mixed. 

 The sucrose medium just referred to was made as follows: 



Monobasic potassium phosphate 1.0 gram 



Magnesium sulphate 0.5 gram 



Sucrose 10.0 grams 



Tapwater 1000.0 cc. 



Agar 10 . or 15 . grams 



At first no attempt was made to adjust reaction, but as the 

 growth on this medium was so slow most of the media used were 

 adjusted to pH 7.0-7.4 using the colorimetric method. 



Several plates were made at each time, thus insuring good 

 distribution of colonies in at least one of the plates. All plates 

 were kept at room temperatures. After the colonies developed 

 transfers were made either to the same sucrose medium or to a 

 similar medium, containing 10 grams of mannitol in place of the 

 sucrose. The mannitol media were used almost exclusively 

 for maintaining the organisms after transfer from the isolation 

 plates. 



METHOD OF STAINING FLAGELLA 



Th3 staining method used was a modification of Loeffler's 

 flagella stain suggested by the writer in a previous paper (1920). 

 Bacteria from a slant on mannitol or sucrose agar were removed 

 and placed in a small quantity of sterile tap water in a test tube. 

 Several small droplets of this suspension were, after a few mmutes, 

 placed on a well cleaned cover glass and allowed to air dry. 

 About five drops of Mordant solution A were placed on the cover 

 glass as soon as the droplets had dried, and this was followed 



