[VOL. 6 



12 ANNALS OF THE MISSOURI BOTANICAL GARDEN 



synthetic media. In culture solutions it manifests itself by 

 the formation of a translucent zoogloea mass at the junction 

 of the solution surface and the walls of the container. At 

 from 4 to 6 days this largely disappears and a fine white sedi- 

 ment appears at the base of the flask. In no case did we ob- 

 serve even the suggestion of the surface scum or pellicle which 

 is widely described in the literature. 



The purity of the culture also was checked by numerous 

 microscopic examinations. The regular method for making 

 microscopic preparations was staining for 5 minutes with 

 dilute 1 :10 aqueous methylene blue, clearing in xylol-alcohol, 

 equal parts, and then in xylol. Preparations were also made, 

 using ordinary carbol gentian violet and clearing as above. 

 The cells from very young cultures (12 hours) show little dif- 

 ferentiation in structure when stained with ordinary carbol 

 gentian violet or with dilute aqueous methylene blue, and tend 

 to be oval or bacillary in form. Slightly older cultures (e. g., 

 60 hours) show some differentiation and the appearance of 

 granules is more frequent. Cells from cultures 4 to 6 days 

 old appear as large, thin-walled, decidedly granular cocci or 

 diplococci, while preparations from old cultures (e. g., 15 days) 

 generally show thick-walled cocci surrounded by considerable 

 slime, the cell contents failing to take either of the above 

 stains appreciably (pi. 1, figs. 1-6). The nature of these cyto- 

 logical differentiations has been discussed by Bonazzi ('15). 

 The culture when obtained was in its twenty-fifth transfer 

 from soil, and transfers and platings made from this time on 

 were designated as Fi, F2, F3, etc. 



Culture medium. — The standard culture medium used in 

 this work was essentially a modified Ashby's medium of the 

 following composition: 



Mannite 20 gms. 



Monopotassium phosphate 0.2 gm. 



Magnesium sulphate 0.2 gm. 



Sodium chloride 0.2 gm. 



Calcium sulphate 0.1 gm. 



Distilled water 1000 gms. 



10% ferric chloride solution 2 drops 



