2o6 



Journal of Agricultural Research 



Vol. XII, No. 4 



In connection with the foregoing investigations concerning the influ- 

 ence of nitrates on pigment formation by Azotobacter, it was thought 

 that some results of cytological interest might be obtained in regard to 

 the effect of varying amounts of nitrates on the volutin bodies. 



Slope cultures of mannit agar were prepared containing the different 

 nitrates as indicated in Table XVIII. These slopes were inoculated 

 with both strains of Azotobacter and incubated at 28° C. for 10 days. 

 At the end of this time each culture was stained and examined micro- 

 scopically. The following method was used for demonstrating the 

 presence of the volutin bodies. The organisms to be examined were 

 air dried on a glass slide and then fixed in the flame of a Bunsen burner. 

 The preparation was then flooded with a i to 10 aqueous solution of 

 methylene blue (Merck's) prepared by adding 10 c. c. of a saturated 

 aqueous solution of methylene blue to 90 c. c. of distilled water. The 

 stain was washed off after five minutes with a i per cent solution of 

 sulphuric acid and immediately rinsed in distilled water. The prepa- 

 ration was dried and examined with the oil-immersion objective. The 

 volutin bodies appeared within the cytoplasm as very dark blue dots, 

 the outline of the cell wall was a lighter blue, while the cell net work 

 was stained a very light blue. 



Guignard's stain ^ was also used to demonstrate the presence of the 

 volutin bodies. Fresh smears on a glass slide were fixed over 10 per 

 cent osmic acid for three minutes. The preparation was then air-dried 

 and fixed to the slide by rapidly passing the latter a few times through 

 a Bunsen burner. The preparation was covered with the stain which 

 was allowed to react for five minutes. The stain was then washed off 

 with distilled water, dried, and examined with the oil-immersion objec- 

 tive. The outline of the cell as well as the net work wdthin-was stained 

 light purple. The granules within the cytoplasm were a reddish purple. 

 The results are given in Table XVIII. 



Tabls XVIII. — Influence of nitrates on the formation of volutin bodies in Azotobacter 



in 10 days 



"Representing an approximate average of two volutin bodies per cell. 



b Representing an approximate average of four volutin bodies per cell. 



* Guignard'sstain. Fifty c. c. of 2 per cent fuchsin in i per cent acetic acid; 40 c, c. of 0.2 per cent methyt 

 green in i per cent acetic acid; i c. c. of glacial-acetic acid. Distilled water was used in making the i per 

 cent acetic-acid solution. 



