juneis, I9IS Azotobacter Chroococcum 231 



The methods used are those proposed by A. Meyer (12) and by his 

 follower Grimme, and are already recognized as valuable in the study of 

 the cytology of bacteria. 



The granules observed by the writer in the cells of this organism might 

 be nuclei, metachromatic granules, fat drops, glycogen, or starch. 



Mencl (11) and Prazmowsky (15) believe them to be nuclei or nucleo 

 equivalents; Jones (9), glycogen granules and nuclei of gonidia; and 

 Fischer (5), metachromatic bodies. None of these authors has at- 

 tempted to prove his point satisfactorily or to disprove other possi- 

 bilities. 



Since the aim of this part of the work is to find the true nature of the 

 granules met with in the organism in question, the methods used will be 

 described and the results of the investigations given. 



Starch. — Smears from cultures on mannit agar, flame-fixed, were 

 mounted in a saturated aqueous solution of iodin, sealed with paraffin, 

 and observed at once and 24 hours later. 



The granules are not to be considered as starch, since they do not give 

 a blue color to iodin or to Meissner's solution.^ 



Even after 24 hours no coloration was visible. The cell diaphanized, 

 and the granules gained in refraction. 



Fat. — Smears from a mannit-agar culture were immersed in ethylic 

 ether for iX hours, dried, stained in methylene blue, and mounted in 

 balsam. 



Since A. Meyer (12) claims that chloroform and alcohol are not good 

 solvents of bacterial fats, on account of the difficulty which they meet 

 in passing through the cell wall and Schleimschicht, the writer tried to 

 avoid any objection that could be raised against the conclusions drawn 

 from the results obtained by the ethylic-ether method. 



With this aim in view tests were made, using the method suggested 

 and recommended by Meyer. It consists in fixing the bacterial smear 

 in formol, immersing in glacial acetic acid, neutralizing, and staining. 

 Methylene blue, i to 10, was used as the stain; it gave very good prepa- 

 rations. The fats are dissolved by this method. Treatment with glacial 

 acetic acid did not dissolve the granules. 



Prolonged treatment in the cold with ether or with glacial acetic acid 

 did not dissolve the granules, since those which had been treated stained 

 just as strongly as the checks. This eliminates the question of their 

 fatty nature, although their deeply staining property should have already 

 led to this supposition. It should be noted that these preparations 

 were not fixed in osmic acid, which treatment would render lipoids, or 

 fatty substances in general, insoluble in fat solvents, and stainable by 

 the ordinary staining solutions. For this reason their myelin nature 

 should not be accepted. 



'Formula for Meissner's solution: Metallic iodin, 7 gm.; potassium iodid, 20 gm. ; water, 100 gtn. 



