juneis, I9I5 Azotobacter Chroococcum 233 



Treatment with the sodium-carbonate solution discolors the granules, 

 leaving the cell network unchanged. 



(d) A cover-glass preparation stained with methylene blue was treated 

 with Meissner's solution and then with a 5 per cent solution of sodium 

 carbonate. After treatment with the Meissner solution the cell should 

 be brown and the granules decolorize if of metachromatic nature; they 

 should remain colored if of chromatic nature. 



This treatment left the granules decolorized and cells stained blue. 



(e) On treating with boiling water and then staining or boiling in a 

 test tube for two minutes and then staining, the metachromatin should 

 dissolve. 



Boiling with water over Bunsen burner for two minutes completely 

 dissolved the granules. Washing with water at 90° to 99° C. for four 

 minutes did not dissolve all the granules, perhaps on account of insuffi- 

 cient temperature; thus, only some granules, the large ones much reduced 

 in size, are to be seen after this treatment. The contents of the net- 

 work took a light-pink color when treated with methylene blue, i to 11. 



(/) Smears were prepared and stained by the Ernst method. Blue 

 coloration of granules after the Bismarck-brown treatment would there- 

 fore indicate metachromatin. 



This method gave a bluish color to the granules, but these prepara- 

 tions do not last when mounted in balsam (dissolved in chloroform). 



(g) On staining unfixed preparations with methyl green the dye 

 should stain only chromatin. 



Staining with methyl green showed a strong metachromacy, since the 

 granules took on a violet color. The staining of unfixed preparations in 

 solutions of Grubler's methyl green gave a violet color to the granules. 

 Later, in trying to explain this metachromacy, tests were made on the 

 purity of the stain. They showed it to contain considerable impurities 

 in the form of violet dyes, probably methyl violet. 



Mounts in 5.A^ potassium hydroxid and in calcium chlorid gave 

 preparations that did not especially change in appearance from the 

 checks. 



RESULTS OF MICROCHEMICAL TESTS 



From what has been said about the granules found in the cells it may 

 be stated that their nature seems to be different from that of the granules 

 found by Mencl (11), Jones (9), Prazmowsky (15), and others in the 

 organism on which they were working, with the exception of those 

 treated by Fischer (5). 



In fact, as has been seen, the granules found by the writer did not give 

 the reaction for glycogen. The results obtained with Meissner's solution 

 must be carefully judged before making a definite statement. If the 

 granules were glycogenic, they would not only give a golden color when 

 treated with the reagent but would also retain it if the excess of the 



