OCCURRENCE IN VIRGINIA OF GREEN-GILLED OYSTERS. 147 



found as a residue, while the Hquid had a greenish opalescense. This liquid was filtered 

 off, and the green substance was subjected to a number of different solvents, both hot 

 and cold, but the pigment still remained insoluble in all reagents used. 



The solvents tried after gastric digestion were: Alcohol, water, 2 per cent sodium 

 chloride solution, ether, chloroform, carbon disulphide, toluene, carbon tetrachloride, 

 acetone, ethyl acetate, 0.5 per cent sodium carbonate, 12 per cent sodium hydroxide, 

 0.2 per cent hydrochloric acid, and 12 per cent hydrochloric acid. 



The green residue of this digestion was then placed in a pancreatic digestion with 

 the addition of a number of other green-gilled tissues. This digestion was carried out by 

 adding a small amount of tested pancreatin to a 0.5 per cent sodium carbonate solution, 

 and allowing the green material to remain in this for three or four days at 37° C. The 

 result in this case was very similar to the gastric digestion, a green residue remaining 

 with a greenish-opalescent supernatant solution. After filtering and washing the residue 

 with disrilled water, the green pigment was still found to be insoluble in the solvents 

 used, whether at boiUng or room temperature. 



The following solvents were used after the pancreatic digestion : Alcohol, water, 

 sodium chloride, 2 per cent; ether; chloroform; carbon tetrachloride; carbon disul- 

 phide; toluene; acetone; ethyl acetate; sesame oil; olive oil; glycerol; cottonseed oil; 

 linseed oil; paraffin oil; gasoline; acetic acid, 0.5 per cent; lactic acid, i per cent; lactic 

 acid, 0.5 per cent; salicyhc acid; and sodium carbonate, 0.5 per cent. 



Putrefaction destroyed the pigment slowly. A solution containing several green- 

 gills allowed to putrefy gave a dark green color to the hquid. Green oysters allowed to 

 die and putrefy showed that the gills turned black, or a very dark green, and finally were 

 thoroughly destroyed, leaving a black mass of foul-smeUing organic matter. 



The green residue of a pancreatic digestion was then subjected to saponification in a 

 I per cent alcoholic sodium-hydroxide solution. This solution was made by distilling 

 absolute alcohol over potash until a distillate was collected that was free from all impur- 

 ities that might give a yellow color with alkaUes. This distillate was then made into 

 a I per cent solution of alcohoUc sodium hydroxide, using chemically pure sodium 

 hydroxide. The green pigment material was thereupon allowed to saponify by boiling 

 two hours, after which the solution was filtered. The saponification yielded a yellow- 

 colored solution. This was neutralized with hydrocholoric acid with the resulting pre- 

 cipitation of sodium chloride. This solution was then evkporated down to about 10 cc, 

 filtered, and studied spectroscopically. 



As a control on this experiment, a like quantity of normal gills was subjected to a 

 similar pancreatic digestion. The solid matter that remained after a three days' diges- 

 tion was then saponified for two hours at boiling temperature in a i per cent alcoholic 

 sodium-hydroxide solution prepared as above. The resulting solution was very nearly 

 colorless, and after neutralizing, filtering, and evaporating, only a suggestion of yellow 

 color could be detected. 



The spectroscopic examination of the green-gilled saponification showed an absorption 

 band covering the violet end of the spectrum. Examination of the normal gill material 

 obtained from saponification showed no shadows whatsoever in the spectrum. 



To establish the fact that the green coloration of the gills in Virginia oysters was not 

 due to the presence of a bacterial pigment, the pigmented gills were subjected to bac- 

 teriological examination. 



