SELIG HECHT 



ii: 



1. Thanks to the work of Ewald and Kiihne, the preparation of 

 visual purple is a simple matter. For my purposes the following 

 routine has been adopted. Thirty frogs are placed over night in 

 complete darkness. The next morning the heads are removed and 

 placed in salt solution. The eyes are then enucleated, cut in two, and 

 the retinas removed as free from the pigment layer as possible. This 

 is much facihtated if the frogs have been kept over night in a warm 

 room. The retinas with some salt solution are now placed in a cen- 

 trifuge tube, and centrifuged at a good speed for 30 minutes. The 

 supernatant Hquid is removed, and a 3 to 4 per cent solution of bile 

 salts is added to the retinas. About 5 cc. are usually added, but this 

 may be varied up to 20 cc. depending on the concentration desired. 

 The retinas are thoroughly stirred up, and are allowed to remain in 

 suspension for half an hour or more. The mixture is then again cen- 

 trifuged. This brings the disrupted retinas and stray pigment granules 

 to the bottom of the tube, whereas the supernatant liquid is a clear 

 purplish pink solution of visual purple. In this form it may be kept 

 on ice for as long as a week or more without deterioration. It can 

 be used directly or after dilution with water or bile salts solution. 



All the manipulations are carried out in the dark, with the aid of 

 the light from a 10 watt ruby lamp. This is hardly a source of error, 

 because the solutions are quite insensitive to such red light. However, 

 even to this light, they are exposed as little as possible. In consider- 

 ing sources of error it may be well to mention the purity of the bile 

 salts. The commercial preparations are hopeless. The bile salts 

 which I used were prepared in our own laboratory, and are a white 

 powder, which when dissolved in water gives an absolutely colorless 

 solution. Only such bile salts give reliable results. 



A solution of visual purple prepared in this manner is extremely 

 sensitive to light. If exposed to sunlight it loses its color in about a 

 second. The solution, however, is not bleached to a completely col- 

 orless condition. The bleached solution is slightly but definitely 

 yellow, and remains so no matter how long the exposure is main- 

 tained. In this I can confirm Trendelenburg (1904) as opposed to 

 Ewald and Kiihne' s original statement (1878, p. 181) that the solu- 



