302 R. R. BENSLEl" 



TECHNIQUE 



1. Methods for the demonstration of all the islets of the pancreas, 



'permitting enumeration 



a. The neutral red method. The animal is killed by bleeding. 

 A cannula is introduced into the aorta and a solution of neutral 

 red in isotonic salt solution containing one part of neutral red in 

 15,000 of solution is injected. Immediately the pancreas takes 

 on a rose tint, and after a sufficient depth of color is obtained, 

 which must be learned by experience, the injection is stopped, 

 the pancreas is removed, and examined. If the injection has 

 proceeded a sufficient length of time, a lobule of the pancreas, 

 placed under a cover glass and examined, will show the islets of 

 Langerhans stained an intense yellow red, while the rest of the 

 pancreas will show a faint rose tint. In a short time after the 

 preparation is mounted, owing to reduction of the dye, the acinus 

 tissue is bleached and the islets remain the only stained elements 

 in the pancreas. If the preparation has been overstained, that 

 is, has been stained for too long a time, exclusion of oxygen from 

 the preparation suffices, in a short time, to reduce the excess dye 

 in the acinus tissue, leaving the islets sharply differentiated as 

 before. In such a preparation it is easy, though somewhat labo- 

 rious, to count the entire content of islets in a pancreas of suit- 

 able size, without missing islets and without duplicating counts. 

 The clearness of such a preparation is sufficiently indicated by 

 fig. 1 which shows such a group of lobules simply pressed apart 

 under cover glass without teasing or sectioning. For counting 

 it is necessary to enlist the help of two or more helpers in order to 

 complete the count before the inevitable reduction of the dye 

 overtakes the islets, and begins to cause the disappearance of 

 the smallest of them. With the help of one man mounting prep- 

 arations, three men can easily count an entire guinea-pig pan- 

 creas in one and a half hours, using- for this purpose counting 

 machines. The procedure is as follows : The pancreas is divided 

 into minute masses 3 or 4 mm. square and of the thickness of 

 single lobules, which are separated as much as time permits by 

 tearing the connective tissue with forceps. These pieces are 



