STORAGE OP FAT IN MUSCULAR TISSUE OF KING SALMON. 77 



in the picture — enough to prevent one from using any but freshly fixed tissues for close 

 comparative estimates. 



In taking samples, in all instances where possible the muscles were quickly cut 

 from the salmon selected while the tissue was yet alive. The muscles were cut in thin 

 pieces with a razor and immersed in a large excess of 10 per cent formalin. Other 

 tissues, such as the liver, caeca, stomach, etc., in which fat studies were to be made 

 were also included. Portions of the material not studied on the collecting grounds 

 were preserved for future use, some of which still retains its fat in approximately the 

 normal quantity and relations (after six months). 



Portions of the muscles and of certain other tissues were fixed in different standard 

 preservatives for future study and comparison. But as these studies are not presented 

 in this paper the detail of fixation will be omitted. 



Fat stain used and its preparation. — Sudan III and scarlet red are the fat stains 

 on which great reliance is placed in the histological identification of the fats. Since 

 the salmon tissues are so filled with fat it was decided to use the scarlet red as giving 

 the sharper differentiation. Bell has made a series of special studies on the micro- 

 chemical staining of fats. It is to him I am indebted for the modifications of the Herx- 

 heimer method which were followed. 



The scarlet red was prepared in saturated solution in alkaline alcohol: Scarlet red, 

 2 grams; sodium hydroxide, 2 grams; 70 per cent alcohol, 100 cubic centimeters. This 

 was ripened in a 4-ounce wide-mouth vaseline bottle in a water bath at about 75° C, 

 for from 20 to 30 minutes. The ripening took place in a closed bottle. But it was found 

 essential not to heat too strongly lest the reactions lead to the production of a die that 

 would stain general protoplasm. This stain is supersaturated while warm and some 

 crystalization will occur on cooling. Bell's recommendation that the stain be filtered 

 just before using was followed, also the precautions recommended by him against evap- 

 oration and sedimentation while staining. This stain retains its properties very well for 

 a very much longer time than one is led to expect from the directions published by Bell. 



Technique of sectioning and staining fresh or formalin-fixed material for fats. — 

 Either fresh tissue or tissue fixed in 10 per cent formalin was frozen with carbon dioxide 

 on a Bardeen freezing microtome. The fixed material can be cut as thin as 25-30// 

 with comparative ease, but the temperature of the block must be just right. The sec- 

 tions were received from the microtome knife directly into 70 per cent alcohol and 

 stained as quickly as possible. 



The sections were handled always with a spatula of proper size. They were stained 

 directly from 70 per cent alcohol. After 5 to 10 minutes in the scarlet red stain the sec- 

 tions were passed as quickly as possible through 70 and 30 per cent alcohol into water. 

 The rinsing was sometimes through the alkaline alcohol of the grade in which the stain 

 was dissolved. This is a safer plan to prevent precipitation of the excess of stain from 

 adhering to the surface of the sections. 



When sections were to be counterstained a short washing in acid water, 0.2 per 

 cent hydrochloric acid, preceded the haematoxylin counterstain. The acid treatment 

 was finally adopted as a routine procedure for all sections. 



■ The stained sections were mounted directly from the wash water into pure glycerin. 

 The glycerine was of course slightly diluted by the adherent water. The glycerin 

 mounts were sealed with paraffin, or better with paraffin-beeswax cement. 

 19371°— vol 33—15 6 



