THE CIRCULATION OP THE BONE-MARROW. 33 



should be run for about 10 minutes. However, experience only can give one com- 

 petent judgment in this, as there are many indications, not reducible to writing, 

 which one learns to recognize and be governed by in individual instances. 

 One may get a complete injection of the superficial vessels of the skin and muscle 

 with practically no penetration of the marrow cavities. The optimum condition 

 is to stop as soon as possible after the maximum complete injection of the smallest 

 capillaries of the bone-marrow, which, being manifestly impossible of direct observa- 

 tion, must be a matter of experience. 



Several injecting solutions were tried. A silver-nitrate solution permeates 

 the vessel walls and, while outlining the larger vessels quite clearly, masks the 

 smaller capillaries completely. Freshly precipitated carmine, even under the best 

 conditions, forms flocculi too large to be carried into the smallest vessels for a 

 complete injection. The best results were obtained from a freshly filtered solution 

 of one part of Higgins india ink diluted with three parts of physiological saline. 

 Very satisfactory injections, which I feel are relatively complete, were secured with 

 this injection mass under the conditions stated above. 



The cannula was placed directly into the heart, into the subclavian artery 

 (making ventral incisions), or into one of the iliacs or the abdominal aorta (with 

 a dorsal incision). This latter procedure was used almost exclusively in the later 

 experiments. The antero-posterior incision was made just to the side of the mid- 

 line; a lateral exposure of the ribs was made and, after removing a section of four 

 ribs, the lung was carefully laid back by blunt dissection, after which the abdominal 

 aorta or common iliac was easily located. The auricle or inferior vena cava was 

 opened for the return-flow outlet. No injections were attempted via the nutrient 

 arteries direct. 



After many methods for fixation had been tried, the best results were found 

 to be obtainable by fixing the "marrow pencils" in Helly's fluid at 38 for from 2 to 

 6 hours and the whole bones in 10 per cent formalin for 24 hours. The former 

 were fixed in the routine manner, dehydrated, cleared, and embedded either in 

 celloidin or paraffin, the celloidin proving better for the study of individual cells 

 when stained. The whole bones were cleared by the Spalteholz (1914) method. 

 As a routine procedure the radius and ulna of one side were fixed and treated for 

 clearing in situ and the "marrow-pencils" of the opposite side were taken out and 

 fixed in Helly's fluid for embedding. 2 It is desirable to fix when fresh and to main- 

 tain the "marrow-pencils" in as perfect form as possible. With reasonable care 

 the fresh marrow may be removed intact, and, except in rare instances, there are no 

 spicules of bone in the marrow calling for decalcification. Danchakoff's (1908) 

 modification for the mounting of celloidin sections was used in making serial sections. 



For staining sections we have used Giemsa's stain, Wright's blood-stain, 

 methylene-blue-eosin, hematoxylin and eosin, and hematoxylin and carmine. 

 The sharpest differentiation was obtained with a slight modification of the ordinary 

 hematoxylin and eosin stain. A two-minute period in a freshly filtered 1 per cent 

 solution of Ehrhch's hematoxylin, diluted one-half, alkalmization in Ba (OH) 2 



2 The humerus in the pigeon contains no blood-forming marrow. 



