2704 Journal of Applied Microscopy 



CYTOLOGY, EMBRYOLOGY, 



AND 



MICROSCOPICAL METHODS. 



AGNES M. CLAYPOLE MOODY. 



Separates of Papers and Books on Animal Biology should be sent for Review to Agnes M. Claypole 

 Moody, 125 Belvedere Street, San Francisco, Cal. 



Flint. J. M. A new Method for the Deti.on- tollowing Spalteholz's method with 

 stration of the Framework of Organs. Johns certain modifications the author ob- 

 Hopkins Hospital Bull. 13: 48-<;2, iqo2. . , . , , ^ 



tained very satisfactory results from 



the new process of digestion by the use of a digestive ferment. Pieces about 3 

 mm. thick of fairly large blocks of hardened tissue are best. These are cut so 

 as to have 2 parallel planes. Van Gehuchten's fluid was perhaps the best as it 

 works rapidly and gives good cytological pictures. Moreover, the chloroform in it 

 loosens the fat. The mixture is: glacial acetic acid 10 parts, chloroform 30 

 parts, absolute alcohol 60 parts. Corrosive acetic gives good results with graded 

 alcohols, but formalin, chromic acid or its salts and osmic acid are unsatisfactory. 

 Trypsin cannot digest tissue hardened in fluid containing these reagents. 

 After fixation dehydration should follow to avoid shrinkage, also gradual changes 

 of alcohol. When dehydration is over the tissue is dropped into a Soxhlet appar- 

 atus and the fat extracted for 5 to 6 days continuously. To avoid possible 

 explosions of the ether in this apparatus it is placed under a hood and heated by 

 means of hot water from a Fletcher heater. After extraction for a week the 

 tissue is removed and slowly passed through graded alcohols, which should be 

 free from bubbles when poured on the tissues. The pieces are then washed in 

 running water for 24 hours, and then put into the digestive mixture. Parke, 

 Davis & Co.'s pancreatin can be used, but Griibler prepares a mixture which is 

 more effective, rendering the sections more transparent. But a small quantity 

 of pancreatin is used, enough to cover the end of an ordinary scalpel handle dis- 

 solved in 100 c c. of a .5 per cent, solution of sodium bicarbonate. Since the 

 products of digestion inhibit the action of the enzyme a dilute solution of trypsin 

 should be used and frequently renewed. Changing the fluid every 48 hours is 

 satisfactory for digestion. Enough chloroform must be used to prevent putrefac- 

 tion, it should cover the bottom of the vessel in which digestion occurs. Thymol 

 prevents decay but stains tissues a dirty brown. The heat required by digestion 

 volatilizes the chloroform and fills the tissues with small bubbles causing it to 

 float in the fluid. By making a small elevated stand of thick paper the tissue 

 cart be kept away from the bottom free from the chloroform and also the falling 

 debris of digestion. The fats present in combination with protein can only be 

 extracted after digestion, so that each process best be carried out at least twice, 

 in some cases four times are required. Organs digesting quickly are thyroid, 

 spleen, lymph gland, lung; while salivary glands, pancreas and adrenal are more 

 resistant. The pancreas yields more slowly to its own ferment than any other. 

 This of course applies only to hardened tissue ; fresh was in no case used. 



