THE CONNECTIVE TISSUES. 1 29 



minutes: Grubler's aniline blue soluble in water, 0.5 gm. ; Grubler's 

 orange G, 2 gm. ; oxalic acid, 2 gm. ; distilled water, 100 c.c. After 

 staining, the sections are washed in water and dehydrated in 95% alcohol, 

 blotted on the slide, and cleared in xylol and mounted in xylol balsam. 

 The connective-tissue fibers and reticulum stain blue. 



Dr. Sabin's modification of this method deserves mention. Fix in 

 Zenker's fluid, cut in paraffin, and fix sections to the slide with the water 

 method. After removing the paraffin, stain sections in ^/c acid fuchsin 

 until red, and without washing fix in a saturated aqueous solution of 

 phosphomolybdic acid diluted ten times for about ten minutes. Wash in 

 95% alcohol and stain for a very short time in the following solution : 

 Grubler's aniline blue soluble in water, i gm. ; orange G, 2 gm. ; oxalic 

 acid, 2 gm. ; boiling water, 100 c.c. Wash in alcohol, blot on the slide, 

 clear in xylol and mount in xylol balsam. 



Digestion Method for Demonstrating the Connective-tissue 

 Framework of Organs and Tissues (Mall, Spalteholz, Hoehl, 

 Flint). For bringing out the framework of white fibrous and reticular 

 fibers of organs and tissues digestion by means of trypsin may be recom- 

 mended. For the account here given we follow Flint. The tissues are 

 fixed in graded alcohol, corrosive acetic, or Van Gehuchten's chloroform - 

 acetic-alcohol mixture. After complete dehydration, small pieces of 

 tissue, not to exceed 3 mm. in thickness, are placed in paper cups and 

 dropped into a Soxhlet apparatus and extracted with ether for a period 

 of six to eight days in order to free the tissue of the fat. After the fat 

 has been removed, the tissues are brought into water, through graded 

 alcohol, and then digested in pancreatin. (Grubler's pancreatin is rec- 

 ommended ; that of Park, Davis & Co. may be used. ) The pancreatin 

 solution to be used is made by adding as much pancreatin as can be taken 

 up on the end of an ordinary scalpel handle to 100 c.c. of a 0.5% 

 solution of bicarbonate of soda. This solution is changed every forty- 

 eight hours. To prevent putrefaction enough chloroform is added to 

 cover the bottom of the dish. The digestion is continued until the cell- 

 ular element has been removed five to ten days. It is often necessary 

 to repeat the fat extraction and digestion several times. After the cellu- 

 lar elements have been removed the tissue is thoroughly washed in flowing 

 water, and may then be mounted in glycerine and studied with a stereo- 

 scopic microscope, or it may be dehydrated and imbedded in celloidin 

 and sectioned. Such sections may then be stained in fuchsin and thor- 

 oughly washed in alcohol ; this removes the stain from the celloidin, 

 leavjng only the connective tissue stained. 



Slide' Digestion. The method may also be applied for digesting 

 tissues on the slide. Fix as above described, imbed in paraffin, and cut 

 very thin sections which are fixed to the slide by the water method. 

 Remove the paraffin and place the sections from alcohol into the Soxhlet 

 apparatus, where they are extracted with ether for a number of hours. 

 Bring the sections through graded alcohol into water, in which they re- 

 main several hours. The sections are now digested in the above-men- 

 tioned pancreatin solution for several hours to several days, or until the 

 cellular elements have been removed. Wash carefully in water. The 

 remaining connective tissue may now be stained in iron-lac hematoxylin 

 or in an aqueous solution of toluidin blue or in an aqueous solution of 

 fuchsin. Dehydrate, clear, and mount. 



