154 



Journal of Applied Microscopy. 



sections, none had been found successful 

 until Cullen ('Beschleunigtes Verfahren 

 zur Farbung frischer Gewebe mittelst 

 Formalins,' Centralblatt fiir allgemeine 

 Pathologie, 1895, Bd. No. 11, pp. 148-450; 

 also Johns Hopkins Hospital Bulletin, 

 April, 1895, and May, 1897) suggested the 

 use of formalin either before oi aft^r 

 cutting, as a partial fixative, when fairly 

 successful results were obtained. 



"A serious objection to the use of this 

 method I have found to be the shrinkage 

 and consequent distortion which always 

 occur to a greater or less extent. At 

 times so great a shrinkage took place in 

 my sections that they were entirely use- 

 less. At Dr. Prudden's suggestion, I 

 have devised a scheme of fixing sections 

 on cover glasses by means of albumen. 

 This may be accomplished by impregnat- 

 ing sections with a solution of egg al- 

 bumen, floating them on cover glasses, 

 and finally coagulating the albumen. By 

 this means shrinkage and distortion, 

 which would otherwise occur from the 

 subsequent action of alcohol, may be 

 largely if not entirely prevented. 



"This modification does not materially 

 increase the time required to prepare 

 stained fresh sections for the microscope. 

 I have repeatedly cut sections of fresh 

 tissues, fixed them on cover glasses, and 

 have hardened, stained, and mounted 

 them in as short a time as nine minutes. 

 Furthermore, it permits the use of hard- 

 ening agents other than formalin. Fair 

 results have been obtained with osmic 

 acid, mercuric chloride, etc. For pur- 

 poses already indicated this modified 

 method seems to be a valuable one, since 

 it enables one to determine the nature of 

 new growths, or the lesions of viscera, 

 etc., within a few minutes — a procedure 

 which would otherwise require hours or 

 days to accomplish by the ordinary 

 hardening and impregnating methods. 

 CuUen's method, as may be imagined, is 

 not applicable for all tissues. The re- 

 sults obtained by the suggested modifi- 

 cation have been entirely satisfactory 

 for diagnostic work, but for fine struc- 

 tural details other methods are still to be 

 preferred. 



"The technique of the method is as 

 follows: 



"1. Almost any form of freezing-mi- 

 crotome may be used. The freezing 

 agent may be ether, carbonic acid, or 

 rhigolene. 



"2. Sections may be cut from perfectly 

 fresh material, but more satisfactory and 

 better results are obtained from material 

 which has previously hardened one or 

 two hours longer in ten-per-cent. forma- 

 lin. (It is convenient to drop bits of tis- 

 sue into ten-per-cent. formalin at the 

 time of the operation or during the post- 

 mortem examination. By the time they 



reach the laboratory they are usually 

 sufficiently impregnated to be cut and 

 stained.) 



"3. Tissues already hardened in for- 

 malin should be soaked in water a min- 

 ute or two to remove the formalin before 

 cutting. 



"4. Sections as they are cut may be 

 dropped directly into the albumen solu- 

 tion, where they remain until needed. A 

 solution of albumen which has been 

 found to answer the purpose is prepared 

 by adding to 50 c.c. of egg albumen 150 

 c.c. of distilled water and sufficient of a 

 solution (usually about 50 c.c.) of salicy- 

 lic acid (saturated), which has been ren- 

 dered slightly alkaline with lithium car- 

 bonate, completely to dissolve the albu- 

 men. The solution may be kept un- 

 changed several weeks by adding a little 

 gum camphor. 



"5. Unhardened sections should be 

 placed in five-per-cent. formalin three 

 or five minutes, after which they are 

 soaked in the albumen solution two or 

 three minutes. 



"6. Float sections on cover glasses. Re- 

 move excess of fluid with filter paper 

 when necessary. Blot sections evenly, 

 taking care not to use pressure enough 

 to cause them to bear the imprint of the 

 cloth. (The best blotting-material seems 

 to be washed cheesecloth used in several 

 layers. The use of filter paper, towels, 

 muslin handkerchiefs, etc., for this 

 purpose has not been satisfactory.) 



"7. Transfer immediately to alcohol, 

 alcohol and ether (equal parts), osmic 

 acid, or mercuric chloride, etc., in order 

 to coagulate the albumen and to fix the 

 section and complete the hardening. 



"8. Sections may be stained on the 

 cover slip in various way. For ordinary 

 diagnostic work staining with haema- 

 toxylin and eosin and mounting in 

 balsam answer well. 



"9. Stain from two to five minutes in 

 haematoxylin (Delafield's or Gage's). 



"10. Decolorize by passing rapidly 

 through acid alcohol— hydric chloride, 1 

 part; eighty-per-cent. alcohol, 99 parts. 



"11. Wash throughly in water. 



"12. Dehydrate and stain in eosin alco- 

 hol. (Eosin which has been precipitated 

 from a saturated aqueous solution by 

 acid stains connective tissue more sharp- 

 ly and gives better results than ordinary 

 eosin. It is prepared by Fischer by add- 

 ing to a saturated aqueous solution of 

 eosin hydric chloride in excess. Filter 

 and wash the precipitate with water 

 until the acid is removed. Dry the 

 precipitate and dissolve in alcohol.) 



"13. Clear in oil of origanum, oil of 

 cloves, creosote, zylol, etc. 



"14. Mount in balsam after having 

 cleaned the upper surface of the cover 

 slip." 



