Journal of 



Applied Microscopy. 



Volume I. 



NOVEMBER, 1898. 



Number 1 1 



Formalin as a Reagent for Blood Studies. 



There is always great difficulty in pre- 

 paring in bulk, for laboratory purposes, 

 those tissues or parts of tissues which 

 are liquid by nature, such as blood and 

 lymph. It is very easy to prepare pieces 

 of muscle, liver, or other solid parts, for 

 detailed microscopical examinations of 

 histological, constituents, when they can 

 be hardened and infiltered. But blooa 

 corpuscles can not easily be thus treated. 

 There are usually several difficulties 

 connected with such a process, as separ- 

 ating the corpuscles from the plasma, 

 mounting the separated corpuscles with- 

 out distortions, and making them take 

 the stain. 



It has been found that almost all re- 

 agents cause some changes in the general 

 shape and outline of corpuscles. Osmic 

 acid is claimed to harden and preserve 

 them without distortion, but as this sub- 

 stance is quite expensive and difficult to 

 operate, it is not adapted to general lab- 

 oratory use among young, inexperienced 

 students. The method of drying fresh 

 blood upon the cover-glass is not usually 

 successful, for the corpuscles are gener- 

 ally distorted by drying, or else they re- 

 fuse to take the ordinary stains after 

 being dried. 



For general laboratory use formalin is 

 an excellent preservative and fixing 

 agent. It is less expensive and more 

 easily operated than osmic acid. It 

 causes no appreciable distortion of the 

 cells and does not interfere with staining. 

 The method used is as follows: 



1. A quantity of freshly drawn blooi, 

 before coagulation has taken place, is 

 mixed with at least three times its 

 volume of a two per cent, formalin 

 solution, 



2, After allowing the mixture to stand 

 at least one hour, a drop from the bottom 

 of the vessel is placed upon a cover-slip 



and a second cover-slip is pressed lightly 

 upon it; then the two are separated and 

 the liquid is allowed to evaporate. 



3. The dried cover-slips are then passed 

 quickly through the flame in order to fix 

 the corpuscles more securely to the glass, 

 so that they may not be removed by 

 subsequent treatment. 



4. When cool, dip once or twice into 

 a five per cent, solution of acetic acid. 



5. After removing the acetic acid with 

 water, stain. If the corpuscles are 

 nucleated, it is best to use some contrast 

 stain, as haematoxylin and eosine or 

 methyl green with eosine or saffranin. 

 If an alcoholic stain is to be used, the 

 films must be washed with alcohol before 

 staining. Non-nucleaterl cells do not 

 require a contrast stain. Human corpus- 

 cles may be stained with Ehrlich's triple 

 stain. 



6. Remove excess of stain with water 

 or alcohol as stain requires. 



7. Remove alcohol with xylol, clove oil, 

 or turpentine. 



8. Mount in Canada balm. 



This method was employed successfully 

 in the laboratory at Purdue University 

 in all blood studies. The bloods used in 

 these studies were those of the cat, the 

 chicken, the ox, the pigeon, and man. 

 The human corpuscles examined seemed 

 to resist all stains for some reason, until 

 the films were treated with a dilute solu- 

 tion of acetic acid. The acid seemed to 

 possess a double function; first, that of 

 clearing the films, and, second, that of 

 causing the stain to become effective. 



This method may be of no particular 

 clinical value, but for general laboratory 

 purposes it promises to be a success. 



Erxest I. KlZER. 

 Biological Laboratories, Purdue Univer- 

 sity. 



