104 



Journal of Applied Microscopy. 



venience and with little loss of time. It 

 should of course be borne in mind that 

 the dishes containing the alcohol and 

 xylol should be covered when not in use. 

 From the distilled water, the cover- 

 glasses with sections fixed to them are 

 transferred to one or the other of the 

 haematoxylin solutions above described. 

 In the stain they remain from fifteen 

 to thirty minutes, are then rinsed in 

 distilled water and transferred to an 

 acid alcohol solution. This solution is 

 made by adding six to eight drops of 

 hydrochloric acid to 100 ccm. of 70% 

 alcohol. As soon as the haematoxylin 

 stained sections are placed in this acid 

 alcohol solution, their purplish-blue 

 color changes to a reddish-brown, and 

 a reddish-brown stain is given off from 

 the section. The washing in the acid 

 alcohol is continued until very little of 

 this stain is given off from the prepara- 

 tions. This step has for its purpose the 

 washing out of the stain from all por- 

 tions of the tissue except the nuclei. 

 After washing in acid alcohol the cover- 

 glasses are transferred to water — not 

 distilled water, but ordinary tap water. 

 This washes out and to some extent 

 neutralizes the acid alcohol in the sec- 

 tions. In the tap-water the sections 

 again assume a purplish-blue color of 

 a lighter hue, however, than before the 

 decolorization. 



The steps for counter staining in eosin 

 are as follows: Transfer the sections, 

 stained in haematoxylin and decolor- 

 ized, from the water into the eosin 

 solution, where they remain for three to 

 five minutes, then wash thoroughly in 

 distilled water and transfer to 95% alco- 

 hol. For counter staining with Van 

 Gieson's solution, place the haematoxy- 

 lin stained and decolorized sections in 

 this solution for twenty to forty sec- 

 onds, wash in distilled water and trans- 

 fer to 95% alcohol. The sections coun- 

 ter-stained in either the eosin or Van 

 Gieson's solutions remain in the 95% 

 alcohol, for two or three minutes, and 

 are then transferred to absolute alco- 

 hol, where the sections are fully 

 dehydrated, about three to five minutes 

 are required. The preparations are now 

 cleared in some clearing agent of which 

 a number are in use. They are all sub- 

 stances which displace the alcohol from 

 the sections and are solvents for the 

 balsam used in mounting them, thus 

 assisting in the penetration of the bal- 

 sam. They are known as clearing 

 agents, however, because they all have 



a high refractive index, and literally 

 make the sections clear. In this labor- 

 atory, oil of bergamot is used for this 

 purpose. Oil of cloves, oil of origanum 

 or oil of cedar may, however, be used. 

 The sections remain in the clearing 

 agent two to three minutes. Cover- 

 glass^ preparations, i. e., sections fixed 

 to a cover-glass and stained as here 

 detailed, are best washed or rinsed in 

 xylol before mounting; this insures a 

 clean surface to the cover-glass when 

 finally mounted. 



Cover-glass preparations are mounted 

 by placing a small drop of Canada bal- 

 sam on a slide and placing the cover- 

 glass, section side down, on the balsam. 



This method of staining may seem 

 somewhat lengthy, and we admit there 

 are shorter methods. The sticking of 

 sections to a cover-glass and the stain- 

 ing of sections fixed to the cover has, 

 however, its advantages. The readiness 

 with which such sections are trans- 

 ferred from one fiuid to another; the 

 fact that portions of the tissue are not 

 likely to be lost; sections cannot fold 

 nor tear; are readily mounted; these 

 are all considerations which go far to 

 compensate for the extra time and 

 extra manipulation required to follow 

 the method. It should also be stated 

 that if the sections are small, a number 

 of sections may be fixed to the same 

 cover-glass by drawing, instead of one, 

 several sections on to the cover-glass 

 after they have been flattened on the 

 distilled water. 



Staining of Celloidin Sections. — Celloi- 

 din sections are stained as are the paraf- 

 fin sections, remembering of course that 

 they need not be fixed to cover-glasses, 

 as the celloidin does not interfere with 

 the staining, and does not, therefore, 

 need to be removed. They are best 

 carried from one solution to another by 

 taking them up on a teasing needle. 

 For clearing celloidin sections, I would 

 recommend the use of carbol-xylol. This 

 is prepared by mixing one part of pure 

 carbolic acid with three parts of xylol. 

 The stained sections are transferred 

 from the 95% alcohol to the carbol-xylol 

 solution, and from this solution in which 

 they need to remain only a few minutes, 

 to a clean slide, this by taking up the 

 section on a section lifter, and drawing 

 it off on to the slide. They are mounted 

 by draining off the excess of carbol- 

 xylol, placing a drop of Canada balsam 

 on the section and covering it with a 

 cover-glass. 



