APPENDIX 243 



paraffin. When cold, thin sections can be cut, the paraffin dissolved out by 

 turpentine, and the sections mounted. 



Preparation of frozen sections. The bichromate solutions are the best 

 fluids to use for preserving tissues which are to be frozen in place of being 

 embedded. The tissue in such cases should not be put into alcohol, but 

 merely requires to be dipped in strong gum before being placed upon the 

 freezing microtome. Portions of the central nervous system need to be 

 soaked in gum to which a little syrup has been added. 



Staining and mounting of sections. The fluids most commonly employed 

 for the staining of sections are : 1. A dilute watery solution of haematoxylm 

 and alum ; 2. A solution of carmine ; 3. A solution of picro-carminate of am- 

 monia. The time of immersion in the staining fluid varies according to the 

 strength of the fluid and the mode by which the tissue has been hardened. 

 The necessity of staining sections may be avoided if the piece of tissue is 

 stained in bulk before embedding. For this purpose a carmine solution is 

 mostly used, on account of its penetrative power, that known as borax-carmine 

 being the best. The tissue must be left in it for twenty-four hours or more, 

 and then placed in acidulated alcohol. An alcoholic solution of magenta can 

 be used for staining in bulk ; from this the tissue goes into a small quantity 

 of oil of cloves or into turpentine, and, after being soaked with this, into the 

 melted paraffin. 



If the tissues have not been stained in bulk, the following is the order of 

 transference of the sections (they are supposed, if cut from paraffin, to have 

 been freed from this by immersion in turpentine) : 



1. From turpentine to absolute alcohol (5 minutes). 



2. From alcohol to distilled water ( minute). 



3. From distilled water to hsematoxylin or carmine (5 minutes or more). 



4. From haematoxylin to distilled water (| minute). 



5. From distilled water to alcohol (2 or 3 minutes). 



6. From alcohol to oil of cloves (1 minute). 



7. From oil of cloves to Canada balsam. 



If the tissues have already been stained in bulk, the sections are simply 

 mounted in Canada balsam after the paraffin used for embedding has been 

 dissolved away from them in turpentine. 



Creosote -shellac method of mounting. Friable sections, such as sections 

 of small embryos, and ribands of sections such as are cut with many micro- 

 tomes, are mounted in the following way : The slide is smeared with a solu- 

 tion of sheilas in creosote, the sections are placed in this and warmed so as to 

 melt their paraffin. They are thus fixed by the shellac, and the slide can be 

 immersed in turpentine to remove the paraffin, and the sections then covered 

 in Canada balsam. For this method the tissue should always have been pre- 

 viously stained in bulk. 



Solutions employed for Staining : 1. Solution of hcematoxylin in water. 

 Rub together in a mortar 10 grammes of powdered alum and 5 grammes of 

 extract of haematoxylin with 25 cubic centimeters of 70 per cent, alcohol, 

 gradually adding 100 cubic centimeters of distilled water. Decant into a 

 bottle and add a drop or two of ammonia. Let the mixture stand a few 

 days, occasionally shaking it. For staining, add two or three drops to a 

 watch-glass full of distilled water, and filter if necessary. 



2. Grenacher's hcematoxylin. To 150 cubic centimeters of a saturated 



