MS 22.0 



METAL STAINS 



537 



meninges. Finally, remove a series of cubes 

 of about one-centimeter side from the 

 cerebral cortex. Stainless steel knives may 

 be used for this purpose, but the author 

 has alwaj's found it more i)ractical to em- 

 ploy the sharp edge of a broken coverslip. 

 If a standard 18-by-22-minimeter cover- 

 slip be broken roughl}'- along a diagonal, it 

 will be found to cut the brain most satis- 

 factorily. This avoids the risk of surface 

 contamination from metals which will in- 

 terfere with subsequent staining. Two or 

 three blocks will be sufficient, and they 

 should be placed in the stoppered bottle 

 containing the fixative, of which at least 

 250 milhhters should be employed for two 

 or three one-centimeter cubes; four times 

 this volume is not an unreasonable 

 quantity. 



The blocks should be removed from the 

 fixatives as soon as they are adequately 

 hardened. The exact degree of hardening 

 necessary can best be gaged by pressing 

 gently on the surface of the block with the 

 rounded end of a glass rod. The block 

 should be springy but not hard, and 

 should not be removed until it can be han- 

 dled with forceps without running the risk 

 of crushing any of the internal structures. 



After removal from the fixative, the 

 blocks are washed in running triple-dis- 

 tilled water for at least a day, or washed 

 in not less than five successive changes of 

 at least 500 milliUters each. After washing, 

 each block is subdivided, preferaljly by 

 the broken edge of a glass coverslip, into 

 five or six smaller blocks, which form a 

 reasonably sized portion for sectioning on 

 the freezing microtome. One of the most 

 fruitful sources of error of this technique 

 is the use of crude commercial mucilages 

 of gum arabic in the course of the section- 

 ing, even after taking stringent precau- 

 tions with the purity of the reagents 

 employed in fixation. It is far better to em- 

 ploy pure sugar solutions than to rely on 

 gum materials which may contain such 

 impurities as will stultify subsequent work. 



Sections are removed, as made, to a 

 watch glass of triple-distilled water. Again 

 it must be emphasized that the watch 

 glass should be chemically clean. Before 

 commencing to section the material, it is 

 desirable to prepare the staining reagent, 



the formula for which is given as MS 22.1 

 Cajal 1916 below. The very greatest care 

 is required in making this solution. All the 

 reagents involved, particularly the mer- 

 curic chloride, should be of the grade sold 

 for analytical analysis; the ordinary com- 

 mercial chloride found in biological lab- 

 oratories is not satisfactory. Cajal does 

 not indicate in his formulas which of the 

 numerous types of gold chloride or mixed 

 gold, potassium, and sodium chlorides he 

 used in the original fornuila. The method 

 of preparing the formula is, however, crit- 

 ical. First, the mercuric chloride is dis- 

 solved in hot triple-distilled water at from 

 70°C to 80°C. It should give a clear solu- 

 tion entirely free from opalescence, and 

 if the faintest trace of a precipitate is seen, 

 the solution must be rejected and a purer 

 batch of the reagent sought. In a second 

 chemically clean beaker dissolve the gold 

 chloride at room temperature in triple- 

 distilled water. The gold solution is then 

 added to the hot mercuric chloride solu- 

 tion with constant stirring; the utmost 

 care is taken to avoid the production of a 

 precipitate, the presence of which will ren- 

 der the solution valueless. The solution is 

 then brought up to 250 with triple-distilled 

 water. 



The sections are taken from the triple- 

 distilled water and transferred to the 

 staining solution for from four to six hours 

 in the dark at room temperature. If time 

 is a consideration, it is possible to warm 

 the solution to about 25°C., which reduces 

 the period required for staining to three 

 hours. It is not difficult to determine when 

 staining is sufficient; the sections should, 

 in any case, be examined at hourly inter- 

 vals and be withdrawn from the stain 

 when they have become dark purple. A 

 Hlac color indicates understaining, and 

 they will be spoiled if they are left until 

 they have become brown. The successful 

 application of this technique depends on 

 the ability to judge the exact shade of 

 purple which indicates a satisfactory ter- 

 mination of the staining process. Sections 

 should be removed from the stain and 

 washed for at least three hours in running 

 trit)le-distilled water, or in at least three 

 changes of a considerable volume of triple- 

 distilled water changed at hourly intervals. 



