600 



METHODS AND FORMULAS 



MS 34.0 



of a day, washed, as has been described 

 above, in successive portions of silver ni- 

 trate solution, notched by a different sys- 

 tem, and transferred to the large bottle 

 of silver nitrate at intervals of a day until 

 all the blocks have been transferred. There 

 will finally be 30 blocks covering 15 peri- 

 ods of immersion in the dichromate solu- 

 tion. It must be emphasized that the suc- 

 cess of the process depends on the length 

 of time the blocks stay in the dichromate. 

 As for the length of time in silver nitrate, 

 it should not be less than 48 hours, but it 

 may be extended for several weeks with 

 the assurance that the impregnation will 

 not be affected. Forty-eight hours after 

 the last of the blocks has been placed in 

 silver nitrate, pour off the solution, add 

 about 50 milliliters of triple-distilled wa- 

 ter, tip the bottle up and down once, pour 

 off the distilled water, and fill the bottle 

 with 90% alcohol. After 24 hours the alco- 

 hol is replaced, and the process repeated 

 as often as the reagent becomes discolored. 



Though it is perfectly possible to obtain 

 adequate sections by freezing techniques, 

 it is usually better to use the celloidin 

 method. An objection has always been 

 that prolonged exposure to strong alcohol 

 tends to remove the stains; but this has 

 not been the author's experience. 



To avoid wasting time preparing sec- 

 tions from unsatisfactory blocks, reject 

 the hopeless cases by an examination of a 

 freshly cut surface under a binocular mi- 

 croscope. For this purpose arrange the 

 blocks in order of their removal from the 

 dichromate. Examine first the block which 

 has been hardened for the longest time. 

 Slice one of the faces of this block with a 

 razor-sharp scalpel and examine it under 

 the surface of 95% alcohol with a binocu- 

 lar microscope. It will almost invariably 

 appear as a uniform sheet of chrome yel- 

 low, at best only slightly granular. This 

 indicates, as anticipated, that the block 

 has been in the dichromate too long. Next 

 take the block which has remained for the 

 least time in the dichromate and slice its 

 edge. It is almost certain to have a pale, 

 transparent, cheesy appearance without 

 any opaque yellow speckling. This indi- 

 cates, as anticipated, that the block has 

 been for too short a time in the dichro- 



mate. Work through the series, taking 

 blocks alternately from groups impreg- 

 nated for a long and for a short time. It 

 soon becomes apparent that the plain 

 opacity of the long-impregnated blocks 

 begins to break up into speckles, whereas 

 golden yellow opaque spots begin to ap- 

 pear in the cheesy, transparent, under- 

 impregnated blocks. Somewhere between 

 these two lies the perfect specimen, prefer- 

 ably one lying intermediate between the 

 two which have been reached as the end 

 points of unsatisfactory material. 



As has been stated, it is customary to 

 section blocks of Golgi-impregnated mate- 

 rial by the freezing technique, on the 

 ground that the prolonged immersion in 

 alcohol necessary for celloidin sectioning 

 tends to degrade the finer details of the 

 preparation. This has not been true in the 

 writer's experience, unless dehydration 

 has been unnecessarily prolonged. It is 

 usually sufficient to put the selected block 

 in about 25 miUiUters of absolute alcohol 

 for about two hours. Next, replace the 

 absolute alcohol with a mixture of equal 

 parts of absolute alcohol and ether and 

 let stand two hours longer. Then place 

 the block for about three hours in the thin, 

 or first, solution of celloidin customarily 

 employed for embedding. Since it will 

 probably now be toward the end of the 

 day, it is the author's custom to transfer 

 the block directly from the thin syrupy 

 solution of celloidin to the thickest solu- 

 tion of celloidin and to leave the material 

 in this overnight. It may be removed from 

 this thick syrup next morning and hard- 

 ened by whatever technique is customary. 

 In this instance immersion in 70 % alcohol 

 is probably simplest. When the celloidin 

 is hardened, sections about 25 microns 

 thick are cut with a knife moistened with 

 70% alcohol and accumulated in 70% al- 

 cohol. Each section is then separately re- 

 moved to a slide and examined under me- 

 dium power of an ordinary microscope. 

 One or two sections will soon be found 

 which will demonstrate to perfection the 

 aborizations of Purkinje cells. These sec- 

 tions should be transferred to a separate 

 watch glass of 70% alcohol. When suffi- 

 cient sections showing the required struc- 

 ture have been accumulated, they may be 



