572 



METHODS AND FORMULAS 



MS 33.0 



brain with the formaldehyde-bromide as a 

 liemostatic measure, and tlien remove 

 small pieces from the white matter of the 

 cerebrum or from such other portions of 

 the brain as it is desired to study. The 

 white matter of the cerebrum is recom- 

 mended as a material because of the rela- 

 tive certainty with which the supporting 

 elements witliin it may be demonstrated. 

 The tissue is best removed in blocks of 

 about 3-2 cm. cube, and as tlie author has 

 elsewhere indicated, it is preferable to use 

 for this purpose the broken edge of a 

 coverslip rather than steel instruments. 

 Three or four of these blocks are removed 

 to the formaldehyde-bromide solution in a 

 chemically cleaned stoppered bottle and 

 permitted to remain there until such time 

 as one is ready to proceed with the prepa- 

 ration. It is stated by McClung 1929, 379 

 that one week in this solution gives excel- 

 lent results; but the original recommenda- 

 tion of Penfield does not place any time 

 limit on the preliminary hardening. 



When one is ready to prepare and stain 

 sections it is simplest to erect a sort of 

 production line of glass dishes containing 

 the successive solutions and water for the 

 intermediate washings. The size and shape 

 of these dishes is not of the slightest im- 

 portance provided that they are chem- 

 ically cleaned. Dishes in which it is in- 

 tended to leave the reagents exposed for 

 any length of time should be made of 

 pyrex glass and furnished with some kind 

 of lid. In the writer's experience the most 

 readily available and useful dish is a deep 

 pyrex petri dish at least 15 milhmeters in 

 depth, even though these dishes require 

 rather large volumes of solution. Twelve 

 such dishes should be chemically cleaned 

 (that is, soaked in dichromate-sulfuric 

 cleaning mixture), washed in tap water, 

 soaked in distilled water, and dried under 

 dust-free conditions. 



The first dish should contain triple-dis- 

 tilled water. The blocks of tissue are 

 placed on a freezing microtome and sec- 

 tioned to a thickness of from 15 to 25 

 microns, and the sections are taken off on 

 a wet knife and accumulated in this dish. 

 When a sufficient number have been ac- 

 cumulated, one may proceed with the 

 staining. 



In another dish place the 1 % ammonia, 

 and transfer to it the sections from the 

 dish of triple-distilled water. Here they 

 may remain overnight to insure the com- 

 plete washing out of formaldehyde and the 

 neutralization of anj^ residual acid which 

 may be piesent. 



The next morning fill another dish with 

 2 % hydrobromic acid and transfer the sec- 

 tions to it one at a time, being careful to 

 pick them up with a glass utensil. It will 

 be found that a 22 X 15 mm. coverslip is 

 excellent for this purpose. As soon as 

 enough sections have been accumulated in 

 the hydrobromic acid, the dish is placed 

 for one hour on the surface of a water bath 

 which is maintained at from 37° to 40°C. 

 While these sections are being treated, fill 

 three more dishes with water and a fourth 

 with the 5% sodium carbonate. The dish 

 containing the sections is now removed 

 from the bath and, again with a glass 

 utensil, the sections are removed to the 

 first dish of wash water. Here they remain 

 for about five minutes while the dish is 

 gently rocked at intervals. All the sections 

 are next removed to the second dish of 

 water for at least 30 minutes of rocking at 

 intervals; and then to the third dish for 

 not less than 15 minutes of similar wash- 

 ing. While the washing in the third dish 

 is being concluded the next dish is filled 

 with 5% sodium carbonate. All the sec- 

 tions are transferred to this and permitted 

 to remain for about one hour, though they 

 may be left two or three times as long 

 without interfering with subsequent stages 

 of the technique. 



Next take four clean dishes. Fill the 

 first of these with triple-distilled water, 

 the second with the diluted silver stain, 

 the third with triple-distilled water, and 

 the fourth with 0.4% neutrahzed formal- 

 dehyde. Take the first of the sections from 

 the sodium carbonate, rinse it in the first 

 dish of distilled water, and pass it to the 

 dish of diluted silver stain. There it should 

 remain for a period of three minutes. It is 

 then removed, rinsed in the next dish of 

 distilled water, and passed into the dish of 

 formaldehyde. It should turn a dull steel- 

 grey color almost immediately. If the first 

 section does not turn this color, take a 

 second section from the sodium carbonate, 



