574 



METHODS AND FORMULAS 



MS 33.0 



in a large volume of the formaldehyde 

 bromide for from two to three days at 

 room temperature before being removed 

 to a fresh batch of bromide in a chemically 

 clean beaker which is then raised to a 

 temperature of 55°C. for about ten min- 

 utes. The bromide is then cooled to room 

 temperature and sections taken from it by 

 the freezing technique described in Chap- 

 ter 15. Sections should be about 25 mi- 

 crons in thickness, and cut with a knife 

 moistened in distilled water. Sections are 

 removed from this knife to a weak solution 

 of ammonia, where they may remain until 

 required ; certainly at least overnight. 



Before proceeding further it is neces- 

 sary to make up the customary solutions. 

 The only one presenting any difficulty is 

 the silver complex of del Rio-Hortega. 

 This is given below as MS 33.1 del RIo- 

 Hortega 1916. All that has already been 

 said about the necessity of the absolute 

 chemical cleanliness of the glassware and 

 the purity of reagents can be repeated at 

 this point. Triple-distilled water, reagent- 

 grade sodium hydroxide, and reagent- 

 grade ammonia are essential. 



Place 50 milhliters of 10% silver nitrate 

 in a chemically clean 500-milliliter beaker 

 and add to it, with constant stirring, 3.5 

 milhUters of 40% sodium hydroxide. Stir 

 until the white curd of silver hydroxide is 

 uniformly distributed. Then while still 

 stirring add about 250 milliliters of triple- 

 distilled water. Allow the precipitate to 

 settle, pour off the supernant liquid, and 

 add a further 250 milliliters of triple-dis- 

 tilled water. This washing by decantation 

 should be repeated at least three times, 

 and the precipitate allowed to settle after 

 the last washing until all but 50 milliliters 

 of the wash water can be discarded. The 

 wet precipitate is then removed to a chem- 

 ically cleaned graduated cylinder and the 

 volume brought up to 75 milliliters before 

 being transferred back to the beaker. 

 Ammonia is added drop by drop until the 

 precipitate is just redissolved. As has been 

 recommended in previous examples, the 

 ammonia should be added slowly. When 

 there are signs that the precipitate is clear- 

 ing up, the (hops shouUl be added at inter- 

 vals of from 10 to 15 seconds, with con- 

 stant stirring between additions. A strong 



light and a dark background assist in de- 

 termining the end point of the reaction. 

 The mixture is now placed in a graduated 

 flask and brought up to a volume of 250 

 milhliters with triple-distilled water. In 

 the present method this staining solution 

 is used at full strength. 



Other solutions also required are : a 4 % 

 solution of neutralized formaldehyde (re- 

 member that reagent-grade formaldehyde 

 neutralized to a pH between 7 and 7.5 

 with reagent-grade borax must of neces- 

 sity be employed), a 2% solution of gold 

 chloride, and the regular fixing solution of 

 sodium thiosulfate. As in the last ex- 

 ample, it is recommended that staining 

 be carried out in chemically cleaned petri 

 dishes. In the present instance not more 

 than half a dozen such dishes will be re- 

 quired. In the first of these place triple- 

 distilled water; in the second, the stain; in 

 the third, triple-distilled water; in the 

 fourth, the neutralized formaldehyde; in 

 the fifth, triple-distilled water; and in the 

 sixth and seventh, the gold chloride and 

 the fixative. Now take sections from the 

 weak ammonia, place them in the first 

 dish of triple-distilled water, and rock 

 them back and forth until they are alkali- 

 free. A trial section is placed in the silver 

 staining solution for approximately three 

 minutes, then removed from the stain, 

 rinsed rapidly in the next dish of distilled 

 water, and placed in the neutralized form- 

 aldehyde. There, in from three to five 

 minutes, it should have assumed a clear 

 grey color, which will not become appreci- 

 ably darker for another five minutes. This 

 reaction time indicates that three minutes 

 in the stain is satisfactory. If the darken- 

 ing is too great or appears too quickly, the 

 time that the section is left in the stain 

 must be reduced. On the other hand, if the 

 section fails to darken, the time must be 

 increased. When the correct time has been 

 established, all the sections may be passed 

 successively through the stain, the dis-- 

 tilled water, and the neutralized formal- 

 dehyde. After a maximum time of approx- 

 imately ten minutes in the formaldehyde, 

 the sections are removed to the next dish 

 of triple-distilled water and thoroughly 

 washed before being toned for ten to 

 twenty minutes and fixed for five minutes. 



