NEUROFIBRILS 491 



per cent. NaOH, shake and again let the precipitate fall to the bottom. 

 Repeat the operation a third and a fourth time till no precipitate is 

 formed on adding NaOH. The supernatant clear fluid is now pipetted 

 off and the precipitate washed with 100 to 150 c.c. of distilled water. 

 Continue washing until the wash water no longer reddens phenol- 

 phthalein. Pipette off the wash-water and add ammonia drop by drop 

 to the precipitate till it is almost but not entirely dissolved. After the 

 addition of 4 c.c. of distilled water the fluid is ready for use. 



Del Rio-Hortega (Trab. Lab. Invest. Biol., Madrid, xiv, 1916, 

 p. 181) has made known a similar method used in those laboratories 

 for preparing the ammoniacal silver nitrate bath. Forty drops of 40 

 per cent, caustic soda are added to 30 c.c. of 10 per cent, silver nitrate, 

 and the precipitate washed ten to twelve times by means of about a 

 litre of distilled water. Fifty cubic centimetres of water are then added 

 to it, and ammonia, drop by drop, until the precipitate is dissolved. 

 The solution, brought finally to 150 c.c. and filtered into a dark brown 

 bottle, keeps well for many months. I find that the ammoniacal silver 

 bath thus prepared can be further diluted with one, two, up to five 

 times its volume of water, and usefully employed for Bielschowsky's 

 method for pieces, particularly for the study of peripheral nerve- 

 endings. 



Agduhr (Ztschr. zviss. Mikr., xxxiv, 1917, pp. 1 — 99), who has 

 exhaustively investigated almost all questions relating to the results 

 obtainable by Bielschowsky's method for pieces, has come to the con- 

 clusion that material is best fixed in neutral or slightly acid 20 per cent. 

 formaldehyde (50 per cent, formalin). Pieces should then be washed in 

 distilled water for many days until the wash-water is free from sub- 

 stances reducible by an ammoniacal silver nitrate solution used as test. 

 For the first silver bath he uses 3 per cent, silver nitrate, and for the 

 second a solution obtained by adding to 10 c.c. of 10 per cent, silver 

 nitrate, first 20 drops of 25 per cent. NaOH, then from 200 up to 600 c.c. 

 of distilled water, and lastly ammonia enough to dissolve the precipi- 

 tate. For the reduction he uses again 20 per cent, formaldehyde. To 

 avoid an excessive impregnation of the connective tissue he also finds 

 it useful to wash pieces in acidified distilled water (see the Bielschowsky's 

 method for peripheral nerve-fibres), but he uses as much as five times 

 the amount suggested by Bielschowsky. 



1023. Da Fano's Modifications. In this series of modifications 

 of Bielschowsky's method it is important that the distilled water 

 should be pure and free from any trace of organic matter. 



Da Fano's first modification {Mod. 1) {Atti. Soc. Lamh. Sc. 

 Med. Biol., Milano, iii, 1914) was meant for the study of reticular 

 tissue of spleen, lymph glands, and other organs, and is to be 

 carried out as follows : (1) Fix small pieces of fresh tissue in 

 10 to 20 per cent, formalin or in Kayserling's first fluid (forty- 

 eight hours at least), or in Orth's fluid (twenty-four to forty- 

 eight hours). (2) Wash pieces in running tap-water for twenty- 

 four to thirty hours, and then in distilled water for another 

 twenty-four hours. (3) Wash sections made by the freezing 

 method in re-distilled water (twenty-four hours), and then place 

 them in filtered 2 per cent, silver nitrate (prepared with re- 

 distilled water) in a Petri dish, taking care that they do not touch 

 each other. Here they are kept in the dark and at room tempera- 



