ZOOLOGY ANL» BOTANY, MICROSCOPY, ETC. 753 



without iodin. lifter 24 hours transfer them to 5 p.c. formalin in 

 water. In this they may remain for an indefinite period, l)ut at least 

 one day must elapse before the next stage is proceeded with. Place a 

 piece of tissue in a bowl of water (which should be changed at least 

 once) for from 2-3 hours. Make the ammonio-silver solution by 

 adding 5 p.c. ammonia to 5 p.c. silver nitrate in distilled water, until 

 the precipitate which at first forms is completely dissolved ; then add 

 more silver nitrate until a distinct cloudiness returns. Filter this solu- 

 tion into a bottle or specimen tube, add the piece of tissue, and put the 

 bottle, tightly corked, in the incubator at 37° C. for 7 days. The 

 silvering may thereafter be continued in the cold if it is not convenient 

 to go on at once to the next stage. Place a piece of the impregnated 

 tissue in a bowl of water (500 c.cm.) to which about 2 c.cm. of 5 p.c. 

 ammonia have been added. Remove the surface deposit as far as 

 possible. This is best done simply with the aid of the fingers whilst 

 the piece of tissue is held under water. Transfer the piece to a 

 second bowl of ammonia and water. Renew the fluid after about an 

 hour, and leave the tissue in this for 3-4 hours longer. Transfer to 

 dextrin solution (dextrin 5 oz. or 140 grm., water 10 oz. or 280 c.cm. ; 

 dissolve by boihng ; filter the solution through cotton-wool while still 

 hot ; after it has cooled add 1 p.c. of carboHc acid) to which ammonia 

 has been added in the proportion of 10 drops of a 5 p.c. solution to 

 1 oz., immediately before use. Allow the tissue to remain in this from 

 12-24 hours. Cut thin sections on the freezing microtome. Transfer 

 them from the knife to a bowl of water to which about 10 drops of 5 p.c. 

 ammonia have been added. After about 5 minutes transfer the sec- 

 tions to another bowl of ammonia and water, and after a similar period 

 give them a thii'd wash. Transfer the sections to a bowl of water to 

 which have been added from 5-10 drops of a saturated solution of 

 citric acid in water, and allow them to remain in this for 4-5 minutes. 

 Place the sections in a bowl of tap water, and after a few minutes 

 transfer them to a second bowl of water. They are now ready for 

 toning. 



The reagents required for the toning and decoloration processes 

 are 1 p.c. solution of gold chloride in distilled water, 1 p.c. solution of 

 pure sodium tungstate in distilled water (it is necessary to effect the 

 solution of the salt by boihng), and 1 p.c. potassium cyanide in distilled 

 water. Using only a clean glass rod or platinum needle, place from six 

 to twelve sections in a watch-glass containing a mixture (carefully filtered) 

 of the sodium tungstate and gold chloride solution in equal portions. 

 Allow the sections to tone for about half an hour, or until they assume 

 a distinct red tint, and then transfer them to a bowl of water in which 

 they should be allowed to wash for some minutes. Next place the sec- 

 tions one at a time in a watch-glass containing a little of the cyanide 

 solution. When the section has lost its deep red colour and assumed a 

 light pink tint, transfer it to a bowl of water and allow it to wash for 

 from 10-15 minutes, giving at least one change of water. Next stain 

 the section for from 5-10 seconds in Loeffler's or Neisser's methylen- 

 blue, wash it well in water, dehydrate with absolute alcohol, clear in 

 equal parts of turpentine and benzol, and mount in benzol-balsam. 



