150-2 Journal of Applied Microscopy 



on a yellowish tinge. Keep the specimens in the dark. The duration of the 

 silver-bath is not of importance ; it may be one to three days in length, or even 

 longer if the solution be renewed. 



When the steps which follow can be carried through without a pause, replace 

 the silver solution with ninety-five per cent, alcohol. Change the alcohol 

 repeatedly during the course of half an hour, in order that all free silver nitrate 

 may be washed away. Follow with absolute alcohol, also renewed, thirty minutes ; 

 alcohol and ether, fifteen minutes ; thin celloidin, thirty minutes ; and thick cel- 

 loidin, five minutes. Then mount the specimen on a wooden block, and harden 

 the celloidin with chloroform. The imbedding has been too hastily done, of 

 course, to permit true infiltration with celloidin, but this cannot be secured with- 

 out destroying the impregnation. 



Clearing of the entire mass, as imbedded, is advantageous. For this purpose, 

 place the block in a mixture of oil bergamot, oil cedar-wood, and melted car- 

 bolic acid crystals, equal parts. This mixture clears rapidly, it may be used 

 over and over again, and it has the additional advantage of allowing the prepara- 

 tions to be kept in it for a little while without impairing the impregnation. 



Sections should be cut as thick as 75;^. In cutting, keep the knife flooded 

 with the clearing mixture. Place the sections on slides, as desired. The sur- 

 plus oil may be removed neatly by pressing on the sections with tissue paper 

 backed with a blotter. Mount in either colophonium, dammar, or balsam, with- 

 out a cover-slip. The mounting medium should cover the section with a thin 

 and even coating. Hasten the drying of the preparation with gentle heat for a 

 few hours. 



Successful preparations should have the neurones, to their finest ramifications, 

 a perfectly opaque black on a nearly transparent field. If the field becomes 

 clouded with fine specks later on, the silver nitrate was not all removed 

 in the washing. 



Under certain conditions, pure formaldehyde may be substituted for the 

 osmic acid in the hardening mixture given above, a good proportion being three 

 parts of commercial formaldehyde to one hundred parts of the bichromate solu- 

 tion. The formaldehyde tends to reduce the potassium bichromate quite rapidly, 

 and so the two should be mixed just before using. The application of formal- 

 dehyde in this way often yields beautiful results with the mammalian nervous- 

 system, but I have not been so successful with it in the field of the lower verte- 

 brates. 



b. Previous Hardening with Formaldehyde. — It is often quite desirable to be 

 able to harden a brain some time in advance of its final use for impregnation, as 

 in planning for classwork, or in the transportation of specimens from the field to 

 the laboratory. This may be accomplished through preliminary hardening with 

 formaldehyde. 



The whole brain is hardened in ten per cent, formaldehyde, and should be 

 kept in this strength of solution until it is wanted. Slices cut from the desired 

 regions are washed with water for half an hour to extract the formaldehyde, and 

 are then to be saturated with potassium bichromate. 



Place the pieces in a three and five-tenths per cent, solution of potassium 



