METHODS OF IMPREGNATION. 49 



Golgi's Chromsilver or Chromsublimate Method. This method 

 depends on the formation of a very fine precipitate, which forms in cer- 

 tain tissue elements or in preexisting space, when treated first with a 

 solution of bichromate of potassium and secondarily with a solution of 

 silver nitrate or bichlorid of mercury. The nature and precise location 

 of this precipitate is not well understood. It is very probable, however, 

 as Kallius suggests, that an albumin-chromsilver compound, of an 

 unknown constitution, is formed in the cells and processes or in spaces 

 filled with the precipitate. This method is especially useful in bringing 

 to view the cellular elements of the nervous system, both central and 

 peripheral ; further, the end- ramifications of gland ducts, and now and 

 then cell boundaries. Usually only a small percentage of the tissue 

 elements or the spaces of any given tissue are colored. This may, how- 

 ever, be regarded as one of the advantages of the method, since it 

 enables a clearer view of the parts colored. The precipitate appears 

 black in transmitted light. It is necessary to state, however, that this 

 method is very unreliable, and that failures are often met with, also that 

 an amorphous precipitate is generally formed, both in and about the 

 tissues, which in part at least destroys the usefulness of the preparations 

 obtained. 



Golgi's methods will perhaps be better understood if we first 

 give a short historic sketch of their development. 



In the year 1875 Golgi applied his method as follows : He fixed (olfactory bulb) in 

 MUller's fluid, and increased the percentage of bichromate on changing the fluid (up to 

 4 %}. Fixation lasted five or six weeks in summer and three or four months or more 

 in winter. He then took out pieces of the tissue every four or five days and treated them 

 experimentally with a 0.5% to \% silver nitrate solution. In summer this process took 

 about twenty-four hours, and in winter forty-eight hours, although a longer treatment 

 was not found to be detrimental. This method must be regarded as very uncertain, since 

 the length of time during which the specimens remain in MUller's fluid must be very 

 closely calculated, as it depends largely upon the temperature of the medium. As soon 

 as the silver reaction was established, the pieces were preserved either in the silver solu- 

 tion itself or in alcohol. The sections were finally washed in absolute alcohol, cleared 

 with creosote, and mounted in Canada balsam. The impregnation disappeared in a 

 short time. In the year 1885 Golgi made a further announcement regarding his method, 

 recommending for fixation the pure bichromate of potassium, as well as MUller's fluid. 

 Pieces of the brain and spinal cord (from I to 1.5 c.c. in size) from a freshly killed ani- 

 mal were used, and the reaction sometimes took place in from twenty-four to forty-eight 

 hours after death. For fixing, potassium bichromate solution in gradually ascending 

 strengths (if to 5^) was employed, large amounts of the fluid being used and placed 

 in well-sealed receptacles. The fluid was repeatedly changed, and camphor or salicylic 

 acid was added in order to prevent the growth of fungi. Since it is difficult to determine 

 exactly when fixation in potassium bichromate reaches the precise point favorable to sub- 

 sequent treatment with nitrate of silver, because the process depends entirely upon the 

 temperature and quantity of the fluid, it becomes necessary, after about six weeks' treat- 

 ment with the bichromate, to experiment every eight days or so to see whether the 

 silver nitrate gives good results. The strength of the latter should be about O.66f and 

 the quantity about 200 c.c. to a I c.c. object. At first a plentiful precipitate is thrown 

 down, in which case the solution should be changed, and this probably repeated once more 

 after a few hours. After twenty-four hours, at the most forty eight hours, this process is 

 usually completed, and the tissues may be sectioned. The sections must then be care- 

 fully dehydrated with absolute alcohol, cleared in creosote and mounted without a cover- 

 glass in Canada balsam (the section is mounted on a cover-glass with Canada balsam, and 

 the cover-slip then fastened over the opening of a perforated slide with the specimen 

 downward). 



In order to obtain a uniform penetration of the objects by the potassium 

 bichromate, the latter may be first injected into the vessels. Golgi uses potassium 

 bichromate-gelatin 12.5% of the salt, based on the amount of the softened gelatin ; com- 

 pare Golgi, 93). After the injection and cooling of the specimen the latter is cut in 

 4 



