544 



METHODS AND FORMULAS 



MS 31.0 



ing the object in the next. Thus, in those 

 techniques in which the term wash is em- 

 ployed, it is understood that all traces 

 of the previous reagent must be re- 

 moved, preferably by running triple-dis- 

 tilled water, before going on to the next 

 step. If running triple-distilled water is not 

 available, it is necessary to make at least 

 ten changes between large volumes of 

 water. Another common cause of failure 



is the employment of too small volumes of 

 reagents, many of which are weak solu- 

 tions, but which are dependent for their 

 success upon the maintenance of an excess 

 of the solute. Even when working with 

 pieces as small as a few-millimeter cube, 

 it is rarely worth while to employ less than 

 50 millihters of a solution, in which the 

 object should, moreover, be gently agi- 

 tated from time to time. 



MS 31 METHODS USING SILVER NITRATE 



31.0 Typical Examples 



Demonstration of the nervous 



elements of the retina by the 



method of Balbuena 1922 



This technique is included for two rea- 

 sons. First, it is the most complex silver 

 nitrate technique which has yet been 

 developed; second, the excellence of the 

 results obtained in a satisfactory prepara- 

 tion justify this complexity. As the tech- 

 nique involves the utilization of a large 

 number of somewhat unusual reagents, it 

 is best to gather these first. They will be 

 given in the numbered sequence in which 

 they are employed: 1. AMS 11.2 Cajal 

 1910a. This is the pyridine alcohol of the 

 well-known Cajal techniques, and is made 

 by adding 50 millihters of pyridine to 200 

 milhliters of absolute alcohol. The solution 

 is stable indefinitely. 2. AMS 11.2 Bal- 

 buena 1922. This is Balbuena's "alcohohc 

 extract of cork crumbs." It is prepared by 

 placing one inch of cork crumbs on the 

 bottom of a 250-miUiliter bottle. The 

 crumbs are best prepared by cutting small 

 pieces (of less than one-millimeter side) 

 from a fresh bottle cork. The bottle is then 

 filled with 70% alcohol and shaken at 

 daily intervals until the solution is a dark 

 yellow-brown color. The reagent is then 

 filtered from the cork crumbs and may be 

 stored indefinitely. 3. MS 31.1 Balbuena 

 1922 (Balbuena's silver stain). This is pre- 

 pared by dissolving 0.13 grams of silver 

 nitrate in 250 milhUters of triple-distilled 

 water. After solution is complete 2.5 miUi- 

 hters of pyridine are added. This solution 

 will remain stable for a considerable time, 

 if it is preserved in the dark. 4. AMS 21.1 



Balbuena 1922. This is Balbuena's devel- 

 oper, which requires the use of two solu- 

 tions. The first of these is an alcoholic 

 extract of amber. This is best prepared by 

 taking 70 milliliters of commercially avail- 

 able oil of amber and adding this to 180 

 milliliters of 80% alcohol. These are 

 shaken together at daily intervals for 

 seven days, at the end of which time the 

 alcoholic extract is separated in a separa- 

 tory funnel. This remains stable indefi- 

 nitely. The second solution required for 

 Balbuena's developer is a solution of 2.5 

 grams of hydroquinone in 250 millihters 

 of triple-distilled water. This should be 

 prepared as it is required, for it is very 

 unstable. 5. AMS 22.1 Balbuena 1922, 

 which is Balbuena's toning solution. It is 

 a buffered solution of gold chloride pre- 

 pared by dissolving 2.5 grams of sodium 

 borate in 250 milliliters of triple-distilled 

 water and then adding to this solution of 

 0.25 grams of gold chloride. This solution 

 also remains stable indefinitely if kept in 

 the dark. 6. A 5% solution of sodium 

 thiosulfate. 



There will also be required for purposes 

 of embedding; absolute alcohol, a mixture 

 of absolute alcohol and ether in equal pro- 

 portions, and the solutions of celloidin, 

 given above as the standard requirements 

 for celloidin embedding in Chapter 13. If 

 the celloidin block is to be set, as is best 

 in this instance, with the aid of anhydrous 

 chloroform, this reagent will also be re- 

 quired. Having made sure that these re- 

 agents are available, one must next secure 

 the material on which to operate. 



The retina of a rabbit is an excellent ma- 

 terial on which to start. The eyeUds are 

 removed from a freshly killed rabbit and 



