532 



METHODS AND FORMULAS 



MS 21.0 



no evidence of the exact reaction pro- 

 duced, nor has it been satisfactorily estab- 

 lished that the resultant precipitate is 

 metallic gold. The ver}^ fugitive nature of 

 many of the gold stains would tend to 

 make one beheve that the metal cannot be 

 involved. It cannot be too strongly em- 

 phasized that absolute cleanliness and the 

 rigorous control of the extent of hght ad- 

 mitted during the reaction are two criteria 

 of success. 



MS 21.0 Typical Example 



Demonstration of the termination 



of the fourth cranial nerve in the 



superior obhque muscle by the 



method of Ranvier 1889 



This method is more than half a century 

 old, but it is one of the simplest and best 

 available, both for training classes in gold 

 techniques and for demonstrating the 

 nerve terminations in muscles. Only two 

 prerequisites are essential to success. The 

 first is that the lemon juice employed shall 

 be from a relatively fresh lemon, shall 

 have been squeezed with as little oil of 

 lemon getting into it as possible, and that 

 it shall then have been filtered through 

 paper; tliis is tedious unless it is done with 

 a filter pump. It is better to prepare the 

 lemon juice immediately before use, and it 

 is not nearly as good, even if it be left 

 overnight. The second essential to success 

 is that the glassware used should be chem- 

 ically clean. In this particular technique 

 four glass-stoppered bottles, or upright 

 glass-stoppered weighing bottles, should 

 be soaked in sulfuric-dichromate cleaning 

 solution, thoroughly washed off in tap 

 water, soaked in distilled water, and then 

 dried under conditions which will leave 

 them as dust-free as possible. In the first 

 bottle place the freshly filtered lemon 

 juice, in the second bottle 1% gold chlo- 

 ride, in the third bottle 0.2% acetic acid, 

 and in the fourth bottle 20% formic acid. 

 There will also be required two glass 

 dishes of triple-distilled water which 

 should be as clean as possible. 



A rabbit is a good subject for this tech- 

 nique; but if a small shark or dogfish is 



available, it will be found to be better. In 

 either case, the animal should be killed, 

 the skin removed from around the orbit, 

 and the eye taken out carefully so as to 

 leave all the muscles intact within the or- 

 bit. The fourth nerve, leading to the supe- 

 rior obhque muscle, should then be identi- 

 fied and severed where it leaves the fora- 

 men. The muscle itself should then be cut 

 off, leaving as much as possible in front of 

 the nerve and about a quarter of an inch 

 behind the nerve. Then take a surgical 

 silk or other fine, clean fiber and tie this 

 round the upper end of the nerve. Using 

 this thread as a suspensor, hang the mus- 

 cle by the nerve in the bottle of lemon 

 juice. It will become transparent within a 

 few hours, and may be passed to the next 

 stage at any period between the time when 

 it becomes completely transparent and 

 about the twelfth hour of immersion. The 

 longer period is, in general, better for 

 mammahan tissue, and the shortest pos- 

 sible period for fish tissues. 



As soon as the muscle has become trans- 

 parent, withdraw it from the lemon juice 

 by the thread, snip off the thread and, hold- 

 ing the muscle by one end in a pair of for- 

 ceps, rinse it thoroughly in one of the jars 

 of triple-distilled water. Then, after having 

 drained off the water by touching the end 

 of the muscle to a clean filter paper, drop 

 the preparation into at least 60 milhliters 

 of 1% gold chloride. Leave it in this solu- 

 tion, with gentle agitation at intervals, 

 for about 20 minutes; it will darken 

 shghtly, from the transparent yellow of 

 the lemon juice to a light brown. 



One certain way to insure failure of this 

 technique is to use metallic forceps to re- 

 move the muscle from the gold chloride 

 and pass it to the acetic acid. It must be 

 picked up on the end of a glass hook or 

 with a glass spoon. The muscle is then 

 rinsed and placed in the bottle of 0.2% 

 acetic acid. The intensity of hght is 

 critical, for if the object be placed in 

 direct sunhght it will become badly over- 

 stained, while in the dark it will not be- 

 come stained at all. It was recommended 

 by Ranvier originally that this technique 

 should be carried out on days when there 

 were clear, white clouds in the sky from 



