546 



METHODS AND FORMULAS 



MS 31.0 



As soon as this condition has been 

 reached, add to the mixture as much of 

 the hydroquinone sokition as one has 

 added extract of amber. The watch glass 

 is then gently shaken backward and for- 

 ward until the hydroquinone solution, the 

 milky precipitate of oil of amber, and the 

 original silver solution are thoroughly 

 homogenized. The sections should be 

 watched in this bath as they darken. If, 

 after about five minutes, the sections have 

 not become a definite dark brown, a fresh 

 bath should be prepared by taking more of 

 the original silver stain, rendering it milky 

 with the extract of amber, and then 

 adding to it the required quantity of 

 the hydroquinone solution. The sections 

 should then be removed from the ex- 

 hausted solution and placed in the fresh 

 one. It is rarely necessary to make more 

 than two changes, or to wait more than 

 10 or 15 minutes, before the sections have 

 become satisfactorily browned. When tliis 

 has occurred they are removed to triple- 

 distilled water where they are very thor- 

 oughly washed in at least five successive 

 changes. The sections are removed from 

 the distilled water directly to the gold 

 toning solution, the dish containing which 

 should be rocked gently backward and 

 forward as though one were developing a 

 photographic plate in a dish. Under this 

 treatment the sections will be seen to 

 change slowly from brown to a dark bluish 

 purple. This change will normally take 

 not more than a minute or two, and if it is 

 not taking place sufficiently rapidly, it in- 

 dicates either an insufficient washing of 

 the section or that the gold has become 

 decomposed through prolonged storage. 

 Additional washing or the substitution of 

 a freshly prepared gold solution will insure 

 satisfactory toning within a few moments. 



When toning is complete the sections 

 are fixed in the 5% sodium thiosulfate 

 bath, which removes from them the last 

 traces of unwanted silver material. The 

 sections are then washed in at least three 

 changes of triple-distilled water. One of 

 the sections may then be taken, rapidly 

 dehydrated in alcohol, cleared, and ex- 

 amined under a coverslip with the highest 

 power of the microscope. If the nervous 

 elements are not stained at all, the whole 



batch of sections may be thrown away, 

 and the operation carefully reviewed to 

 decide the point at which the mistake oc- 

 curred. If, alternatively, the entire section 

 is found to be too greatly blackened, the 

 situation may often be improved by pro- 

 longed soaking in thiosulfate solution. If, 

 however, directions have been followed 

 carefully, it is probable that the nervous 

 structures of the retina will be displayed 

 better than they can be by any other 

 technique. If this is the case, the sections 

 may be removed, dehydrated, cleared, and 

 mounted in balsam in the ordinary 

 manner. 



Demonstrations of neuroblasts and 

 axons of the developing spinal cord 

 of a three-day chicken embryo by 



the method of Cajal 1910b 

 This technique may be divided into 

 three stages: first, the removal of the 

 chicken embryo from the yolk, and its 

 fixation; second, staining in silver nitrate; 

 third, developing the stain. For the first 

 operation it is necessary to assemble three 

 fingerbowls, two Syracuse watch glasses, 

 a four-ounce, chemically clean, glass- 

 stoppered bottle, scissors, and forceps. It 

 is, of course, also necessarj^ to have an egg 

 which has been incubated for 72 hours. 

 The reagents required are a liter of normal 

 saline heated to around 37°C.; about 4 

 ounces of chemically pure alcohol, which 

 may be either the ordinary commercial ab- 

 solute alcohol or the latter diluted to 95 % 

 as specified in the original formula; and a 

 solution of 1.5% silver nitrate in triple- 

 distilled water. A chemically clean eye- 

 dropper type pipet is also required. 



One technique for removal of chicken 

 embryo from the yolk has been described 

 in Chapter 20, but a variation of this tech- 

 nique is required in the. present case. First 

 of all, fill three fingerbowls to within about 

 one inch from tlie top with the normal 

 saline. Break tlie egg into one of the 

 fingerbowls. Breaking an egg, after 72 hours 

 of incubation, into a fingerbowl in such a 

 manner as to avoid also breaking the yolk, 

 is an art to be learned only -with practice. 

 The inexperienced should submerge the 

 egg in saline, break out the air space so as 

 to cause the embryo to drop away from 



