548 



METHODS AND FORMULAS 



MS 31.0 



pared by dissolving 2.5 Gm of p3a-ogallic 

 acid in 250 milliliters of water and 15 

 milliliters of 40% formaldehyde. 



The hardened embryo is removed from 

 the bottle of alcohol, and drained by 

 touching the corner to a filter paper to re- 

 move as much of the alcohol as possible. 

 It is then dropped into the silver nitrate 

 solution which has been heated to 35°C. 

 It will immediately turn slightly brown, 

 and will float at the top for some time. 

 While it is still floating, place it in an 

 oven at 35°C., in the dark, and leave it for 

 four or five days. It should, however, be 

 examined at daily intervals, in case some 

 material has been carried in which is caus- 

 ing the precipitation of the silver. This 

 will immediately become apparent if the 

 inside of the bottle or the floor of the 

 bottle becomes covered with a brownish 

 or black stain. Should this be observed, 

 immediately remove the embryo to a 

 fresh 1.5% silver nitrate. 



When the embryo has been sufficiently 

 impregnated, it is washed in triple-dis- 

 tilled water. Damage from shrinkage will 

 be minimized if the wash water be heated 

 to about 25°C., on the assumption that 

 the developing solution which will be used 

 next will be at about 20°C. It is not neces- 

 sary, nor indeed desirable, to remove 

 much of the silver nitrate, the main pur- 

 pose of the rinse being to avoid carrying 

 over the silver nitrate solution to the de- 

 veloping solution. This latter should be in 

 a chemically clean, wide-mouthed, stop- 

 pered bottle, and at least 100 milliliters 

 will be required for the development, 

 which is carried out at room temperature, 

 preferably in the dark, for about 24 hours. 

 The embryo will become black in the 

 developer, and there is no means of 

 finding out whether the impregnation 

 has been successful until it has_been 

 sectioned. 



Paraffin sections about eight microns 

 thick are now prepared, mounted on 

 slides, dewaxed, and graded down through 

 alcohol into water in which they can be 

 examined under a low-power objective. 

 They should at this point sliow^ neuro- 

 blasts and neurofibrils completely; black- 

 ened, with little or no blackening of other 

 portions of the embryo, save the periphery 



on which some deposition of black mate- 

 rial is inevitable. 



If the sections on examination should 

 prove to be uniformly blackened through- 

 out, indicating either overimpregnation or 

 overdevelopment, or far more probably 

 the utilization of impure reagent, nothing 

 can be done about it save to throw the 

 sections away and start with a fresh 

 embryo. If, however, the impregnation is 

 not sufficiently heavy — that is, if the 

 neuroblasts and neurofibrils are perfectly 

 apparent but are only stained a light 

 brown rather than the dense black which 

 should be observed — they may be toned, 

 and at the same time increased in contrast, 

 with Cajal's gold toning solution, which 

 will be found in Chapter 24 as AMS 22.1 

 Cajal 1910. SHdes are taken directly from 

 distilled water and placed in this solution, 

 and examined at intervals. They will turn 

 from a brown to a purplish shade, and will 

 become darker in so doing. The reaction 

 may be stopped at any point by removing 

 the sections from the toning solution and 

 washing them in distilled water. 



When the desired degree of intensity 

 has been reached, the sections are thor- 

 oughly washed in distilled water, up- 

 graded through alcohols, and mounted 

 under a coverslip in balsam after the 

 usual reagents. 



Demonstration of spirochetes in sec- 

 tions by the method of Dieterle 1927 



This is a simple method for the demon- 

 stration of spirochetes in post-mortem 

 material. It can be used after many kinds 

 of fixation, though it is usually preferable 

 to take the formaldehyde-fixed material. 

 This is sectioned either by the paraffin or 

 the freezing technique, the latter being so 

 customary in pathological work, that it 

 has given rise to the supposition that this 

 method alone can be used. Better demon- 

 strations for teaching, as distinct from 

 diagnostic, purposes are, however, ob- 

 tained from paraffin sections of about 

 eight to ten microns in thickness. These 

 sections are mounted on slides in the 

 normal way and the slides are accumu- 

 lated in distilled water. Four solutions are 

 required, two of which must be main- 



