16 THEOPHILUS S. PAINTER 



The other was the cold Flemming method, with urea, as recom- 

 mended by Hance ('17). The modified Bouin's fluid gave superb 

 preparations for all stages of maturation, and was the more 

 satisfactory of the two preservatives. However, it is my experi- 

 ence that for spermatogonia! and somatic divisions, no preserva- 

 tive can surpass the results obtained by cold Flemming to which 

 urea is added. ^ 



A great deal of care was used in handling the material after 

 preservation. When modified Bouin was used, the suggestions 

 of Allen ('19) for washing and dehydration were closely followed. 

 As an agent for securing rapid mixing of fluid, I used air which 



* For the convenience of the reader, the exact technique employed is given 

 below. The modified Bouin fluid is made up as follows : 



A. Picric acid, saturated aqueous solution 75 cc. 



Formol, C. P 25 cc. 



Glacial acetic acid 10 cc. 



B. Chromic acic, crystals 1.5 grams 



Urea, crystals 2.0 grams 



The ingredients under A may be mixed and kept as a stock solution. Just 

 before using the chromic acid and urea should be added, in the order named, and 

 the solution heated to about 38°C. 



In operating for the removal of tissue, the fixing fluid is kept in a water-bath 

 on the same table. Immediately on removal, the testis is slit open and a small 

 mass or two of the tubules are cut out with scissors and dropped into the fixing 

 fluid. The tubules are quickly teased apart so that each of them will be exposed 

 to the action of the fixative. Fixation at 38°C. is carried on for about an hour. 

 The tissue is then placed and washed in 5 per cent alcohol. Using the drop 

 method throughout (an ordinary burette is useful), the tissue is brought through 

 the following solutions: 10 per cent alcohol, to which a few drops of a saturated 

 solution (aqueous) of lithium carbonate has been added. It is kept in this 

 solution until most of the picric acid has been removed. Then 50 per cent alcohol 

 is added until the strength of the solution is about 35 per cent. Then a mixture 

 of equal parts of aniline oil and 50 per cent alcohol is dropped in. This is fol- 

 lowed by a similar mixture of 70 per cent alcohol and aniline oil. Next, pure 

 aniline oil is added and the tissue cleared in this. The aniline oil is replaced by 

 wintergreen oil. From this it is embedded in paraffin, but the tissue is passed 

 through some six or eight changes in which there is an increasing proportion of 

 paraffin. The time taken for embedding is around three hours. 



In the second method, urea is added to ordinary strong Flemming solution to 

 the extent of 0.5 gram per 100 cc. The solution is then chilled with ice to about 

 4°C. The tubules are teased out in this fluid and kept in it on ice for twenty- 

 four hours. Subsequently it is washed and treated in the ordinary way. From 

 95 per cent alcohol one may pass to oil of cedar (using the drop method), then 

 zylol, and finally paraffin. 



