440 



METHODS AND FORMULAS 



DS 22.20 



plied in three separate stages. The first 

 solution contains 3 grams of potassium 

 dichromate, 1.15 grams of chromic acid, 

 and 1.25 grams of osmic acid in 250 milh- 

 hters of water. This is poured over the 

 pancreas on the sUde and placed in a dark 

 cupboard for about 24 hours. At the con- 

 clusion of this period the fixative is poured 

 off (it may be used many times), the dish 

 filled with distilled water, and rocked 

 gently backward and forward for about 

 half an hour. The pancreas and mesentery 

 will usually become detached from the 

 glass at this stage, and the glass may be 

 withdrawn. The distilled water is then re- 

 placed with the second solution of 

 Champy, which contains 2.5 grams of 

 chromic acid dissolved in a mixture of 35 

 millihters of pyroligneous acid and 175 

 milhhters of water. Pyroligneous acid, it 

 may be said, is the product of the dry 

 destructive distillation of wood, and con- 

 tains a mixture of various creosotes and 

 tars in a weak solution of acetic acid. It is 

 unfortunately not at present known which 

 of its various constituents exercise the re- 

 quired effect, but it may be stated quite 

 categorically that weak solutions of acetic 

 acid cannot be substituted for it. This 

 solution should be allowed to act for about 

 20 hours and the specimen again washed in 

 distilled water for about 30 minutes. 



At this point the pancreas may be cut 

 into small pieces with a pair of sharp scis- 

 sors. Pieces of about 3-millimeter side 

 should be selected and removed from the 

 distilled water in which the cutting is done 

 to another vial of distilled water. After 

 enough pieces have been selected, the dis- 

 tilled water is poured off and replaced 

 with 3% potassium dichromate for three 

 or four days before being washed in run- 

 ning water for 24 hours. Fixation is now 

 complete. Many of the modifications 

 which have been suggested are for the 

 purpose of diminishing the time in the 

 dichromate bath, by using various di- 

 chromate fixatives at higher temperatures. 

 The method of Champy, however, is reli- 

 able, and though it is slow and cumbrous, 

 it yields far more certain results than 

 many of its modern, more complicated 

 variants. 



After washing in running water for 24 



hours, the specimens are dehydrated and 

 embedded in paraffin in the customary 

 manner before being sectioned. It is 

 recommended that sections about five 

 microns in thickness be employed, and 

 that these be mounted on the shdes in the 

 customary manner. 



It is now necessary to make the acid 

 fuchsin stain of Altmann 1890 (DS 22.2 

 Altmann 1890) about which there has been 

 considerable controversy. This solution is 

 prepared by dissolving 20 grams of acid 

 fuchsin in 100 cc. of "aniline water," 

 which is itself a saturated solution of 

 aniline in water. It is better that the solu- 

 tion be made when a little free aniline is 

 present, since the solubihty of acid fuchsin 

 in anihne is far higher than in water. Take, 

 therefore, 100 grams of water, add to it 

 about five milhliters of anihne, and shake 

 vigorously for a few moments. Throw in 

 20 grams of acid fuchsin, shake vigorously 

 for a few moments, and lay on one side in 

 a warm place. Shake the bottle at inter- 

 vals during the next 24 hours, at the end 

 of wliich time the acid fuchsin (despite 

 statements to the contrary in the Utera- 

 ture) will be found to have dissolved. The 

 failure of some investigators to cause more 

 than a few grams of acid fuchsin to go into 

 solution (see for example the remarks of 

 Gatenby and Painter 1937, 305) is prob- 

 ably due to tlie fact that they first pre- 

 pared a saturated solution of anihne in 

 water, and then endeavored to dissolve 

 the acid fuchsin into it. A mixture of ani- 

 line and water will dissolve, possibly by a 

 process of mutual soluliihty, the required 

 quantity of acid fuchsin. This point must 

 be insisted upon, since tlie entire success 

 of the preparation depends upon the main- 

 tenance of a very high concentration of 

 acid fuchsin in the solution first used for 

 staining. The other staining solutions re- 

 quired in this method are a 0.5% solu- 

 tion of toluidine blue in water and a 

 0.5% solution of aurantia in 70% alco- 

 hol; neither present any difficulty of 

 preparation. 



Now take one of the shdes, drain off 

 the water so far as possible, and blot 

 round the section with filter paper before 

 flooding the slide with acid fuchsin. The 

 draining and blotting are necessary, since 



