294 



METHODS AND FORMULAS 



DS 11.20 



almost impossible to overstain in them. 

 The picric acid, moreover, acts as a fixa- 

 tive. It is possible to take a small living 

 invertebrate, throw it into the stain for 

 ten minutes or so, and remove it fixed and 

 stained. The original formula of Ranvier 

 1889 called for a preparation of a dry 

 stock and the preparation of a working 

 solution from this. Until quite recent 

 times the dry stock could be purchased. 

 It undoubtedly makes a better solution 

 and keeps better than do the formulas 

 prepared as solutions directly. 



Iron carmines (DS 11.25) had a brief 

 vogue in the first decade of the present 

 century and then again fell into disfavor. 

 They are nuclear stains strongly re- 

 sembling the reactions of the iron hema- 

 toxyhns. The formula of De Groot 1903 is 

 the one most usually recommended. Am- 

 monia carmines (DS 11.26) and hydro- 

 chloric carmines (DS 11.26) are no longer 

 very well known, though the formula of 

 HoUande 1916 gives excellent nuclear 

 staining. The final class, which contains 

 those formulas that cannot reasonably be 

 fitted into the previous classes, are rarely 

 used today. The only formula finding any 

 great acceptance is the "hthium carmine" 

 of Orth (1892), which was rediscovered in 

 Germany in the 1920's. It is most warmly 

 recommended by Spielmeyer, 1924, for 

 counterstaining sections of the nervous 

 system. 



11.20 TYPICAL EXAMPLES 



Preparation of a wholemount of a 



liver fluke using the carmalum 



stain of Mayer 1897 



Though many persons will be forced to 

 rely for their material on a supply house, 

 better preparations can be made if the 

 living flukes are secured from a slaughter 

 house. In this case the flukes should be 

 removed from the liver (where they will 

 mostly be found crawling upon the surface 

 if the animal has been dead for some time) 

 to a vacuum flask containing physio- 

 logical saline solution at a temperature of 

 about 35°C. to which has been added a 

 small quantity (approximately one tenth 

 of a gram per liter) of gelatin. Flukes can 

 be transported ahve for relatively long 

 distances in this solution, and every possi- 



ble effort should be made to keep them 

 alive until they have been brought to the 

 laboratory. In the laboratory the con- 

 tents of the thermos flask should be 

 poured into a dish and the worms trans- 

 ferred individually to another large dish 

 containing warm physiological saline, 

 where the last of the blood will be washed 

 from them. Better preparations will be 

 secured if time be taken to anesthetize 

 the worms before fixing them. Most of 

 the thick and opaque mounts which one 

 seens in laboratories result from an en- 

 deavor to fix an unanesthetized worm 

 which has contracted during the course 

 of fixation. Liver flukes are easy to anes- 

 thetize, the simplest method being to 

 sprinkle a few crystals of menthol on the 

 surface of the warm saline and leave them 

 for about half an hour. One should not, of 

 course, permit them to die in this solu- 

 tion, but should watch them carefully, 

 terminating the process when their mo- 

 tions become exceedingly slow and con- 

 sist only of an occasional feeble contrac- 

 tion rather than the active movements in 

 wliich they were indulging when removed 

 from the liver. 



While the worms are being anesthetized 

 preparations for fixing them should be 

 made. Take two sheets of quarter-inch 

 plate glass, each of such a size as will en- 

 able one to lay on them the number of 

 worms which are to be fixed, and place 

 upon the lower plate two or three thick- 

 nesses of a rather coarse filter paper or 

 paper toweUng. Blotting paper is too soft 

 for tliis purpose; a good filter paper is 

 much to be preferred to a paper towel. 

 The selection of a fixative must, of course, 

 rest in the hands of the operator, but the 

 author's preference is for the mercuric- 

 acetic-nitric mixture of Gilson 1898 

 (Chapter 18, F 3000.0014). This has all 

 the advantages in sharpness of definition 

 given by mercuric fixatives, while the 

 addition of nitric acid appears to render 

 the flattened worms less brittle in sub- 

 sequent handling. Whatever fixative is 

 selected, the sheet of filter paper is now 

 saturated thoroughly with it and the 

 anesthetized worms are removed from the 

 physiological saline and laid out one by 

 one about an inch apart on the blotting 

 paper. This must be done as rapidly as 



