ZOOLOGY AND BOTANY, MICROSCOPY, ETC. 311 



and Fredericella. Chloretone was used for narcotization and 3 p.c. 

 formalin for killing and subsequent preservation. The colony was 

 placed in a tube or beaker of convenient size and covered completely 

 with water. When the lophophores of the individual polypides were 

 well extended the chloretone solutions were added in the following- 

 order :— (1) A few drops of sat. sol. chloretone in water ; (2) 1 part 

 ditto to 4 parts water ; (3) 2 parts ditto to 3 parts water ; (4) 3 parts 

 ditto to 2 parts water ; (5) 4 partsditto to 1 part water ; (6) saturated 

 solution of chloretone. 



The amount of each solution was equal to the amount of water con- 

 taining the colony. Each solution was added drop by drop very slowly. 

 Gradually some of the solution containing the colony was removed in 

 order to keep the amount constant. 



The time required for the application of each solution varied from 

 15 to 30 minutes. After the colony had been in the saturated chloretone 

 solution for 15 minutes the killing agent was added. A 3 p.c. solution 

 of formalin was diluted with a saturated solution of chloretone, and the 

 following grades were used :— (1) 1 part 3 p.c. formalin to 2 parts sat. 

 sol. chloretone : (2) 1 part ditto to 1 part ditto ; (3) 2 parts ditto to 

 1 part ditto ; (4) 3 p.c. formalin. 



These solutions were added drop by drop in the same manner as for 

 narcotization, and 15 to 30 minutes were allowed for the application of 

 each grade. Two and a half to five hours are necessary for the entire 

 procedure. For Crist atella the minimum time is sufficient, but for 

 Plumatella and especially for Fredericella the maximum time is necessary. 



(4) Staining- and Injecting. 



Staining Methods for Microfilaria.* — F. Fiilleborn discusses in 

 considerable detail various methods of staining these parasites. For 

 simple clinical diagnosis, he recommends the dry method ; two or three 

 drops of blood are spread on a clean side, dried, dehasinoglobinized with 

 distilled water, fixed with absolute alcohol, stained with Bohmer's 

 hematoxylin, differentiated with weak acid, blued in tap-water, dried 

 and mounted in cedar-wood oil. For diagnosis between Microfilaria 

 bancrofti and M. loa, he recommends wet-fixation. In this case, the 

 preparation is dehsemoglobinized with saline, fixed and passed through 

 mounting alcohols to absolute alcohol and down again to distilled water, 

 stained with hematoxylin and mounted. For anatomical study, fresh- 

 stained (vital-stained) preparations treated with azur-eosin give good 

 results. Finally, as a simple and satisfactory method, pyronin-methyl- 

 green is recommended. A thick film is dried and dehasrnoglobinized 

 with saline. This is stained for half-an-hour or longer with Unna- 

 Pappenheim's carbol methyl-green-pyronin, and differentiated and de- 

 hydrated simultaneously by rapid alcohol passage. It may then be 

 mounted and examined For many important practical details and 

 precautions to be observed in the following of any of these procedures, 

 the original paper should be consulted. 



* Centralbl. Bakt., lte Abt. Orig., lxxiii. (1914) pp. 427-44. , 



