793 



APPENDIX TO CHAPTER XIII 



THE preparation and preservation of Rotifers well extended as in life to 

 serve as type specimens is now possible, and the following is an outline of 

 Mr. C. F. Rousselet's method, which consists of three stages: narcotising, 

 killing and fixing, and preserving. The whole operation is necessarily 

 performed under a dissecting microscope. 



The first step in the preparation of Rotifers is to isolate the animals 

 by transferring as many as may be available by means of a very 

 fine pipette to a fresh watchglass full of perfectly clean water until all 

 [(articles of foreign matter have been eliminated. This is necessary 

 because when the animals are dead these particles adhere to the cilia of 

 the Rotifers, from whence it is very difficult to remove them. In the case 

 of fixed Rotifers, such as Melicerta, Lirnnias, Stephanoceros, &c., it is 

 necessary to cut off and trim a very small piece of the plant to which 

 they are attached ready for mounting, so as not to have to do this when 

 the animals are killed and prepared. It is also necessary to separate the 

 different species, as most of them require a little different, more or less 

 prolonged, treatment under 1 the narcotic. The great difficulty with 

 Rotifers has always been to kill and fix them whilst fully extended as in 

 life. The most rapid killing agents are too slow to prevent complete re- 

 traction ; recourse, therefore, has been had to narcotising, and after many 

 experiments a satisfactory narcotic has been found in the following 

 mixture : 



2 per cent, solution of hydrochlorate of cocaine . 3 parts 



Methylated spirit .... . . 1 ,, 



Water ... . . . 6 



The Rotifers then, separated as to species, and in a watchglass full of 

 perfectly clean water, are ready for narcotising. One or two drops of the 

 above solution are added to the water and mixed. The effect of the 

 narcotic is most varied in different species. Some will not mind it at 

 all and continue to swim about, others will contract at once but soon 

 come out again and swim about at a diminishing race until they finally 

 sink to the bottom with the cilia beating but feebly. Then is the right 

 time for killing and fixing. In the case of more vigorous species, ai'n T 

 three or four minutes another dose of two or three drops of the narcotic 

 is added, and then repeated again if necessary until it is seen that the 

 animals can move but very slowly. At this moment the animals are 

 killed quickly and suddenly by adding one drop of very weak ($ to | per 

 cent.) solution of osmic acid. 



The different species of Rotifers vary so much in their behaviour imder 

 the-narcotic that it is by no means easy to always hit the exact moment 

 for- killing the animals fully extended ; repeated failures and practice 

 alone can guide one in this respect. It is very essential that the animals 

 be still living when the osmic acid is added, as when a Rotifer 'is quite 

 dead various post-mortem changes begin immediately to take place in 

 the tissues, whilst it is desired to fix and preserve the tissues as in life. 

 The word ' fixing ' implies rapid killing and at the same time hardening 

 of the tissues to such an extent as to prevent their undergoing any 

 further change by subsequent treatment with preserving fluids. The 

 action of osmic acid is very rapid, half a minute being quite enough ; if 



Ass. 1896, p. 830, and compare with them the suggestion of Dr. Plate in Zeitm-Jir. f. 

 wiss. Zvol. xlix. (1889), pp. 1-41. 



