56 University of California Publications in Zoology [Vol. 13 



iodine color persists. The mixture is then gradually run down to water 

 when the process of staining is begun. In the case of formalin as a fix- 

 ative it is best to use it at about 36° C. as the boiling formalin has a 

 tendency to contract the endoplasm and ectoplasm and so leave the 

 cuticle somewhat wrinkled. When other fixatives are used the methods 

 of handling are in accordance with those usually given for protozoan 

 fixation. 



For in toto staining the most satisfactory results are obtained by 

 the use of Heidenhain 's iron-alum haematoxylin solution, as follows : 

 From water the organisms are subjected to a 1 per cent iron-alum 

 solution for twenty-four to thirty-six hours. Then thoroughly washed 

 in distilled water, stained in a 0.3 per cent solution of haematoxlyn 

 ( Heidenhain 's) for twenty- four hours and then washed in tap water. 

 It is necessary then to differentiate with a 1 per cent iron-alum solution 

 under the microscope to be sure that differentiation is carried on to the 

 right degree. After differentiation the animals are again washed in tap 

 water and distilled water, then passed up through the alcohols, xylol, to 

 cedar oil, and mounted either in Canada balsam or styrax. Freshly 

 made up iron-alum and freshly made up haematoxylin solutions have 

 given uniformly better results than the ' ' ripened ' ' solutions. 



Eberlein (1895) suggests the freeing of the stomach fluid from 

 excess food particles by straining through a warmed linen cloth. Such 

 a method might result in the loss of many of the organisms. We have 

 foiuid that by careful manipulation of the centrifuge an almost pure 

 culture of the animals may be obtained. By rotating the centrifuge at 

 the proper speed the food particles heavier than the organisms will be 

 thrown down first while those lighter than the organisms will remain 

 nearer the top. The top and bottom portions may then be thrown 

 away. This process is of course carried on simultaneously with that of 

 washing and staining and if the top and bottom portions are removed 

 each time the fluid is changed the remainder will soon consist mainly 

 of the desired organisms. 



Segregating. — Both in making whole mounts and in sectioning the 

 animals it has been desirable to segregate the species. This is accomp- 

 lished by means of the following apparatus : A small hypodermic 

 syringe, fitted with a glass tube drawn out to an almost microscopic 

 cross section is clamped to the microscope stage in such a manner that 

 the end of the glass tube is in the field of vision. The glass tube is ad- 

 justed and held in position by a screw clamp. Then with the organisms 

 in a cedar-oil medium and the glass tube and svringe filled with cedar 



