204 BULI^ETIN OF THE BUREAU OF FISHERIES. 



fish examined have been those which are abundant and easily taken. While only a 

 limited number of fish belonging to rarer species have been examined the absence of 

 Myxosporidia in these fish can not be considered of any particular significance, since it is 

 comparatively rare to find all the fish of any species infected with the same parasite. 

 For these reasons it has not been considered advisable to include in this paper a 

 list of the fish examined, since such a list might easily lead to very erroneous conclusions. 



METHODS. 



The description of the trophozoites and spores have been made almost entirely 

 from living material. They were removed to the sHde from the recently killed fish 

 and studied at once. To prevent evaporation the cover glass was ringed with paraffin 

 of a low melting point. In the case of the trophozoites it is essential that they be 

 studied without delay, for they almost invariably undergo degenerative changes within 

 a few hours after the death of the host. 



Some efforts have been made to grow the trophozoites on culture media, but so far 

 without success. 



While the spores are naturally much more resistant to unfavorable conditions than 

 the trophozoites, it is nevertheless important that all descriptions be based on perfectly 

 fresh material. There is a marked tendency in most species of Myxosporidia for sporula- 

 tion to take place much more rapidly under unfavorable conditions, and spores formed 

 under such conditions are often smaller and may show other striking variations from the 

 typical form. 



Although the descriptions in all cases have been based primarily on living specimens, 

 fixed and stained material has been utilized in many instances. For the study of the 

 nuclei smears made by the Giemsa method have been of great value. They were pre- 

 pared as follows : 



A small amount of material was spread on the cover glass to form a very thin 

 film, which was then exposed to osmic vapor for about one-half minute. The film was 

 then allowed to dry, fixed in absolute alcohol about one-half hour, and after drying 

 stained by Giemsa's method for several hours. After staining they were decolorized 

 in acetone, dried, and mounted in damar. Damar has been found to be much more 

 satisfactory than Canada balsam for this purpose. Films mounted in damar have faded 

 very slightly, if at all, after several years, while those mounted in neutral balsam are 

 so badly faded after a year or two as to be of no value. 



The success of this method varies greatly in different species. In some cases the 

 nuclei are brought out with great clearness, while in others the results are very un- 

 satisfactory. 



Wet smears were also used, but in general were not as valuable as those prepared 

 by the Giemsa method. In making these the material was spread in a thin film over 

 the cover glass, exposed to osmic vapor for one-half minute, and then placed at once 

 in Worcester's formol-corrosive-acetic fixing fluid for one to three hours. They were 

 stained with Delafield's hematoxylin, iron hematoxylin, or Mayer's acid hemalum. 



In the preparation of sections the best results were obtained with material fixed 

 in Worcester's formol-corrosive-acetic fluid and stained with iron hematoxylin and 

 Congo red. 



