194 BULLETIN OF THE BUREAU OF FISHERIES. 



catch. Sometimes 50 per cent of the Fundulus that have been roughly handled, as 

 when stripped for eggs, will become diseased in 24 hours. Of these, half may be dead 

 within 12 hours. If a few crabs happen to be confined in the same aquarium with a 

 large number of Fundulus, they inflict injuries upon practically all the fish and all are 

 soon diseased. Uninjured Fundulus develop the disease infrequently. (See p. 196.) 

 Roughly speaking, 3 to 4 per cent of the Fundulus that are brought into the laboratory 

 at this season (July and August) and confined in small aquaria having but a liter or 

 two of water for each fish, will be found diseased within two days. Within another 

 day or two some of these fish die and a large number die in the course of a week. Dis- 

 eased Fundulus are therefore almost constantly available. 



METHODS OF STUDY. 



Both fresh and preserved tissues were examined microscopically, the method of 

 handling the tissues being as follows: The scales having been removed with forceps, 

 the edge of a slide is drawn over a diseased area with a little pressure, and the mucus 

 and cellular material thus obtained is spread evenly over the surface of another slide; 

 or, a portion of integument or muscle which has been removed with a scalpel is ground 

 between two thick slides by giving to the upper slide a circular motion. It is necessary 

 to use considerable pressure, and at times cut tough fragments with the sharp edge of 

 the upper slide. Under these conditions, both slides may be preserved for observation 

 and still others made from the ground-up material. Some of the smear preparations 

 made in this manner were examined while fresh and others were fixed and stained. 

 Altogether about 85 fish were examined microscopically. Fresh smears which were 

 sometimes supplied with bile and serum were sealed with vaseline, and could then be 

 examined from time to time, during a period of 24 hours. 



For sectioning, tissues were fixed in a saturated solution of corrosive sublimate in 

 35 per cent alcohol with 0.2 per cent acetic acid and 6 per cent formaldehyde; also in 

 the ether-formalin-alcohol mixture given below. Some of the smears were fixed in the 

 same sublimate mixture; others in a solution of corrosive sublimate in 2 parts abso- 

 lute alcohol and i of ether; still others in a mixture of absolute alcohol (60 per cent), 

 ether (35 per cent), and strong formaldehyde (5 per cent). The mercury preparations 

 are stained with a modification of Mayer's hsematein. (See Hahn, Archiv fiir Protis- 

 tenkunde, bd. xvii, no. 3, p. 316, footnote.) Usually the alcohol and formalin prepa- 

 rations are stained in methylene blue or Giemsa's stain. The methylene blue was 

 extracted in a saturate alcoholic (70 per cent) solution of both eosin and orange G. 

 The Giemsa was washed in water, allowed to dry, and decolorized in carbol-xylol, 

 without the use of alcohol. Some of the more recent preparations fixed by either of 

 the above fluids have been more successfully stained by first treating with haematein 

 for several hours, then decolorizing in 70 per cent alcohol with i per cent HCl, returning 

 through the alcohols to water, and staining in methylene blue or toluidin blue. After 

 dehydration they were left in a contrast stain (eosin and orange G) for a few minutes 

 and rapidly run into 95 per cent alcohol, carbol-xylol, and two changes or xylol. Both 

 smear preparations and sections are mounted in Canada balsam without cover glasses. 



Searching is most satisfactorily carried on with an ocular of i inch and an objective 

 of one-fifth inch focal distance. A one-twelfth inch oil immersion objective combined 

 with the same ocular for ordinary observation is supplemented, when occasion requires, 

 with a no. 2 compensating ocular. The one-fifth inch objective is not too high to be 



