26 MARINE AND FISHERIES 



5 GEORGE v., A. 1915 



in a "car" to the laboratory, where they were kept alive either in the car or in 

 tanks supplied with running water. The study of the living parasites was made 

 during the months of July, August and September, 1912, and all the preserved 

 material was collected during the same period. 



In searching for parasites of the gall bladder, the bile duct of the fish was 

 ligatured and the gall bladder removed to a carefully cleaned watch glass where 

 it was cut open. Into a pipette freshly made from new glass tubing a small quan- 

 tity of the bile was drawn. If a fresh preparation was desired this was dropped 

 on a slide and covered with a coverglass. Both slides and coverglasses were 

 prepared as follows: After being cleaned in a mixture of one part bichromate of 

 potash and one part concentrated sulphuric acid to ten parts water they were 

 washed first in tap water and then in distilled water and stored in 95% alcohol. 

 When required for use, the alcohol was burned from them by passing them through 

 the flame of an alcohol lamp. If fixed and stained smear preparations were 

 desired the bile was dropped from the pipette on a coverglass and then sucked 

 back again so that only a very thin film of bile remained on the coverglass. The cov- 

 erglass was then inverted and allowed to drop on the fixing fluid in such a way that 

 it was supported by the surface tension of the liquid. In this manner the prep- 

 arations were given no opportunity to dry. This is practically the method of 

 Doflein ('98), with the exception that in all cases no blood was added to the gall. 

 The fixing fluids were Schandinn's fluid, consisting of two parts saturated aqueous 

 solution of corrosive sublimate to one part absolute alcohol used either hot or cold 

 and Hermann's fluid consisting of 75 cc. of 1% platinic chloride, 4 cc. of 2% osmic 

 acid and 1 cc. of glacial acetic acid. These fluids were allowed to act for from 

 five to ten minutes and the coverglasses were then transferred (after Schandinn's 

 fluid) to 60% alcohol containing iodine, or (after Hermann's fluid) to distilled 

 water. The stains used were Giemsa's azar-eosin or Dalafield's haematoxylin. 

 Both were diluted before use to one or two per cent and allowed to act for from 

 twenty-four to forty-eight hours. After staining in Giemsa's mixture the smears 

 were washed in tap water and destained in a mixture containing 95% acetone 

 and 5% xylol. When sufficiently destained they were passed in succession through 

 the following mixtures: (1) acetone 70% and xylol 30%; (2) acetone 50% and 

 xylol 50% ; (3) pure xylol, and were finally mounted in Canada balsam. For the 

 details of this method of using Giemsa's stain, Kisskalt and Hartmann ('10, p. 14) 

 may be consulted. After staining in Dalafield's haematoxylin, smears were 

 either first destained in acid alcohol or mounted directly in Canada balsam. 



For the study of attached stages, the wall of the gall bladder was sectioned . 

 Pieces of the bladder, opened in a watch glass as described above, were fixed in 

 Schandinn's fluid, imbedded in paraffine, and cut into sections from four to seven 

 microns in thickness. The sections were stained in Giemsa's mixture or in Dala- 

 field's haematoxylin, diluted as for the smear preparations, or in Heidenhain'a 

 iron haematoxylin. In the case of Giemsa's stain the best results were obtained 

 by washing in water rapidly, for twenty seconds or so, and then destaining in a 

 mixture of acteone 95 cc. and xylol 5 cc. for eight to ten minutes. 



