CARMALUM-STAINED LIVER FLUKE 97 



the aid of a potassium-permanganate, oxalic-acid bleach in the following 

 manner. Prepare a solution of potassium permanganate so weak that it 

 appears only a very faint pink. This is best done by adding a few drops of a 

 strong solution to a beaker of distilled water. Then each worm is dropped 

 individually into the solution and allowed to remain there until such time as 

 it has turned a bronzy brown on the outside. This appearance can best be 

 detected in reflected light. Just as soon as the first bronze sheen appears on 

 the outside of the worm, it must be removed to fresh distilled water where 

 it can remain until all the other worms in the batch have been treated simi- 

 larly. It will be necessary to renew the potassium permanganate from time 

 to time, which is done by adding a few more drops of the stock solution to 

 the beaker. The strength of oxalic acid which is used to bleach the worm is 

 quite immaterial; 2 or 3 per cent, arrived at by guess work rather than by 

 weighing, is an adequate solution. Since the bleaching of the surface is not 

 nearly as critical as is the deposition of the potassium permanganate, all the 

 worms may be bleached at the same time by pouring off the distilled water 

 and substituting the oxalic acid for it in the beaker. One or two twists of the 

 wrist to rotate the worms in the beaker will result in their turning from a 

 bronze sheen to a dead white. The oxalic acid is poured off without any waste 

 of time, and the worms are washed in running tap water for an hour or so 

 before being dehydrated in the ordinary way, cleared, and mounted in balsam. 

 Little trouble will be experienced with curling of the worms if they have been 

 fixed and treated as described. If they should curl, they should undergo the 

 final dehydration in 96 per cent alcohol while being pressed loosely between 

 two sheets of glass, which will be sufficient to hold them flat. 



Summary 



1. Anesthetize flukes in warm saline solution with menthol. 



2. Place a layer of filter paper saturated with Gilson's fluid on a glass plate. 

 Have ready another sheet of fixative-saturated paper and another glass plate. 



3. Lay worms about an inch apart on paper on glass, cover with second sheet 

 saturated with fixative, lay second glass on top, and add sufficient weight 

 to flatten but not squash worms. 



4. After about 12 hours remove flattened worms to jar of fixative. Fix 1 to 6 

 days. Wash in running water overnight. 



5. Transfer to 1 part Mayer's carmalum diluted with 100 parts 5 per cent potas- 

 sium alum. Leave until internal structures are stained clearly (1 to 3 weeks). 



6. Wash in 5 per cent potassium alum. 



7. Bleach surface with permanganate-oxalic acid. 



8. Wash thoroughly, dehydrate, clear, and mount. 



