Carmalum-stained Liver Fluke 191 



tional layer of paper is placed on top, saturated with fixative, and the 

 second sheet of glass placed on top of this. Assuming that the sheets of 

 glass employed are of the size of a sheet of typewriter paper, it is sug- 

 gested that about a 2-lb weight be placed on the upper sheet of glass. 

 The whole should now be left for at least 12 hr before removing the 

 glass and upper paper, picking the worms up one by one on a glass section 

 lifter (metal cannot be used because of the presence of mercuric 

 chloride), and transferring them to a large jar of fixative, where they may 

 remain for another day to another week at the discretion of the technician. 

 At the conclusion of fixation the worms should be washed in running 

 water for at least 24 hr. It has not been the author's experience that 

 this fixative, followed by such washing, requires the use of iodine for 

 the final removal of the mercury. At this stage, moreover, iodine tends to 

 render the worms brittle, and the author would strongly recommend its 

 omission. After thoroughly washing in water, the worms may be stained; 

 the formula selected for the purpose of this example is the well-known 

 carmalum of Mayer. Objects of this type are better stained by the additive 

 process than by a process of differentiation, that is, they are better placed 

 in an exceedingly weak solution and allowed to absorb the stain slowly 

 than placed in a strong solution that requires subsequent differentiation. 

 The best diluent for the stain is a 5 per cent solution of potassium alum. 

 The extent of the dilution is dependent upon the choice of the operator 

 and the size of the object to be stained. In the present instance a dilu- 

 tion of about 1 part of the stain to 100 parts of 5 per cent potassium alum 

 would be correct. It is far more dangerous to have the solution too strong 

 than it is to have it too weak, and, since it is an excellent preservative, the 

 worms can remain in it for an indefinite period. The worms are merely 

 placed in this diluted stain and left there until their internal structures 

 have become clearly visible. It is suggested that they be examined at the 

 end of a week, and subsequently every three days, until examination with 

 a low-power binocular microscope, using a bright light from beneath, 

 shows the testes to be darkly stained. At this point the worms are removed 

 to a fresh clean solution of 5 per cent potassium alum and rinsed for a 

 short time to remove all the adherent color. However, they will still be 

 pink on the outside. Since the purpose of the stain is to demonstrate the 

 internal organs, it is obviously desirable to bleach this outer layer in 

 order to produce bright scarlet internal organs against a white back- 

 ground. In the experience of the author this may be done most readily 

 with 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 



