DS 11.20 



DYE STAINS OF GENERAL APPLICATION 



295 



possible to prevent fixation taking place 

 before an additional layer of i)aper (satu- 

 rated with fixative) is placed on the top, 

 and the second sheet of glass placed on 

 top of this. Assuming that the sheets of 

 glass are the size of a sheet of typewriting 

 paper, it is suggested that about a two- 

 pound weight then be placed on the upper 

 sheet of glass. The "sandwich" should 

 now be left for at least 12 hours before 

 removing the glass and upper paper. Then 

 the worms should be picked up one by one 

 on a glass section lifter (metal cannot be 

 used because of the presence of mercuric 

 chloride) and transferred to a large jar 

 of fixative, where they may remain from 

 another day to another week at the discre- 

 tion of the technician. 



At the conclusion of fixation the worms 

 should be washed in running water for at 

 least 24 hours. 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 iodine tends to render the 

 worms brittle and the author strongly 

 recommends its omission. After being 

 thoroughly washed in water, the worms 

 may be stained. The formula selected for 

 this example is the very well-known 

 carmalum of Mayer (DS 11.21 Ma5^er 

 1897 below). Objects of this type are 

 better stained by the additive process 

 than by a process of differentiation. That 

 is, they are better placed in weak solution 

 and allowed to absorb the stain slowly 

 than if placed in a strong solution which 

 will require subsequent differentiation. 

 The best diluent for the stain is a 5 % solu- 

 tion of potassium alum. The extent of the 

 dilution is dependent upon the choice of 

 the operator and the size of the object 

 which is to be stained. In the present in- 

 stance a dilution of about one part of the 

 stain to 100 parts of 5% potassium alum 

 would be correct. It is far worse 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 

 indefinitely. The worms are placed in this 

 diluted stain and left there until their 

 internal structures have become clearly 

 visible. It is suggested that they be ex- 

 amined at the end of a week and sub- 



sequentl}^ every three days, until such 

 time as 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 solution of 5% 

 potassium alum and rinsed for a short 

 time until all the adherent color has left 

 them. They will still, however, be pink on 

 the outside. Since the purpose of the stain 

 is to demonstrate the internal organs it is 

 desirable to bleach this outer layer in 

 order to produce bright scarlet internal 

 organs against a white background. In the 

 experience of the writer 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 b}^ adding a few drops of a strong 

 solution to a beaker of distilled water. 

 Each worm is then taken individually and 

 dropped into the solution until such time 

 as it has turned a bronzy brow^n on the 

 outside. This appearance can best be de- 

 tected in reflected light and just as soon as 

 the first bronze sheen appears on the out- 

 side, the worm must be removed to fresh 

 distilled water, where it can remain until 

 all the other worms in the batch have been 

 similarly treated. It will be necessary, of 

 course, to renew the potassium permanga- 

 nate from time to time by adding a few 

 more drops of the stock solution to the 

 beaker. The strength of oxalic acid used to 

 bleach the worm is quite immaterial. Two 

 or 3%, arrived at by guess work rather than 

 by weighing, is an adequate solution. As 

 the bleaching of the surface is not as critical 

 as is the deposition of the potassium jier- 

 manganate, all the worms may be bleached 

 at the same time by pouring oflf from the 

 beaker the distilled water in which they 

 have been accumulated, and substituting 

 for it the oxahc acid. 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 then poured off without any waste 

 of time, and the worms washed in running 

 tap water for an hour or so, before being 

 dehydrated in the ordinary way, cleared, 

 and mounted in balsam (see Chapter 6). 



