DS 12.30 



DYE STAINS OF GENERAL APPLICATION 



331 



surface presents an opaque white appear- 

 ance, the worm may be considered dead 

 and only fit to be thrown away. If it con- 

 tracts strongly and develops lateral twist- 

 ings, it is insufficiently narcotized. Any 

 stage between these two may be con- 

 sidered sufficient. 



It is much more convenient if the worm 

 be killed in an extended condition. The 

 simplest way to do this is with the aid of 

 two blocks of glass lying on top of each 

 other. Capillary attraction will hold a 

 small worm quite satisfactorily into the 

 angle of these two blocks, or it may be 

 forced against the angle by a heavy piece 

 of glass rod, if capillary attraction proves 

 insufficient. The selection of the fixative 

 is a matter of some dispute; the author 

 himself prefers the antique picro-sulfuric 

 acid of Kleinenberg 1879 (Chapter 18, 

 F 5000.0050) which was developed for the 

 purpose of fixing earthworms and is ad- 

 mittedly worthless for any other purpose. 

 It lends itself excellently, however, to 

 afterstaining, and leaves the embedded 

 worm less brittle than any other fixative 

 which the writer has ever tried, with 

 the possible exception of the irrational 

 chromic-nitric mixture of Perenyi (F 6000.- 

 0040 Perenyi 1882). This also has the 

 advantage of preventing the worm from 

 becoming brittle. Whichever fixative is 

 chosen, the worm should now be immersed 

 in it to just about its own depth, until 

 such time as it has partially hardened, 

 i.e. enough to hold it straight, and should 

 then be placed in a large volume of the 

 fixative for whatever period is desired. 

 If Kleinenberg is employed, 24 hours will 

 be sufficient, but the solution of Perenyi 

 should be allowed to act for from one to 

 three days. Kleinenberg's fixative should 

 be washed out in large volumes of 70% 

 alcohol until no more color comes away. 

 If Peren}^i's fixative is employed, running 

 water may be used for the washing proc- 

 ess. In either case, mercuric fixatives 

 should be avoided, since considerable 

 tampering with the specimen with steel 

 instruments remains to be done, and this 

 cannot be satisfactorily conducted after 

 mercuric fixatives. 



After it is washed, the worm should be 

 divided into pieces about three-eighths of 



an inch long. It is usually desirable to 

 retain about half an inch of the anterior 

 region for sagittal sections. The repro- 

 ductive regions need only be saved in the 

 unlikely event that they may be required 

 for advanced classes. The segments should 

 now be dehydrated in alcohol, or any 

 other selected reagent, in the customary 

 manner and cleared in oil of cedar. This 

 reagent is selected because it does not 

 render the segments brittle, yet docs make 

 them sufficiently transparent for exami- 

 nation by transmitted light with a dis- 

 secting microscope (to disclose such sand 

 particles as may still remain in the intes- 

 tine). These show up clearly as brightly 

 refractive objects, and must be removed 

 by being pushed out of position with the 

 bent end of a sharp needle. The experi- 

 enced technician does not need to be told 

 that the retention of a single one of these 

 sand grains within the body of the worm 

 will result in the destruction not only of 

 the microtome knife but of a considerable 

 volume of material which might otherwise 

 be used for good sections. If any con- 

 siderable mass of sand grains remains on 

 the inside, it may often be removed by 

 filling a hypodermic syringe with cedar 

 oil and directing the jet of cedar oil against 

 the inside of the intestine. If this is done, 

 several changes of cedar oil should be used 

 to make sure that the particles of sand 

 dislodged from the inner surface of the 

 intestine do not become reattached to the 

 epidermis. The cedar oil should now be 

 thoroughly removed in benzene (xylene 

 tends to render the muscles brittle) since 

 cedar oil itself tends to dilute the wax or 

 to require too long a period of embedding 

 for such muscularized tissues as those of 

 the earthworm. 



The pieces are then embedded in paraffin 

 and cut into 10-micron sections, Avhich 

 may then be mounted, either individually 

 or in such groups as may be desired on 

 clean slides, and then flattened and dried. 

 When the sections are dried they are 

 deparaff^inized as usual and taken down 

 through the various reagents to water, in 

 which they may be accumulated in 

 batches to be stained together. 



The selection of tlie iron hematoxylin 

 of R^gaud (DS U.IU R^gaud 1910) in 



