113] COMPARATIVE STUDIES ON NEMATODES— HETHERINGTON 9 



organization after which they may be removed to 95 or 80 per cent alcohol 

 for storage. If the larger specimens do not clear sufiSciently at first, almost 

 any degree of clearing may subsequently be obtained by allowing the fluid 

 in which the worms are placed to evaporate, the degree of clearing being 

 proportional to the amount of evaporation. Still greater transparency is 

 obtained by adding glacial acetic acid and phenol to the worms. 



With specimens now in the fluid they may be prepared either for sec- 

 tioning or for mounting in balsam with equal ease. Oils of synthetic 

 wintergreen and oleum cidri ligni (Merk) or chloroform may be dropped 

 slowly into the dish with the specimens and mixed thoroughly by agitation. 

 The progress of this clearing should be watched carefully under a binocular 

 or compound microscope and if the slightest shrinkage is observed it indi- 

 cates that the clearing is being rushed. The change of liquids must be 

 very gradual especially at first when the tissues are hardening but as soon 

 as the mixture is three quarters clearing fluid, the greater part may be 

 drawn off and the pure liquid added more rapidly and allowed to remain 

 for 10 to 15 minutes or longer. If now infiltration by paraffin is desired, 

 the wax is shaved into the dish with the specimens in clearing fluid and the 

 whole set aside in a slightly warm place for 2 hours when the worms may 

 be placed in pure paraflBn, melting at 58 C. and after proper infiltration 

 imbedded and sectioned. 



Following Carnoy-phenol fixation numerous stains work nicely on 

 sections, preferably those stains which do not require taking the sections 

 to water because in such cases the cuticula is likely to swell and tear loose 

 the sections or parts of them. Delafield's or Ehrlich's hematoxylin in 

 50 or 70 per cent alcohol stain well followed by Orange G or some other 

 counterstain. Among the iron-hematoxylins Dobell's works splendidly 

 and is, for the reason mentioned previously, preferable to Heidenhain's, 

 it is also more selective and does not require a watery mordant. 

 Staining is accomplished in the following manner: 



Sections are freed of paraffin and run down to 70 per cent alcohol in 

 the usual way. At this point they are mordanted in 1 or 2 per cent solution 

 of iron alum (ammonium ferric sulphate) in 70 per cent alcohol for one- 

 half to one hour (or longer), rinsed in 70 per cent alcohol and placed in a 

 1 per cent solution of hematein in 70 per cent alcohol for a period as long 

 as that of mordanting or longer. The sections after this treatment and 

 rinsing in 70 per cent alcohol are ready for destaining which may be done 

 rapidly in 0.5 per cent acid (HCl) in 70 per cent alcohol, but preferably, 

 for more even results, in the mordant itself until satisfactory differentia- 

 tion has taken place. The sections may be counterstained or not as one 

 desires, cleared, and mounted in damar or in what is an excellent medium 

 — cedar immersion oil. 



