Nematodes 



FLUID WHOLEMOUNTS IN NONAQUEOUS MEDIA 



35 



kept dry, and many of the neutral niount- 

 ing media described in Chapter 20, under 

 the heading M 23.1, are less trouble to 

 use, and probably just as good. Liquid 

 petrolatum is difficult to seal, though the 

 author has had most success with the hot- 

 resin method described in the last para- 

 graph. Even with this material, however, 

 there is a slow diffusion of the brown 

 resin through the liquid petrolatum which 

 ultimatel}' damages the slide. Since liquid 

 petrolatum does not evaporate, it is some- 

 times preserved by holding a covershp in 

 place with a drop of cement at each corner. 

 The quantity of cement used is thus so 



small that diffusion through the mounting 

 medium is negligible, while the degree of 

 adherence is sufficiently good for all nor- 

 mal handling. Bromonaphthalene is used 

 only for mounting diatoms, when a 

 medium of high refractive index is re- 

 quired. The only satisfactory cement for 

 sealing is a de-waxed shellac prepared by 

 the method of Hitchcock (Chapter 28, 

 V 11.2). Both the preparation of the cell, 

 and the process of mounting, are special- 

 ized procedures which are described in 

 considerable detail in the second of the 

 typical preparations which terminate this 

 chapter. 



Specific Examples 



Preparation of Nematodes in Glycerol 



Nematodes are awkward objects from 

 which to make v.holemounts, for their 

 thick cuticle permits only slow diffusion of 

 reagents, and it is almost impossible 

 to get them into either resinous or gelati- 

 nous media. The objection to shrinkage is 

 not on aesthetic grounds, but on the 

 basis that the folds and ridges of cuticle 

 render it almost impossible to make out 

 clearly those internal organs upon which 

 classification depends. Nematodes are, 

 therefore, almost invariably mounted in 

 glycerol. 



No difficulty will be experienced in 

 collecting small nematodes from the blood, 

 or when they are free-swimming (as 

 Anguillula). The standard method of se- 

 curing nematodes and their eggs from 

 feces, however, is by flotation from a 

 strong salt solution. Fresh specimens are 

 collected and flooded with 10 or 15 times 

 their volume of a 20% solution of sodium 

 chloride. This may be added directly 

 to the cardboard containers customarily 

 used for such samples, and the unpleasant 

 odor may be diminished by adding small 

 quantities of nitrobenzene both to the 

 salt solution anfl to the feces themselves. 

 After the sohds have settled, the top 

 layer, on which tlie nematodes will be 

 floating, is jerked into another dish with a 

 quick movement of the wrist. It is almost 

 impossible to pick the worms or eggs from 

 the surface in a pipet so that, when a 

 sample has been thus isolated, it should be 



diluted to a salt concentration of about 

 1 % which allows the specimens to sink to 

 the bottom. They may then be washed 

 with weak saline until free of fecal matter. 

 This method of collection cannot be used 

 with fecal specimens which have been 

 mixed with animal charcoal as a deodor- 

 ant, because the charcoal also floats on 

 the surface. Worms may, however, be 

 collected from such samples by a modified 

 Berlese funnel (see Chapter 4, Fig. 21). In 

 this technique a plug of glass wool is 

 placed at the bottom of an ordinary glass 

 funnel and the fecal material poured in. 

 The bottom of the funnel is then lowered 

 into a tube of 1 % salt solution until the 

 liquid rises just to the lower edge of the 

 fecal matter. A lamp, or some other heat 

 source, is then placed above the feces. 

 The worms endeavor to escape from the 

 heat and, burrowing down through the 

 feces, ultimately pass through the glass- 

 wool plug and accumulate at the bottom 

 of the tube of salt solution. 



The collection of small nematodes from 

 soil samples is much more difficult than 

 from feces. The flotation method is 

 practically impossible because in most 

 soil samples there are large quantities of 

 organic matter which will also float, 

 while the modified Berlese funnel usually 

 permits enough clay to sift down to make 

 it difficult to sepaiate' the worms. Pro!)- 

 ably the best procedure is to dilute soil 

 samples with a 1 % salt solution and then 



