36 



THE ART OF MAKING MICROSCOPE SLIDES 



Nematodes 



survey small aliquots by strong trans- 

 mitted light under a dissecting micro- 

 scope. The nematodes may be recognized 

 by their activity, picked out with a fine 

 pipet, and transferred to fresh saline. 



Whatever method has been employed, 

 one is left with a collection of nematodes 

 in salt solution. The solution should 

 be changed frequently, until the worms 

 are clean; for satisfactory wholemounts 

 cannot be made if either dirt or mucus 

 adheres to the outside. Heat is the only 

 fixative which will penetrate a nematode 

 rapidly. It is conventional, therefore, to 

 fix worms in hot 70 % alcohol, though hot 

 water will, in point of fact, do equally 

 well. The exact temperature is immaterial 

 and usually 100 times as much 70% alco- 

 hol as there is saline around the worms is 

 warmed until bubbles appear. This is usu- 

 ally at about 55° to 60°C. The hot alcohol 

 is then rapidly flooded over the living 

 worms, which are again collected by being 

 allowed to settle to the bottom of the 

 dish or tube. Most of the worms fixed by 

 this method will be found to have straight- 

 ened out, and the few which have not 

 had better be thrown away. 



The worms must next, very carefully 

 and slowly, be transferred to absolute 

 alcohol, in which they must remain until 

 they are completely dehydrated. This 

 transfer is best effected through 5 % grades 

 of alcohol; that is, from 70 to 75 to 80 to 

 85, etc. In the case of worms with very 

 tough cuticles, a faster schedule may be 

 employed. The reason the worms must 

 be transferred to absolute alcohol before 

 passing to glycerol is that it is almost 

 impossible to get rid of water once it has 

 got into the glycerol, and the high re- 

 fractive index of the glj^cerol is lost if it is 

 diluted. It is easy to find out how fast a 

 schedule may be emploj'^ed by taking one 

 of the worms from 70% and throwing it 

 directly into, say, 95% alcohol. If, after 

 two or three hours in this, there is no sign 

 of tlie colla])se of the wall, the rest may 

 follow it, but if the wall collapses one 

 must experiment with 80% and so on until 

 one has found the most rapid transfer 

 which may be made. When the worms are 

 all accumulated in absolute alcoliol, a 

 little glycerol is added. Assuming that the 



worms are in 100 milliliters of absolute 

 alcohol, it would be safe to add about 10 

 drops of glycerol, being very careful to 

 shake rapidly and continuously so as to 

 disperse the glj^cerol rapidly. The worms 

 are left in this mixture for about 24 hours 

 before a further 10 or 20 drops of glycerol 

 are added and mixed. This schedule is 

 continued until about 10 milliliters of 

 glycerol have been added. The solution is 

 then concentrated by evaporation, in a 

 desiccator, at a rate which leaves the 

 worms in concentrated glycerol at the end 

 of about a week. It is easiest to suck air 

 through with an aspirator, being careful 

 that the air itself passes through a de- 

 hydration column before entering the 

 desiccator. This method of preparation is 

 laborious in the extreme, but it yields a 

 product which looks exactly like a glass 

 model. The author knows no other 

 method which will produce clear nema- 

 todes without causing the collapse and 

 wrinkUng of the cuticle. 



To make these cleared nematodes into 

 permanent mounts, one now secures the 

 necessary slides, coverslips, a metal tool 

 of the type shown in Fig. 20, and a can 

 either of Noyer's or Fant's cement. It is 

 unnecessary to make a cell, or to use a 

 concave slide, because the viscosity of the 

 glycerol will hold the cover a reasonable 

 distance away from the slide while the 

 mount is being made, and the method of 

 mounting embeds the coverslip so firmly 

 that it does not subsequently shift. The 

 slide is taken, cleaned by any preferred 

 means, and the required specimen or 

 specimens placed in the center in a drop of 

 glycerol. Square coverslips should be 

 employed and a little experience will 

 soon show what amount will fill the cover- 

 sUp to the edge. The coversHp should be 

 lowered vertically to avoid displacing the 

 worms, and, as soon as the glycerol has 

 reached the edge, the heated metal tool is 

 plunged into the cement and used to 

 seal one edge. The success of tlie process 

 depends on having the cement hot enough 

 at the moment when it is applied. The 

 opposite edge of the coverslip is then 

 sealed, and these two seals connected by a 

 third. The application of cement to the 



