298 



METHODS AND FORMULAS 



DS 11.20 



As a clearing agent, tlu^ auilior ))iefers 

 benzene, for the double reason that it can 

 be used for the solution of balsam in the 

 next stage and also because it leads to less 

 hardening than almost any other reagent. 

 The acetone or alcohol is replaced with 

 benzene by the same slow flow process: 

 passing through the stages of 20% ben- 

 zene, 40% benzene, 60 7o benzene, 80% 

 benzene, the percentage figures referring to 

 the amount of benzene in mixture with 

 fresh acetone or alcohol. These benzene- 

 acetone mixtures should be run through in 

 quantities of a Uter each, but they need 

 not, of course, be wasted, for only the first 

 fraction passing through will be seriously 

 diluted and the remaining material may 

 be led from the outlet pipe of the bottle 

 in which the specimen is to another 

 bottle in wliich it may be stored for future 

 use. Though it is perhaps pointing out the 

 obvious, attention should be drawn to the 

 fact that either synthetic rubber tubing 

 or alternatively solid glass joints must be 

 employed. 



Transference of the perfectly cleared 

 specimens to Canada balsam is a more 

 difficult project than even the clearing 

 and dehydrating already undertaken. The 

 author prefers the method of evaporation, 

 even though it is most tediously slow, 

 because it has the advantage of complete 

 safety. In this niethod the benzene in 

 which the specimens are now resting is re- 

 placed, using the same flow method which 

 has been used for the application of all the 

 other reagents, by a 1 % solution of 

 Canada balsam in benzene. Unfortunately 

 it is impossible to evaporate the benzene 

 directly from the balsam, unless a tre- 

 mendous volume of solution is employed, 

 for even the relatively thin solution used 

 for mounting sections should be at least 

 40% by weight balsam and for the mount- 

 ing of wholemounts, 60 or 70% is not 

 excessive. A solution stronger than 1%, 

 even when apphed by the drip method, 

 will inevitably result in the collapse of the 

 specimen; therefore one is forced to sub- 

 stitute increasingly stronger solutions be- 

 fore the final evaporation is made. The 

 simplest way to do tliis is to pass the 

 specimens as described into a 1 % solution 

 and then to transfer the specimens in this 



solution to a beaker of the 1% solution. 

 The height of the 1% solution in the 

 beaker is then measured with a millimeter 

 scale and a red mark made halfway be- 

 tween the top of the liquid and the bottom 

 of the beaker. The beaker is then placed 

 in a large desiccator, the lid of which is 

 removed at daily intervals to liberate the 

 benzene, and the whole is permitted to 

 evaporate until the halfway mark is 

 reached. A fresh solution of 2% balsam 

 is then taken and a drop of it placed care- 

 fully in the presumable 2 % solution which 

 is now in the beaker. If this drop shows by 

 its refractive index that it is now of the 

 same concentration as that in the beaker, 

 the solution which is to be added should 

 be adjusted, either with the addition of 

 balsam or with the addition of benzene, 

 until there is httle or no difference be- 

 tween the two solutions. The beaker is 

 then filled up with tliis fresh 2% solution 

 to the original level and again permitted 

 to evaporate down to the halfway mark. 

 At this time an additional portion of 4% 

 solution is added and so on until the 

 beaker is filled with a 32% solution. This 

 is now permitted to evaporate until it is 

 just hquid enough to permit the removal 

 of each medusa with a considerable por- 

 tion of the balsam either to a hollow- 

 ground slide or a deep cell. No coverslip is 

 placed on it at this stage, but additional 

 solution from the beaker is piled on top 

 of the specimen as it evaporates so that a 

 Uttle mount of semiliquid balsam is con- 

 stantly kept over the specimen. The ut- 

 most care must be taken to exclude dust 

 during these proceedings. When the 

 balsam has, by evaporation, been thick- 

 ened to the point where it no longer flows, 

 a coverslip should be dipped in clean 

 benzene and placed on top of the mound 

 of balsam and the shde slowly heated on a 

 hotplate until it becomes sufficiently fluid 

 for the coverslip to settle of its own accord 

 onto the specimen. The specimen is then 

 cooled, permitted to set for a day or two, 

 and then cleaned up in the ordinary 

 manner. 



Though it must be admitted that the 

 preparation of mounts of this type oc- 

 cupies several months, only a very few 

 hours per week are required to look after 



