BOTH THE SEXES IN DIPTERA. 379 



Methods of Work. 



For the convenience of those who wish to examine genitalia or to test my statements, 

 and are nnacquainted with the usual procedure, I shall now explain my methods of 

 work on these very minute parts. I will presume that the enquirer has the usual 

 outfit of the worker in microscopy. Fresh or newly-killed insects are undoubtedly the 

 best subjects ; but specimens preserved in alcohol are nearly as good, and old pinned dry 

 ones arc often extremely useful. The genitalia, protected in a cavity, are as a rule 

 intact, though the insect may have lost every other appendage. If a species is to be 

 thoroughly studied, at least three specimens of each sex ought to be procured. I assume 

 that they are newly-killed insects. (1) Immerse one of each sex in 15 per cent, solution 

 of caustic potash ; 10 per cent, solution will do but takes longer. This is to clear the 

 insects, 'and the length of the process will depend on the density of the chitinous 

 structure ; a day may be enough, or it may take over a fortnight. In this process all 

 the muscles and nerves are dissolved and destroyed, leaving tlie membranes, the exo- 

 skeleton, and all the chitinous structures. (2) After this is accomplished the prepara- 

 tions must be thoroughly washed in water ; and (3) then placed in glacial acetic acid for 

 24 hours to get rid of the potash crystals and any fat that may have escaped the action 

 of the chemical. (1) They must again be washed in water. (5) Afler this they are in 

 a flaccid state, and can be readily arranged on a 3-inch slip in any desired position. 

 The wings will be found to flatten better if the specimen is floated on to the slip. In 

 the case of the male a lateral arrangement is best, as it more clearly shows the genitalia 

 in their natural positions ; but where possible it is well to have a preparation showing 

 the ventral surface as well. A pair of strong cHps, such as are used to hold papers, is 

 useful in the next process. (6) After the insect has been arranged on the slide, another 

 slip is placed above it ; it is squeezed flat by applying a clip at one end, and the process 

 is completed when the second clip is applied at the other end. There is now an oppor- 

 tunity for roughly examining the preparation with low powers to see that the organs 

 are properly displayed, and that (for instance) a claw does not cover the hypopygiura, or 

 the trophi of the head are not hidden. If unsatisfactory, undo the clips, separate the 

 slides (care is required in this), drop some water on the insect, and rearrange it. If, 

 however, the initial effort is successfid, tie the slips tightly together with fine twine, 

 remove the clips, and (7) immerse in methylated spirit for at least 21 hours. In cases 

 where the preparations have been over cleared, a stain is useful. Aniline blue is best in 

 my experience ; f uchsin is also good, but care must be taken not to overstain. A drop 

 can be placed on the insect before the upper slide is applied. In the spirit the prepara- 

 tions will be dehydrated, and on the result of this process the success of the preparation 

 as a microscopic object depends. Al'ter that they will be ready for transference to spirit 

 of turpentine, wliich should be of the best quality procurable. (8) To do this, the slips 

 must be withdrawn from the spirit with a pair of forceps, the string untied, the slips 

 slowly and carefully separated, and the preparations taken from the slide on a section- 

 lifter. (9) After being at least 24 hours in turpentine they are ready for inounting in 

 Canada balsam, though many microscopists recommend a further clearance in clove-oil. 



SECOND SERIES. — ZOOLOGY, VOL. IX. 53 



