DS 11.40 



DYE STAINS OF GENERAL APPLICATION 



311 



The most essential precaution to be ob- 

 served in the dissection of testes from 

 small insects for cytological investigations 

 is that the insect should be dissected 

 under the surface of physiological sahne. 

 For this purpose the most convenient ves- 

 sel is a deep dish or fingerbowl, to the 

 bottom of which is fitted a circle of cork 

 or linoleum weighted on the underside 

 with lead sheet to which it is attached by 

 rivets. It is undesirable in cytological in- 

 vestigations to kill an animal by an anes- 

 thetic. In the case of the grasshopper, the 

 simplest method is to remove the head 

 with a pair of sharp scissors before pinning 

 the body down in place, after the removal 

 of the wings and legs from one side, with 

 the aid of four pins placed at the anterior 

 and posterior margins of the body. Sufh- 

 cient physiological saline is then added to 

 cover the specimen to a depth of about a 

 quarter of an inch, and the dissection is 

 then conducted under a low^-power binocu- 

 lar microscope. Before commencing the 

 dissection it is desirable to have available 

 the necessary fixative, which in the pres- 

 ent instance should be the osmic-chromic- 

 acetic of Flemming 1882 (Chapter 18, F 

 1600.0010) or some other fixative of the 

 same general composition. It is also neces- 

 sary that the slides to be employed be 

 rigorously cleaned and that the fixative 

 be placed in a petri dish of a diameter suffi- 

 cient to permit the slide to be laid flat. 

 Smears of the type which are going to be 

 prepared cannot satisfactoril}' be fixed in 

 such a manner that they will adhere to the 

 slide, if the whole of the latter is immersed 

 in the fixative. It is therefore necessary to 

 secure two short glass rods, the ends of 

 which are bent at right angles to prevent 

 them from roUing, and to place them in 

 the bottom of the petri dish into which the 

 fixative is poured until it just reaches the 

 upper surface of the rods. The depth of the 

 fixative should be tested before any dissec- 

 tion is undertaken by laying a clean slide 

 across the two glass rocls. The whole of 

 the lower surface of the slide should be in 

 contact with the fixative, which should 

 not at any point cover the ui)per surface. 



Three or four shdes are now laid at hand 

 and the dissection commenced by the re- 

 moval of the cliitinous exoskeleton from 

 the whole side of the abdomen. It is very 



difficult to find the testes unless one is 

 accjuaintcd witli the species of grasshopper 

 under dissection, for they may either be 

 fused into a single central mass or may be 

 composed of a series of tubules joined to- 

 gether at the base and lying across the 

 upper part of the intestine at right angles 

 to the direction of the vas deferens. It is 

 probably easiest first to identify the in- 

 testine, then to identify the heart, and 

 then to look between the intestine and 

 heart at about the central portion of the 

 body for a series of small, curly objects 

 attached posteriorly to a tube. These curly 

 objects (or object, according to the spe- 

 cies) wall be the testes. They should be 

 removed by cutting away the adherent 

 connective tissues and trachea, and should 

 then be thoroughly washed in the physio- 

 logical sahne which remains in the dish. A 

 piece is now cut from one of the testes of 

 about one-quarter-millimeter side and is 

 removed with a pair of fine forceps to the 

 surface of one of the specially cleaned 

 slides. Excess normal saline should be re- 

 moved from it with chemically clean blot- 

 ting paper, and a second slide crushed 

 down on it with a twisting movement in 

 such a way that the smear is distributed 

 over an area approximately equivalent to 

 a IS-millimeter circle. The two slides are 

 then rapidh^ pulled apart and each is laid 

 face down on the glass rods in the fixative. 

 The petri dish should be covered with the 

 customary lid which one should remove 

 only when adding slides to the dish to 

 avoid inhaling the irritating vapors of the 

 osmic acid. Usually, two or three shdes 

 can be placed in a dish at one time. The 

 smears should not remain in contact with 

 the fixative for more than about 30 min- 

 utes before being carefully removed and 

 placed in distilled water. They may re- 

 main in distilled water for at least several 

 days but must, in any case, be thoroughly 

 washed in several changes with not less 

 than two or three hours in each change. 

 When all the slides have been accumulated 

 in distilled water, and are known to be free 

 of both chromic and osmic acids, one may 

 then proceed to the staining technique. 

 In the technique originally described by 

 Henneguy (D8 11.43 Henneguy 1891) al- 

 most any of the synthetic nuclear stains 

 listed in this section were recommended. 



