APPENDIX 407 



since sperm from one mating are stored for a considerable time. If all 

 flies are removed from a culture, the females which are found four to 

 six hours later> having emerged from the pupae in the interval, will 

 still be virgin. Virgins can also be obtained by taking newly hatched 

 flies from ordinary cultures; they are recognized by their crumpled 

 and unstretched wings, and elongated bodies with pale soft chitin. 



The male (which need, of course, not be virgin) and female for a 

 cross should be placed gentiy into a vial, care being taken that they 

 do not get stuck on the food. It is best to put them on a piece of paper, 

 and push this into the vial which is laid on its side until the flies have 

 recovered from the anaesthetic, when it can be stood on end. Always 

 label all crosses immediately. 



SPECIAL TECHNIQUES: CYTOLOGICAL 



The ordinary chromosomes of Drosophila are so small that they 

 cannot be well used even for demonstrations. On the other hand, the 

 nuclei of the salivary glands contain giant chromosomes which can be 

 seen with a low-power microscope (very well with ^xh objective) 

 and are so easy to prepare that they could be stained by fairly advanced 

 classes. 



In making the preparation it is first necessary to obtain the glands. 

 They Ue just behind the head of the larva. Take old, fat larvae, which 

 have crawled up on the side of the bottie to pupate. Hold the body of 

 the larva with a needle or fine forceps, in the left hand, and pull off 

 the head into a drop of water or salt solution. The two glands will be 

 seen as rather long, very transparent sacs attached along their sides to 

 highly refringent fat bodies. It will probably be necessary to use a 

 lens to isolate them. As much as possible of the debris should be 

 removed, and the salt solution sucked off with a pipette or filter paper. 

 The glands are then simultaneously fixed and stained in aceto-carmine 

 (boil excess carmine with 40 per cent acetic acid, filter). Leave the glands 

 in acetocarmine for up to two hours, not allowing it to dry up. Then 

 blot off excess carmine, put on a cover glass, lay blotting paper on the 

 over glass and squeeze firmly to flatten the glands and expel fluid. 

 The cells are then quite disrupted, and the tangles of chromosomes, 

 which are stained red, are spread out. The usual fault is not to squeeze 

 the preparation hard enough. The time of staining, which also hardens 

 the tissue and affects the flattening and spreading, varies with different 



