270 CHROMATIN, ANIMAL CHROMOSOMES, NUCLEOLI 



with equal parts of water seems better. Prokofieva {Cytologia, 

 V, 1934, p. 498) uses equal parts of 5 per cent, chromic acid and 

 50 per cent, formol as a fixative. 



634. Methods for Invertebrate Chromosomes. As in all chromo- 

 some work, the key to successful fixation is bringing the unaltered 

 living tissue into direct contact with the fixing agent. As a rule, 

 metaphase chromosomes present no great difficulty, but for early 

 meiotic stages, in general, and especially for ova which carry large 

 amounts of yolk, or are surounded by dense envelopes, special 

 methods must be employed. 



In the case of small animals, such as fleas or lice, the end of the 

 abdomen may be cut off and the viscera squeezed out on a glass 

 slide which is quickly plunged into a jar of the fixative. For larger 

 animals, one may open up the body cavity and pipette fixative over 

 the viscera before the gonads or nervous tissue are separated away. 

 It is generally better, however, to dissect out the desired tissue in 

 normal body fluids, or in some medium like Ringer's solution 

 (cold-blood formula) or an isotonic salt solution. (Isotonic salt 

 solution ranges from 0-70 to 0-75 per cent, of sodium chloride). 

 Sometimes a 2 per cent, solution of urea is used as the dissecting 

 medium. With any of these methods great care should be taken 

 not to allow either the tissue or the dissecting medium to undergo 

 any evaporation. In the past the favourite fixatives have been 

 chromo-aceto-osmic mixtures, especially Flemming's solution, and 

 picro-formol-acetic combinations, such as Bouin's fluid. For 

 many types of chromosome work, the iron-aceto-carmine 

 technique is quite adequate, and in cytogenetic laboratories, it is 

 being extensively employed. 



For eggs surrounded by chitinous, or otfier more or less impervious, 

 membranes, one must use fixatives with great penetrating power. 

 One will l)e well repaid, however, for the time spent in removing or 

 puncturing the envelopes, when this is possible. The fixatives generally 

 employed are the fluids of Carnoy, either the G : 3 : 1, or the 1:1:1 

 formula, Gilson and Tetrunkeviteh. For the eggs of many insects, 

 Kahle's fluid is recommended, but the egg envelopes must be punctured 

 before its use. It is usually necessary to take special measures to ensure 

 the easy sectioning of such material (§ 172). 



635. Illustrative Examples. In this section the methods 

 currently employed for the study of Drosophila chromosomes will 

 be described in detail and will serve as a guide for the study of 

 various types of insect tissues. Following this, will be found 

 information about the fixatives commonly used for the study of 

 the chromosomes of other types of invertebrates. 



636. Oogonial Divisions. These are found in the ovaries of late 

 pupte or in adult flies up to twelve hours after hatching. The 

 ovaries are dissected out either in Ringer's solution (cold-blood 

 formula) or in 2 per cent, urea, or in a 0-73 per cent, solution of 

 sodium chloride. 



