CHROMATIN, ANIMAL CHROMOSOMES, NUCLEOLI 273 



dissection in Ringer's solution, the glands are placed on a clean 

 slide, the excess fluid removed, and iron-aceto-carmine is added 

 from one side. After a minute or two the stain is removed and 

 replaced by fresh fluid, thus ensuring that there will be no dilution 

 due to the dissecting medium. The stain is allowed to act for ten to 

 twenty minutes, depending on the sample of stain, room tempera- 

 ture and, perhaps, the condition of the larvae. When the glands 

 are a deep red in colour the coverslip is added (it may be placed 

 over the glands when they are first mounted, if desired), and 

 then the excess stain is drained off with a pipette and filter paper. 

 Next the preparation is blotted with filter paper, using a good 

 deal of pressure, both to crush the glands and to remove more 

 stain. It is essential that the coverslip is not allowed to move 

 during the blotting, for this will break the chromosomes and other- 

 wise badly distort them. The nuclei are usually freed from the 

 surrounding cytoplasm, when the glands are crushed, but the 

 walls generally remain intact. The next step is to rupture the 

 nuclear walls so that the chromosomes can spread out. This is 

 best done under a wide field binocular by pressing on the cover- 

 slip with a blunt needle directly above a nucleus. Do not allow 

 the coverslip to move about. Any excess stain is now removed 

 from the preparation and the coverslip is sealed with melted 

 paraffin, vaseline, or in some other way. Such a slide will last 

 for several days or longer, if it is kept cool. The reader should 

 note that the success of the aceto-carmine method depends upon 

 having a good stain with just the right amount of iron. Read 

 carefully the directions given in § 257. 



642.* For making permanent aceto-carmine slides several very 

 good methods are in current use among Drosophila workers, and 

 it is too soon to say which should be standard practice. The 

 preliminary steps are described in the preceding paragraph; 

 further treatment consists of dehydrating the preparation and 

 mounting in euparal, Venetian turpentine or balsam or damar 

 The writer has found that the methods used by Bridges and 

 others (see Drosophila Information Service No. 6) give unusually 

 clear preparations. The larvae are reared at a low temperature, 

 and given food very rich in yeast. When they crawl up on the 

 paper, or the walls of the bottle, preparatory to pupating, they are 

 removed and the glands are dissected out in cold Ringer's solution 

 (or in a 0-73 per cent, salt solution) and transferred to a dish of 

 plain (without iron) aceto-carmine which has been chilled with ice, 

 where they are left for twenty minutes to several hours. The 

 glands are then mounted as described above, but instead of sealing 

 the coverslip the slide is placed in a dish containing alcohol fumes 

 (any convenient dish may be lined with filter paper and saturated 

 with 95 per cent, alcohol). The slides are allowed to " season " 

 in alcohol fumes up to twenty-four hours. An hour is said to be 



* See also p. 277. 



