82 



Journal of Applied Microscopy. 



(c) Rinse in water and differentiate by 

 dipping- into the ferric-alum solution for 

 a few seconds and then rinsing in tap 

 water, repeating the operation until the 

 right degree of differentiation is attained 

 as determined by examination under the 

 microscope. The chromatin should be 

 stained a blue-black or black, the spindle 

 gray or light blue. Wash well in water 

 for about twenty minutes and dehydrate, 

 clear and mount in balsam, or, if it is 

 preferred, stain after washing for a 

 minute or so in a strong- one-half satu- 

 rated aqueous solution of orange G. 



One of the best safranin stains to 

 employ is Babes' — equal parts of concen- 

 trated aqueous and alcoholic solutions. 

 Stain in this three to twenty-four hours, 

 wash with ninety-flve per cent, alcohol, 

 clear, and mount in balsam. No othei 

 differentiation than that of the ninety- 

 five per cent, alcohol is needed with this 

 formula. 



Picro-acetic mixture, of which there 

 are several formulas, is possibly even 

 better than the two standard fixers just 

 mentioned. It is a saturated aqueous 

 solution of picric acid, or (possibly 

 better) a half saturated solution (satu- 

 rated solution one part, water one part) 

 of picric acid with one or two per cent, 

 of glacial acetic acid added. Place the 

 testis in this for six to twelve hours, soak 

 in seventy per cent, alcohol one day and 

 in eighty-two per cent, alcohol several 

 days, changing until the picric acid is 

 almost entirely removed, when it may be 

 carried on for imbedding. The most sat- 

 isfactory stain with tissue fixed in this 

 way is Heidenhain's iron hematoxylin, 

 as above. The time of mordanting and 

 staining may be much shorter than with 

 Hermann's or Flemming's; one-half hour 

 in the ferric alum, and half an hour in 

 the stain. Safranin is not as satisfactory 

 with this fixer as with Flemming's or 

 Hermann's. Any one of these fixers and 

 stains gives good figures of cell-division, 

 suitable for demonstration. 



The testis of the crayfish, so common 

 in our rivers and streams, likewise is a 

 very good subject for the demonstration 

 of karyokinesis, the only objections 

 being the small size of the cells and the 

 large number of chromosomes. Their 

 division, however, is very easily demon- 

 strated, as is also the centrosome. The 

 testis will be found immediately beneath 

 the heart, on the dorsal side, under the 

 carapace, and is easily distinguished as 

 a three-lobed white organ. It may be 

 removed from a five to eig-ht cm. male 

 in the summer or fall and fixed and 

 stained in one of the ways mentioned 

 above. The sections should not be more 

 than five or seven // thick. 



The larvae of Amphibia, especially the 

 tailed forms, are very suitable objects 



for the demonstration of cell-division. 

 Just hatched specimens are most suit- 

 able, although rapidly growing forms, 

 such as Amblystoma, should be suitable 

 throughout the spring. While division 

 figures may be found readily in all parts 

 of the body, the epidermis and oral epi- 

 thelium are the most favorable regions. 

 By fixing but a short time (one to two 

 minutes) it is possible to remove large 

 pieces of the epidermis by scraping, and 

 these may be washed, examined, stained 

 if found suitable, and mounted without 

 further treatment. It will be found 

 more satisfactory, however, to fix the 

 caudal portion in one-third per cent, 

 platinum chloride or chromo-formic (four 

 or five drops of strong formic acid in 

 two hundred cc. of a one-third per cent, 

 aq. solution of chromic acid, added just 

 before using). Leave in either of these 

 twenty-four hours, wash in water four 

 to six hours, and harden in fifty, seventy, 

 and eighty-two per cent, alcohols. Sec- 

 tions parallel to the surface should be 

 made so as to cut the epidermis very 

 obliquely and have more cells in each 

 section. Amblystoma is most favorable, 

 since the young- larvae are not densely 

 pigmented and grow very rapidly. Spe- 

 lerpes larvae, although they may be 

 found during summer and winter, are not 

 serviceable, apparently because of their 

 slow growth. 



The Amphibia have one disadvantage, 

 in that the achromatic portion of the 

 figure is not as strongly developed as is 

 desirable; the testis seems the least 

 objectionable from this aspect. On the 

 other hand, in invertebrate eggs gener- 

 ally, and especially Ascaris and Echi- 

 noderm eggs as most availaible, the 

 spindle and polar radiations are strongly 

 developed. These forms are not as gen- 

 erally available as are tne Amphibia. 

 Those who are so located that they have 

 access to freshly killed horses may obtain 

 from the coecum or ileum, the parasitic 

 worm, Ascaris megalocephala, from 

 which the uterus filled with developing 

 eggs may be removed and fixed in either 

 of the three folloiwing fiuids: (a) Glacial 

 acetic acid one part, absolute or ninety- 

 five per cent, alcohol three parts; (b) 

 absolute alcohol one part, glacial acetic 

 acid one part, chloroform one part, and 

 mercuric chloride to saturation; or (c) 

 seventy per cent, alcohol eight parts, 

 glacial acetic acid two parts, which 

 formula Professor Conklin of the Univer- 

 sity of Pennsylvania has stated to be 

 very satisfactory. In formula (a), wash 

 out with strong alcohol until all odor 

 of acetic acid has disappeared; in 

 formula (b), wash thoroughly in fifty per 

 cent, alcohol until all trace of the acid 

 has been removed, and in seventy and 

 eighty-two per cent, alcohols changing 



