March 13, 1914] 



SCIENCE 



895 



chromosomes sharply from the other cell mate- 

 rial and at the same time produce a chromo- 

 some stain translucent enough so that each 

 chromosome in a crowded equatorial plate will 

 stand out by itself. In the synapsis condition 

 of the spireme, a translucent but extremely 

 " contrasty " diiferentiation between the tightly 

 massed thread and other cell materials was 

 also thought desirable. Flemming's safranin, 

 gentian violet, orange G combination and iron 

 hematoxylin were usually unsatisfactory in 

 attempting to secure the sought-for results. 



Magdala-red was tested out after the meth- 

 ods given by Chamberlain,^ but with unsatis- 

 factory results. Both strong and weak solu- 

 tions of the stain were used, but for chromatin 

 and chromosome staining it never appeared to 

 possess " body " enough to give a sharp con- 

 trast when used with anilin-blue or other blue 

 cytoplasmic stains. After a preparation had 

 been in the stain bath for 24 hours, the red 

 color easily washed out during the hurried 

 passage through acid alcohol, 95 per cent, 

 neutral alcohol, etc., to x^dol. Occasionally 

 fair preparations were obtained, but when 

 these were examined by artificial light, the red 

 stain, through lack of " body," lost its bril- 

 liancy and contrast value. 



The thought occurred to try mixing magdala- 

 red with safranin O, and thus remedy the 

 " lack of body " defect of the former and in- 

 crease the brilliancy and translucency of the 

 latter. Accordingly, a solution of safranin, 

 made up of equal parts saturated solution of 

 safranin O in 50 per cent. alcoBol and a satu- 

 rated solution of safranin in a .3-per-cent. 

 solution of anilin oil in water, was mixed with 

 a strong (saturated) solution of magdala red 

 in 9 per cent, alcohol. The precipitate thrown 

 down upon mixing the two stains was elimi- 

 nated by filtering and the red solution remain- 

 ing was used as the stain. Serial section prep- 

 arations on the slide were allowed to remain 

 in this stain for varying lengths of time — 24 

 hours in most cases, after which they were 

 treated with anilin-blue, etc., after the method 

 as given by Chamberlain, p. 48. Many varia- 



1 ' ' Methods in Plant Histology, ' ' 2d ed., 1905, 

 pp. 42, 44, 47-48, 79-83. 



tions in method were practised; the main 

 thing being to allow the red stain to act for a 

 long time and the blue a very short time. 

 The necessity of using the hydrochloric acid 

 to bring- out the brilliancy of the anilin-blue 

 and to produce the desired contrast effect made 

 the process undependable where good prepara- 

 tions were constantly desired. In other words, 

 where only a small amount of material was to 

 be had and every slide preparation must be 

 made to count, as in much research work, the 

 method was not a practicable one. 



In order to eliminate the use of acid, the 

 anilin-blue was discarded, and a saturated 

 solution of azure II was substituted. This 

 combination of magdala-red-safranin and 

 azure II gave in most cases good results. A 

 brilliant red and blue were both obtainable 

 without the iise of acid. From the solution of 

 magdala-red-safranin, the slide was dipped 

 directly into the azure II, allowed to remain 

 about a second, and then rushed through the 

 higher alcohols to xylol and neutral balsam. 

 Variations in practise were common and it 

 has been my intention here merely to indi- 

 cate the general method. In many cases, 

 satisfactory results were obtained by shorten- 

 ing the bath in the red stain to an hour or 

 even to 30 minutes. 



Preparations of reduction division stages 

 in the pollen-mother-cells of Lilium canadense 

 L. were stained as follows : cytoplasm, deep 

 brilliant royal blue or greenish blue; spindle 

 fibers dark blue and sharply outlined against 

 the cytoplasm; nucleoli, usually blue in active 

 and red in " resting " nuclei, chromatin sub- 

 stance (spireme, etc.) brilliant deep ruby red. 

 Preparations of pollen-mother-cells of Nico- 

 tiana in early prophase have been obtained in 

 which the vacuolated nucleoli were stained blue 

 and little beadlike globules within the nucleolar 

 vacuoles shown brilliant red. The chromatin 

 is aways stained a brilliant red and against 

 the background of dark blue cytoplasm the 

 contrast is extremely sharp. Chromosomes in 

 those plants where their numbers are large are 

 ordinarily counted with extreme difficulty, 

 even under the most favorable conditions, 

 owing to the crowding of the chromosomes on 



