NUCLEAR STAINING. 455 



sequent washings out, must be found by experiment for each object. 

 It differs for each according to the nature of the object and the 

 method of fixing. While, for example, for staining the contents 

 of certain pollen mother-cells in process of division the duration 

 recommended above for staining, washing, dehydrating and clear- 

 ing holds good ; on the other hand, with sections through the 

 oogonia of F-ucus we have obtained the best results with safranin 

 staining for only half an hour, gentian ten minutes, and orange G. 

 staining for one minute. Staining in orange G. must follow 

 washing in water, and cannot come directly after treatment with 

 alcohol. If absolute alcohol is used at this stage the complete 

 removal of the gentian-violet will result. If this triple staining 

 has been successful, the different constituents of the protoplasm 

 are stained in different fashions, most materially aiding their 

 differentiation : e.g., the chromatin will be purple-red, and the 

 threads of the achromatic spindle, or " spindle fibres " grey-brown, 

 grey, or violet. 1 As vessels for staining, small beakers can be used, 

 each of suitable size and depth to take one object-slide, and this 

 independent treatment of each object-slide has advantages. [Or 



1 " I have tested the safranin-gentian- violet, and the saframn-gentian- 

 violet-orange methods upon the root tips of Vicia Faba and on various other 

 vegetable objects, and can most heartily recommend them for staining vege- 

 table nuclei. I obtained the most instructive preparations by leaving the 

 sections (I worked with microtome sections) from one-half to two hours in 

 Hermann's aniline-water-safranin. [This is 1 gram safranin in 10 c.c.m. alcohol 

 and 90 c.c.m. aniline water.] I then washed them successively in acid alcohol 

 and in alcohol, added aniline-water-gentian-violet [1 gram gentian- violet to 10 

 c.c.m. alcohol and 90 c.c.m. aniline water], which I left on the sections for two 

 to four minutes at most ; washed off the dye with water, immersed them for 

 five minutes or longer in Gram's iodine solution [1 part iodine, 2 parts potas- 

 sium iodide, in 300 parts water], and then washed either wiih alcohol or suc- 

 cessively with alcohol and clove oil, and mounted in Canada balsam ; or I also 

 stained subsequently with orange. In the latter case I washed the sections, 

 after they came from gentian-violet, but a very short time in alcohol, and then 

 placed them in a concentrated aqueous solution of orange, which was allowed 

 to act for a few minutes, then washed them in alcohol and transferred, in the 

 usual way, to Canada balsam. In good preparations where orange was used 

 the Karyokinetic figures from aster to diaster were deep red, the nuclear 

 framework of resting nuclei and the spirem and dispirern were violet or blue, 

 the achromatic nuclear figure and the cytoplasm were yellow-brown. Gjurasin 

 has used these methods with much success, after many others had failed, for 

 bringing out the Karyokinetic figures in the asci of Poziza, after the material 

 had been fixed for two days in Flemming's chrom-osmic-acetic acid." Zimmer- 

 mann's Botanische Microtechnik, Eng. ed., 1896, p. 187. 



