352 TRAXSACTIOXS AND PROCEEDINGS OF [Sess. lvi. 



alcohol superadded to avoid as much as possible the 

 hydration with the subsequent dehydration of tissues ; for 

 I find that sections treated with watery stains show when 

 mounted in balsam, a shrinkage of delicate structures which 

 is absent if aniline dyes dissolved in absolute alcohol are used 

 as staining reagents, and which is very slight if reagents be 

 employed containing a high percentage of alcohol. To obtain 

 good results with Kleinenberg's ha?matoxylin, place glass- 

 slides with the serial sections in a vessel containing the dye, 

 and keep the vessel for twelve hours at a temperature of 

 30° C, then remove the slides to a second vessel containing 

 a saturated solution of bismarck-brown in methylated spirit ; 

 leave in this vessel also for twelve hours at 30° C. In this 

 way a double staining is produced, as the nucleus and 

 nucleolus are stained a very transparent violet, while the 

 cell-walls appear brown. The method, employed in the 

 laboratory of the Eoyal Botanic Garden, of using glass 

 vessels (3i inches high with a 2 inches diameter at the base 

 and a 3 inches diameter at the brim) as the receptacles for 

 dyes, absolute alcohol, &c., is very handy, for a dozen slides 

 can be stained simultaneously, and both hrematoxylin and 

 bismarck-brown be used again and again. Ehrlicli's acid- 

 hfematoxylin I have also used, but find that it does not give 

 the same transparency to nucleoli, &c. Here a word of 

 warning may not be out of place to all who intend to 

 investigate the nucleolus. Beware of overstaining ! A 

 section with deeply stained nuclei looks beautiful under a 

 low magnifying power, but is of no use whatever for minute 

 investigations. Other stains used for differentiating the 

 nucleolus were heliocin and metliylene blue, nigrosin and 

 eosin, nigrosin and hematoxylin.* Sections stained as 

 indicated should be very thoroughly dehydrated, then cleared 

 in resinified turpentine (not in clove oil, as the latter causes 

 considerable shrinkage) and mounted in caiuxda-balsam 

 dissolved in turpentine, as this balsam has a considerably 

 lower refractive index than either chloroform-ljalsam or 

 benzol-balsam. 



If these instructions be followed in all their details I 

 believe my results will be confirmed, in the main points at 

 any rate, if not entirely so. 



* Trans. liot. Soc. Ediii., vol. .xix. (January 1891) p. 46. 



