64 ANILIN COLOURS GIVING INDIRECT NUCLEAR STAINS. 



solution and concentrated aqueous solution (this is very much 

 to be recommended), and (B) a concentrated or supersaturated 

 aqueous solution made with the aid of heat. 



Some people still employ simple aqueous solutions. 



Lastly, there is the anilin solution of BABES (Zeit. f. wiss. 

 Mik., iv, 4, 1887, p. 470). It consists of water 100 parts, 

 anilin oil 2 parts, and an excess of safranin. The mixture 

 should be warmed to from 60 to 80 C., and filtered through 

 a wet filter. This solution will keep for a month or two. 



ZWAARDEMAKER (Zeit. f. wiss. Mik., iv, 2, 1887, p. 212) 

 makes a mixture of about equal parts of alcoholic safranin solu- 

 tion and anilin water (saturated solution of anilin oil in water).* 



Any of these stains may be used with any of the following 

 washing-out processes. Of course you will have to stain longer 

 in the weaker solutions. As to the anilin solutions, see ante, 

 96. 



Washing-out and Clearing. 



For general directions for washing out and clearing, see 

 above, 97 and 99. 



FLEMMING'S first method (1. c. in last ). Wash out with 

 pure alcohol, followed by clove oil. This method stains 

 resting chromatin as well as " mitoses." 



FLEMMING'S second method (Zeit. f. wiss. Mik., i, 3, 1884, p. 

 350). Wash out until hardly any more colour comes away, in 

 alcohol acidulated with about 0*5 per cent, of hydrochloric 

 acid, followed by pure alcohol and clove oil. (You may use 

 the HC1. in watery solution if you prefer it.) The strength 

 given appears unnecessarily high, and I therefore generally 

 use with good results an alcohol of about 0'2 per cent, of HC1. 

 (Objects are supposed to have been well fixed twelve hours 

 at least in the strong chromo-aceto-osmic mixture, and stained 

 for some hours). PODWYSSOZKI (Beitr. z. path. Anat. v. Ziegler 

 u. Neuwerk, i, 1886; Zeit. f. wiss. Mik., iii, 3, 1886, p. 405) 

 prefers to stain for half an hour only, and wash out with O'l 

 per cent, of HC1. in alcohol. In each of these ways you get 

 " mitoses " and nucleoli alone stained (if the fixation have been 

 performed as above directed) . 



* To make " anilin water " shake up " anilin oil " (which is nothing but 

 pure anilin) with water, and filter. 



