198 BOTANICAL MICROTECHNIQUE 



as much longer as convenient, to remove all trace of the iron alum. The 

 material is now ready to be mounted in balsam (Sec. 187) or glycerin 

 (Sec. 188). If mounted in balsam it must be carried through xylol (never 

 oil of cloves, which fades hsematoxylin). 



The haBmatoxylin can be extracted to a point where the nucleus is prac- 

 tically the only structure stained, and is consequently one of the best of 

 the nuclear stains. Such material may be counterstained (that is, stained 

 in addition) with safranin (Sec. 184), thus differentiating the nucleus (gray 

 or black) from the rest of the protoplasm (red). Iron-alum hsematoxylin is 

 probably on the whole the most satisfactory of all the staining methods for 

 protoplasmic structures. It is subject to great latitude in the time limits, 

 which may be set for the different stages of the process except that of extrac- 

 tion, which must of course be watched carefully ; but these are soon learned 

 with experience. It is perhaps the least uncertain of the stains, and although 

 the process is somewhat long it can always be depended upon to give 

 good results. 



188. Delafield's hsematoxylin. This stain reacts very differently from iron- 

 alum hsematoxylin. It stains cell walls sharply, but does not differentiate 

 protoplasmic structures as well as the latter. It is one of the best stains for 

 tissues of higher plants, and may be combined very effectively with safranin, 

 as described below and in Sec. 185. 



Delafield's hsematoxylin is made as follows : a solution of 1 gram haema- 

 toxylin in 6 cc. absolute alcohol is added drop by drop to 100 cc. of a 

 saturated solution of ammonia alum. Filter after exposing for a week to 

 the air and light. Then add 25 cc. of glycerin and 25 cc. of methyl alcohol. 

 Allow the mixture to stand for several hours (4-7), until the color is dark, 

 and then filter. The solution should then remain two months in a tightly 

 stoppered bottle to "ripen." The prepared stain may be purchased from 

 dealers (Sec. 218). 



Material is transferred to Delafield's hsematoxylin from water or 25% 

 alcohol. Staining will take place rather rapidly, requiring from a few min- 

 utes to an hour or more. The stain may be diluted to half or a fourth of 

 the above strength, and the staining, although longer, is frequently better. 

 Wash the material in tap water until a rich purple color develops. If the 

 sections or other subjects are overstained, or if a precipitate is formed when 

 the material is placed in alcohol, rinse in acid alcohol (y'j cc. hydrochloric 

 acid in 100 cc. 70% alcohol). The acid alcohol takes out the color, which 

 may thus be extracted until the nucleus alone remains stained. When 

 washed in acid alcohol the material must be placed in tap water until the 

 purple color returns. Then run up in the grades of alcohol through 95% 

 and absolute alcohol, clear in xylol, and mount in balsam, as described in 

 Sec. 187, or pass from water into glycerin, as outlined in Sec. 188. 



