102 



Journal of Applied Microscopy. 



Notes on Microscopical Technique. 



G. Carl Hubee, M. D. 

 Fourth Paper. 



Perhaps no branch of microscopical 

 technique presents so great a variety of 

 methods as tliat of staining. Nearly all 

 known stains have some time or other 

 been used and recommended as general 

 or special stains. It is not to be sup- 

 posed, however, that it is necessary to 

 know even a major portion of these 

 methods in order to do useful work. In- 

 deed, even in the histological labora- 

 tories, many of the methods are never 

 used, others only in rare and very special 

 instances. The great bulk of the work is 

 carried on with very few methods, and, 

 as it seems to me better to be able to 

 follow one or two methods with some 

 degree of confidence than to have a 

 casual acquaintance with a number of 

 methods, I shall confine my account to 

 only a very few of the many staining 

 methods known. Before detailing, how- 

 ever, the methods to be discussed, a few 

 general remarks on the staining of tis- 

 sues may not be out of place. The 

 majority of stains used in microscopical 

 technique may roughly be divided into 

 "nuclear stains" and "protoplasmic or 

 general stains." The former stain more 

 particularly the nucleus, the latter shows 

 a selective action toward the proto- 

 plasm. In explanation of these facts, 

 the following considerations are offered: 

 — it is well known that not all the con- 

 stituents of the nucleus show a selective 

 action toward nuclear stains, but only 

 that portion which, owing to its affinity 

 to these stains, is known as the chro- 

 matin, and the chromatin is, roughly 

 speaking, composed largely of nucleinic 

 acid. Now, it is well known that the 

 nuclear stains belong, as a general rule, 

 to that group known as basic stains. 

 Many of the stains may, however, be 

 regarded as salts. In some of these salts, 

 the acid radicle is instrumental in bring- 

 ing about the stain, and such stains are 

 grouped under the head of acid stains. 

 They show an affinity toward the pro- 

 toplasm of cells, or toward cell products, 

 while in other stains the base does the 

 staining; these are the basic stains. 

 These show an affinity toward the 

 nucleus, that is its chromatin, because 

 the nucleinic acid forms a chemical com- 

 pound with the stain, which has some 

 stability and which is not broken down 

 or materially changed by the further 

 manipulation of the section. Nuclei rich 

 in chromatin stain deeply, and vice 

 versa. The nuclear configuration depends 

 on the arrangement of the chromatin in 

 the nucleus. It is not to be supposed, 

 however, that the nuclear stains color 

 only the nucleus. Nearly all of them 



color the protoplasm more or less, but 

 they do not form so firm a compound 

 with the protoplasm, if any is formed at 

 all, and are therefore more readily 

 washed out of it than out of the nucleus, 

 either with water or alcohol, some acidu- 

 lated solution or with an acid stain. 



Of the nuclear stains none have a wider 

 applicability and are more generally used 

 than the solutions of haematoxylin. For 

 general work the following solutions are 

 recommended: • 



Boehmer's Haematoxylin Solution. 

 Sol. No. 1— 



Haematoxylin crystals, 1 gram. 



Absolute alcohol, 10 ccm. 



Sol. No. 2— 



Potash alum, 10 grams. 

 Distilled water, 200 ccm. 



Solution No. 1 is prepared by mixing 

 the haematoxylin crystals and the alco- 

 hol in a well stoppered bottle, shaking 

 occasionally and allowing it to stand for 

 twenty-four hours. 



Solution No. 2 is prepared by dissolving 

 the alum in warm distilled water, and 

 allowing this solution to cool. 



Mix solutions one and two in a large 

 open dish, stirring constantly while mix- 

 ing. Allow to stand for about a week 

 and filter into a clean bottle. The stain 

 is then ready for use. 



Practically the same stain may be 

 made after the following formula, known 

 as Meyer's Hemalum Solution: 



Hematein, 1 gram. 



90% alcohol, 50 ccm. 



Potash alum, 50 grams. 



Distilled water, 1000 ccm. 



And a small crystal of thymol. 



Dissolve the hematein in the alcohol 

 by the aid of heat. Dissolve the alum in 

 the water; hastening by warming the 

 water. Mix the two solutions, stirring 

 while mixing. Add the thymol crystal. 

 This solution has the advantage of being 

 ready for use at once, and does not pre- 

 cipitate as does Boehmer's haematoxylin 

 after standing some weeks. Either solu- 

 tion will, however, stain readily for many 

 months. It is well, however, to filter the 

 stain before using. 



Acid or Protoplasmic Stains. — Of the 

 acid stains which may be used as counter 

 or double stains to the nuclear stain 

 obtained with haematoxylin, none are 

 more useful than a solution of eosin, 

 and a solution made of picric acid and 

 acid fuchsin. 



Solution of Eosin. — As there are a num- 

 ber of preparations of eosin in the mar- 

 ket, some of which will hardly stain at 

 all, while" others stain very readily, it 

 may be well to state that in this labora- 

 tory we have used for some time an eosin 



