MASSON'S TRICHROME STAIN 



193 



MASSON'S TRICHROME STAIN 



after fixation by picro-formol (Bouin) 

 or formalin. So altered, the collagen 

 does not properly absorb the dyes and 

 differentiates badly. Trichrome stains 

 with iron haematoxylin. All trichromic 

 methods are based upon a common first 

 step: the staining of nuclei by iron alum 

 haematoxylin, followed by differen- 

 tiation with picric alcohol. 



Three solutions are required: A. Iron 

 alum, violet crystals, 5 gm., aq. dest. 

 100 cc. B. Regaud's hematoxylin solu- 

 tiin made up by dissolving 1 gm. hema- 

 toxylin in 60 cc. hot aq. dest. Cool and 

 add 10 cc. glycerin and 10 cc. of 95% 

 alcohol. This stain is ready for use at 

 once. C. Differentiating mixture con- 

 sisting of 2 parts sat. picric acid in 95% 

 alcohol and 1 part of 95% alcohol. 



Step 1. Place iron alum solution and 

 the hematoxylin solution in staining 

 jars in a water bath heated to 40-45°C. 

 Mordant deparaflined sections in heated 

 iron alum, 15 min. Rinse with aq. 

 dest. Stain in heated hematoxylin, 15 

 min. or more. Rinse the uniformly 

 black sections with 95% alcohol and 

 immerse in picric alcohol. Control the 

 progress of differentiation under the 

 microscope. As soon as the nuclei alone 

 remain colored, wash with running 

 water for 15 min. 



If after washing, the background of 

 the preparation, particularly the col- 

 lagen remains gray, rinse with alcohol 

 and complete picric differentiation. 

 The chromatin is electively black and 

 opaque. 



Various authors have proposed to 

 "simplify" this method by staining the 

 nuclei with Weigert's iron perchloride 

 hematoxylin or with alum hematoxylin. 

 I must say that I have long ago tried 

 such modifications before adopting the 

 above method and that I have aban- 

 doned them entirely. The red of the 

 ponceau-fuchsin solution superposes 

 itself on the gray or blue color imparted 

 by hematoxylin so that chromatin takes 

 a dull color lacking in specificity. 

 Rather than to use such modifications 

 it would be better to omit all nuclear 

 staining and start with the next step 

 (ponceau-fuchsin and so forth) : the 

 results are thus comparable to those ob- 

 tained by Mallory's original method. 



Step 2. Stain in a mixture of acid 

 fuchsin and Ponceau ofxylidin BS (J. R. 

 Geigy, S. A. Basel). No other Ponceaux 

 I have used, French, German or Amer- 

 ican give results comparable to that of 

 Geigy's. 



Three solutions are required: A. 

 Ponceau, BS (Geigy), 1 gm., aq. dest. 

 100 cc, glacial acetic acid 1 cc. B. Acid 

 fuchsin (National Anilin Co., New 



York), 1 gm., aq. dest. 100 cc, glacial 

 acetic acid 1 cc. 1 part of A with 2 

 parts of B. C. Aq. dest. 100 cc, glacial 

 acetic acid 1 cc. D. Aq. dest. 100 cc, 

 phosphomolybdic acid 1 gm. 



Place sections stained with the iron 

 hematoxylin in the A B mixture, 5 min. 

 Rinse them with C. Transfer them to 

 Z> in a Copeland or Borrel jar at 40°C. 

 5 min. or more. The collagen should 

 remain perfectly colorless. 



Step 3. Stain the collagen with 

 anilin blue or Fast green. To make the 

 former dissolve 2 gms. anilin blue (Na- 

 tional Anilin Co., New York) in 100 cc. 

 warmed aq. dest. Cool and add 1 cc. 

 glacial acetic acid. To make the latter 

 dissolve 1 gm. fast green (National 

 Anilin Co., New York) in 100 cc. aq. 

 dest. and add 1 cc. glacial acetic acid. 

 After the phosphomolybdic differenti- 

 ation (Step 2 jD) rinse the sections in 

 acetic water C Pour on them 8 to 10 

 drops of the anilin blue or fast green 

 solutions 2 to 3 min. Wash in acetic 

 water C 2 to 3 min. Dehydrate with 

 absolute alcohol, clear in toluol or xylol, 

 mount in balsam or permount. Total 

 length of this technique is approxi- 

 mately 60 minutes. Chromatin is 

 black, cytoplasms stain in various 

 shades of red, granulations of eosino- 

 philes and mast cells stain ruby red, 

 erythrocytes are black, elastic fibers 

 stain red, collagenic fibers and mucus 

 stain dark blue (with anilin blue) or 

 green (if Fast green is used). 



To this relatively fast technique, I 

 prefer a slow one, based on the use of 

 diluted solutions of Ponceau-fuchsin, 

 anilin blue or fast green. 



After nuclear staining with iron alum 

 hematoxylin (Step 1) wash and immerse 

 the slides in 1 part A B mixture and 9 

 parts 1% acetic acid, 30 min. Rinse in 

 acetic water C, 1 min. Transfer the 

 sections into D at 40°C. 5 min. or more. 

 Rinse in acetic water C. Stain Anilin 

 blue solution 1 part {or Fast green solu- 

 tion 1 part) with acetic water C 9 parts, 

 15 to 30 minutes. Rinse in acetic water. 

 Dehydrate with absolute alcohol, clear 

 in toluol or xylol, mount in balsam or 

 permount. 



The results are grossly the same as 

 after the rapid method, but more deli- 

 cate and precise. Moreover, the stain- 

 ing of the collagen is slow and progres- 

 sive and can be stopped at the most 

 favorable step; in many circumstances 

 an excessive staining of collagen masks 

 some fine details, for example the fine 

 prolongations of the connective tissue 

 cells. 



N.B. After staining of collagen with 

 Fast green, it is preferable to mount in 



