322 



METHODS AND FORMULAS 



DS 12.16-DS 12.20 



12.16 Pearson 1941 Tech. Bull., 3:16 



preparation: To 100 0.5% eosin Y add, drop by drop, 4 hydrochloric acid. Wash ppt. 

 by decantation. dry, and dissolve in 40 95% ale. 



12.16 Smith 1926 see DS 21.14 Smith 1926 



12.16 Tonkoff 1900 test. 1907 Bohm and Oppel Bohm and Oppel 1907, 120 



formula: abs. ale. 100, spirit blue 0.1, ADS 12.1 Gram 1884 0.03 



12.16 Unna 1895 orange G-tannin 14352,21:540 



formula: water 100, tannin 33, orange G 2 

 use: see DS 13.22 Langeron 1942b and DS 22.2 Volkonsky 1928. 



12.2 Double Contrasts from 

 One Solution 



Double contrasts from one solution are 

 just as easy to use as are single contrasts. 

 In those cases in which there is no histo- 

 logical or cytological difference between 

 the elements which it is desired to stain, 

 they present no improvement over the 

 more conventional techniques, but unless 

 one is dealing with homogenous organs, or 

 with a very young embryo, there seems to 

 be no possible reason why the double-con- 

 trast material should not be employed. 

 The division of these stains into two 

 groups is based entirely on the color of the 

 nucleus which they are designed to set off. 

 The first group, contrasting with red 

 nuclei, can therefore be used either after 

 magenta, carmine, or safranin, the picro- 

 contrasts being by convention more com- 

 monly employed after carmine than after 

 the other reagents. The picro-indigo- 

 carmine of Cajal 1895 is the oldest and 

 best known of these mixtures, but it is 

 probably less effective than the formula of 

 Masson used for the same purpose. The 

 formula of Grosso 1914 was originally 

 developed for staining blood, but it is of 

 much wider application. The picro-spirit 

 blue of Smith 1912, if used after magenta 

 as a nuclear stain, is, in the opinion of the 

 present writer, undoubtedly the best tech- 

 nique which has yet been developed for 

 staining sections of heavily yoked mate- 

 rial. The non-picric formulas, which are 

 included in the next division of con- 

 trasts for red nuclei, contain two of the 

 best counterstains (Chatton 1920 and 

 Kostowiecki 1932) which have ever been 

 developed. The formula of Kostowiecki 

 in particular has all the advantages of 

 the better known Mallory-Masson poly- 

 chrome stains, but may be applied from a 



single solution. Roux's blue here given in 

 Roux's formula of 1894 was at one time 

 well known but has now fallen into disuse. 

 Contrasts for blue nuclei present more 

 difficulties than do those for red since 

 nuclei for these techniques are custom- 

 arily stained by hematoxyhn, which is 

 very sensitive to acid. Though many of 

 the picro-contrasts can be employed with 

 hematoxylin, it is strongly recommended 

 that one of the oxazine nuclear stains 

 given above under DS 11.42 be employed 

 in its place. The best known of these 

 picro-contrasts is van Gieson 1896. The 

 non-picric contrast formulas, given under 

 DS 12.222 below, may be safely employed 

 after either hematoxylin or methylene 

 blue stains. Probably the best known, and 

 certainly one of the easiest to use, is the 

 saffron-erythrosin of Masson 1911. 



12.20 TYPICAL EXAMPLES 



Preparation of a transverse section of 

 a Squalus embryo using the picro- 

 carmine stain of Ranvier 1899 fol- 

 lowed by the picro-indigo-carmine of 

 Cajal 1905 



This simple little preparation is very 

 old-fasliioned, but it may well be placed 

 in the hands of a beginning student as his 

 introduction to the art of section cutting 

 and staining. Moreover, it utiUzes the 

 embryos of Squalus acanthus, which are 

 frequently found in elementary labora- 

 tories where this form is dissected. There 

 is often grave doubt as to what shall be 

 done with such embryos and they are usu- 

 ally lost. It is presumed, of course, that we 

 are dealing with an inland laboratory in 

 which preserved specimens are brought 

 from a biological supply house, so that 

 the small embryos themselves will already 



