280 



METHODS AND FORMULAS 



DS ll.lO-DS 11.11 



are entirely dry before they are dewaxed in 

 xylene and brought down to 90% alcohol 

 through absolute alcohol in the usual 

 manner. 



Ehrlich's acid alum hematoxylin (DS 

 11.123 Ehrhch 1896) has been selected for 

 this typical example because it is one of 

 the best, though at the same time one of 

 the most frequently misused, of the hema- 

 toxylin stains. The method given for its 

 preparation should be rigorously followed, 

 that is, the hematoxylin should be dis- 

 solved in a mixture of acetic acid and 

 absolute alcohol, and the glycerine, water, 

 and ammonium alum should be added to 

 the bottle. The bottle should then be 

 shaken vigorously and the mixture al- 

 lowed to ripen with the bottle stopper 

 loose for some months. "Artificially" rip- 

 ened hematoxyhn does not give as good a 

 preparation, but there is no reason why 

 this stain should not be prepared in half- 

 gallon lots at routine intervals so that a 

 sufficiently ripened solution is always 

 available. When it has once been ripened, 

 which can be told both by the "fruity" 

 smell and by its dark color, it remains in a 

 fit condition to use for many years. One of 

 the most frequently omitted precautions 

 is that of maintaining the concentration 

 of the ammonium alum by adding about 

 100 grams per liter to the bottle in which 

 the hematoxylin is kept after it has been 

 sufficiently ripened. This stain should 

 never be diluted but should always be used 

 full strength by the method now to be 

 described. 



Each slide, or all the shdes together in a 

 glass tray, are taken from the 95 % alcohol 

 and placed in full-strength Ehrhch's hema- 

 toxyhn solution for a few minutes. The 

 exact time is not important, but they 

 should be examined at intervals to make 

 sure that they are not becoming over- 

 stained. If the technician is inexperienced, 

 it is recommended that a period of one 

 minute be used, and that they should then 

 be examined under a low power of the 



microscope. The nuclei should appear 

 quite densely stained, the background be- 

 ing only hghtly stained. Each shde is then 

 removed individually from the tray, wiped 

 on the underside with a clean cloth, and 

 then differentiated with 95% alcohol 

 (never with acid alcohol) dropped onto it 

 from a drop bottle or from a pipet. It will 

 be observed at once that the drops of the 

 viscous hematoxylin solution are rolled 

 back from the section by the 95% alcohol, 

 and that after this has continued for a 

 short time the nuclei become more distinct 

 and the background less distinct. The ex- 

 act point at which differentiation should 

 cease is determined by the operator, but it 

 is better, in general, since the sections are 

 not to be counterstained, to discontinue 

 differentiation when the nuclei are clearly 

 defined against the background. Each sec- 

 tion is then transferred directly to a 

 saturated solution of lithium chloride in 

 70% alcohol, in which it turns from pink 

 to blue. If the conventional method of 

 differentiating these stains with acid alco- 

 hol is followed, it results in a hopelessly 

 diffuse stain. The purpose of the 95% alco- 

 hol is to utilize the surface tension of the 

 stain to hold it in the nuclei. If the slide 

 is placed in acid 70% alcohol, it will be 

 found that the stain diffuses out from the 

 nuclei which, instead of appearing clear and 

 sharp, appear blurred around the edges 

 as does an out-of-focus photograph. 

 Differentiation by rolhng back the stain 

 with 95% alcohol gives a clear, sharp 

 stain which is as well differentiated as 

 any of the ferric alum mordant stains, but 

 which has the advantage of giving a 

 greater transparency and also of staining 

 the background sufficiently to render it 

 apparent for class demonstration purposes. 

 The slides may remain in the saturated 

 solution of lithium chloride in 70 % alcohol 

 for as long as is required. They are subse- 

 quently passed directly through the higher 

 alcohols to xylene and mounted in balsam 

 or some synthetic substitute. 



11.11. MORDANT HEMATOXYLIN STAINING 



Mordant staining with hematoxylin results, in general, in black nuclei, heavily and densely 

 stained, from which the stain is with difficulty removed by subsequent treatment. It is 

 therefore to be recommended in those cases in which it is desired to follow with a complex 

 counterstaining, particularly those which involve the use of an acid rinse. The method most 



