Demonstration of 72-hour Chick Sections 243 



down to 90 per cent alcohol through absolute alcohol in the usual 

 manner. 



Ehrlich's acid alum hematoxylin has been selected for this typical 

 example because it is one of the best, although at the same time one of 

 the most frequently misused, of the hematoxylin stains. The method given 

 for its preparation should be rigorously followed; that is, the hematoxylin 

 should be dissolved in a mixture of acetic acid and absolute alcohol, and 

 then the glycerin, water, and ammonium alum should be added to the 

 bottle, which should be shaken vigorously and allowed to ripen with the 

 stopper loose for some months. "Artificially" ripened hematoxylin 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 its "fruity" smell and dark color, it 

 remains in a fit condition to use for many years. One of the most fre- 

 quently omitted precautions is the maintenance of the concentration of 

 the ammonium alum by the addition of about 100 g per liter to the 

 bottle 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 

 given. 



All the slides, gathered together in a glass tray (each slide may be 

 treated individually), are taken from the 96 per cent alcohol and placed 

 in full-strength Ehrlich's hematoxylin 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 overstained. In the absence of ex- 

 perience, it is recommended that a period of 1 min be used and that then 

 they be examined under a lower power of the microscope, at which time 

 the nuclei should appear rather densely stained, the background being 

 only lightly stained. Each slide is removed individually from the tray, 

 wiped on the underside with a clean cloth, and then "differentiated" by 

 dropping 96 per cent alcohol (never acid alcohol) on it with a drop 

 bottle or a pipette. It will be observed at once that the drops of the 

 very thick hematoxylin solution are rolled back from the section as the 

 96 per cent alcohol drops on it and that, after a short time, the nuclei 

 become more distinct and the background less distinct. The exact point at 

 which differentiation should cease is at the will of the operator, but it is 

 better, in general, since the sections are not to be counterstained, to dis- 

 continue differentiation when the nuclei are clearly defined against the 

 background. Each slide is transferred directly to a saturated solution of 

 lithium chloride in 70 per cent alcohol, where it passes from a pinkish 

 color to a deep blue. If the conventional method of differentiating these 

 stains with acid alcohol is followed, it results in a hopelessly diffuse stain. 

 The purpose of the 96 per cent alcohol is to utilize the surface tension of 



