DEMONSTRATION OF 72-HOUR CHICK SECTIONS 133 



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 that this stain should not be prepared in half-gallon 

 lots at routine intervals, so that a sufficiently ripened solution is always avail- 

 able. 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 frequently omitted precautions is the maintenance of the concen- 

 tration of the ammonium alum by the addition of about 100 Gm. 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 the 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 experience, it is recom- 

 mended that a period of 1 minute be used and that then they be examined 

 under a lower power of the microscope, at which time the nuclei should appear 

 quite 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 dif- 

 ferentiation should cease is at the will of 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 slide is trans- 

 ferred 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 hope- 

 lessly diffuse stain. The purpose of the 96 per cent alcohol is to utilize the 

 surface tension of the stain to hold it in the nuclei, and, ii the slide is placed 

 in acid-70 per cent alcohol, it will be found that the stain diffuses out from 

 the nuclei which, instead of appearing clear and sharp, appear, as it were, blurred 

 around the edges as in an out-of-focus photograph. Differentiation by rolling 

 back the stain with 96 per cent alcohol gives a clear, sharp stain which is as 

 well differentiated as any of the iron alum mordant stains, but which has the 

 advantages of giving a greater transparency and also staining the background 

 sufficiently to render it apparent for class demonstration purposes. 



