274 



METHODS AND FORMULAS 



DS 11.10 



come covered with a brownish deposit 

 which must be scraped off with a knife 

 before the solution is prepared for stain- 

 ing. If the brown powder on the outside 

 of the crystal forms a layer of any thick- 

 ness, it is better to reject the whole and 

 secure a fresh supply of the reagent. He- 

 matoxyhn itself has little staining effect, 

 the color being produced by the formation 

 of lakes with hematin, an oxidation prod- 

 uct of hematoxylin. It was customary in 

 former times to prepare considerable 

 quantities of solution, which was kept 

 with the stopper loose in the bottle for a 

 period of at least one month before use. 

 For the purpose of Heidenhain's tech- 

 nique, however, it is far more important 

 that a small quantity of the ferric alum 

 be carried over into the hematoxylin solu- 

 tion than that the latter should be aged. 

 The staining will be both simpler and 

 more effective if a few drops of hematox- 

 ylin be placed in the iron alum solution 

 and a few drops of the iron alum solution 

 be placed in the hematoxyhn. Both solu- 

 tions should, of course, be filtered immedi- 

 ately before use if the finest shdes are re- 

 quired for the reason that chromosome 

 figures in a rat can be obscured by even a 

 very small particle of dust. 



The slides are now taken from distilled 

 water and placed in the mordant solution. 

 It matters very little how long they remain 

 in this solution, although the usual direc- 

 tions call for overnight. The ideal time 

 varies with every type of tissue studied 

 and is greatly dependent on temperature. 

 If the solutions be heated to 50°C., with 

 the understanding that this will cause a 

 swelhng of the section and a general ob- 

 scuring of the finer details, the period may 

 be shortened to as httle as 10 minutes. 

 But the finest stains are those secured by 

 leaving the sections in the mordant solu- 

 tions at room temperatures. On removal 

 from the mordant solution the sections 

 should be rinsed very briefly in distilled 

 water. The purpose of the rinse is te re- 

 move the surplus mordant from the sur- 

 face of the slide without extracting it from 

 the tissues. The shdes are then placed in 

 the hematoxylin solution, in which they 

 should remain for approximately the same 

 length of time as they have been in the 



mordant. It is not of importance how long 

 this be though from three to 24 hours is 

 customary period. Sections may be re- 

 moved from time to time from the staining 

 solution and examined with the naked eye. 

 A successful preparation shows the whole 

 section to have become completely black- 

 ened, although a slight bluish tinge in the 

 black is permissible. If the sections have 

 not become completely blackened in 24 

 hours, it is only necessary to replace 

 them, after a brief rinse, in the mordant 

 solution and leave them there, say a fur- 

 ther period of 24 hours, before returning 

 them to the stain. 



If, however, the sections are sufficiently 

 blackened on removal from the staining 

 solution, it remains only to differentiate 

 them, that is, to extract the color from 

 all portions of the sections except the 

 chromosomes. This is customarily done 

 with the same solution in which they 

 were mordanted, though, of course, a 

 fresh solution or a stronger solution may 

 be employed if desired. Differentiation 

 at the commencement of the process goes 

 relatively slowly so that all of the slides, 

 which are presumable being carried in 

 a glass rack, may be removed and 

 placed in the ferric alum solution. The 

 actual time in which differentiation takes 

 place cannot be forecast because it de- 

 pends on a large number of uncontrollable 

 factors. But it is never less than five 

 minutes nor very often more than a few 

 hours. Sections should therefore be with- 

 drawn from the ferric alum every four or 

 five minutes and examined briefly under 

 the low-power of a microscope. It is a mat- 

 ter of great convenience in controUing dif- 

 ferentiation of chromosomes in this type 

 of preparation if an ordinary student mi- 

 croscope can be fitted with a glass plate 

 over the stage so that a slide wet with 

 ferric alum can be placed, without fear of 

 damage to the instrument, on the surface 

 of the stage for examination. It is not un- 

 common for a beginner to place the slide 

 upside down on the surface of the stage, 

 with the subsequent loss of all the sections. 

 This can readily be avoided if the worker 

 will make it a matter of routine, as he lifts 

 the slide from the mordant, to hold it at 

 an angle between himself and a light 



