IRON-HEMATOXYLIN-STAINED TESTIS 123 



to hold it at an angle between himself and a light source so that the light is 

 reflected from the surface. If the sections are, as they should be, on the upper 

 surface of the slide when it is so placed, they will appear to be double through 

 the reflection from the undersurface as well as the uppersurface of the glass. 

 A good rule is never to place a slide which has just been taken from a fluid on 

 the stage of the microscope until one has seen the double reflection. 



If a low-power examination of the section shows the nuclei to be standing 

 out clearly, the entire tray should be removed to distilled water because from 

 this time on differentiation is very rapid, and each slide must be controlled 

 separately. If, however, the nuclei are not sharply defined and a considerable 

 degree of black or bluish color remains in the background, then the entire tray 

 may be left in the iron alum for as long as is necessary. When this preliminary 

 differentiation down to the distinction of the nuclei under low-power exam- 

 ination has been completed, it is necessary to continue differentiation while 

 examining the slides at frequent intervals under a very high power of the 

 microscope. It is convenient to have available a water-immersion objective. 

 It is obviously impossible to place immersion oil on a wet slide, while short 

 working distance of a high-dry objective renders it particularly liable to cloud 

 up from the evaporation and recondensation of the water. Water-immersion 

 objectives are usually of 3 mm. equivalent focus, and this gives a sufficiently 

 wide field to permit differentiation to be observed, while at the same time it 

 has sufficiently high magnification for satisfactory control. Each slide should 

 be taken separately, returned to the 2.5 per cent iron alum for a few minutes, 

 and then reexamined. The various phases of mitosis and meiosis do not retain 

 the stain to the same degree, and care must be taken that the color is not 

 washed completely out of the other chromosomes as a result of examination 

 only of metaphase figures in which the color is retained longer than in any 

 other. A great deal of practice is required to gauge accurately the exact mo- 

 ment at which to cease differentiation, which may be stopped almost instantly 

 by placing the slides in a slightly alkaline solution. In Europe most tap waters 

 are sufficiently alkaline for the purpose and are generally specified, but in the 

 cities of the United States it is often best to add a very small quantity of lith- 

 ium carbonate or sodium bicarbonate to the water used to stop differentiation. 

 Slides may be left in this for any reasonable period of time; the process is 

 complete when the slide turns from a brown to a blue color. 



Then the slides are rinsed in distilled water, graded up through the various 

 percentages of alcohols, dehydrated, cleared, and mounted in balsam in the 

 usual manner. A section on a slide, which is required for examination over a 

 long period of time, should be some distance from the edge of the coverslip, 



