DS 11.10 



FORMULAS AND TECHNIQUES 



275 



source so that the hght is reflected from 

 the surface. If the sections are, as they 

 should be, on the upper surface of the shde 

 when it is placed on the stage, they will ap- 

 pear to be double through the reflection 

 from the luider surface as well as the upper 

 surface of the glass. A good rule is never 

 to place an unmounted slide on the stage 

 of the microscope until the double reflec- 

 tion has been seen. 



If a low-power examination of the sec- 

 tion shows the nuclei to be standing out 

 clearh', the entire tray should be removed 

 to distilled water, because from this time 

 on dififerentiation is very rapid and each 

 slide must be controlled separately. If, 

 however, the nuclei are not sharply de- 

 fined 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, has been completed, it is necessary 

 to continue differentiation while examin- 

 ing the slides at frequent intervals under a 

 very high power of the microscope. It is a 

 matter of convenience if a water immer- 

 sion objective is available. It is obviously 

 impossible to place immersion oil on a wet 

 slide, while the short working distance 

 of a high-dry objective renders it particu- 

 larly liable to cloud from the evaporation 

 and recondensation of the water. Water 

 immersion objectives are usually of three 

 millimeter equivalent focus. This provides 

 a sufficiently wide field to permit differ- 

 entiation to be observed, while at the same 

 time it has a magnification sufficiently 

 high for satisfactory control. Each slide 

 should now be taken separately and re- 

 turned to the ferric 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 com- 

 pletely out of the other chromosomes 

 by examining only metaphase figures in 

 which the color is retained longer than in 

 any other. Considerable practice is re- 

 quired to gauge accurately the exact 

 moment at which to cease the differentia- 

 tion, which may be stopped almost in- 

 stantly by placing the sUdes in a sUghtly 



alkaUno solution. In ]']urope most tap 

 waters are sufficiently alkafine for this 

 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 which is used to stop differentia- 

 tion. Slides may be left in water for any 

 reasonable period of time; the process is 

 complete when the slide turns from a 

 brown to a blue color. 



The sHdes are then rinsed in distilled 

 water, upgraded through the various per- 

 centages of alcohols, dehydrated, cleared, 

 and mounted in balsam in the usual man- 

 ner. SUdes which have on them sections 

 required for examination over a long pe- 

 riod of time should have the sections some 

 distance from the edge of the coversfip 

 because, as the balsam oxidizes inward 

 from the edge, it tends to remove the color 

 of the stain from the chromosomes, leav- 

 ing them a rather unpleasant shade of 

 brown. If this happens to a valuable slide, 

 however, the matter can be remedied by 

 the utilization of a green fight which will 

 make the chromosomes again appear 

 black. 



Preparation of a wholemount 



of a 48-hour chicken embryo, 



using the alum hematoxylin 



stain of Carazzi 1911 



Fertile eggs are relatively easy to secure 

 and should be incubated at a temperature 

 of 103°F. for the required period of time. 

 The term 4S-hour chick is relatively mean- 

 ingless, because the exact stage of devel- 

 opment which will have been reached after 

 two days in the incubator depends not 

 only on the temperature of the latter, but 

 also on the temperature at which the egg 

 was stored prior to its incubation, and 

 even on the age of the hen. It is therefore 

 desirable, if any very specific age of devel- 

 opment be required, to start a series of 

 eggs in the incubator at three- or four-hour 

 intervals and then to fix and mount them 

 at the same time. 



For the removal of the embryos from 

 the egg there are required first a number of 

 fingerbowls, or any kind of circular glass 

 dishes of five to six inches diameter and 



