types was also effected. It was sometimes pos- 

 sible to take a solution that produced too much 

 red and too little blue coloration of the cells and 

 follow this with a stain that was equally out of 

 balance in the opposite direction and so procure 

 a reasonably satisfactory slide. Much the same 

 result could be obtained by using a mixture of the 

 two solutions. 



Wright's stain was obtained from various com- 

 mercial sources. One product was found that, 

 as a single staining solution, gave satisfactory 

 results. Anyone seeking a Wright's stain for 

 use on avian blood should test out samples from 

 different sources in the hope that one of them will 

 be properly balanced. Buffers of various sorts 

 were tried with samples of Wright's stain. These 

 manipulations still gave results that were too 

 red or too blue. 



The slides are spread on a staining rack over 

 a sink or tray and flooded with stock Wright's 

 stain. Use enough stain. If the stain is dry 5 

 minutes after it is applied, too little was used. 

 Usually after this interval of time the surface of 

 the solution has a metallic sheen and an equal 

 quantity of distilled water is added. The slides 

 are allowed to stand for another 5 minutes. 

 There is wide variation in the periods of time 

 that can be allowed for the concentrated stain, 

 and the water that follows it, to stand on the slide. 

 Different standing periods should be tried before 

 concluding that the stain is unsatisfactory. The 

 following are some of the periods that have been 

 used in this study for the first and second solu- 

 tions: 5 minutes and 2.5 minutes, 5 minutes and 

 5 minutes, and up to 9 minutes and 9 minutes. 

 It was found that as the stock solution of stain 

 aged, longer periods were necessary. 



After the staining time has been completed, 

 the slides are drained and blotted. Before the 

 coverglass is put on, they are again warmed under 

 the infrared lamp and the coverglass is heated 

 over an alcohol lamp. 



Many investigators do not cover blood smears 

 but it has been a procedure followed in these 

 studies on blood, and it has seemed that doing so 

 makes the colors slightly more brilliant and the 

 small details a little sharper. 



Bulk staining with Wright's stain 



When many slides are to be done at one time, 

 the job is usually speeded up by using bulk stain- 



ing methods. Stainless steel racks that hold 25 

 to 100 slides are used, and 2 stainless steel trays 

 with covers, or glass dishes with covers, are ar- 

 ranged conveniently. Wright's stain is poured 

 into one tray or dish and distilled water into the 

 other. The slides are left in the stain 5 to 15 

 minutes and then dipped slowly 2 or 3 times in 

 the water. The rack is shaken to remove the 

 excess water and then placed on a towel in a 

 warm place (such as on a radiator or in an in- 

 cubator) to dry. Small drops of water that ad- 

 here to the surface of the smear and afterward 

 dry slowly often produce faded round spots. 

 These spots usually do not interfere with the 

 study of the smear. 



The bulk staining method causes greater dam- 

 age to water-soluble components of cells than 

 does the rack method. Damage is less if the sec- 

 ond solution is stain and water, equal parts. 



May-Griinivald Gienisa 



Although May-Griinwald Giemsa (M. G. G.) 

 is a more vigorous stain than Wright's, the colora- 

 tion that it gives to the cytosome and nucleus is 

 much like that given by Wright's. Wright's 

 stain is preferred for circulating blood of the 

 hatched chicken. The azurophilic substances 

 in the monocyte are differentiated better with it 

 than with M. G. G., and the heterophil rods are 

 preserved better. But if the blood is leukemic 

 or for some other reason contains numerous im- 

 mature cells, use M. G. G. or (if two slides are 

 available) both stains. Because M. G. G. 

 stains intensely, it is recommended for all im- 

 pression smears from hematopoietic organs, both 

 adult and embryonic, as well as for the circulat- 

 ing blood of the embryo. Both May-Griinwald 

 and Giemsa were purchased as prepared solu- 

 tions and have been entirely satisfactory. The 

 following procedure was used: 



1. Dry slides under the infrared lamp and place on a 

 staining rack. 



2. Cover with May-Griinwald solution, 5 minutes. 



3. Dilute with equal quantity of distilled water and 

 mix 1-2 minutes. 



4. Pour off and without rinsing add diluted Giemsa," 

 15-20 minutes. 



5. Methyl alcohol. 1-3 dips. 



6. Blot immediately. 



7. Put on coverglass. 



' Add 40 drops of stock Giemsa solution to 40 cc. of distilled 

 water. If the color is weak, 80 drops to 40 cc. may be used. 



229 



