Wright-Gienisa 



The technic for Wright-Giemsa was suggested 

 to us by Dr. C. J. Hamre of the University of 

 North Dakota, who has found this stain satis- 

 factory for the study of myelopoiesis in birds. 

 It is a vahiable method and for any critical study 

 on myelopoiesis should be used in addition to 

 May-Griinwald Giemsa. 



1. Clean slides in concentrated HNO3, 3 hours. 



2. Wash slides in running tap water, in distilled water, 

 and in 95-percent alcohol. 



3. Make smear of blood and dry in air. 



4. Use Wright's stain on rack, 4 minutes. (Proper 

 length of time is determined by appearance of me- 

 tallic sheen. ) 



5. Add distilled water, 4^5 minutes. 



6. Wash in stream of running water and without drying 

 add Giemsa,^ 15-20 minutes. 



7. Wash in running distilled water. If precipitate is 

 present run Wright's stain over the slide. 



8. Wash off with running water. 



9. .Stand slides on edge — do not blot. 



chiefly to show nuclear structure of heterophils 

 for making Arneth counts. After the blood was 

 spread, it was held for a second or two until a 

 dull effect, which comes with the drying of the 

 slide, began to replace the high gloss of the wet 

 surface. At that instant the slide is put quickly 

 into a Coplin jar of fixative. Since the fixative 

 contains ether, it is better to use a Coplin jar 

 with a screw top. Fix, 1 to 24 hours. Wash in 

 several changes of 80-percent alcohol and run up 

 to absolute methyl alcohol. Staining with May- 

 Griinwald Giemsa is done in a Coplin jar rather 

 than on a rack and the staining times for one or 

 both dyes should be increased, sometimes as much 

 as 50 to 100 percent over that given under the 

 technic for staining the usual dry smear with 

 M. G. G. On one set of slides, May-Griinwald 

 was applied for 10 minutes and Giemsa (80 drops 

 in 40 cc. of distilled water) for 20 minutes. 

 When used this way on blood from the early 

 embryo, sharply defined chromosomes are ob- 

 tained in mitotically dividing cells. 



Petrunhevitch ISo. 2 and M. G. G. 



Petrunkevitch No. 2 is primarily a tissue fix- 

 ative. It penetrates rapidly and since it is made 

 up in alcohol it does not require washing. The 

 original formula as given by Petrunkevitch 

 (1933) has been modified slightly to adapt it for 

 use with avian tissues. 



80-percent alcohol 3 liters 



Nitric acid 90 cc. 



Cupric nitrate 60 gms. 



Paranitrophenol 150 gms. 



Mix and filter 



Ether 150 cc. 



Combine the chemicals in the order given. Store in a 

 cold place and in a well-sealed bottle. 



In handling the paranitrophenol, do not inhale 

 the dust and do not allow it to remain on the wet 

 skin. Wear rubber gloves when weighing out 

 the material and when handling the prepared fix- 

 ative; this precaution is especially pertinent if 

 there is danger that the prepared fixative may be 

 splashed on the skin. 



The paranitrophenol should be chemically 

 pure. Before using it. note whether there is an 

 indication of deterioration — brownish discolora- 

 tion of the clumps of powder. 



Petrunkevitch No. 2 fixative was used here 



" Use 15 drops of Giemsa stock to 10 cc. of distilled water. 



230 



Methyl alcohol and thionin 



The chief use in this study for the combina- 

 tion of methyl alcohol fixative and thionin stain 

 has been to hold the granules of basophils in their 

 normal size and relationships within tlie cell. 

 This requires that the film of blood or impression 

 smear be fixed before it dries. 



1. Fixation in absolute methyl alcohol, 5 minutes. 



2. Stain in thionin.^ 24—48 hours. 



3. Differentiate in 95-]iercent alcohol. 



4. Clear in 100-percent alcohol, xylene, and mount 



with cover glass. 



Reticulocyte stain 



One-percent brilliant cresyl blue was made up 

 in 25 cc. of 0.85-percent salt solution or in avian 

 Ringer's solution— NaCl, 8.5 gms., KCl, 0.42 

 gms., CaClj, 0.25 gms.; and water, 1,000 cc. 

 The solution should be kept cold and should be fil- 

 tered before using. Various methods were tried, 

 including (1) mixing a drop of blood and a drop 

 of stain on the slide for periods varying from 1 to 

 5 minutes and (2) drying a film of dye made 

 with 0.3-percent solution of dye in 95-percent 

 alcohol and making tlie blood smear over this. 

 In the second method, the films of blood dried 



' Thionin. saturated solution in 50-percent alcohol, 



