228 Hematologic Elements and Related Issues (chap. 16) 



3. Wash in running water: 5-10 minutes. 



4. Wash in distilled water: 5 or more minutes. 



5. Stain in eosin-azure overnight. 



6. Differentiate in 95% alcohol: may require 2-3 hours. In stubborn 

 cases of differentiation (old solution) a brief immersion in colo- 

 phonium alcohol (page 225) may help to sharpen the colors, but 

 not as well as a new solution. The over-all appearance of the slide 

 should be blue with a slightly greenish tinge, but differentiation 

 must be done under the microscope. 



7. Dehydrate, absolute alcohol; clear and mount. 



results: 



nuclei — -dark purple blue 

 erythrocytes — light pink 

 eosinophilic granules — brilliant red 

 cytoplasm — pale blue 



comments: 



1. After 2 hours fixation, cells in center are not as well fixed as 

 those at periphery, but fixation longer than 2 hours produces 

 granular cytoplasm in eosinophilic erythrocytes and erythroblasts. 



2. Block embeds in celloidin and mounts with clove oil, page 77 

 and stains with a weak solution of Delaefield's hematoxylin, 2 

 drops/ 100 ml. distilled water, overnight, or in a mixture of 10-15 

 drops/ 100 ml. for approximately 5 minutes, or till nuclei are faint 

 blue; check under microscope. The author has had good results 

 with the above-scheduled method (step 2), but take care not to 

 stain too long, never over 60 seconds. 



3. Bone marrow when collected should have an anticoagulant added 

 to it. Gardner [1958) and many other workers mix the bone mar- 

 row in a tube containing 0.5 mg. heparin powder. Kniseley^ wets 

 his syringe with d-potassium EDTA as an anticoagulant. Smears 

 can be made with some of the aspirated (drawn out by suction) 

 marrow. The remaining material is poured into a small funnel 

 lined with 'Tiltrator Coffee Paper" {Zbar and Winter, 1959). 

 Rinse marrow, while in funnel, several times with saline, or until 

 the marrow has lost most of its color. This also helps to wash the 

 material into one mass at the apex of the funnel. Partially clotted 

 marro^v will be broken up and washed free of blood, leaving excel- 

 lent clear particles of marrow. Fold in the fiher paper, place in 



^ Ralph M. Kniseley, Chief of Chnical Research and Training, Oak Ridge Institute of 

 Nuclear Studies. Personal connnunication. 



