62 MICROSCOPIC TECHNIQUES 



HEMOGLOBIN 



Of the many tests for hemoglobin in tissue, smears and blood cells 

 the more recent procedures of Ralph ( 1941) , Goulliart ( 1939, 1941) , 

 and Dunn ( 1946) will be given. Previously Dunn and Thompson 



(1945) had modified the Van Gieson stain, and later these authors 



( 1946) adapted the patent blue method of Lison ( 1938) for the 

 staining of hemoglobin. The cyanol method of Dunn given below is 

 a simplification of the technique of Fautrez and Lambert ( 1937) . 



Ralph Method for Hemoglobin 



SPECIAL REAGENTS 



Benzidine Reagent. 1% benzidine in absolute methanol. 

 Peroxide Reagent. 25% Superoxol in 70% ethanol. 



PROCEDURE 



1. Flood the dried blood or tissue smear on a glass slide with the 

 benzidine reagent for 1 min. 



2. Drain off and flood the smear with the peroxide reagent for 1.5 

 min. 



3. Wash in distilled water for 15 sec. 



4. Dry and mount in neutral balsam. 



Result. Hemoglobin will appear dark brown. 



Goulliart Method for Hemoglobin 



SPECIAL REAGENTS 



Glacial Acetic Acid Containing a Few Crystals of Potassium Iodide. 

 Do not use after a week. 



PROCEDURE 



1. Treat a dried smear or frozen section on a slide with a drop of 

 reagent. 



2. Examine after 30 min. with a polarizing microscope for groups 

 of very small boat-shaped birefringent crystals of protoiodoheme. 

 These crystals slowly change into square tabular Teichmann crys- 

 tals. The reaction may be speeded by warming. 



