Azo bilirubins and azohematoidins resist alcohol dehydration and mounting in resinous media after adequate coupling reactions. The azo colors are resistant to hydrochloric acid extraction (0.1 N 30 min routinely) but are bleached at varying rates according to the specific dye by sodium dithionite solution. Fresh dithionite is required. The azo coupling reaction of bile casts is prevented by ethanol acetic anhydride acetylation, restored by alcoholic KOH saponification and prevented completely by oxidation with 0.2% CrO3 (4 hr) or by a diammine silver, iodine, thiosulfate sequence. These oxidations do not alter the alkaline azo coupling reactions of tissue protein. Azo coupling of bile casts is unaltered by a 24-hr 10% iodine-methanol, 2-min thiosulfate sequence or by a 24-hr 0.1 M bisulfite reduction. The reactive bilirubin is resistant to hot methanol chloroform primary fixation as well as to exhaustive extraction by this solvent after formol fixation, although both of these procedures extract readily visible amounts of yellow material into the solvent. It appears that the azo-reactive material must be protein bound. Alkaline azo coupling with diazosafranin is weakened by lesser amounts and completely prevented by addition of equimolar or greater amounts of uric acid to the diazo.
5-((8-Hydroxyquinolin-5-yl)diazenyl)-3-methyl-1H-pyrazole-4-carboxylic Acid