THE pH RANGE OF THE DIAZOSAFRANIN REACTION OF RAT AND OTHER MAST CELLS

R. D. LILLIE ¹, PHILIP PIZZOLATO ¹, LINDA L. VACCA ¹, ROY A. CATALANO ¹, and PATRICIA T. DONALDSON ¹

¹ Departments of Pathology, Louisiana State University Medical Center and U.S. Veterans Administration Hospital, New Orleans, Louisiana 70112

5-Hydroxytryptophan and 5-hydroxytryptamine (5-HT) give strong red azo coupling colors with fresh p-nitrodiazobenzene (fast red GG) at coupling pH levels of 3-9. Tyrosine, histidine, histamine, adrenaline, noradrenaline, dopa, dopamine and tryptophan give strong red colors at pH 7-9, weakening to orange and yellow at pH 5-6 and negative below. Tryptophan gives weak orange-yellow to about pH 4. Rat mast cells color deep red with diazosafranin at pH 3-8. Protein colors pink to red at pH 7-8, weaker at 5-6, and remains almost uncolored below that. Preoxidation with 10 min 1% H₅IO₆, 2 hr 0.1 M FeCl₃, 3 hr 5% K₂Cr₂O₇ or 1 hr 3°C 10% I₂/CH₃OH does not prevent the diazosafranin reaction of rat mast cells. Periodic acid does not inhibit in vitro azo coupling of 5-HT or 1-naphthol, that of noradrenaline is prevented, and can be restored by reduction with Na₂S₂O₅ or Na₂S₂O₃. Azo coupling of enterochromaffin and adrenal medulla is prevented by these oxidations and restored by Na₂S₂O₄ reduction. Diazosafranin staining of mast cells is not extracted by 24 hr 0.24 N HCl/70% alcohol. Safranin and other cationic dye staining of rat mast cells resists aqueous 0.1 N HCl some hours, largely disappearing at 24 hr, and is removed by 5-15 min in 0.12 N HCl/7O% alcohol. At pH 1 0.1% toluidine blue colors rat mast cells deep violet; when superimposed after acid diazosafranin the red mast cells assume a deep purple, intermediate color. Since in extended use of the method pH 3 diazosafranin colors only bilirubin casts, hematoidin and dog, rat, mouse and gerbil mast cells, and not monkey, most human or lead and mercury fixed guinea pig mast cells, it is suggested that the method is showing 5-HT in rat mast cells. The occasional reaction of human mast cells may be due to pathologic presence of that substance in these cells.

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