Improved Technique for the Detection of Acid-fast Bacilliby Fluorescence.

Hagemann (Muench. Med. Wochschr. 85:1066, 1938) discovered that tubercle bacilli have selective affinity for phenol-auramine stain. Since then, several investigators (Richards, Kline, and Leach, Am. Rev. Tuberc. 4:255, 1941; Bogen, Am. Rev. Tuberc. 4:267, 1941; Lempert, Lancet 2:818, 1944; Gray, Am. Rev. Tuberc. 68:82, 1953; Kuper and May, J. Pathol. Bacteriol. 79:59, 1960) have advocated its use in fluorescence microscopy for the demonstration of acidfast bacilli. However, it has had little acceptance as a clinical laboratory procedure. This may be accounted for by: (i) the danger of false-positive findings due to fluorescing artifacts often present in smear preparations, and (ii) primary and secondary fluorescence of tissue components. This report describes a modified method based on the fact that ferric chloride blocks or reduces most of the interference fluorescences (Clark and Hench, Am. J. Clin. Pathol. 37:237, 1962). Two solutions were required: 10% aqueous ferric chloride and phenol-auramine. The stain was made of 3 g of auramine 0 dye, 40 ml of phenol, and 60 ml of glycerol in 900 ml of distilled water. It was used for staining mycobacteria in body fluids, sputum concentrates, tissue smears, fixed tissues, and culture smears. Smears were fixed in dry heat at 82 to 86 C for 8 to 10 min. Deparaffined tissue sections were brought down to water. Smears and tissue sections were stained for 10 min in phenol-auramine at room temperature and washed well in tap water; ferric chloride