A Rapid Silver Staining Method for Identification of Mycobacterium Leprae in Histologic Sections

Tohoku J. exp. Med., 1986, 150, 363-364

Short Report A Rapid Silver Staining Method for Identification of Mycobacterium leprae in Histologic Sections

MASACHIKASENBA, NORIKO FUKUSHIMA* and TAKAYOSHI TODAt Department of Pathology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852, *Department of Central Laboratory, Fraternity Memorial Hospital, Tokyo 130 and 'Department of Laboratory Medicine, Ryukyus University Hospital, University of the Ryukyus School of Medicine, Okinawa 903- 01

SENBA,M., FUKUSHIMA,N. and TODA,T. A Rapid Silver Staining Method for Identification of Mycobacterium leprae in Histologic Sections. Tohoku J. exp. Med., 1986, 150 (3), 363-364 A few methods have been reported for the purpose of staining Mycobacterium leprae in paraffin sections, including Fite oil fuchsin method, auramine-rhodamine method, and Blanco-Fite silver method. Among these staining techniques, Fite oil fuchsin method and auramine-rhodamine method are popular. However, the Blanco-Fite silver method takes approximate- ly 20 days. Therefore, we developed a new procedure for rapid identification of M. leprae in paraffin sections using another silver solution and found that the procedure gave stable and satisfactory results. This new method has proved superior to others in demonstrating a reliable staining for M. leprae. Mycobacterium leprae ; silver staining method

Leprosy is a chronic infectious disease primarily of the skin, nerves and certain mucous membranes caused by Mycobacterium leprae, an acid-fast organism. In the world, there are more 10 million patients with leprosy according to the World Health Organization estimates. The highest endemicity occurs in Africa where known cases number 6 per thousand of the total population, but the estimated incidence is nearly twice this. The majority of these patients reside in Southeast Asia, India, sub-Saharan Africa, and Latin America. The cutaneous specimens of M. leprae infection at Ryukyus University Hospital were used. The materials were fixed in 10% formalin, embedded in paraffin, and cut at 4 micron. The steps involved in a new silver staining technique are as follows : 1) Deparaffinize and hydrate to distilled water. 2) Treat with 0.5% periodic acid solution for 20 min. 3) Wash in distilled water. 4) Treat with ammoniacal silver nitrate solution and keep at 60°C in a water bath for 30 min. Ammonical silver nitrate solution : To 20 ml of 10% silver nitrate solution add 0.4 g sodium hydroxide, and add dropwise 28% ammonium hydroxide, until only a few granules of the resulting precipitate remain on the bottom of the cylinder. And add distilled water to make 100 ml. Dilute 1 part ammoniacal silver nitrate solution with 4 parts distilled water for use. Store in a refrigerator and use as needed. 5) Wash in distilled water. 6) Treat with 0.5% gold chloride solution for 3 min. 7) Wash in running water. 8) Treat with 10% sodium thiosulfate solution for 5 min. 9) Wash in running

Receive d September 8, 1986; accepted for publication October 3, 1986. 363 364 M. Senba et al.

Fig 1. Lepromatous leprosy. The granulomatous infiltrate consists predomi- nantly of lepra cells with foamy cytoplasm. Numerous lepra bacilli are present. Silver stain. Oni ginal magnification x 180.

water. 10) Treat with nuclear fast red solution for 5 min. Nuclear fast red solution : Dissolve 0.1 g nuclear fast red in 100 ml of 5° solution of aluminum sulfate with aid of heat. Cool, filter, and add grain of thymol as a preservative. 11) Wash in running water. 12) Dehydrate, clear and mount. M. leprae bacilli were stained intense black against light pink background and nuclei were counterstained in red with nuclear fast red (Fig. 1). M, tuberculosis, and M. avium- intracellulare were stained blackish lighter than M. leprae. Melanin and lipofuscin granules stained black. These were distinguished easily by hematoxylin and eosin staining, since they were pigmented (brown to yellow) and M. leprae were not. Connective tissue was stained blackish. As the staining methods for M. leprae in paraffin sections, oil fuchsin method (Fite et al. 1947), auramine-rhodamine method (Truant et al. 1962), and silver method (Blanco and Fite 1948) have been reported. The author's silver procedure is more rapid than Blanco- Fite silver method. Auramine-rhodamine method could not identify all bacilli of M. leprae because the bacilli were less fluorescent than M. tuberculosis and M. avium-intracellulare. However, excellent results have been obtained in our silver method because all orgainsms of M. leprae were clearly observed. The mechanism of silver staining for M. leprae is not clear. A possible explanation may be that M. tuberculosis, M. leprae, M. ulcerans, M. chelonei, and M. kansasii contain periodic acid Schiff (PAS) positive materials (Wear et al. 1985) and lipid which are reacted with silver particles.

References 1) Blanco, F.L. & Fite, G.L. (1948) Silvering of lepra bacilli in tissues. Arch. Path., 46, 542-549. 2) Fite, G.L., Cambre, P.J. & Turner, M.H. (1947) Procedure for demonstrating lepra bacilli in paraffin sections. Arch. Path., 43, 624-625. 3) Truant, J.P., Brett, WA. & Thomas, W. (1962) Fluorescence microscopy of tuber- cle bacilli stained with auramine and rhodamine. Henry Ford Hosp. Bull., 10, 287-296. 4) Wear, D.J., Hadfield, T.L., Connor, DH., Neafie, R.C., Meyers, W.M. & Binford, C. H. (1985) Periodic acid-Schiff reaction stains Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium ulcerans, Mycobacterium chelonei (abscessus ), and Mycobacterium kansasii. Arch. Path. Lab. Med., 109, 701-702.