Levaditi Method

LEVADITI METHOD (Levaditi) consists in the impregnation of tissues with silver for the purpose of detecting spirochetes in them, particularly the spirochete of syphilis. Fixation for 1 or several days in a 10% solution of commercial formalin, with the pieces of tissue to be examined not being thicker than 1-2 mm*. Transfer for 24 hours to 90% alcohol and then to distilled water, until the pieces sink. After this, transfer to an aqueous 1.5-3% solution of silver nitrate and keep in it for 3-6 days at 37°. After rapid rinsing in distilled water, place for 24-48 hours in a reducing solution: pyrogallic acid-2.0-4.0, 40% formalin-5 cm* and distilled water-100 cm³. Washing in water. Embedding in paraffin. The silvering and reduction procedures are carried out in the dark (dark glass bottles). Sections should be made as thin as possible; for examination, the most suitable are usually not the most superficial layers of the piece (abundant silver deposits), but the deeper ones; sections are stored in the dark. If the pieces were not fixed in formalin, it is advisable to pre-treat them with the latter for several days. Spirochetes treated by the L. method appear black, tissues-brownish-yellow. When applying the L. method to stain spirochetes in the central nervous system, it is necessary to keep in mind the possibility of silver impregnation of the finest nerve fibers, which may lead to confusion with spirochetes. According to Nakano, the L. method can be reduced to the following stages. 1. Fixation in formalin for 10-20 minutes at 37°. 2. Hardening for 3-5 hours in 95% alcohol. 3. Washing in running water for 10 minutes and then in distilled water for 10-15 minutes. 4. Treatment with a 1.5% solution of silver nitrate for 4-5 hours at 50°. 5. Reduction in the above-mentioned pyrogallic acid solution for 4-10 hours at 50°. 6. Rinsing in water and embedding in paraffin. This original L. method is the best method for detecting spirochetes in tissues. There is also a L. method with pyridine, as well as various modifications of the L. method, but they give less reliable results. (See also Syphilis.)