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Letters to the editor: Chelated bismuth as a stain in electron microscopy.

Salts of heavy metals have been employed widely in biologic electron microscopy to enhance specimen contrast, although specificity and binding mechanism in many instances remain obscure (see reference 1 1 for a review). Among the most commonly used are lead (7) and uranium (10) salts applied either to thin sections, or, as in the case of uranium, to tissue blocks (4). Bismuth was employed by Albersheim and Killias (3) as an en bloc stain for nucleic acids; these investigators were unsuccessful in staining Epon thin sections. Bismuth iodide was found useful as an impregnating agent following glutaraldehyde fixation in studies of the synaptic cleft by Pfenninger et at. (6); these investigators reported that poststaining sections of bismuth-impregnated tissue with uranium and lead promoted electron contrast in nervous tissue. Also, Ainsworth and Karnovsky have demonstrated the usefulness of bismuth chelated with tartrate in increasing the visible size and electron opacity of ferritin in thin sections (2) and, with Ito (1), have used bismuth as the final reaction product in the ultrastructural demonstration of periodate-reactive sites. These investigators reported some increase in contrast in tissue sections treated with bismuth alone, and mentioned briefly that uranium and lead treatment of sections following bismuth gave adequate contrast to tissues studied. Most recently, alkaline bismuth stain has been used by Schwartz and Venkatachalam (8) to identify ferritin-injected blood vessels in the inner medulla of the rat kidney; however, these investigators did not comment upon concomitant enhancement of tissue contrast. Shinji et al. (9) have also investigated the use of alkaline bismuth reagent in demonstrating 1,2-glycol groups. However, none of the above investigators explored combining bismuth-, uraniumand lead-staining procedures with respect to obtaining optimal tissue contrast. This study reports our observations on the use of bismuth as a routine stain for electron microscopy, employing chelated bismuth as the staining reagent in thin sections, both alone and in combination with uranium and lead.