TREPONEMA PALLIDUM: A BIBLIOGRAPHICAL REVIEW 1 5 Schereschewsky, J. (1909a) Zuchtung der Spirochaeta Volpino, G. & Fontana, A. (1906) Einige Verunter- pallida (Schaudinn). Dtsch. med. Wschr., 35, 835 suchungen uber kunstliche Kultivierung der Spiro- Schereschewsky, J. (1909b) Weitere Mitteilung uber die chaeta pallida (Schaudinn). Zbl. Bakt., L Abt. Orig.. Zuchtung der Spirochaeta pallida. Dtsch. med. Wschr., 42, 666-9 35, 1260-1 Wassermann, A., Neisser, A. & Bruck, C. (1906) Eine Schuberg, A. & Schlossberger, A. (1930) Zum 25. Jahres- serodiagnostische Reaktion bei Syphilis. Dtsch. med. tag der Entdeckung der Spirochaeta pallida. Klin. Wschr., 32, 745-6 Wschr., 9, 582-6 Wenyon, C. M. (1926) Protozoology, London, Bailliere, Sequeira, P. J. L. (1956) The morphology of Treponema Tindall & Cox, vol. 2 pallidum. Lancet, 2, 749 Willcox, R. R. (1960) The evolutionary cycle of the Stokes, J. H. & Beerman, H. (1934) The fundamental treponematoses. Brit. J. vener. Dis., 36, 78-91 bacteriology, pathology and immunity of syphilis. Wilson, G. S. & Miles, A. A. (1955) The spirochaetes. In: Modern clinical syphilology, 2nd ed., Philadelphia, In: Topley & Wilson's Principles of bacteriology and Saunders, pp. 17-57 immunity, 4th ed., London, Edward Arnold, chapter 38, Swain, R. H. A. (1955) Electron microscopic studies of pp. 1031-56; Syphilis, rabbit syphilis, yaws and pinta... the morphology of pathogenic spirochaetes. J. Path. chapter 81, pp. 2027-4 Bact., 69, 117-28 Zabolotnyj, D. K. (1905) [Spirochaetes in syphilis]. Thiel, P. H. van (1959) Are spirochaetes bacteria or Russk. Vrac, 23 protozoa? Antonie van Leeuwenhoek, 25, 161-7 Zabolotnyj, D. K. (1906) [The pathogenesis of syphilis]. Turner, T. B. & Hollander, D. H. (1957) Biology of the Russk. Vra6, 42 treponematoses, Geneva (World Health Organization: Zabolotnyj, D. K. (1909) [The pathogenesis of syphilis]. Monograph Series, No. 35) Arh. biol. Nauk, 14, 263-82, 389-438 Vanoye, M. (1840-41) Note sur un animalcule trouve Zabolotnyj, D. K. & Maslakovec, N. N. (1907) [Observa- dans le pus syphilitique. Ann. Soc. Sci. nat. (Bruges), tions on the movement and agglutination of Spiro- 2, 39-42 chaeta pallida]. Russk. Vrac, 11 3. MORPHOLOGY: I. METHODS OF EXAMINATION The morphology of T. pallidum may be examined der (1957) succeeded in staining T. pallidum with no in slide preparations of stained smears, by dark- less than 402 different dyes. field under the light microscope, and by phase- contrast and electron microscopes. With the light Impregnation methods microscope without dark-field and with the electron Contrary to general belief treponemes are easily microscope, the treponemes are examined in the stained (Turner & Hollander, 1957; Wheeler, 1960) dead state: under the dark-field and phase-contrast but they are difficult to see under the light microscope microscopes, live organisms can be studied. Using because of the small amount of protoplasm possessed the light microscope, the unstained organism is by the organism and also because the suspending extremely difficult to see. medium frequently contains too much tissue debris which also takes up the stain (Wheeler, 1960). Early STAINED SMEARS 1 investigators used aniline dyes or coal tar derivatives (e.g., methylene blue, azure eosinates, Victoria blue, Stained smears are examined under the con- cresyl violet, Giemsa and similar mixtures). A ventional light microscope, and two basic techniques mordant (i.e., a protein precipitant such as phenol, of staining are used: (a) the treponeme is im- tannic acid, acetic acid, phosphotungstic acid, etc.) pregnated with a dye, or a metallic ion such as has to be used to make the stain effective. Schaudinn silver, to render the organisms visible against a pale & Hoffman (1904-05) employed a modified Giemsa background; or (b) the background may be stained stain (see Wilson & Miles, 1955), with which T. palli- black, leaving the unstained treponeme pale by dum and other pathogenic treponemes stain rose-red. comparison (see Bessemans et al., 1936; Yamamoto, Noguchi (1918) found that treponemes fixed with 1929a, b). Matsumoto (1930) and Turner & Hollan- Fontana's fixative and Fontana's mordant could be stained by carbol fuchsin. This was further con- I See Campbell & Rosahn (1950). firmed by DeLamater, Wiggall & Haanes (1950), 16 R. R. WILLCOX & T. GUTHE who used a modification of Fontana's technique for Beamer & Firminger, 1955). This was used by photographic study, and to examine the so-called Levaditi & Manouelian (1906) in Metchnikoff's life-cycle of the Nichols pathogenic rabbit-adapted laboratory soon after the discovery of T. pallidum strain of T. pallidum (DeLamater, Wiggall & Haanes, in the histological examination of monkeys infected 1950b). The slides were first placed in Ruge's solu- with syphilis. tion (glacial acetic acid and formalin), then in In general the dye stains are more suited to Fontana's mordant, and subsequently stained with smears, and silver impregnation methods to tissue gentian violet. Takahashi (1922) used a potassium section. Stroesco & Vaisman (1936)-in Levaditi's permanganate stain. laboratory-developed a silver impregnation method Rose & Morton (1952) found Fontana and Giemsa applicable to frozen sections (see Stokes & Beerman, stains useful, especially for virulent organisms. 1948). Tannic-acid-mordanted fuchsin, as in Leifson's flagella stain (Leifson, 1930), was found convenient. Background stains and other staining methods These authors described another modification useful Of the background relief stains, Indian ink, congo for cultured treponemes. A drop of centrifuged red and nigrosin have been used for T. pallidum culture was placed on a cover-slip and immediately (Campbell & Rosahn, 1950). The dark-field, using exposed to osmium tetroxide vapour. Then, after an optical method, is on the same principle. Lenn- prolonged immersion in acetone, the organisms were hoff (1948) described an in vitro method of staining. *' mordanted " in formalin, stained in basic fuchsin It was noted that, if a colloidal mercury preparation and examined in the wet state. was injected into the chancre of a rabbit, and Victoria blue and a carbol gentian violet stain immediately followed by an intravenous injection of have also been reported as satisfactory (Golds- sodium thiosulfate, treponemes stained black would worthy & Ward, 1942; Jordan & Burrows, 1945) be found in the chancre within two minutes. and Rizk et al. (1951) used carbol gentian violet (and also silver impregnation and other methods) to study T. pallidum from cases of bejel; Wright's IMMUNOFLUORESCENCE stain was preferred. Crystal violet and basic fuchsin More recently, staining by immunofluorescence were used by Levine (1952) for a variety of trepo- has been applied to T. pallidum. A new specific nemes including T. pallidum, while Vago (1947a, b,c) technique has been evolved by Deacon et al. (1964) used a method employing mercurochrome. which requires but 30 seconds for staining. The Coutts, Silva-Inzunza & Valladares Prieto (1952) reagent consists of human or rabbit syphilis anti- observed that T. macrodentium and T. micro- serum from which non-specific antibody has been dentium absorbed a dilute carbol fuchsin stain removed by Reiter treponeme sonicate. Yobs et al. rapidly but T. pallidum did so less easily. If T. palli- (1964) have proposed a technique for staining dum and the dental treponemes were stained with T. pallidum in tissues. carbol fuchsin and observed under the dark-field microscope, T. pallidum appeared with an argentine brilliancy whereas the dental treponemes showed THE DARK-FIELD with only a pinkish brilliancy. The use of stained The dark-field, which uses the light microscope smears under the dark-field was earlier described with transmitted rather than direct light, has definite by Hoffman (1921) (see DeLamater, Haanes & advantages in that it permits the examination of Wiggall, 1950). A more recent stain is an ink-blue live motile organisms (see Clarkson, 1956). For the dye originally found in " Parker 51 " blue-black recognition of T. pallidum fixed specimens have ink (Gomes, 1953; Turner & Hollander, 1957; definite limitations. Not only may there be agonal Goldman & Sawyer, 1959). Yamamoto (1929a, b) or later distortion of the organisms but also the found that two blue stains would stain T. pallidum study of motility-an essential point of recognition and T. pertenue but not T. cuniculi. -is not possible. Coles (1909) described the use of Methods other than the use of aniline dyes were dark-ground illumination, which was also used by developed because of some lack of consistency in Antoni (1921) and by Hoffman (1921a) (see Camp- the results obtained with stained smears. One was bell & Rosahn, 1950). Hoffmann (1921b) employed the method of silver impregnation improved by the dark-field for the examination of stained and Fontana (1912) (see Campbell & Rosahn, 1950; unstained smears as did Coutts, Silva-Inzunza & TREPONEMA PALLIDUM: A BIBLIOGRAPHICAL REVIEW 17 Valladares-Prieto (1952) and Wheeler (1960). Coutts, ELECTRON MICROSCOPE Silva-Inzunza & Morales-Silva (1952a, b), although the study of motility was sacrificed, found the The limits of resolving power of the light micro- examination of unstained smears useful by this scope have been extended over 100 times by the method as it could be done at leisure. electron microscope: enormous magnification may Sequeira (1956) made careful dark-field observa- be obtained. With an instrument working with an tions of T. pallidum, T. pertenue and T. refringens.