tapered ends and were not double contoured. In this they resembled the oral

Transcription

1 MORPHOLOGIC CHARACTERISTICS OF CERTAIN CULTURED STRAINS OF ORAL SPIROCHETES AND TREPONEMA PALLIDUM AS REVEALED BY THE ELECTRON MICROSCOPE EDWARD G. HAMPP,1 DAVID B. SCOTT,2 AND RALPH W. G. WYCKOFF3 National Institutes of Health, Bethesda, Maryland Received for publication August 16, 1948 Knowledge of spirochetal morphology has been gained for the most part by systematic studies of material from infected hosts and in certain instances from pure cultures maintained in the laboratory on artificial culture media. In this regard the hanging drop preparation, the stained smear or section, and dark-field illumination of wet preparations have been used to advantage. The last method has been more universally employed by the exacting investigator to demonstrate the fine structural differences that exist between the various types of spirochetes. The members of the genera Treponema and Borrelia present morphologic and structural features that have been the subject of considerable speculation and controversy for many years. In this laboratory the electron microscope has been used to demostrate some of these controversial structures, and the present paper describes certain results thus far obtained. Eight strains of spirochetes have been used. Two of these were strains of the small oral treponemes identified as FM and MRB; four were strains of Borrelia vincentii, designated as N9, N19, N37, and CON; and two were cultured strains of Treponema pallidum denoted as Nog and Ni (the Noguchi and Nichols strains). The small oral treponemes were morphologically and culturally similar in all details and representative of our collection of this type of spirochete. Under dark-field optical illumination, they showed uniform and closely spaced spirals and had long tapered ends. They were not double contoured. The four strains of B. vincentii were morphologically and culturally similar to one another and were approximately one and one-half times the size of the smaller oral treponemes. By dark-field examination these spirochetes had large, uniform, and widely spaced coils. The extremities were abruptly tapered and the organisms were not double contoured. The strains of T. pallidum were culturally similar to one another; the organisms had uniform and closely spaced spirals and long tapered ends and were not double contoured. In this they resembled the oral treponemes, but were on the average about one and one-half times as long. All spirochetes were grown in a clear liquid medium which is a modification of a "hormone" agar described by Huntoon (1918). This modified broth is essen- 1 Research Associate, National Institute of Dental Research, National Institutes of Health, USPHS, and Fellow, Research Commission, American Dental Association. 2 S. A. Dental Surgeon,. USPHS, National Institute of Dental Research, National Institutes of Health. 3 Scientist Director, USPHS, Laboratory of Physical Biology, National Institutes of Health, Bethesda, Maryland. 755

2 756 E. G. HAMPP, D. B. SCOTT, AND R. W. G. WYCKOFF [VOL. 56 tially a veal heart infusion in which "thio-peptone" is substituted for "bactopeptone" and agar omitted. Prior to use, the medium was filtered through a Berkefeld "N" filter to remove particulate matter. At the time of use, 100-ml portions of the basic medium were enriched with 10 ml of a 1 per cent solution of glutathione and 10 ml of ascitic fluid. Volumetric 25-ml flasks, filled to within 2 ml of the top with this finished medium, were inoculated with spirochetes. The flasks were rubber-stoppered and the cultures incubated at 37 C. At the expiration of the desired incubation period, the cultures were centrifuged and the sedimented organisms washed and recentrifuged five to seven times in distilled water to remove salts, soluble proteins, and particulate matter. This treatment has been found necessary in order to provide sufficiently clean preparations for electron microscopy. After the last washing the spirochetes were suspended in 0.1 per cent formaldehyde in distilled water and placed in the refrigerator overnight. The stock suspensions of spirochetes were diluted to arbitrary concentrations and microdrops dried on the usual collodion-covered specimen screens. Approximately 300 preparations were made from more than 30 separate cultures. These were examined with an RCA type EMU microscope after being "shadowed" by oblique evaporation of 40 mg chromium from a filament placed 18 cm behind and 3 cm above the specimens. The following typical electron micrographs demonstrate the morphologic characteristics, fine structures, and "granules" typical of these strains of organisms. General morphology. Figures 1, 2, 3, and 4 are micrographs of the small oral treponemes, Borellia vincentii, and the Nichols strain of Treponema pallidum. The oral treponemes of figures 1 and 2 represent the larger organisms of this strain, and exhibit shallow, closely spaced spirals. A short form of B. vincentii (figure 3) was selected in order that the entire organism might be included within a single microscopic field. Its spirals are larger and deeper and its ends are abruptly tapered. In addition, one end possesses a hook-shaped structure that is often observed in shadowed preparations but has not been observed during dark-field examination of living organisms. Spirochetes of the Nichols strain of Treponema pallidum (figure 4) are approximately as wide as the small oral treponemes and their ends are gradually tapered. The organism in the upper part of figure 4 shows spirals with about the same amplitude as those of the small oral spirochetes. Spirochetes in most of the preparations for electron microscopy appear straighter than the corresponding living organisms seen by dark-field illumination. Such changes have been observed in stained smears of these organisms and they have been shown to be a result 'of drying in air and heat fixation (Hampp, 1945). Surface structure. The external appearance of all types of spirochetes studied is strikingly similar (figures 1, 2, 3, 4). Definite cell membranes have not been observed; if they exist they must be tightly adherent to the organisms. Most surfaces are not smooth and their irregularities, as indicated by the shadows cast, may be an expression of uneven distribution of the protoplasm.

3 1948] SPIROCHETAL MORPHOLOGY AND ELECTRON MICROSCOPE 757,C<S Figure 1. Small oral treponeme (strain FM). X 11,000. Figure 2. Small oral treponeme (strain MRB). X 11,000.

4 758 E. G. HAMPP, D. 13. SCOTT) ANI) R. W. G. WYCKOFF [Vol,. 56 Figure 3. Borretia vincentii (strain N19). X 11,000. Figure 4. Treponema pallidum (Nichols strain). X 11,000.

5 1948] SPIROCHETAL MORPHOLOGY AND ELECTRON MICROSCOPE 759 Figure 5. Small oral treponeme (strain MRB). X 11,000. Figure 6. Small oral treponeme (strain FM). X 11,000.

6 760 E. G. HAMPP, D. B. SCOTT, AND R. W. G. WYCKOFF [VOL. 56 Filamentous structures. Terminal filaments have been a constant finding by dark-field examination of young, rapidly proliferating cultures of spirochetes. A large percentage of individual cells have shown these appendages at one or both ends. They have usually been coiled in the same manner as the spirochete, and have appeared to be single fibers. The appearance of these filaments under the electron microscope is illustrated in figures 5, 6, and 7. Some are short; others are longer than the spirochetes themselves. At the high magnifications of the electron micrographs it is clear that terminal filaments are not single fibers, but consist of intertwined fine strands (figures 6 and 7). Smith (1932) has postulated that these structures represent a continuation of the cell wall or "periplast," but no evidence has been found to support this assumption. In addition to the terminal filaments there are many other flagellar processes attached to the spirochetes of all strains studied. The presence of such flagella has also been observed by other investigators using the electron microscope (Wile et at., 1942; Morton and Anderson, 1942; Wile and Kearney, 1943; Mudd et al., 1943). Evidently the previously held opinion (Bergey, 1939; Topley and Wilson, 1936) that spirochetes are devoid of flagella is incorrect. Flagella occur anywhere along the spirochetes. They are found singly and in tufts and are sometimes intertwined to form cords (figures 1 through 10). When granules are present, many flagella are often seen in association with them (figures 16, 20). Internal granules. Internal granules have been frequently observed within the cytoplasm of spirochetes, both by dark-field illumination and by stained smears. They have often been seen in the aging cultures of the strains of oral spirochetes and T. pallidum (Hampp, 1946; Hampp, unpublished data), which have been used in the present investigation. Their typical appearance under the electron microscope is illustrated by figures 11 and 15. The granules produce irregularities in the surface of the organism, as evidenced by their shadows, and seemingly represent local accumulations of cytoplasm. End granules. Another type of granule is occasionally seen that is different from either the internal granules or the external granules described below. It is small, occurs at the end of an organism, and lacks visible internal structural detail (figure 12). Mudd et al. (1943) have referred to this structure as an "end granule." External granules. Periodic dark-field examination of spirochetal cultures indicates that about 2 to 3 weeks after inoculation another type of granule appears (Hampp, 1946); in the early stages of their formation they are intimately associated with the organisms and may occupy middle, subterminal, or terminal positions. Later they are "shed" and are found free in the culture medium. Typical granules associated with spirochetal cells are shown in figures 13, 14, 15, 16, and 17. In figures 13 and 14 a portion of each organism is enclosed within a spherule. In the case of the middle granules of figures 16 and 17 there is a different relationship between these structures and the somatic substance of the organisms. In figure 16 an opaque region can be seen within the granule surrounded by a homogeneous substance, which is sharply delineated. A profusion of flagella are exhibited. Figure 17 demonstrates a granule, the content of which

7 19481 SPIROCHETAL MORPHOLOGY AND ELECTRON MICROSCOPE 761 Figure 7. Borrelia vincentii (strain N37). X 15,000. Figure 8. Borrelia vincentii (strain CON).- X 14,000.

8 762 E. G. HAMPP, D. B. SCOTT, AND R. W. G. WYCKOFF [VOL. 56 Figure 9. Borrelia vincentii (strain CON). X 11,000. Figure 10. Borrelia vincentii (strain CON). X 11,000.

9 1948] SPIROCHETAL MORPHOLOGY AND ELECTRON MICROSCOPE 763 Figure 11. Borrelia vincentii (strain 19). X 11,000. _'_ 8~~~~~~~~~ Figure 12. Borrelia vincentii (strain CON). X 11,000.

10 764 E. G. HAMPP, D. B. SCOTT, AND R. W. G. WYCKOFF [VOL. 56 Figure 13. Borrelia vincentii (strain N37). X 11,000. Figure 14. Borrelia vincentii (strain N19). X 11,000.

11 1948] SPIROCHETAL MORPHOLOGY AND ELECTRON MICROSCOPE 765 Figure 15. Borre-lia vincentii (strain N19). X 11,000. -~~~~~~~~~~~~M.''. Figure 16. Borrelia vincentii (strain N19). X 11,000.

12 766 E. G. HAMPP, D. B. SCOTT, AND R. W. G. WYCKOFF [VOL. 56 Figure 17. B5orrelta vincentii (strain IN37). X 11,0uuu. Figutre 18. (Top) Rorrelia vincentii (strain N37). X 11,000. (Bottom) Treponema pallidurn (Nichols strain). X 11,000.

13 19481 SPIROCHETAL MORPHOLOGY AND ELECTRON MICROSCOPE 767 Figure 19. Borrelia vincentii (strain N9). X 11,000. Figure 20. Borrelia vincentii (strain N19). X 11,000.

14 768 E. G. HAMPP, D. B. SCOTT, AND R. W. G. WYCKOFF [VOL. 56 is seemingly like the cytoplasm of the organism and blends with it. None of the granules examined was attached to a spirochete by a stalk, as indicated by Mudd et al. (1943). The content of the grinule of figure 15 more nearly resembles that of free granules and may represent;a more advanced stage in granule development. Typical free granules, the end products of granule "shedding," are shown in figure 18. They are roughly circular in outline and sharply bounded. They consist for the most part of what appear to be short sections of spirochetes closely packed together. The contents of these granules are probably responsible for the fine lacelike appearance and the bright white, highly refractile bodies described by Hampp (1946) under the dark-field microscope. Examples of another type of free granule repeatedly observed are shown in figures 19 and 20. These granules consist of tangled masses of spirochetes or spirochetal segments. The significance of granules in the life history of the spirochetes is unknown but certain investigators have suggested that they may be germinative units (Balfour, 1911; Noguchi, 1911; Noguchi, 1917; Leishman, 1918; Mudd et al., 1943; Hampp, 1946). Others are undecided or hesitant in accepting this hypothesis (Fantham, 1916; Akatsu, 1917; Wenyon, 1926; Warthin and Olsen, 1930). Topley and Wilson (1936) have indicated that they are probably particles of culture medium adhering to the sides of the spirochetes. The electron micrographs demonstrate that this explanation is wrong, and that free granules are definitely a phase in the development of spirochetes. Although it is not possible to determine from these micrographs that the granules are germinative units, their constant rhythmic occurrence in living cultures suggests this possibility. Further support of this hypothesis is provided by the fact that cultures up to 31 months old, showing only refractile granules by dark-field examinfation, have invariably given normal growths on transfer to fresh medium (Hampp, 1946). SUMMARY Shadowed preparations of pure cultures of two strains of the small oral treponemes, four strains of Borrelia vincentii, and the Nichols and Noguchi cultured strains of Treponema pattidum have been studied with the electron microscope. Morphological characteristics, filamentous and flagellar appendages, and granules of various types have been described and illustrated. REFERENCES AKATSU, S Influence of carbohydrates on cultivation of spirochetes. J. Exptl. Med., 25, BALFOUR, A Infective granule in certain protozoal infections as illustrated by spirochaetosis of Sudanese fowls. Brit. Med. J., 1, 752. BERGEY, D. H., et al Bergey's manual of determinative bacteriology. 5th ed. Williams and Wilkins Co., Baltimore. FANTHAM, H. B Spirochaetes and their granular phase. Brit. Med. J., 1, HAMPP, E. G Comparative study of dark-field and stained smear technics for identification of oral spirochetes on the basis of morphologic characteristics. J. Am. Dental Assoc., 32,

15 19481 SPIROCHETAL MORPHOLOGY AND ELECTRON MICROSCOPE 769 HAMPP, E. G Morphologic alteration of smaller oral treponemes during aging of cultures; effect of age on viability of spirochetal cultures. J. Am. Dental Assoc., 33, HAMPP, E. G Unpublished data. HUNTOON, F. M "Hormone" medium. Simple medium employable as substitute for serum medium. J. Infectious Diseases, 23, LEISHMAN, W. B Note on "granule-clumps" found in Ornithodorus moubata and their relation to spirochetes of African relapsing fever (tick fever). Ann. inst. Pasteur, 32, MORTON, H. E., AND ANDERSON, T. F Some morphologic features of the Nichols strain of Treponema pallidum as revealed by the electron microscope. Am. J. Syphilis Gonorrhea Venereal Diseases, 26, MUDD, S., POLEVITZKY, K., AND ANDERSON, T. F Bacterial morphology as shown by the electron microscope. J. Bact., 46, NOGUCHI, HIDEYO 1911 Method for pure cultivation of pathogenic Treponema pallidum (Spirocheta pallida). J. Exptl. Med., 14, NOGUCHI, HIDEYO 1917 Spirochetes. Am. J. Syphilis Gonorrhea Venereal Diseases, 1, SMITH, D. T Oral spirochetes and related organisms in fuso-spirochetal disease. Williams and Wilkins Co., Baltimore. TOPLEY, W. W. C., AND WILSON, G. S The principles of bacteriology and immunity. William Wood and Co., Baltimore. WARTHIN, A. S., AND OLSEN, R. E Granular transformation of Spirocheta pallida in aortic focal lesions. Am. J. Syphilis Gonorrhea Venereal Diseases, 14, WENYON, C. M Protozoology. Bailliere, Tindall and Cox, London. Refer to 2, WILE, U. J., AND KEARNEY, E. B The morphology of Treponema pallidum in the electron microscope: Demonstration of flagella. J. Am. Med. Assoc., 122, WILE, U. J., PICARD, R. G., AND KEARNEY, E. B The morphology of Spirochaeta pallida in the electron microscope. J. Am. Med. Assoc., 119,