TREPONEMA PALLIDUM: A BIBLIOGRAPHICAL REVIEW 9

Kast, C. C. & Kolmer, J. A. (1943) A note on the culti-vation of Treponema pallidum with the preservation ofvirulence. Amer. J. Syph., 27, 309-13

Kolmer, J. A. (1930) Failure of vaccination of rabbitsagainst syphilis with a note on selective localization ofSpirocheta pallida. Amer. J. Syph., 14, 236-40

Lancet, 1926, 1, 882 (Annotations-Berlin: Cultivation ofSpirochaeta pallida)

Levaditi, C. (1908) Les spirochetes pathogenes. In:Bericht iiber den XIV. Internationalen Kongressen farHygiene und Dermographie, Berlin... 1907, Berlin,Hirschwald, vol. 2, pp. 160-78

Levaditi, C. (1913) Presence du treponeme dans le sangdes paralytiques generaux. C. R. Acad. Sci. (Paris),157, 864-6

McLeod, C. P. & Magnuson, H. J. (1951) Developmentof treponemal immobilizing antibodies in mice follow-ing injection of killed Treponema pallidum. J. vener.Dis. Inform., 32, 274-9

McLeod, C. P. & Magnuson, H. J. (1953) Production ofimmobilizing antibodies unaccompanied by activeimmunity to Treponema pallidum as shown by injectingrabbits and mice with the killed organisms. Amer.J. Syph., 37, 9-22

Magnuson, H. J., Halbert, S. P. & Rosenau, B. J. (1947)Attempted immunization of rabbits against syphiliswith killed Treponemapallidum and adjuvants. J. vener.Dis. Inform., 28, 267-71

Manteufel, P. & Herzberg, K. (1929) Zur Syphilis-Framboesiefrage. Zbl. Haut- u. GeschlKr., 30, 299-301

Mizumoto, R. & Hayashi, 0. (1956) Influences of P32upon the antibody titer of late rabbit syphilis. TohokuJ. exp. Med., 64, 21-26

Musumeci, V., Cantone, B., Chiarenza, A. & Auri-sicchio, A. (1958) Ricerche di terapia sperimentaledella infezione sifilitica; ricerche sperimentali, sull'in-fluenza della penicillina sul comportamento delbismuto nell'organismo del coniglio, trattato consalicilato marcato con isotopo 210 Bi. G. ital. Derm.Sif., 99, 143-55

Nakajima, T. & Hayashi, 0. (1956) Influences of P32upon the antibody titer of incurable latent syphilis.Tohoku J. exp. Med., 64, 17-19

Noguchi, H. (191 la) A method for the pure cultivationof pathogenic Treponema pallidum (Spirochaetapallida). J. exp. Med., 14, 99-108

Noguchi, H. (191lb) A cutaneous reaction in syphilis.J. exp. Med., 14, 557-68

Noguchi, H. (1912) The luetin reaction. J. Amer. med.Ass., 59, 1262-3

Noguchi, H. (1913) Paralysie generale et syphilis. Pressemid., 21, 805-7

Noguchi, H. & Moore, J. W. (1913) A demonstrationof Treponema pallidum in the brain in cases of generalparalysis. J. exp. Med., 17, 232-8

Rosahn, P. D. (1948) Radioactive tracer techniques andtheir possible application to studies in syphilis. Amer.J. Syph., 32, 307-16

Schaudinn, F. & Hoffmann, E. (1904-05) VorlaufigerBericht fiber das Vorkommen von Spirochaeten insyphilitischen Krankheitsprodukten und bei Papillo-men. Arb. GesundhAmte (Berl.), 22, 527-34

Schereschewsky, J. (1909) Zuchtung der Spirochaetapallida (Schaudinn). Dtsch. med. Wschr., 35, 835

Schobl, 0. (1930) The duration of anti-treponematousimmunity in Philippine monkeys originally conveyedby immunization with killed yaws vaccine. Philipp.J. Sci., 43, 599-601

Schobl, O., Tanabe, B. & Miyao, 1. (1930) Preventiveimmunization against treponematous infections andexperiments which indicate the possibility of anti-treponematous immunization. Philipp. J. Sci., 42,219-37

Tani, T., Inoue, R. & Asano, 0. (1951) Studies on thepreventive inoculation against syphilis. Jap. med. J.,4, 71-86

Turner, T. B. & Hollander, D. H. (1957) Biology of thetreponematoses, Geneva, World Health Organization(WorldHealth Organization: Monograph Series, No. 35)

Volpino, G. & Fontana, A. (1906) Sulla cultivazioneartificiale della S. pallida (Schaudinn). Riv. Ig. San.pubbl., 17, 462-6

Waring, G. W., jr, & Fleming, W. L. (1951) Furtherattempts to immunize rabbits with killed Treponemapallidum. Amer. J. Syph., 35, 568-72

Wheeler, A. H. (1960) Preliminary studies on activeimmunization in experimental syphilis in rabbits. In:Eleventh Annual Symposium on Recent Advances in theStudy of Venereal Diseases. Digest of proceedings,Item 20, Atlanta, Ga., United States Public HealthService

Willcox, R. R. (1960) The evolutionary cycle of thetreponematoses. Brit. J. vener. Dis., 36, 78-91

World Health Organization, Expert Committee onVenereal Infections and Treponematoses (1960) WldHlth Org. techn. Rep. Ser. 190

2. TAXONOMY 1

The name " spirochaete " was first given byEhrenberg in 1838 to large, free-living, flexible

1 See Wilson & Miles (1955), Wenyon (1926), Jordan& Burrows (1945).

organisms floating in fresh and marine water, e.g.,Spirochaeta plicatilis. Spirally-shaped organismswere for a long time grouped under the commonname of Spirillum, Spirochaeta or Vibrio. Later

R. R. WILLCOX & T. GUTHE

some were shown to be bacteria, and these are nowgrouped under Vibrio and the various species ofSpirillum. The remainder are actively motile not byflagella, but by means of a screw-like rotation of theorganism. All these spiralled organisms havecommonly been loosely termed " the spirochaetes ".This term, however, is now strictly confined tomembers of the family Spirochaetaceae.There are four main genera of " spirochaetes " in

the loose usage of the word: Spirochaeta, Cristispira,Leptospira and Treponema; from the last-namedBorrelia have now been separated. The first twogenera are the Spirochaetaceae; the others form thefamily Treponemataceae.

SPIROCHAETA

The various species of this genus are large slenderorganisms 200F-500,u long and 0.5,u-0.74,u thickwith 100-250 spirals, and they are not capable ofcausing disease. The term Saprospira is used forsmaller free-living forms which may be encounteredin foraminiferous sand (Jordan & Burrows, 1945).

CRISTISPIRA

Members of this group are characterized by aspirally arranged veil-like membranous appendageor crista round the body of the cell. The crista maysplay out to resemble the undulating membrane ofa trypanosome-in fact, it was first named Trypano-soma balbiani (Bradfield & Cater, 1952). Cristispiraare saprophytic in certain molluscs (e.g., Cristispirabalbiani, found in the crystalline style of the oyster,which is 45u-lOO1 long and l1,-1.5,u thick; its bodyis divided into chambers by septa of thickenedcytoplasm).

LEPTOSPIRA

Some members of this group are pathogenic toman and are the cause of Weil's disease, or infectiousjaundice, and also of certain fevers. They have alarge number of closely-wound spirals, and the endsare frequently turned round at a sharp angle; atrest they are characteristically hooked. They arethe thinnest of all spirochaetes, being only 0. l,u-0.2,thick and 5,u-18,u long. Included in the group areL. icterohaemorrhagiae, the cause of Weil's disease,or infectious jaundice; L. canicola, a pathogen ofdogs; and L. biflexa, an easily-cultured, free-livingleptospire said to inhabit ponds, gutters and drippingtaps (Bradfield & Cater, 1952).

TREPONEMATA

Treponemata are widely distributed in nature.Numerous species have been described in water, inthe gut of certain insects (e.g., the ant and the cock-roach) and in the large gut of the toad. In man theyare found in the mouth, alimentary tract, bronchi,around the urethral orifice, in certain ulceratingconditions of the skin, in condylomata, in the bloodof patients with relapsing fever (although these arenow classified as Borrelia), and in many of thelesions of syphilis. They are considerably thickerthan leptospirae, are less closely wound, and varyconsiderably in size. T. termitidis is 20,u-60,u long,while T. parrum may be only 3,u in length. Asaprophytic water form, T. elusum, has been de-scribed.

Borrelia

Most treponemata have a low refractive indexand stain poorly with aniline dyes. Those associatedwith Vincent's angina and relapsing fever in manand with avian spirochaetosis have a refractiveindex like that of bacteria, and stain more readily.On these and other less distinctive grounds they areassigned by many authors to a separate genus,Borrelia (Wilson & Miles, 1955; Swain, 1955;Breed et al., 1948; Eagle, 1948).Those now classified as Borrelia include B. vincentii,

the cause of Vincent's angina, B. anserinum whichaffects geese and turkeys (McNeil et al., 1949), andthe relapsing fever borreliae. The latter includeB. recurrentis, agent of cosmopolitan relapsing feverwhich is transmitted by lice and is found in Europe,Africa, Asia and South America, and other speciestransmitted by soft ticks (B. duttoni, found inAfrica, B. hispanica, in Europe, B. turicata, B. par-keri, B. venezuelensis, B. mazzotitii and others in theAmericas). (Some authors still describe these astreponemes with the generic prefix " T." instead of" B.".) Some writers include also as Borrelia certainof the saprophytic genital treponemes.

B. vincentii is 7,u-18p in length and 0.23,u-0.64,u(average 0.42,u) thick. It has 3-8 shallow spirals ofvaried amplitude and, unlike B. duttoni and B. re-currentis, is stated to have no external amorphouslayer but is enclosed by a clearly-marked cellmembrane (Swain, 1955).

B. duttoni is 9,u-16,u thick and 0.24p-0.60,u wide.It is tapered at the end and is not of constant thick-ness. Short forms with up to four spirals of in-constant amplitude and longer forms of 5-8 spirals

10

TREPONEMA PALLIDUM: A BIBLIOGRAPHICAL REVIEW I I

are encountered. There is an outer covering ofstructureless material. B. recurrentis closely re-sembles B. duttoni in size and general morphology(Swain, 1955).

Treponemata of man and animalsThose remaining for classification as treponemata

include T. cobayae, found in the blood of guinea-pigs; T. microdentium, T. macrodentium and T. muco-sum (Noguchi, 1912c), which are apparently non-pathogenic and are found in the mouth of man(being smaller than the pathogenic B. vincentil alsofound in that site 1); a number of saprophytic genitaltreponemes found in increased numbers in septicconditions (T. refringens, T. phagedenis (T. balaniti-dis), T. pseudopallidum, T. gangrenosa nosocomialis,T. minutum (T. genitalis), and T. calligyrum);T. cuniculi, which is responsible for a venerealdisease in rabbits; T. pallidum, the cause of humansyphilis, and T. pertenue and T. carateum, theorganisms responsible for yaws and pinta, respec-tively.Of those infecting man, T. microdentium, the small

mouth treponeme, resembles T. pallidum in morpho-logy and is also cultivable with least difficulty.According to Rosebury & Foley (1941) it is the onlymember of the oral group which can be acceptedwithout doubt as a distinct species. Of the humangenital treponemata, Schaudinn & Hoffmann (1904-05) also described T. refringens, at the time of theiroriginal description of T. pallidum, as a broader,less regular, more refractile organism, but did notdecide whether it was a separate species (see alsoLevaditi, 1906). It has a right-handed spiral (Sequeira,1956). T. phagedenis, with its variety of alternativenames, was described by Noguchi (1912a), whoalso (1913) described T. calligyra (calligyrum) andT. minutum or T. genitalis (see also Noguchi, 1918;Noguchi & Kaliski, 1918): all may be obtained fromgenital sores or condylomata (see Moreau & Giuntini,1956; Coutts et al., 1952).Moreau & Aladame (1957), who studied three

oral treponemes (including T. microdentium) andfour genital treponemes (T. refringens, T. phagedenis,T. calligyra and T. minutum) noted that theirfermentative pattern in culture differed accordingto species. It has been suggested from electronmicroscope studies of these organisms (Moreau &Giuntini, 1956) that although T. calligyra andT. minutum may be considered to be treponemes,

I T. buccalis, T. dentium and T. spirillum have also beendescribed-see Wilson & Miles (1955, page 2252).

T. refringens and T. phagedenis should be classifiedas Borrelia.Treponemes will resist trypsin digestion for many

days, but 10% bile salts (sodium taurocholate)cause complete disintegration (von Prowazek, 1907)and all, with the exception of leptospirae (Noguchi,1917; Wilson & Miles, 1955) are eventually brokenup by 10% saponin (Jordan & Burrows, 1945).

Relationship of treponemes to protozoaAn argument which has recurred from time to

time is whether treponemes should be classified asprotozoa. McDonagh (1912) so classified spiro-chaetes and described an apparent life-cycle re-sembling that of the malaria parasite. At the sameperiod, on the other hand, Dobell (1912), amongothers, pointed out that the one important featurewhich distinguished protozoa from bacteria wasmotility without flagella. When early workers withthe electron microscope claimed to have foundflagella on T. pallidum even this was in doubt(Wilson & Miles, 1955). However, it seems that theapparent " flagella " in T. pallidum arose fromrupture of the treponeme before examination, but,even knowing this, van Thiell (1959) still inclinedtowards protozoa, stating that the presence offlagella could no longer be used as an argument forits relationship to bacteria: neither, he considered,did the movement of T. pallidum fit in with that ofspirochaetes. Bessemans & de Geest (1933), pointingout the difficulty of culture of T. pallidum in vitro,also inclined to protozoa. Most workers, however,like to consider spirochaetes as representing aphase of life midway between the bacteria andprotozoa (Jordan & Burrows, 1945; van Thiell,1959).

DISCOVERY OF PATHOGENIC TREPONEMES

At least four different varieties of pathogenicorganisms of the genus Treponema are known:T. pallidum (syphilis), T. pertenue (yaws), T. carateum(pinta) and T. cuniculi (venereal treponematosis ofrabbits). They all resemble each other morpholo-gically.

T. pallidumDonne, in 1837, is stated (Campbell & Rosahn,

1950) to have been the first to describe a spiralledmicro-organism in the exudate of primary syphiliticlesions of the genitalia, and he believed he hadfound the causative organism of syphilis. His work

R. R. WILLCOX & T. GUTHE

was confirmed by Vanoye (1840-41), who consideredthat these organisms could be used in the diagnosisof syphilis.According to Stokes & Beerman (1934), Metch-

nikoff stated that Bordet & Gengou in 1903 undoub-tedly saw the " spirochaete " of syphilis. Klebs isalso stated to have seen it in 1875-77 in syphiliticmaterial, and also to have transmitted the diseaseto the monkey (see Klebs, 1932; Wilson & Miles,1955). Credit, however, is usually given to Metch-nikoff and Roux in Paris for having infected mon-keys and apes experimentally with syphilis in 1903(Metchnikoff & Roux, 1903, 1904, 1905; see Jordan& Burrows, 1945). They also showed that calomelointment applied to chimpanzees 1-2 hours afterlocal inoculation would prevent infection.The final confirmation of T. pallidum as the

causative agent of syphilis-indeed, its discovery-is attributed to Schaudinn & Hoffmann (1904-5,1905a, b) (see also Hoffmann, 1905, 1906; D'Ago-stino, 1957) who observed the organism in chancresof syphilitic patients. Other early workers includeMansurov (1885), who described a vegetable parasiteof syphilis, and Zabotolnyj (1909), who apparentlysaw T. pallidum two years before Schaudinn &Hoffmann but attached no importance to his dis-covery. After the discovery by Schaudinn &Hoffmann, numerous confirmatory papers appearedwithin a very short time all over the world (includingRussia-e.g., Zabolotnyj, 1905, 1906, 1909; Ivanov,1905). See also Minouflet (1906).The discovery of T. pallidum added enormous

impetus to experimental research. The followingyear (1906) claims were advanced that the organismhad been cultured (Volpino & Fontana, 1906;Schereschewsky, 1909a, b), although the culturesobtained were not shown to be virulent. In thesame year Bertarelli (1906) showed that syphiliscould be passed from the eye of one rabbit to thatof another and Parodi (1907) first transmitted thedisease to the testicle of a rabbit. Virulent cultureswere, however, claimed by Noguchi (1911 a)-butthis also proved a disappointment as the virulencesoon disappeared.Around this time, too, the first diagnostic serum

test for syphilis was evolved by Wassermann(Wassermann et al., 1906) and the dark-field methodof diagnosis was soon introduced (Coles, 1909).Zabolotnyj & Maslakovec (1907) described thecessation of movement and the agglutination ofT. pallidum under the effect of serum from a syphi-litic patient. Ehrlich & Hata (1910) discovered

salvarsan, or 606, to provide what was hoped to bean effective and rapid treatment for the disease(and also for yaws). From the cultured treponeme,Noguchi (191lb) prepared luetin, which was testedin many hospitals and which would produce apositive skin reaction in most cases of late syphilis(less often in early cases). Moreover, it was con-sidered to be specific for the disease (Noguchi, 1912b).

Thus, during less than a decade before the FirstWorld War, most of the essential problems ofsyphilis appeared to have been solved-at least, itmust have appeared so to syphilologists at the time.

T. pertenue, T. carateum and T. cuniculi

T. pertenue, the causal organism of yaws, was alsodiscovered in 1905, by Castellani. It is morpholo-gically indistinguishable from T. pallidum (Wilson& Miles, 1955). T. carateum, the causative organismof pinta, although suggested by Menk (1927) andby Gonzalez-Herrejon (1927) on the basis of a highincidence of positive Wassermann reactions inpinta patients (see Turner & Hollander, 1957) wasfirst demonstrated by Saenz et al. (1938) (see Fox,1939; Jordan & Burrows, 1945). Leon y Blanco(1940), who called it T. herrejoni, showed thistreponeme also to be morphologically indistin-guishable from T. pallidum. For further history seeSchuberg & Schlossberger (1930).Although T. pertenue was soon shown to be

transferable to rabbits (Nichols, 1910, was alreadyworking on a strain obtained from a coloured soldierreturning from the Philippines), T. carateum, onthe other hand, is not easily passed to animals andonly one such claim has been made, that of Leony Blanco & Oteiza (1945) (see Holcomb, 1942).

T. cuniculi, the cause of a venereal treponematosisin rabbits first described by Ross (1912), was identi-fied by Bayon (1913) and is also morphologicallysimilar to T. pallidum. It causes a complaint knownas " rabbit syphilis ", or sometimes as pallidoidosis.Affected rabbits develop superficial small, scaly,eroded, sometimes crusted, lesions on the genitaliaand adjacent perineal region: sometimes the nostrilsand eyelids may be affected (see section 17).

EVOLUTION OF PATHOGENIC TREPONEMES

The evolution of the pathogenic treponemes canonly be a matter of speculation. It is considered bysome (e.g., Cockburn, 1961) that they arose aeonsago from free-living water forms which have sincebecome adapted to their human or animal hosts,

12

TREPONEMA PALLIDUM: A BIBLIOGRAPHICAL REVIEW 13

having been subjected to all the influences concernedin natural selection. Although it is suggested thatthe four treponemes may have come from a singlesource (Hoffmann-Bonn, 1953) speculators maydiffer as to how far back in the evolutionary scalethe separation between them occurred. Hudson(1946) considered the treponemes pathogenic tohumans (T. pallidum, T. pertenue and T. carateum)all to be T. pallidum, the disease picture in man beingmodified by various external environmental factorsand by factors within the host. This author (1963)believed that a common organism became adapted toman in Africa in palaeolithic times. Hackett (1963)suggests that the human treponematoses probablyarose long ago from an animal infection. Mutationssubsequently occurred-T. carateum probably beingthe earliest-resulting in the diseases now known asyaws, pinta and venereal syphilis. That the distri-bution of the disease syndromes of yaws, pinta andendemic and non-venereal syphilis conforms to someconsiderable extent to the environmental backgroundis well known (Guthe & Luger, 1957; Guthe& Willcox, 1954). Willcox (1960) has indicated theevolutionary cycle which may occur in the diseasesyndromes caused by human treponematoses result-ing from pressures in the extrinsic environment,and in the intrinsic environment of the host.

STRAINS OF T. PALLIDUM

Those strains of T. pallidum which have beenmaintained in animals have remained virulent toboth animals and man. These include: the Nichols(Nichols-Hough or Nichols pathogenic), Truffi,Gand, Gent, Ami, Fan, Tho, Est, Rei and Pourstrains (see section 15). Strains maintained inanimals for a period of years may undergo modifi-cation (Turner & Hollander, 1957).

Although virulent treponemes have not beenestablished in culture outside the body, there are anumber of strains-usually designated T. pallidumwhich have been maintained in artificial media formany years. These include the Reiter, Noguchi,Nicholsnon-pathogenic, Kazan, Krooand Vasarhelyi.Details of these are given below (see section 19).

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Bertarelli, E. (1906) Sulla trasmissione della sifilideal coniglio. Riv. Ig. San. pubbl., 17, 646-60; (Zbl.Bakt., 1906, 41, 320-6; Trans.)

Bessemans, A. & de Geest, B. (1933) Treponeme paleet culture tissulaire. C. R. Soc. Biol. (Paris), 114,530-2

Bradfield, J. R. G. & Cater, D. B. (1952) Electron-microscopic evidence on the structure of spirochaetes.Nature (Lond.), 169, 944-6

Breed, R. S., Murray, E. G. D. & Hitchens, A. P. (1948)Order V. Spirochaetales Buchanan. Family II. Trepo-nemataceae Schaudinn. In: Bergey's Manual ofdeterminative bacteriology, 6th ed., Baltimore, Williams& Wilkins, pp. 1058-79

Campbell, R. E. & Rosahn, P. D. (1950) The morphologyand staining characteristics of Treponema pallidum.Review of the literature and description of a newtechnique for staining the organism in tissues. YaleJ. Biol. Med., 22, 527-43

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Coutts, W. E., Silva-Inzunza, E. & Valladares-Prieto, J.(1952) Comparative study of certain treponematafound in human genitalia, specially referring toT. pallidum, T. macrodentium and T. microdentium.Dermatologica (Basel), 105, 79-84

D'Agostino, M. (1957) Anecdotario de la sifilis.Schauddin y el descubrimiento del Treponema palido.Dia med. (B. Aires), 29, 2820-1

Dobell, C. C. (1912) Researches on the spirochaetes andrelated organisms. Arch. Protistenk., 26, 117-240

Donne, A. (1837) Nouvelles experiences sur les animal-cules spermatiques, sur quelques-unes des causes de lasteritite chez la femme, suivies de recherches sur lespertes seminales involontaires, et sur la presence dusperme dans l'urine, Paris, C. Chevalier

Eagle, H. (1948) The spirochetes. In: Dubos, R. S., ed.,Bacterial and mycotic infections of man, Lippincott,Philadelphia, chapter 27, pp. 527-55

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TREPONEMA PALLIDUM: A BIBLIOGRAPHICAL REVIEW 1 5

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3. MORPHOLOGY: I. METHODS OF EXAMINATION

The morphology of T. pallidum may be examinedin slide preparations of stained smears, by dark-field under the light microscope, and by phase-contrast and electron microscopes. With the lightmicroscope without dark-field and with the electronmicroscope, the treponemes are examined in thedead state: under the dark-field and phase-contrastmicroscopes, live organisms can be studied. Usingthe light microscope, the unstained organism isextremely difficult to see.

STAINED SMEARS 1

Stained smears are examined under the con-ventional light microscope, and two basic techniquesof staining are used: (a) the treponeme is im-pregnated with a dye, or a metallic ion such assilver, to render the organisms visible against a palebackground; or (b) the background may be stainedblack, leaving the unstained treponeme pale bycomparison (see Bessemans et al., 1936; Yamamoto,1929a, b). Matsumoto (1930) and Turner & Hollan-

I See Campbell & Rosahn (1950).

der (1957) succeeded in staining T. pallidum with noless than 402 different dyes.

Impregnation methodsContrary to general belief treponemes are easily

stained (Turner & Hollander, 1957; Wheeler, 1960)but they are difficult to see under the light microscopebecause of the small amount of protoplasm possessedby the organism and also because the suspendingmedium frequently contains too much tissue debriswhich also takes up the stain (Wheeler, 1960). Earlyinvestigators used aniline dyes or coal tar derivatives(e.g., methylene blue, azure eosinates, Victoria blue,cresyl violet, Giemsa and similar mixtures). Amordant (i.e., a protein precipitant such as phenol,tannic acid, acetic acid, phosphotungstic acid, etc.)has to be used to make the stain effective. Schaudinn& Hoffman (1904-05) employed a modified Giemsastain (see Wilson & Miles, 1955), with which T. palli-dum and other pathogenic treponemes stain rose-red.Noguchi (1918) found that treponemes fixed with

Fontana's fixative and Fontana's mordant could bestained by carbol fuchsin. This was further con-firmed by DeLamater, Wiggall & Haanes (1950),