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Journal of Experimental Medicine

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T H E CULTIVATION OF TRICHOMONAS OF T H E HUMAN
MOUTH (TETRATRICHOMONAS HOMINIS).
BY TOKUZO OHIRA, M.D., AND HIDEYO NOGUCHI, M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research.)
P~s34 To 37.

(Received for publication, October 15, 1916.)

Few attempts have been made to cultivate the parasitic flagellatesof man. The first report on the cultivation of the trichomonas waspublished by Lynch (1) in 1915.
Lynch found numerous trichomonades in the vagina and gums of a negress
who was suffering from catarrhal vaginitis and acute gingivitis. He placed
the material in neutral and acid bouillon and kept it alive for 3 days at30°C. Then, as the organisms began to be affected by bacterial growth hetransferred them to new media and was thus able to carry the culture throughseveral generations.

The experiment described in this communication was carried out
at first without the knowledge of Lynch's report and differs considerably from his.
MateriaL--The tartar of the mouth which has long been known toharbor trichomonades was used. I n most instances they are few innumber. ~Culture Media.--A number of different media were employed.
Acid bouillon, neutral bouillon, Ringer's solution, ascitic fluid, asciticbouillon (equal parts), ascitic fluid-isotonic salt solution (equal parts),
ascitic fluid-Ringer's solution (equal parts), bouillon-Ringer's solution,
sheep serum water, sheep serum water-Ringer's solution (equal parts),
rabbit sennn water, nutrient agar, ascitic agar, ascific Ringer's agar,
sheep serum-glucose agar. Fresh tissue was placed in both the fluid
and solid media.
Most of the media were unsuitable; only a few gave relatively goodresults; the mixture of ascitic fluid and Ringer's solution in equal
We wish to express here our indebtedness to Dr. M. I. Schamberg for his
courtesy in providing us with some of the materials used for the present study.
341

342

TRICHOMONAS OF THE HUM_A~ MOUTH

portions and in its natural alkalinity gave entire satisfaction. Samples of ascitic fluid from different sources were used and gave equally
good results. The bouillon employed by Lynch was not so suitableas our ascitic Ringer's solution, the chief reason probably being thatit enables the contaminating bacteria to multiply so rapidly andprofusely that the trichomonades no longer find it a suitable medium.
Undiluted ascitic fluid was also found to be too rich in protein material, although in this respect it is more suitable than bouillon.
Culture.--When the culture medium is inoculated with a small
amount of dental tartar, the trichomonades are so few that it takessome time to find one. But by placing the inoculated tubes at atemperature of 37°C. 2 and then transferring a small quantity of theculture into new medium every 24 hours for 4 or 5 days in succession, the number of the organisms gradually increases and no difficulty was experienced in finding quantities of agglomerating flagellates as well as free individuals in every field. The organisms group
together in clusters of varying size around the masses of bacteria(Fig. 1). At lower temperatures (23-27°C.) the growth of bacteria
as well as of protozoa proceeds much more slowly than at higher temperatures, and the transfer may be made every 48 hours.
The method of transfer is simple. As the trichomonas grows at
the bottom of the tube with bacterial sediments, it is necessary tosuck up this precipitate with a capillary pipette and transfer it to
new tubes. In both sucking up and transferring precaution should
be taken not to stir the culture too much. In this way strains werecarried for over twenty generations, and the loss of the strain wasusually due to an accident or negligence in the transfer.
A brief note on the Entamtrba gingivalis Gross (Entama~ba buccalis
Steinberg), which is almost always encountered in this medium, may
be made here. In this medium the entameba can easily be kept
alive over 24 hours, and with a little precaution sometimes over 48
hours; that is, b y keeping the medium always at about 37°C. and byhaving a sufficient amount of culture fluid. If the alkaline reactionof the medium is reduced by adding drops of a 1 per cent solutionof acetic acid to almost the neutral point indicated by litmus paper,
2Lynch could not get cultures at 37°C.

TOKUZOOHIRA ANDHIDEYO NOGUCHI

343

the rapidity of the growth of the trichomonas is somewhat reduced,
whereas that of the entamebm is accelerated. By transferring thesedimentJ into a new, already heated medium once every 24 hours,
the a~eba~ can be kept alive over a week; in one instance they werekept alive as long as 10 days. Judging from the fact that only asmall amount of the sediment is carried from one culture tube intotwo, three, or more new tubes each time, the ameba~ remain alive
and multiply, though not so strikingly as in the case of the trichomonades.

Morphological and Biological Properties of the Culture Trichomonas.
The body of the trichomonas is uniformly pear-shaped; but being
highly flexible it can take any shape according to circumstances, espedally at the time when it passes through the narrow spaces in thebacterial masses, as observed in a fresh preparation. The protrusionof any definite pseudopodium as drawn by Kuczynski (2) in the caseof Trlchomonas augusta has not been seen in our culture, but a prolongation of the protoplasm near the posterior end (Fig. 2) was often
observed. This is often used as an anchor, while the main part hasa lively oscillating movement (,con Prowazek's haftpseudopodialer
Fortsatz). This prolongation of the protoplasm, however, has nothing to do with a real pseudopodium as we understand it in thecase of the ameba, and there is no evidence that it is used for
obtaining food. On the other hand, the presence of a cytostome(peristome) is probable, though we could not discern it clearly. In
one instance a long chain of bacilli was gradually taken in through apoint near the beginning of the flagella. After it became embeddedin the protoplasm the chain broke into two parts, one of which wassoon cast off from the body through the same point by which it had
first entered, while the other seemed to be digested.
The size of the culture trichomonas varies within wide ranges, but it
usually measures 10 to 15/~ in length and 4 to 8/z in width (Fig. 11).
Sometimes particu]arbr large forms are encountered, about 25 # longand 12/z wide (Figs. 12 and 13). Flagella at the anterior end are uniformly four in number, radiating out from a basal granule situated justin front of the nucleus. They are unequal in length, in most instances

344

TP~ICHOMONAS OF "£11J~ HUMAN" MOUTII

two of them being a little longer than the other two. The length of
the flagella averages 14 to 16 #. An undulating membrane rising froma blepharoplast, which lies close to, but differs from the basal granules,
is distinctly seen (Fig. 8). The distal end of the limiting line of themembrane usually lies within the body, while its terminal end sometimes projects beyond the margin as a short, free flagellum (Fig. 3).
The axostyle usual]y seems to take its origin at the distal end of
the spindle-shaped nucleus, but sometimes its course may be traced
through the nucleus as far up as the basal granule, or very close toit. In its distal course the axostyle runs downward through the
body and projects beyond the margin at the posterior end of theorganism, forming a free tail 5/z or less in length. The nucleus issingle, oval, more or less elongated in shape, has a distinct karyosome, and measures 1 to 1.5 # by 2 to 3 #.
The protoplasm shows no differentiation of ectoplasm and endoplasm, and no contractile vacuoles or chromidial apparatus could beobserved other than fine granulation with scattered, coarse particles.
In some specimens the protoplasm is filled with ingested bacteria.
In preparations stained with Giemsa's solution two or more characteristic purple rings are found, not urdike the forms described bysome authors as a stage of so called cyst formation (Fig. 6).
The common method of reproduction seems to be a binary, longitudinal fission (Figs. 14, 15, 16, 17, and 18); multiple fission (or ratherbudding) may also occur in the culture. Figs. 2 to 10 show a seriesof successive stages of division, which is complete within from 30
minutes to 1 hour in the hanging drop. The number of daughter
cells of multiple fission is not great (4, 6, 8, etc.); they do not seemto separate from each other at the same time, but usually bud off
one after another in succession, so that the gbneral impression differsconsiderably from the usual mode of schizogony as in other protozoa(Figs. 19 and 20). The division of blepharoplast is of the mitotictype (Fig. 19), while that of the nucleus itself is promitotic (Fig. 21).
In cultures no form has been observed which may be interpreted
as a cyst.

TOKUZOOIIIEAANDIIIDEYODlSC~rSSlON A ~ D

NOGUCHI345s~Y.

Trichomonades from the mouth were studied by Steinberg 3 who
proposed to group them into three distinct types; namely, Tridmmonas
elongata, Trichomonas caudata, and Trichomonas flagellata. Doflein(3) regards them as probably identical with Trichomonas hominis.
Opinions differ as to whether or not Trichomonas vaginalis Donn~and Trichomonas hominis Grassi are the same species. Lynch, forinstance, believes that they are the same species, while yon Prowazek(4), Bensen (5), and others (6, 7) insist that they are different types.
Bensen's view seems to be well supported by the difference alleged to
be found between the mode of encystment in the two trichomonades,
were it not for the fact that our knowledge about the so called cyst oftrichomonades is still obscure. According to Alexeieff (8) many of
the so called cysts were evidently blastomyces contained in the cellbody of the trichomonas. An autogamy alleged to take place in cysts
as described by Bohne and yon Prowazek (9) has not been confirmedby Dobell (10). And Wenyon (11) contends that it has never beenfound possible to produce any development of these cysts outside thebody on the warm stage as can be done with the cysts of Entama~bacoli. Therefore, it is still premature to take the process of encystmentinto consideration as far as the classification of trichomonas is concerned. On the other hand, Rodenwaldt (12) seems to think that
there are many species of trichomonas in the human intestines, andWenyon has described a new trichomonas from the human intestines(Macrostoma raesnili Wenyon).
Further cultural studies in the morphology and biology of theseorganisms must be carried out in order to solve these problems.
In the light of modern investigations there are five subgenera to
be included under the genus Trichomonas Donn& They are as follows:(1) Protrichomonas Alexeieff, with three anterior flagella, without an undulating membrane.
(2) TrichomasHx Biitschli, with three anterior flagella and a trailLug flagellum (Schleppgeissel) without an undulating membrane.
' Steinberg, quoted by Doflein, F., in Lehrbuch der Protozoenkunde, Jean,
3rd edition, 1911, 492.

346

TRICHOMONAS O1~ THE H U M A N MOUTH(3) Trichomonas Donn6, with three anterior flagella and an undulating membrane.
(4) Macrostoma Alexeieff, Amend, Wenyon (11), with three anterior flagella and an undulating membrane wedged in a deep groove(peristome).
(5) Tetratrichomonas Parisi (13), with four anterior flagella andan undulating membrane.
As far as our culture trichomonas from the h u m a n m o u t h isconcerned, it has been shown t h a t it is not strictly a trichomonas
and t h a t it should be classed under the subgenus Tetratrlchomonas.
BIBLIOGRAPHY.
1. Lynch, K. M., Trichomoniasis of the Vagina and the Mouth. Cultivationof the Causative Organism and Experimental Infection. (A PreliminaryCommunication.), Am. J. Trop. Dis., 1914-15, ii, 627.
2. Kuczynski, M. H., Untersuchungen an Trichomonaden, Arch. Protistenk.,
1914, xxxi/i, 119.
3. Doflein, F., Lehrbuch der Protozoenkunde; eine Darstellung der Naturgeschichte der Protozoen mit besonderer Berficksichtigung der parasitischen
und pathogenen Formen, Jena, 3rd edition, 1911, 492.
4. yon Prowazek, S., Notiz fiber die Trichomonas hominis (Davaine), Arch.
Prot~stenk., 1902, i, 166.
5. Bensen, W., Untersuchungen fiber Trichomonas int~st~nalis und ~ginaUs
des Menschen, Arch. Protistenk., 1910, xviii, 115.
6. Freund, H. A., Trichomonas hominis inlestinalis; Study of Its Biology andIts Pathogenicity, Arch. Int. Med., 1908, i, 28.
7. Kofoid, C. A., and Swezy, O., Mitosis and Multiple Fission in Trichomonade
Flagellates, Proc. Am. Acad. Arts and Sc., 1915, li, 289.
8. Alexeieff, A., Un nouveau trichomonas ~ quatre flageU6s ant6rieurs, Corapt.
rend. Soc. b/o/., 1909, lxvii, 712; Snr la sp6cification dans le genre trichomonas Donn6, 1911, lxxi, 539.
9. Bohne, A., and yon Prowazek, S., Zur Frage der Flagellatendysenterie,Arch. Protistenk., 1908, xii, 1.
10. Dobe11, C. C., On the Intestinal Protozoan Parasites of Frogs and Toads,
Proc. Cambridge Phil. Soc., 1906-08, xiv, 428.
11. Wenyon, C. M., A New Flagellate (Macrostoma mesnili, N. Sp.) from the
Human Intestine with Some Remarks on the Supposed Cysts of Trlchomonas, Paraslt., 1910, iii, 210.
12. Rodenwaldt, E., Flagellaten (Trichomonas, Lamblia), in "con Prowazek's
Handbuch, 1911, Liefg. 1, 78.
13. Parisi, B., Su alcuni flageIlati endoparassiti, Arch. Protistenk., 1910, xix, 232.

TOKUZO OHIK&ANDHIDEYONOGUCHI347EXPLANATION OF PLATES.
All the specimens were obtained from cultures of Tetratrichomonas hominis.
PLATE 34.
FIG. 1. Dark-field illumination of a colony of Taratrichtnmmas hom~is in culture. X 1,000.
PLATE 35.

Air-dried, fixed in methyl alcohol, and stained with Giemsa's solution.
with camera lucida, oc. 4, obj. ~ , oil immersion. Tube 160.
FIG. 2. Typical shape.
FIos. 3 to 9. Different shapes.
FIG. 10. A phase of binary fission.

Drawn

PI.AT~. 36.
Moist, fixed in Schaudinn's sublimate alcohol with a few drops of acetic acid,and stained with Heidenhain's iron and hematoxylin.
FIG. 11. Small type.
FIGs. 12 and 13. Large types.
FIGs. 14 and 15. Preliminary phases of binary fission.
FIG. 16. Beginning of binary, longitudinal fission of the protoplasm.
FIGs. 17 and 18. Final phases.
PLATE 37.
Fro. 19. A phase of multiple fission, showing six daughter nuclei and a mitotic
spindle of a basal granule.
Fro. 20. Final stage.
FIO. 21. Promitotic division of the nucleus.

THE JOURNAL OF EXPERIMENTAL MEDIOINE VOL. XXV,

PLATE 34.

(Ohira and Noguchi: Trichomonas of the Human Mouth.)T H E JOURNAL OF EXPERIMENTAL MEDICINE VOL. XXV.

Tokuzo Ohira fec.

PLATE 35.

(Ohira and Noguchi: Trichomonas of the Human Mouth.)THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XXVTokuzo Ohira fec.

PLATE 36.

(Ohira and Noguchi: Trichomouas of the Human Moutb.)

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XXV.

Tokuzo Ohira fec.

PLATE 37.

(Ohira and Noguchi: Trichomonas of the Human Mouth.)