野口英世著

Journal of Experimental Medicine

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E X P E R I M E N T S ON T H E C U L T I V A T I O N O F SO C A L L E D
T R A C H O M A BODIES.*
BY HIDEYO NOGUCHI, M.D., ANn MARTIN COHEN, M.D.
(From the Laboratories of The Rockefeller Institute for Medical Research,New York.)

PLATE46.
In 190 7 yon Prowazek and Halberstaedter 1 descri'bed certain
cell inclusions in the conjunctival epithelia of persons suffering fromtrachoma. The inclusions were composed of coarse coccoid andmore numerous minute granular bodies that were partially embedded in an irregular~ indefinite, and homogeneous substance ormatrix. The coarser bodies stained bluish and the minute ones
reddish in Giemsa solution. When the scrapings from the conjimctiva showing the cell inclusions were introduced into the eye oforangoutangs there appeared after several days in the conjunctiva
similar inclusions accompanied by a mild form of conjunctivitis.
The two disappeared together, yon Prowazek and Halberstaedter
concluded that the inclusions are the etiological agents of trachoma;
and yon Prowazek has proposed for them the name of chlamydozoa.
This discovery was speedily confirmed, and the bodies wereviewed as the etiological cause of trachoma when von Prowazek andHalberstaedter 2 detected similar inclusions in the conjunctiva incases of uncomplicated blennorrhea neonatorum. Thus a discussion
arose as to the specificity of the trachoma bodies, and Herzo~advanced the theory 3 that under certain conditions the gonococcusundergoes transformation into small forms which, when accumulated within the protoplasm of epithelia, assume the appearance ofso called trachoma bodies. Herzog believed that this transformation was accomplished by a rapid succession of transplantations of* Received for publication, July I, 1913.
z Halberstaedter, L., and yon Prowazek, S., Deutsch. reed. T4/chnschr., 19o7,xxxiii, 1285; Arb. a. d. k. Gsndhtsamte., 19o7, xxvi, 44.
2 Halberstaedter, L., and yon Prowazek, S., Berl. klin. H/ehnschr., 19o9, xlvi,IIIO.
3 Herzog, H., Arch. f. Ophth., 191o, lxxiv, 52o.
579.

Hideyo Noguchi and Martin Cohen.

573

the gonococcus in vitro, and he stated that trachoma is caused by
infection with the transfoi-med or mutated gonococcus (his microgonococcus). The present authors 4 studied.cell inclusions in various
conjunctival affections, and came to view the trachoma bodies as a definite pathogenic organism which can bring about a fairly well defined
clinical condition peculiar to itself. They regarded the occasional
occurrence in gonorrheal conjunctivitis of the gonococcus and thetrachoma inclusion bodies as accidental and coincidental; and they
considered that this coincidence could not be explained by the transformation of the gonococcus into the trachoma bodies or that thetwo separate etiological factors operate to produce trachoma. Williams, 5 on the other hand, has come to regard the inclusion as degenerated forms of the Koch-Week's bacillus.
Hence i.t appears that the so called trachoma bodies have beentreated as definitely living organisms by yon Prowazek who regards
them as a new protozoan; by Herzog who considers them a mutatedgonococcus; and by Williams who regards them as degenerated
Koch-Week's bacilli. Others consider them to be parasitic without
definite orientation, and still others as products of degeneration, due
to the invasion of cells by a still undetected etiological agent, analogous to those found in carcin.oma, ~variola, rabies, and some otherdiseases. It has not been shown that the inclusions are the parasites
themselves, and in order to settle this point proof must be broughtthat they are actually living, for which cultivation outside the body
supplies the clearest evidence. What follows is in the nature of apreliminary report of our studies, in which .the clinical part has
been contributed by Cohen and the cultural by Noguchi.
Sources of Materials.--Three different groups o f conjunctival
affections were selected: ( I ) cases of conjunctivitis combined withso called trachoma body inclusions; 6 (2) cases of typical trachoma
without ,the so called trachoma bodies; ( 3 ) c a s e s of acute catarrheal
conjunctivitis in which neither trachoma nor the cell inclusion affection occurred. To obtain the material the lid was inverted and carefully rinsed wi,th sterile physiological salt solution,' after which the4 Noguchi, H., and Cohen, M., Arch. Ophth., 1911, xl, I.
5 Williams, A. W., Arch. Ophth., 1913, xlii, 506.
6 Cohen, M., Arch. Ophth., 1913, xlii, 29.

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Cultivation of So Called Trachoma Bodies.

surface of the conjunctiva was scraped with the edge of a sterile slide.
The scraping was collected with a sterile platinum spatule andmixed in a tube with a small quantity of sterile ascitic fluid. In oldtracboma cases where the conjunctivae and tarsi were removed thetissues were immersed in a small amount of ascitic fluid to prevent
desiccation. The materials were carried to the laboratory without
delay.
Cultivation.--The usual culture media and methods were first
employed without success. Then the procedure used for cultivatingspirochmtm was adopted. 7 The scraping or piece of tissue wasplaced in a culture tube in which a piece of fresh sterile animal tissue(rabbi.t kidney) and a large quantity of sterile ascitic fluid werealready present. The tube employed measures 1.2 by 20 centimeters, and the amount of ascitic fluid used is about ten cubic centimeters in each tube. After the inoculation a layer of sterile paraffinoil (two or three centimeters high) is poured upon the ascitic fluid
and the tube is placed in an anaerobic jar. Usually two or threetubes are made with each specimen of scraping or tissue, andseveral specimens cultured on each occasion. Cultivation is conducted at 37 ° C.
The organism to be described was isolated in all cases of conjunctivitis with cell inclusions, and in one of three cases of trachoma
without the inclusions. It was absent from all the control cases.
The fact should be emphasized that the organism could not be cultivated at each trial, but at times repeated attempts were necessary forsuccess. The failure with old cases of trachoma may be due to theremoval of conjunctivae and tarsi, so .that further attempts at cultivation could not be made.
The tubes remain in the anaerobic apparatus about ten days, atwhich time films from each tube are made by removing the fluidwith a sterile capillary Pasteur pipette. The air-dried films are
fixed in methyl alcohol (Merck's reagent) for thirty minutes andstained with Giemsa solution for one hour. Films from cultures inwhich the inclusion ~bodies are growing show many extremely minutecoccoid organisms, mostly in pairs, but some in heaps of varying• numbers. The cultures contain, of course, ordinary bacteria, such
7 Noguchi, H., Jour. Exper. Med., 1912, xvi, 199, 211.

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575

as staphylococci, streptococci, diplococci and certain bacilli; but theminute size of the inclusion bodies and their reddish stain enables
one to recognize them without much difficulty.
The purification of the culture is the most tedious phase of thework, and much patience is required to accomplish it. The cultures
usually contain many organisms growing profusely on the surface
of ,the ascitic fluid agar, while the colonies of the inclusion organism
are slow to develop and extremely minute. They do not attain asize easily visible to the naked eye, and as they are almost transparent are easily overlooked. They do not grow on the surface of
the agar unless ascitic fluid or other suitable serum components arepresent The medium found best suited thus far consists of twoparts of slightly alkaline beef infusion agar and one part of ascitic
fluid with o.5 per cent. of glucose. To obtain colonies sufficiently
isolated for fishing many dilutions of the original culture must bemade; and inoculation may be made in the anaerobic apparatus orwithout it, but the former gives better results, since certain aerobic
organisms are partially or completely prevented from developing.
The minute organism is a facultative anaerobe which during thefirst two or three generations grows better in the absence ofoxygen, while in later generations it is unaffected by the air.
In order to obtain pure growth a culture showing discrete, almost
invisible colonies is selected, and by means of a fine platinum needle
a single colony is touched and transferred to a new tube of medium
containing a piece of sterile kidney and ascitic fluid. The sterile
paraffin oil havingbeen added, theembe is placed at 37 ° C. From now
on the anaerobic apparatus is not employed.' When transfers are
made into various other media without the addition of fresh tissue
the result is usually negative. For some generations the organism
fails to grow on the surface of agar even when ascitic fluid andglucose are present.
After three or four days the fluid culture shows slight turbidity
about the tissue, but no gross change takes place. Films made fromthe bottom of the tube and stained with Giemsa solution reveal theorganism, usually in two main and many intermediate forms. The
first form consists of rather large, lanceolate, or ovoid bodies, someoccurring singly, others in pairs and groups or rosettes (figures 3,576

Cultivation of So Called Trachoma Bodies.

4, and 5)- The second variety are extremely minute, round or
somewhat ovoid in shape, arranged usually in heaps or pairs (figures17 and I8). The coarse forms take a blue, ,the finer a red stain.
The two forms may be separate, but usually occur mingled in asingle mass, the coarser occupying the outer and the minuter bodies
the inner zone. A few coarse forms may lie among the minuteones, the two thus representing a typical trachoma body. Intermediate forms are always present, but they appear merely to constitute a stage during which the coarse forms are undergoing transformation into minute granules (figures 9, IO, 13, and 14). By
starting with a single colony and following its development day by
day it can easily be demonstrated that the different morphologicalelements are not due to contamination one with another. Thecoarse bodies predominate during the first two days, and are replaced by the minute ones. A comparison between the cultivatedorganism and the cell inclusions in corresponding developmental
stages reveals a striking similarity (figures I, 2, 6, 7, 8, II, 12, 15,and 16).
Although when freshly isolated the organism grows best underanaerobic conditions, after several generations a good growth may
be obtained in the presence of oxygen. During the anaerobic period
the organism develops only in media containing animal protein, but
after it has become aerobic it will grow without the addition ofserum, although development is less free than when the latter ispresent. The colonies developed on the surface of the agar slant
vary according to the degree of removal from the parent culture.
Thus, during the anaerobic phase the colonies assume the appearanceof minute, transparent, and almost invisible specks, whereas in theaerobic phase they are more distinct, though still extremely minute.
In the depth of the agar they develop throughout the entire column
within forty-eight hours as minute specks of greyish white color.
No turbidity is produced in serum agar and no gas in glucose media.
In neutral or slightly alkaline bouillon (beef infusion, peptone) theorganism grows slowly in fine, powdery, slightly opalescent masses.
The tissue at the bottom (if such has been added) becomes pale andgrey. No odor is produced in any medium. The best medium is
ascitic fluid tissue, covered with a layer of paraffin oil, in which theHideyo Noguchi and Martin Cohen.

577

organism grows well after forty-eight hours, the organisms remaining transplantable for a period of two months at 37 ° C. Blood is
not hemolyzed.
Morphological Proper~l;es.--This organism is Gram amphiol andthrows off the stain completely if decolorized long enough. It isdistinctly stained by carbolized fuchsin or gentian violet, and only
faimly by Loeftler's methylene-blue.
Various strains obtained from different cases present slight morphological deviations, and a strain obtained from one patient may
appear somewhat coarser than that derived from another. The
initial body forms may be arranged in rosettes or in~rregular heaps,
or even in short chains. The individuals in some s~ains are moreround or oval in shape, and in others preponderatingly spindleshaped, triangular, or crescentic. When the organism enters upon
the elementary body stage some strains form much more minutegranules than others. Similar variations occur among the cell inclusions which exist in cases of trachoma, blennorrhea neonatorum,and inclusion conjunctivitis.
Animvd Inoculation.--Repeated attempts to produce the cell inclusions in various kinds of monkeys (Macacus and Papio) by meansof pure cultures of this organism failed. In some instances atransient papillary conjunctivitis lasting about one week was produced, but the films showed only initial body forms, without cellinclusions.
SUMMARY.

I. An organism was isolated and studied in pure cultures fromcases of conjunctivitis accompanied by the so called trachoma, bodyinclusions, as well as from a case of old .trachoma without inclusions.
2. This organism was not found in the cultures made from otherforms of conjunctivitis in which the inclusions were absent.
3. The organism presents the morphological features characteristic of so called trachoma bodies. It undergoes an early transformation, during which the forms known as ini,tial bodies appear, anda later change, during which forms resembling elementary granules arise, while certain intermediate forms between these occur
simultaneously.
4- No definite cell inclusions could be produced in monkeys by
inoculating pure cultures of the organism.

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Cultivation of So Called Trachoma Bodies.

5. T h e c u l t i v a t i o n o f t h i s o r g a n i s m f r o m a c a s e o f t r a c h o m aw i t h o u t t h e cell i n c l u s i o n s , t o g e t h e r w i t h ~the p r e v i o u s l y a s c e r t a i n e df a c t t h a t t h e i n c l u s i o n s c a n 'be p r o d u c e d in s u i t a b l e a n i m a l s b y i n o c u l a t i n g s u c h m a t e r i a l d i r e c t l y f r o m a h u m a n case, s u g g e s t s t h e p o s s i b i l i t y o f t h e o r g a n i s m b e i n g still p r e s e n t in s u c h c a s e s o f t r a c h o m a ,a l t h o u g h n o t in t h e f o r m o f cell i n c l u s i o n s .
6. T h e f a c t s p r e s e n t e d j u s t i f y t h e s t a t e m e n t t h a t b y a s u i t a b l emethod an organism resembling the various important stages of thetrachoma bodies and totally distinct from the gonococcus has beeno b t a i n e d in a l i v i n g c o n d i t i o n c a p a b l e o f i n d e f i n i t e c u l t i v a t i o n f r o mcases of human trachoma and inclusion conjunctivitis. Whether
the organism and trachoma bodies are identical cannot be positively
stated at present.
B u t t h e w a y is n o w o p e n e d t o d e t e r m i n e t h i spoint, as well as the specificity of the trachoma bodies.
:EXPLANATION OF PLATE 46.
All the figures are from film preparations stained with Giemsa solution. Magnification M I,OOO.
FIG. I. Initial stage of the so called trachoma bodies in a conjunctival epithelium from a case of trachoma.
FIG. 2. Similar inclusion from a case of so called inclusion conjunctivitis.
FiGs. 3, 4, and 5. Initial stage of the cultivated organism, forty-eight hours
old in ascitic fluid tissue media.
FIGS. 5 and 7. Intermediary stages of the trachoma bodies from a case oftrachoma.
:FIG. 8. Intermediary stage of similar inclusion from a case of so called inclusion conjunctivitis.
FIGS. 9 and IO. Intermediary stage of the cultivated organism, three to fourdays old in ascitic fluid tissue media.
FIGS. II and 12. Inclusions consisting of intermediary and elementary formsof the trachoma bodies, from a case of trachoma and one of inclusion conjunctivitis.
FIGS. 13 and 14. The cultivated organism showing stages where intermediaryand elementary forms are mingled, four to five days in ascitic fluid tissue media.
FIGS. I5 and 16. Inclusions consisting exclusively of minute (elementary)
granules from similar cases.
FIGS. I7 and i8. The cultivated organism which entered the stage corresponding to the elementary granules of the inclusions, seven days in ascitic fluid
tissue media.T H E JOURNAL OF EXPERIMENTAL MEDICINE VOL. XVIII.

PLATE 48.

(Noguchi and Cohen : C u l t i v a t i o n of So Called Trachoma Bodies.)