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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 OF T H E
MICRO61~GANISM C A U S I N G E P I D E M I C
POLIOMYELITIS.*BY S I M O N F L E X N E R , M.D., A~D H I D E Y O N O G U C H I , M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research,
New York.)
PLATES 38 TO 4 I.

Several attempts have been made to demonstrate under the
microscope and to develop in artificial cultures the microSrganism
causing epidemic poliomyelitis, but hitherto unsuccessfully. Giersvold1 cultivated certain micrococci from the cerebrospinal fluid, andFox 2 a bacillus from the circulating blood of poliomyelitic patients,
but both have now been discredited as causes of the disease. The
discovery of the filterable nature of the causative agent independently by Flexner and Lewis 8 and Landsteiner and Levaditi 4
not only disposed finally of the claims made for the above mentioned bacteria, but also discouraged renewed efforts at cultivation.
Flexner and Lewi# noted and Levaditi 6 confirmed a clouding ofserum bouillon by an aqueous Berkefeld filtrate of the central
nervous tissues of poliomyelitic monkeys, but the phenomenon
proved to be due to protein precipitation and not to multiplicationof a living parasite. Proescher 7 has recently stained certain
coccus-like bodies in films prepared from the central nervous organsof monkeys infected experimentally with the virus of poliomyelitis.
The precise nature of these bodies has stil,1 to be worked out; ap* Received for publication, June 2o, I9I 3.
1 Giersvold, M., Norsk Mag. f. Lcegevidensk., I9O5, iii, I28o.
Dixon, S. G., Fox, H., and Fucker, J. B., Pennsylvania Department of
Health, Laboratory Report, March 2, 1911.
Flexner, S., and Lewis, P. A., ]our..4m. Med. Assn., 19o9, liii, 2o95.
4 Landsteiner, K., and Levaditi, C., Compt. rend. Soc. de blol., I9o9, lxvii, 592.
Flexner, S., and Lewis, P. A., ]our..4m. Med. Assn., 191o, liv, 45.
6 Levaditi, C., Presse todd., 191o, xviii, 44.
r Proescher, F., New York Med. ]our., 1913, xcvii, 741.

461

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Microgrganism Causing ElMdemic Poliomyelitis.

parently they have not yet been demonstrated in human nervous
tissues, and similar bodies have been found in the nervous organsof animals experimentally infected with rabic materials. The comparatively large size of many of these coccoid bodies leaves it
doubtful whether they can bear relation to filterable viruses.
The successful cultivation of spiroch~et~e, both parasitic andsaprophytic, by Noguchi s provided the impulse for a renewed
attempt at the cultivation of the micro6rganismal cause of poliomyelitis. The application by Noguchi of his method to the central
nervous tissues derived both from human and from experimental(monkey) examples of poliomyelitis has yielded results of an entirely different order from those previously obtained. The results
have already been described briefly in our Fifteenth Note 9 and they
will be described more fully in this place.
NATURE AND SOURCES OF MATERIALS.

During the several years that epidemic poliomyelitis has prevailed
in the eastern United States we have collected many specimens from
affected human beings and monkeys. Portions of nervous and otherorgans were regularly set aside in 50 per cent. sterile glycerin in the
refrigerator. Hence there were available for the cultivation tests
both fresh specimens yielded by recent autopsies upon human beingsand monkeys and material preserved for periods ranging from a
few days to many months. The new experiments upon cultivation
were begun during the summer of I912, at which time many acutecases of poliomyelitis were 'being cared for in the Hospital of theRockefeller Institute, where a number of autopsies came to be performed upon fatal cases. Thus the cultivation was carried out
simultaneously upon fresh, sterile, or practically sterile ( f o r ordinary bacteria) specimens derived from human beings and frommonkeys experimentally inoculated, and upon glycerinated specimens from both sources.
The material subjected to cultivation should be taken as soonafter the death of the person or animal as possible, and should beobtained when possible in an aseptic condition. While any parts Noguchi, H., ]our. Exper. Med., 1911, xiv, 99; 1912, xv, 90; xvi, 199, 211.
t~Flexner, S., and Noguchi, H,, Jour. Am. Med. Assn., 1913, Ix, 362.

Simon Flexner and Hideyo Noguchi.

463

of the central nervous system is suitable, the brain is to be preferred
for the reason that it can be obtained more readily than the spinal
cord or the intervertebral ganglia in an aseptic state. The dura
covering one of the hemispheres having been seared with a hot
instrument is incised with sterile instruments and the underlying
cortex scrupulously exposed. From this .a piece of about I:wo cubic
centimeters extending into the white matter is excised and placed atonce into a sterile dish. If desired, portions of the cerebellum
may be obtained in a similar manner, and by removing the cerebral
hemispheres and then lifting up the cerebellum the medulla may be
exposed in an aseptic manner, so that portions may also be taken.
It is advisable to make the initial inoculation with fragments aswell as with emulsions or with filtrates of the nervous tissues. The
fragments are to be preferred since they require almost no manipulation. When, however, the original material is suspected or
known to be contaminated, it is advisable to employ filtrates at thesame time. The filtrates are prepared by grinding the nervous
tissue in strength of about 5 per cent. with sand in distilled wateror physiological salt solution. The resulting emulsion is shaken
in the machine for about thirty minutes, after .which the suspended
matter is removed by centrifugalization and the supernatant fluid
is pressed through a Berkefeld filter, N. or V.
M E T H O D OF C U L T I V A T I O N .

The culture medium consists essentially of human ascitic fluid,
to which has been added a fragment of sterile fresh tissue. In
contradistinction to the spirochmtae, for which sheep serum watersupplies a suitable medium, the micro6rganism isolated from thenervous organs of cases of poliomyelitis demands ascitic fluid. In
obtaining the initial culture, the exclusion of oxygen is necessary,
but it is not, as in the case of Treponema pcdlidum, essential thatthe inoculated tubes should be placed in an anaerobic jar; it suffices
t o cover the liquid with a deep layer of sterile paraffin oil. In someinstances, however, the initial growth can be obtained more certainly
when the anaerobic jar is employed.
Test-tubes measuring 1. 5 by 20 centimeters are chosen. Into464

MicroSrganism Causing Epidemic Poliomyelitis.

each of a series a fragment of the usual size of sterile fresh
kidney of the normal rabbit is placed. Next is added a fragmentof corresponding size of the cerebrum or o'ther part of the brain or
spinal cord. Upon these are poured about fifteen cubic centimetersof sterile ascitic fluid and finally about four cubic centimeters of
the sterile paraffin oil. The ascitic fluid must have been originallysterile, since sterilization by fractional heating or filtration renders
it unsuitable. The control tubes consist of the ascitic fluid andkidney fragment wi.thout brain tissue and aseitic fluid plus brain
tissue without the kidney fragment. It is best to prepare two setsof the cultures, one to be placed in the anaerobic jar and the otherkept outside. The incubation is conducted at the temperature of
the thermostat, namely, at 37 ° C.
The culture tubes in the anaerobic jars should not be disturbed
for from seven to twelve days, while those not within the jar may
be inspected daily. Any .tube showing marked turbidity, coagulation, or gas production within one or two days may be set aside
as grossly contaminated. The tubes which remain clear or show
only slight turbidity may none the less be contaminated. To exclude contamination small quanti.ties of the medium are removed
with pipettes and stained for bacteria in the ordinary way andcultivated upon the usual solid or fluid media. Obvious bacteria
in the films as well as any growth taking place in the ordinary
media in the presence of a~r may be put down as contaminations
and the. tubes containing them discarded.
Gross contaminations having been excluded, the peculiar micro-organism obtained from the poliomyelitic nervous tissues exhibits
the following growth characters. At the expiration of about fivedays' incubation a faint opalescence appears about the fragmentsof tissue at the bottom of the tube. This opalescence can be
gradually diffused through the tube by gentle shaking (agitation),in the course of which it is observed that the turbidity about thetissues was really greater than was at first apparent (figure I, fluid
culture). The control tubes when not grossly contaminated either
remain perfectly clear or a slight granular precipitate of washed out
particles of tissue forms about them. The two appearances arequite distinct and become more pronounced after another period ofSimon Flexner avd Hideyo Noguchi.

465

three to five days, during which time the opalescence first described
extends into the upper portion of the medium, while in the controls
the precipitate gathers more and more at the lower end of the tubes.
At the expiration of ten or twelve days the diffuse opalescence ofthe medium in the tubes in which growth has been observed to takeplace begins to diminish as sedimentation sets in, during which
minute, irregular particles form, heavy enough to fall slowly to thebottom of the tube.
The corresponding tubes contained in the anaerobic jar when
examined on the seventh day show a similar opalescent growth but
slighter than that just described. When replaced in the jar andreturned to the thermostat the opalescence increases so that at the
expiration of another period of five or six days it approximatesthat present at the end of one week in the tubes kept outside the jar.
Cultivation may also be performed in a solid medium, consistingof ascitic fluid and sterile rabbi.t tissue, to which a suitable quantityof 2 per cent. nutrient agar has been added in order to produce asolid mixture. The melted agar, cooled to a temperature of 4o °C., is mixed with the ascitic fluid in the tube containing the fragment of tissue, also previously heated to 4 °o C. After cooling andsolidification the medium should be sufficiently firm to maintain its
stability. In this medium an initial growth, aside from contaminations, from poliomyelitic material has never been obtained, but once
growth has been secured in the fluid medium it is possible, but notin all instances, to transmit it to the solid medium. Of.ten successful transfer from the fluid to the solid medium is not accomplisheduntil about the third generation of the growth has been obtained in
the fluid medium. The changes indicative of growth in the solid
medium begin about the fragment of tissue and appear first as adiffuse opalescence which gradually aggregates into visible grains ofminute size .that are recognized as colonies. Several days areusually required before this initial change in the medium becomesapparent. The rapidity with which it does become apparent isaffected by the generation of the culture and hence by its adaptation
to the medium. Gradually the opalescence rises in the medium
within the tube, until i't reaches within ~bout three centimeters o f
the surface of the medium, at which a sh,arp line of demarcation

466

MicroSrganism Causing Epidemic Poliomyelitis.

appears. When the number of colonies developing is large theindividuals are so minute as to give a diffuse grayish appearance
to the medium, which requires a lens to bring out the separatecolonies. When the number of colonies developing is smaller they
may reach a diameter of about one th'ird of a millimeter 'and hence
become visible as such to the n'aked eye, to which they appear roundin shape and grayish white in color. Under favorable conditions
the opalescence indicating growth about the tissue fragment becomes apparent at the end of forty-eight hours and distinct after
seventy-two to ninety-six hours, although several days may be required before the ascent of the growth to the line of demarcation,
at which the presence of oxygen brings about a cessation ofdevelopment (figure 2, solid culture).
The description just given relates to cultivation with fragmentsof fresh central nervous organs in the ascitic fluid and in the presence of sterile rabbit tissue. It is, however, possible to secure
cultivation even in the absence of the rabbit tissue. For thispurpose a somewhat larger fragment of the poliomyelitic brain isplaced in the tubes containing the ascitic fluid, which is then covered
with the layer of paraffin oil. After incubation at 37 ° C. the initial
growth takes place, but with less constancy than when the rabbit
tissue is also present. When it occurs the growth is usually soslight that the opalescence remains absent or nearly so, and it
becomes necessary to transplant into a medium containing fresh
rabbit tissue to establish its existence.
Just as it is possible to eliminate the fresh rabbit tissue so is it
possible to dispense with the ascitic fluid, which may be substitutedby an extract prepared from the brain tissue or even sheep serum
water. The extract is prepared by grinding in a porcelain mill andthen afterwards shaking for half an hour in a machine twenty gramsof fresh normal brain of a Macacus rhesus monkey in 5oo cubic
centimeters of distilled water or Ringer solution. The emulsion iscentrifugalized and the supernatant fluid passed through a Berkefeld filter, V. The clear sterile filtrate is used to replace the ascitic
fluid, but in order to obtain growth in this fluid or in serum water
it is necessary to employ a fragment of rabbit tissue. A successful
result has never been obtained with either, in the absence of theSimon Flexner and Hideyo Noguchi.

467

rabbit tissue, from which it follows that they are inferior to theascitic fluid as a medium of cultivation.
In sever,al parallel experiments an emulsion of the brain tissue
was compared with the brain fragment as the source of the culture
and it w,as found that growth was obtained with greater difficulty
and somewhat less frequency from the emulsion than from thewhole tissue fragment. While growth was detected during thefirst week in the corresponding tubes of fragment and emulsion, aweek later the growth may have disappeared from the emulsion
tubes, from which it was concluded that certain constituents of thenervous tissue, liberated by the emulsion, exerted an injurious effectupon the developing micro6rganism. In support of this supposition
it was found that when an emulsion or extract of the brain wasadded to an active culture prepared in the usual way the microorganisms previously readily demonstrable in stained preparations
became indistinguishable. That they do not entirely disappear
under these circumstances is shown by the fact that when transplanted into the ascitic fluid tissue medium a growth reappears.
The Berkefeld filtrates of emulsified tissue also give rise to cultures, but less constantly than the original nervous material, fromwhich the filtrates were prepared. In cultivating with the filteredextract, .the ascitic fluid rabbit tissue medium should always be
employed, and two sets of tubes prepared, one to .be placed in theanaerobic jar and the other to be kept outside. The initial appearance of growth is somewhat delayed, so that an average of ten daysof incubation may be necessary before the characteristic alterationof the medium becomes apparent. In no instance was success attained with the filtrate in the absence of the rabbit tissue.
Glycerinated human and monkey poliomyelitic tissues were employed as fragments, and, in the case of monkeys, as filtrates forcultivation. The specimens had been kept in 5° per cent. glycerin
at a temperature of 2 to 4 ° C. for periods varying from twentyfive days to one year. Portions of the brain or spinal cord sopreserved were cut off with sterile scissors, washed thoroughly insterile distilled water, and placed in the ascitic fluid tissue medium.
The resulting growths, when they occurred, produced the characteristic appearances already noted. On the whole, the glycer-

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Micro6rganism Causing Epidemic Poliomyelitis.

inated tissues gave somewhat more inconstant results than the freshmaterial.
Once the initial growth from any of the sources mentioned has
been secured it is, as a rule, possible to obtain subcultures by transplanting about o.2 of a cubic centimeter of the original culture pertube into the next series. Since the initial growth begins about
the tissue at the bottom of the tube it is advisable to take thefluid for transplantation from this location. Subcultures are obtained more constantly in the fluid than in the solid medium.
Hence growth will sometimes appear in the former and not in thelatter. Once the micro6rganism has been adapted to the conditionsof artificial cultivation, transplantation to the solid medium may beeffected. The adaptation is indicated by a more rapid and abundant growth in the fluid medium, and its occurrence is promoted by
the presence of fresh sterile tissue and is prejudiced by its absence. Among the monkey tissues was one specimen which had
been glycerinated for nine months, and from which a growth was
obtained with a Berkefeld filtrate.
In the course of the growth the sterile kidney turns first pink
and then gray in color, but no obvious solution or hemolysis of the
red corpuscles contained in the tissue occurs.
The addition of various carbohydrates and alcohols, such as
glucose, saccharose, lactose, maltose, gelactose, levulose, mannite,
glycogen, dextrin, inulin, dulcite, and arabinose, to the ascitic fluidor sheep serum water in proportion of o. 5 per cent. did not affectthe cultivation, and the growths when pure had no apparent effectupon them, since neither acid nor gas are produced.
Litmus milk, to which fragments of the sterile fresh tissue havebeen added, affords a medium in which growth will take place without producing any alteration in the appearance of the medium.
N U M E R I C A L R E S U L T S OF C U L T I V A T I O N .

In a series of thirty-three experiments with the nervous tissue ofmonkeys, comprising thirteen specimens of brain tissue, two of brain
emulsion, and eighteen filtrates of brain and spinal cord fromtwenty-four animals infected from two different strains of humanpoliomyelitic virus, an initial growth was obtained in nineteen in-Simon Flexner and Hideyo Noguchi.

469

stances, of which sixteen proved to be pure and three mixed cultures. Of these, pure subcultures were obtained thirteen times,
namely, eight times from brain tissues, four times from filtrates,and once only from emulsion. In a second series in which humannervous tissues were directly employed, eight successful cultivations
were secured. One was from fresh tissue, three were from recently
glycerinated tissues, and four from tissues which had been glycerinated for many months. In two specimens of the last four, initial
cultures but no subcultures were obtained. In all the others subcultures resulted and were maintained alive for an indefinite period.
MORPHOLOGICAL CHARACTERS.
Uncontaminated cultures of the microSrganism from the various
human and monkey sources already mentioned possess common
morphological and staining characters. Fluid cultures viewed
under the dark-field microscope exhibit among the innumerable
dancing protein and other granules present minute bodies, globular
in form, hanging together in short chains, pairs, and small masses,
devoid of independent motility and distinguishable with difficulty
as a special class among the indefinite granules present. Stained
preparations, on the other hand, bring out unmistakable microorganisms grouped in the three ways stated and of very minutesize. Staining may be accomplished with more or less difficulty inseveral ways, but the two methods which have thus far yielded themost satisfactory results are those of Giemsa and Gram.
The Giemsa stain may be applied in two ways according as a
rapid or a somewhat slower but better result is desired. In thefirst, the film, either upon a slide or a cover-glass, is allowed to
become air-dry, when it is fixed over the flame, and several drops ofGiemsa solution are dropped upon it and diluted with about twenty
drops of distilled water. After gentle mixing of the dye andwater the preparation is held over the flame until a trace of vapor
rises. Care is exercised to avoid boiling. The preparation is thenwashed in a stream of distilled water, pressed between blotters, anddried. This method does not yield uniform staining or constancyof shade, but is useful for rapid examinations. The second method
is the one usually employed. The film having been air-dried is

470

Micro6rganism
Causing Epidemic Poliomyelitis.

fixed in methyl alcohol for about an hour, washed in distilled water,
and is then immersed in the solution containing one drop of thestain per cubic centimeter of distilled water, in which the fihn remains for from two hours to twelve to fourteen hours (over night).
Gram's stain is applied in the usual way, using gentian violet as thedye and safranin as the counterstain. The micro6rganism behavesvariably towards the .Gram stain. When the medium in which itis grown contains peptone, it retains the stain more assiduously thanwhen it is devoid of that substance. Then the in,tensity with which
the Gram stain is retained varies with the age of the culture. The
older the culture the more strongly the gentian violet is held. While
the grouping is of course identical whichever stain is employed, theapparent size of the micro6rganism is affected by the dye. Whenthe gentian violet is retained the individuals appear larger than they
do with either the safranin or the Giemsa stain. Loeffler's alkaline
methylene-blue, carbol-fuchsin, and' carbol-gentian violet all stain
the microgrganism slightly or without affording sharp differentiation. The microsc<~pical appearances of the mi.cro6rganism vary
somewhat according to whether the growth is in a fluid or solid
medium and peptone is present or absent. Peptone and possibly
other constituents of the bouillon render the microSrganisms somewhat larger as well as enable them to retain more firmly the Gram
stain.
Films prepared from the lower layer of the ascitic fluid culture
about the sterile rabbit tissue and stained by Giemsa solution
reveal the presence of a variable number of minute globoid bodies,arranged in pairs or short chains, or in small aggregated masses.
In the fluid culture the pairs and chains predominate (figures 3, 4,
and 5)- According as the staining operation was carried out by the
rapid or slower method, the globoid bodies assume a bluish or aviolet color; rarely they present a red tint. Since the most uniform
results are obtained by the slower method of staining, it may beconsidered that the violet coloration is the normal. The individual
microSrganisms average about o.2 of a micron in diameter, the limitsof visible bodies being o.I 5 to o.3 of a micron. Possibly still smaller
individuals exist that exceed the limit of visibility. For purposes ofcomparison a group of chain forms has been compared by photography with Streptococcus pyoge~ws (figure 6).

Simon Flexner and Hideyo Noguchi.

471

Considerable difficulties may be encountered in demonstrating the
microSrganism in initial cultures not older than four or five days.
Either nothing whatever that ,can be recognized as mieroSrganisms
may be detected, or merely roundish bodies the nature of which it
may be difficult to decide. When the same tubes are examined on
the sixth or seventh day the unmistakable minute globoid bodies
are usually detected. Although the original growth lends to appear
slowly, the final multiplication takes place rapidly, and is completed
from about the eighth to the twelfth day, after which the cultures
remain stationary for a variable period. Kept in the thermostat
the microSrganisms may remain largely unchanged for many days,
and in the refrigerator for many weeks.
Within the solid cultures the micro/Srganism develops not as
chains but as pairs and aggregated groups. The arrangement isvery readily ascertained by compressing a fragment of the agar
between two slides, which are afterwards separated. The resulting
films having been stained in the usual manner by Giemsa or Gram
solution, the minute particles of agar adhering to the glass will frequently exhibit colonies, of which the members are sufficiently
separated to expose the grouping (figure 7). In the Gram-stained
films the bodies are somewhat coarser than in the Giemsa-stained
ones. In young agar cultures the Gram-stained bodies are partly
purple and partly pink, while in older cultures almost all appear
purple.
After a somewhat variable period, and generally from ten days tothree or four weeks after the maximum growth has been obtained,
certain metamorphoses or degenerations set in. Probably the enlarged and irregular stained bodies are to be viewed as degenerations, while the minute fragments just at the limit of visibility may
possibly represent a metamorphosis into a minuter variety withwhich submicroscopic forms may ,be associated. When the conditions of culture are unfavorable, as, for example, when the brain
extract or other medium without sterile tissue is employed, this
supposed metamorphosis appears to arise earlier, and indeed within
the first few days of growth. Subcultivation from one of thealtered cultures or from their Berkefeld filtrates yields frequently
but not uniformly new developments of the visible microSrganisms.

472

MicroSrganism Causing Epidemic Poliomyelitis.

The important question, as ~co whether the cultures just mentioned
contain .actual ultramicroscopic forms of the micro6rganism, orwhether the failure to detect the visible forms is due .chiefly to dilution, must remain for the present undecided. Wha~t is certain,
however, is that the globoid organisms are capable of passing
though Berkefeld filters which exclude the usual test bacteria(e. g., Micrococcus prodigiosus). Cultures from the Berkefeld
filtrates should always be made in the asci,tic fluid tissue medium, as
in the case of the initial growth.
The film preparations stained b y the Giemsa stain and especiallyby the rapid method sometimes show the globoid organisms surrounded by a zone either faintly tinted or colorless, that may equalin width the diameter of the individual miero6rganisms. Single
bodies, aggregated groups, or chains may be thus enveloped. The
nature of the material .composing the zone is unknown, as isalso whether it represents an independent part of the organism(capsule?) or is derived from the culture medium.
I N F E C T I O N OF M O N K E Y S .

The microSrganism described is present so constantly in thecentral nervous tissues of affected human beings and monkeys thata strong presumption exis~ts to the effect that it bears some relationship to epidemic poliomyelitis. It is sufficiently obvious that whatever they may ultimately prove to be, as far as we now know the
globoid bodies are not common pathogenic or even saprophytic
microgrganisms. Whether ~the bodies are limited to the central
nervous organs or occur more widely distributed in the body of
infected human beings and monkeys is still to be determined. The
question which presses at the moment is whether the cultures arecapable of setting up in monkeys clinical and pathological statescorresponding with those characteristic of experimental poliomyelitis. Before proceeding to describe the inoculation experiments, it may be well to point out tha:t apparently the globoid bodies
are highly parasitic micro6rganisms. In view of this circumstanceit may readily happen that they may quickly lose pathogenic properties in artificial cultures. There are of course several instances
known among the bacteria and spirochmt~e of which this circum-

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473

stance is true. We were therefore prepared to encoumer difficulties in the way of infecting monkeys with cultures. It is necessary to recall the difficulties surrounding the original implantationof highly infected human poliomyelitic material upon monkeys inorder to have presented emphatically the prolbable obstacles thatstand in the way of the successful accomplishment of the infectionof monkeys with the cultures.
Two series of inoculations were made. In one the culturesderived from the nervous tissues of monkeys and in the other fromhuman beings directly were separately inoculated into Macacus
rhesus monkeys. The inoculations were made either into the brain
or into the sciatic nerve and peritoneal cavity simultaneously.
Before describing ~he several instances of successful inoculation,certain important points should be presented. It is known that thevirus in an emulsion or filtrate prepared from the nervous tissuesof infected monkeys survives in the thermostat and remains infectious for at least thirty days, and, also, that the filtrate soon
becomes inactive after being transferred to a new fluid medium
consisting of serum bouillon. Possibly the inactivity arises chiefly
from the great dilution which the virus has undergone. Whence
it follows that dilutions of the virus beyond the point at which infection through intracerebral inoculation of several cubic centimeters of suspension can be produced are easily secured. We havefrequently had occasion to confirm this general fact, since second
transfers into the fluid medium from a supposed culture derived
from monkeys previously inoculated with the highly active M Avirus have in many instances failed to cause infection even when
injected intracerebrally. Dilution ,beyond the point of infectivityof the virus, even in the culture medi,a, thus is readily accomplished.
Bearing these facts in mind it follows that successful inoculation
with culture generations still more remote than the second dilutiono~[ the original virus is to be interpreted as representing, probably,
an implantation of the artificially cultivated micro6rganism upon
the susceptible monkeys. Moreo.ver, from the monkeys in which
experimental poliomyelitis has been thus produced the micro/brganism introduced can be recovered in culture and even implanted successfully again.

MicroSrganism Causing Epidemic Poliomyelitis.

474

T h e i m p l a n t a t i o n u p o n m o n k e y s o f the v i r u s c o n t a i n e d in theo r i g i n a l h u m a n n e r v o u s tissues is a d m i t t e d l y difficult.
When
filtrates are i n o c u l a t e d failure usually follows. W h e n e m u l s i o n s
are e m p l o y e d , i n f e c t i o n o c c u r s in f r o m a b o u t 5o t o I o o per cent.
o f the instances, p r o b a b l y d e p e n d i n g u p o n v a r i a t i o n in the d e g r e e o f
i n f e c t i v i t y .of the original virus. T h e h u m a n strains o f t h e v i r u s
are less i n f e c t i o u s f o r m o n k e y s t h a n the a d a p t e d o r so calledm o n k e y strains, a fact t h a t is the m o r e significant as respects the
successful inoculation o f c u l t u r e s d e r i v e d directly f r o m h u m a ntissues.

Cultivated H u m a n gtrains.--Fluid a n d solid c u l t u r e s

in thet h i r d g e n e r a t i o n f r o m f o u r h u m a n sources, n a m e l y , D., Kr., Ko.,a n d F., w e r e m i x e d .
E a c h c u l t u r e h a d been o b t a i n e d f r o m recently g l y c e r i n a t e d specimens o f the central n e r v o u s system.

Experiment A.--Macacus rhesus. December 27, I912. 0.5 c.c. of the mixture was injected into the left sciatic nerve and 0.8 c.c. into the left cerebral
hemisphere.
December 30, 1912. Animal less active than normally; legs and arms weak.
December 31, I912. Excitable; uses legs awkwardly. Etherized.
Autopsy.---At the site of intracerebral inoculation punctiform hemorrhages;
the left lateral ventricle contains a small, dark clot; the cervical, dorsal, andlumbar spinal cord appear congested; the left sciatic nerve is slightly swollen
at site of injection. Film preparations from the two inoculation sites show no
ordinary bacteria. T h e microscopical examination of the medulla, cervical,
dorsal, and lumbar levels of the spinal cord, and intervertebral ganglia from thecervical and lumbar regions shows in all typical and marked characteristic
lesions of experimental poliomyelitis. The lesions consist of degeneration of
the nerve cells, associated with neurophagocytosis, infiltration with mononuclear
cells of the ground substance, and perivascular and meningeal infiltration(figures 8, 9, and IO).
Experiment B.~Macacus rhesus. December 31, 1912. A suspension of the
medulla and spinal cord of monkey A was injected into left sciatic nerve andleft cerebral hemisphere.
January I to January 4, I913. Increased excitability.
January 6, 1913. Excitable; vision defective; ataxia.
January 7, I913. Arms weak, right more than left.
January 8, 1913. 9.30 A.~t., prostrate; respiratory paralysis. Death at IO a.~.
Autopsy.---A small cyst with clear contents at the site of injection. Sciatic
wound healed. Congestion of gray substance of spinal cord.
The microscopical examination exhibits marked lesions of experimental
poliomyelitis in the spinal cord, medulla, and intervertebral ganglia. Portionsof the nervous system were placed in 50 per cent. sterile glycerin.
Experiment C.--Macacus rhesus. May I4, 1913. Inoculated intracerebrallywith emulsion of glycerinated spinal cord and medulla ot~ monkey B.

Simon Flexner and Hideyo Noguchi.

475

May 18, I913. Excitable.
May 19, I913. Arms weak.
May 20, 1913. Arms and legs paralyzed.
May 21, 1913. Etherized.
-/lutopsy.--A small cyst at the site of inoculation. At various levels of the
spinal cord are visible lesions.
Microscopical examination shows marked and characteristic lesions of experimental poliomyelitis in the medulla, spinal cord, and intervertebral ganglia.
Cultures in the ascitic fluid tissue m e d i u m were m a d e at autopsy
f r o m m o n k e y s A, B, and C. F r o m m o n k e y s A and B the original
m i c r o 6 r g a n i s m injected was recovered in pure culture. T h e culturef r o m m o n k e y C was unsuccessful. H e n c e a n o t h e r portion o f theoriginal glycerinated m a t e r i a l was inoculated as follows :Experiment D.---Macacus rhesus. June 7, 1913. Intracerebral injection ofemulsion of glycerinated brain and cord from monkey B.
June 12, 1913. Excitable; tremor; prostrate.
June 13, 1913. Etherized.
Autopsy.--A small cyst at the site of inoculation; visible lesions in the spinal
cord.
Microscopical examination shows typical lesions of experimental poliomyelitis
in the medulla, spinal cord, and intervertebi'al ganglia.
Cultures in the ascitic fluid tissue m e d i u m f r o m this animal g a v e
a pure g r o w t h o f the m i c r o 6 r g a n i s m originally injected into m o n k e y
A, the beginning of this serie's, m o r e t h a n six m o n t h s previously.
T h e successful culture in this instance indicates that the failure tor e c o v e r the m i c r o 6 r g a n i s m f r o m m o n k e y C was due to a technicalfault.
Cultivated Monkey Strains.--These strains consisted o f cultivations f r o m the highly adapted M A virus which had been longp r o p a g a t e d in m o n k e y s f r o m emulsions or filtrates o f the nervous
organs. H e n c e the cultures, while representing the s a m e original
virus, varied in precise generation according to the period at which
they were isolated. All w e r e f a r r e m o v e d f r o m the original hum a n strain.

Experiment E.--Macacus rhesus. December 3, 1912. Two cultivated strains,
one in the third and the other in the fifth generation, were combined, and of the
mixture I e.c. was injected into each sciatic nerve, and IO e.¢. were injected into
the peritoneal cavity.
December 6, 1912. Excitable; coat roughened.
December 9, 1912. Somewhat less excitable.

476

MicroSrganism Causing Epidemic Poliomyelitis.

December 23, I912. Again excitable. The monkey remained excitable continuously, and on March 19, I913, was found in convulsions, which recurred at
frequent intervals, until on March 25 death occurred.
The microscopical examination reveals focal lesions in the medulla, consistingof minute hemorrhages, small aggregations of mononuclear cells, partly in the
ground substance and partly about the small vessels, and a few small vascularthrombi. Small hemorrhages also occur in the crura cerebri, and in the cervicalregion of the spinal cord, in which slight perivascular infiltration with mononuclear cells is also present.
Cultures yielded the original micro6rganism.£ :T h e i n t e r p r e t a t i o n o f e x p e r i m e n t ; ! ~ s n o t p e r f e c t l y clear.
Thelong intervening period between the inoculation and the developmento f c o n v u l s i o n s a n d o c c u r r e n c e o f d e a t h is n o t s t r i c t l y p a r a l l e l e d b yt h e effects, o f d i r e c t i n o c u l a t i o n o f e i t h e r e m u l s i o n o r f i l t r a t e o f t h e
n e r v o u s tissues. M o r e o v e r , . t h r o m b i d o n o t o c c u r in u n c o m p l i c a t e d
i n s t a n c e s o f e x p e r i m e n t a l p o l i o m y e l i t i s . O n .the o t h e r h a n d t h e
i n f i l t r a t i v e l e s i o n s a r e s i m i l a r to t h o s e p r e s e n t in e x p e r i m e n t a lpoliomyelitis.

Experiment F. Macacus rhesus. December 27, 1912. Injected into left
cerebral hemisphere o.8 c.c. of cultivated virus, fifth generation.
January I, 1913. Excitable; arms weak.
January 2, 1913. Excitability continues; arms and back partly paralyzed;
tremor of head.
January 3, 1913. Legs weak.
January 4, 1913. Animal prostrate. Etherized.
Autopsy.--A small cyst at the site of injection. Spinal cord edematous.
No visceral lesions.
Microscopical examination shows marked characteristic degenerative andinfiltrative lesions of experimental poliomyelitis in the medulla, spinal cord, andinter'vertebral ganglia.
Cultures yielded the original micro6rganism.
Experiment G.--Macacus rhesus. January 27, 1913. Injected into left cerebral hemisphere 2.5 c.c. of culture in fifth generation from monkey E.
February I, 1913. Excitable; movements uncertain; vision appears to be
defective.
February 2, 1913. Neck and back paralyzed; unable to sit up; convulsions incourse of which death occurs.
Autopsy.--February 3, 1913. The general viscera are apparently normal. A
small cyst the size of an almond is present at the point of inoculation in left
hemisphere. Gray matter of medulla and spinal cord is congested.
The microscopical examination of the medulla, spinal cord, and intervertebral
ganglia shows typical lesions of experimental poliomyelitis.
Cultures yielded the original microSrganism.
Experiment H.--Macacus cynomolgus. February 5, 1913. Injected into theleft cerebral hemisphere 2.5 c.c. of a Berkefeld filtrate prepared from the siteof inoculation of monkey G.

Simon Flexner and Hideyo Noguchi.

477

February 6, 1913. Shows tendency to incline toward right side; does not
use the right arm, which is spastic, the result probably of injury from theinj ection.
February 7, 1913• Death at IO A.~.
Autopsy.--At the site of inoculation there is a cyst containing blood stained
fluid communicating with the lateral ventricle.
Microscopical examination shows marked lesions in the intervertehral ganglia
and lesions of the blood vessels adjacent to the site of inoculation resembling
those characteristic of experimental poliomyelitis. The spinal cord also reveals
fine lesions 6f the same nature. Hence it appears that within the short periodof forty-eight hours the specific lesions developed throughout a wide area of
the nervous system of this animal.

~J ~ - . Experiment I. Macacus rhesus.

February 8, 1913. Repetition of experim e n t ~ . Injected into the left cerebral hemisphere I c.c. of filtrate from site of
inoculation of monkey F.
February 14, 1913. Prostrate; legs, back, and arms paralyzed. Etherized.
./tutopsy.--A small, blood-containing cyst at the site of inoculation. Spinal
cord throughout shows gross lesions of the gray matter.
The microscopical examination confirms the character of these lesions. Spinal
cord, intervertebral ganglia, and medulla all show marked degenerative andinfiltrative lesions of poliomyelitis, and identical lesions occur in the crura
cerebri and in the cerebral hemispheres.
Experiment J.--Macacus rhesus. February 13, 1913. Suspension of glycerihated brain and spinal cord of monkey F injected intracerebrally.
February 18, 1913. Death.
./tutopsy.--A cyst the size of an almond containing clotted blood occurs at
the site of inoculation. Throughout the spinal cord the gray matter is congested.
Microscopical examination shows marked characteristic lesions of experimental poliomyelitis.
Experiment K.--Macacus rhesus. May 14, I913. Injected intracerebrallywith an emulsion of glycerinated spinal cord and medulla obtained frommonkey :]~ F.
May 18, 1913. Distinctly weak.
May 19, 1913. Ataxic.
May 2o, 1913. Paralysis of arms, legs, and back.
May 21, 1913. Etherized.
./tutopsy.--At the site of inoculation there is a ~small cyst containing blood.
Visible lesions in the spinal cord.
Microscopic examination reveals marked characteristic poliomyelitic lesionsof the medulla, spinal cord, and intervertebral ganglia.
Culture yielded microSrganism identical with that inoculated in monkey F
almost six months previously. This culture in the fourth generation was inoculated intracerebrally on June II, into a Macacus rhesus. On June 16 themonkey was weak, slow, and ataxic; it was etherized on July 2. The microscope reveals typical lesions of the brain and spinal ganglia, or, in other words,
evidences of the encephalitic type of poliomyelitis.
Experiment L.--Macacus rhesus. January 13, 1913. Injected into left cerebral hemisphere I c.c. of cultivated virus, sixth generation.

478

Micro6rganism Causing Epidemic Poliomyelitis.

January 16, 1913. Movements irregular; tends to move in circles.
January 17, 1913. Apparently normal.
February 6, I913. Ataxic; excitable, arms and legs weak.
February lO7 1913. Weakness of arms and legs increased. Etherized.
~tutopsy.--No lesion at the site of inoculation, and no gross changes in the
spinal cord.
Microscopical examination shows fine infiltrative and degenerative lesionsof the medulla, spinal cord, brain, and intervertebral ganglia.
Culture yielded micro6rganisms identical with that inoculated.
Experiment M. Macacus rhesus. February II, 1913. Injected emulsion of
the spinal cord and brain of monkey K into the sciatic nerve and peritoneal
cavity.
February 14, 1913. Excitable; convulsions.
February 15, 1913. Legs weak.
March I, 1913. Excitability continues; movements slow and restricted.
March 5, 1913. Condition unchanged. Etherized.
Microscopical examination shows typical poliomyelitic lesions of the intervertebral ganglia in the lumbar and cervical regions, and similar but inconspicuous lesions of the spinal cord.
Experiment N.--Macacus rhesus. February 27, 1913. Inoculated into left
hemisphere with 2 c.c. of monkey culture from two sources in the eighteenthand twentieth generation respectively.
March i, 1913. Inclines slightly towards the right side.
March 2, I913. Death.
Autopsy.--At the site of inoculation is a cyst the size of an almond containing clear fluid and communicating with the lateral ventricle; gray matter ofthe spinal cord is congested.
Microscopical examination of the cyst shows the presence of a Gram-positive
coccus (mixed infection).
Experiment O.--Macacus cynomolgus. March 4, 1913. 2 c.c. of Berkefeld
filtrate from the site of inoculation of monkey N are injected intracerebrally,
and IOO c.c. intraperitoneally.
March 17, 1913. Left arm weak; legs somewhat spastic.
March 18, 1913. All extremities weak and spastic.
March 19, 1913. Head retracted; convulsi0ns; unable to rise. Etherized.
Autopsy.--No lesion at the site of inoculation. A placque jaune 1.5 by 2 cm.
occupies the surface of the cortex on the left side, posterior to the motor area.
General viscera normal
Microscopical examination reveals characteristic focal lesions of experimental poliomyelitis in the intervertebral ganglia, and vascular and interstitiallesions of the spinal cord, crura cerebri, and cerebral hemispheres.
B e c a u s e o f c o n t a m i n a t i o n w i t h m i c r o c o c c i a t t h e i n o c u l a t i o n sitein m o n k e y N, a f i l t r a t e p r e p a r e d f r o m t h e l o c a l site w a s i n j e c t e d
i n t o m o n k e y O, r e s u l t i n g in c o m b i n e d flaccid a n d s p a s t i c p a r a l y s i s .
However, the sections of the nervous tissues of monkey O byshowing lesions of experimental poliomyelitis indicate the existenceof that disease.

Simon Flexner and Hideyo Noguchi.

479

The preceding experiments show unmistakably that the inoculation of the cultures is followed by the appearance of the clinicalsymptoms and pathological effects characteristic of experimental
poliomyelitis in the monkey. The two methods of inoculation now
chiefly practised with the usual suspensions or filtrates of nervous
tissues were employed successfully with the cultures. The essential
results are identical whether the cultures are inoculated into thebrain or simultaneously into sciatic nerve and peritoneal cavity.
The inoculation of the cultures produces in monkeys no direct
effect. Immediately after injection the animals appear normal andthey remain apparently in this .condition for an incubation period
which may be as brief as forty-eight hours, but commonly enduresfor one to several weeks, at the expiration of which the usual
symptoms of excitement, tremor, weak and then paralyzed muscles
succeed one another. Death may arise from prostration, convulsions, or paralysis o f the respiration, although in most of theinstances related it was artificially hastened by etherization.
The central nervous tissues of the monkeys may be employed forrecultivation of the micro6rganism inoculated, or for transmitting
the experimental affection to still other and probably an indefinite
series of monkeys. This transmission can be accomplished eitherwith enmlsions or Berkefeld filtrates of the fresh or glycerinatedorgans. Since the experimental disease is accurately reproducedin this manner, it follows that not only the cultures but the nervous
tissues infected with them possess the distinctive disease-producing
properties of the virus of poliomyelitis as commonly understood.
The local effects of the cultures vary with the inoculation site
and the dosage. Unless secondary infection arises from the outside, the intrasciatic and intraperitoneal inoculations cause no considerable reaction, while the intracerebral injection leads to theformation of a small cyst, in which the introduced micro6rganism
may or may not be readily discoverable in film preparations. I f
relatively few of the micro/Srganisms are inoculated, they tend todisappear from view; while if many are introduced, they are readily
detected, either lying free or enclosed in emigrated polynuclear ormononuclear cells (figure I I ) . When pyogenic micrococci enterfrom the skin, the local lesion tends to be larger, and to contain480

MicroSrganism Causing Epidemic Poliomyelitis.

many pus cells, and the two micro6rganisms may appear side byside, both without and within cells. When an excess of the mixed
fluid and solid cultures is injected into the brain, the local effect isalso more severe and many leucocytes may be called out by the
positively chemotactic elements contained within the medium. The
action of contaminated cultures is less readily interpreted than are
the effects of pure cultures; but any doubt may be ~'emoved by preparing a Berkefeld filtrate from the local lesion and portions of the
medulla, spinal cord, and intervertebral ganglia, which is to be
employed for inoculation.
The disappearance of the micro6rganism from the inconspicuous
local lesions probably results from a general diffusion throughout
the nervous organs and restricted multiplication at the site of injection. The micro6rganism can indeed be recovered in culturesfrom any part of the central nervous system, and is best recoveredfrom the intact cerebral hemisphere, where it usually exists in apure state and is readily accessible under aseptic conditions.
R E L A T I O N OF T H E MICRO(~RGANISM TO NERVOUS TISSUES.

The micro6rganism the cultures of which have been describedcan be detected in film preparations and in sections prepared fromthe central nervous organs of human beings who have succumbedto poliomyelitis and of monkeys subjected to inoculation with theordinary virus, or with cultures. A special technique which has
been devised by Noguchi is required for the demonstration.
Staining Method for Film Impressions.--I. Cover-glasses ofsuitable size and thinness (No. I or o) receive a succession of impressions from the selected piece of nervous tissue.
2. After becoming air-dry the cover-glass is placed, film side
down, in a mixture which is freshly prepared at each operation,
consisting of one part of Grfibler's Giemsa solution, and two partsof Merck's methyl alcohol reagent, where it remains for two
minutes. In order to prevent the film surface from contact withthe bottom of the staining dish, it is well tosupport one end upon adrawn out glass capillary tube. The staining vessel may conveniently be a small Petri dish .and cover.
3. Twenty parts of a i to io,ooo potassium hydrate solution areSimon Flexner and Hideyo Noguchi.

481

poured into the dish at the expiration of the two minutes, and thewhole is thoroughly mixed by gentle agitation. At the expirationof one hour the cover-glass is removed.
~. The film side is washed in distilled water for a few seconds,
after which it is differentiated in a solution of tannic acid, consisting of one or two drops of a 2o per cent. solution, added to forty
cubic centimeters of distilled water. This operation requires
several seconds and careful observation.
5. The cover-glass is now washed in distilled water for two
minutes, dried in the air, and mounted in cedar oil.
Staining Method for Sections.--The nervous tissues having been
hardened in Zenker's solution or sublimate alcohol are embedded ,inparaffin and the sections are mounted upon slides in small series.
I. The sections are treated with Lugol's solution and afterwards with a 0. 5 per cent. solution of sodium hyposulphite, after
which they are thoroughly washed in distilled water.
2. The sections are passed successively through 95 per cent.
alcohol, absolute alcohol, chloroform, aceton, benzol, ether, absolute
alcohol, 95 per cent. alcohol, and finally distilled water, in each ofwhich reagents they remain about one hour.
3- The staining solution consists of three cubic centimeters ofconcentrated Giemsa solution to forty-five cubic centimeters of
distilled water, in which the sections remain for twenty-four hours.
4. After brief washing in distilled water, the sections are rapidly
passed in Succession through aceton, ace'ton thirty parts plus xylo]
seventy parts, aceton fifty parts plus xylol fifty parts, aceton thirty
parts plus xylol seventy parts, and finally pure xylol.
5- The cleared sections are mounted in cedar oil.
With these methods the minute microSrganism has been demonstrated in the films in eighteen out of nineteen specimens ofglycerinated human nervous tissues. It was detected regularly
lying between cells and in some instances within the cytoplasm ofcells, some of which were nerve cells (figures I2 and I3). The
micro6rganism appears invariably in pairs or small aggregatedgroups and never in chains. The demonstration of the microorganism in sections is far more difficult, but it has been accomplished in a few instances in the interstices of the tissues and in
the protoplasm of cells.

482

MicroSrganism Causing Epidemic Poliomyelitis.

The detection of the microgrganism in film preparations and insections has been accomplished also with nervous tissues derived
from monkeys inoculated at one time with the ordinary virus, atanother with cultures. In this case also it is more readily brought
out in film preparations than in sections. The grouping is identicalwith that present in the specimens of human origin.
DISCUSSION.

From the facts presented it follows that by employing a speciallydevised method there has been cultivated from the central nervous
tissues of human beings and monkeys the subjects of epidemic
poliomyelitis a peculiar minute organism that has been caused toreproduce the symptoms and lesions of experimental poliomyelitis.
The microSrganism consists of globoid bodies measuring from o.I 5
to o. 3 of a micron in diameter, and arranged in pairs, chains, andmasses, according to the conditions of growth and multiplication.
The chain formation takes place in a fluid medium, the other groupings in both solid and fluid media. Within the tissues of infected
human beings and animals che chains do not appear.
No statement is ventured at present as to the place among living
things to which the bodies belong. It is obvious that the cultural
conditions are those that apply more particularly to the bacteria.
On the other hand, the micro6rganism is associated with the production of an acute disease in which suppuration does not form a
prominent part. No special attention at the present time has been
given to the solution of the question as to whether the microorganism actually belongs to the bacteria or to the protozoa. In
the manner of evolution of the symptoms, and in the appearanceof the lesions, the experimental disease caused by the inoculation of the cultures resembles that produced by the virus ofpoliomyelitis as ordinarily employed. The central nervous organsof monkeys infected with the cultures bear preservation andglycerinization as do the infected human tissues, or the monkey
tissues infected directly from human tissues. Cultures to which
glycerin is directly added survive in the refrigerator at least eightdays.
The micro6rganism passes through Berkefeld filters and theSimon Fl~xner and Hideyo Noguchi.

483

filtrates yield upon recultivation the particular microdrganism contained within the filtered culture. Moreover, Berkefeld filtrates
prepared from the nervous tissues of infected human beings andmonkeys yield also in culture the identical microSrganism.
By employing a suitable staining method the microSrganism has
been detected in film preparations and sections prepared fromhuman nervous tissues, and from the corresponding tissues ofmonkeys inoculated with the usual virus or with cultures or filtrates
prepared f r o m monkeys previously injected with cultures. F r o m
all the infected materials mentioned, irrespective of the manner of
their origin, the microSrganism has been recovered in cultures. As
would be expected it is more uniformly recoverable from theoriginal nervous tissues than from filtrates, and doubtless for thereason that in the former it exists in greater abundance.
T o obtain the initial culture is difficult, and this irrespective ofwhether the tissues submitted to cultivation have come immediatelyfrom man or from monkeys previously inoculated with the ordinary virus or even with the cultures. Once the microdrganism
adapts itself to a parasitic state it is developed with greater difficulty under saprophytic conditions. Whenever the nervous tissues
have been shown to be infectious, the microBrganism has been
recoverable, notwithstanding l o n g preservation and glycerination. In other words, infectivity of the nervous organs and thepresence of the microSrganism exist together. It has indeed
happened that a .specimen of infected nervous tissue has at the firstattempt not yielded the initial growth, although it has yielded itupon the second attempt. Persistence will usually lead to a successful cultivation, provided no technical fault is committed. An important factor in the technique of cultivation is the sample ofascitic fluid. Not all samples are suitable, and a prelimi.nary testis necessary, using for the purpose a growing culture, in selecting
samples for culture purposes. Once a suitable ascitic fluid is obtained it should be carefully husbanded in the refrigerator. Even
with this precaution failure may still occur. In such an instance
repetition, using the same materials but in two series, one of which
is prepared for enclosure in the anaerobic jar, while the other isallowed to remain outside, may yield the desired result; or the484

Micro6rganismCausing Epidemic Poliomyelitis.

result may come on a second trial that appears to be an exact
repetition of the first.
Only the exceptional cultures possess the degree o.f pathogenicity
sufficient to cause specific infection, and the production of experimental poliomyelitis. A pathogenic strain may be effective at different and even remote generations, and a non-pathogenic strain
may lack pathogenicity even 'in the second generation. This important fact indicates strongly that the pathogenic effect is not due
to mere mechanical carrying over into the cultures of an invisible
parasite or virus with which the cultivated microSrganism is accidentally associated. If such accidental association were the causeof the experimental disease produced by the cuttures in monkeys, itwould display itself preferably in the first gene, ations and without
reference to the strain of the visible micro6rganism. On the other
hand in this fluctuation of pathogenicity the cultures imitate theaction of the virus as contained in human materials, namely,
nervous tissue, secretions from the nasopharynx, and intestinal
washings, in which ~he virus, either known or believed to be present,
may yet fail to be demonstrated by reason of the want of infectious
power for monkeys or for the particular monkey inoculated in agiven instance. Moreover, it is a common .experience in bacteriology to find even among the ordinary bacteria lack or rapid lossof virulence among saprophytic cultures, while virulence is not only
retained, ,but may be increased in rapid passages from animal toanimal.
In view of these considerations it would appear that an etiologicalrelationship has been shown to exist between the cultivated microorganism and epidemic poliomyelitis as it occurs in human beings
or in monkeys. There remains merely a single other possibility,
already, indicated, namely, that two factors are present in the cultures, the one an invisible because ultramicroscopic organism, theother the globoid bodies described. On this basis it would haveto be supposed that the former but hypothetical factor is theessential agent of infection. As against this supposition it may beurged that an instance of symbiosis of this nature is not known toanimal pathology. Regarding the cultivated minute but visible
microSrganism itself, it may .be held on the basis of the data presented that it fulfills the conditions hitherto demanded for theSimon FIexner and Hideyo Noguchi.

485

establishment o f causal relation between an e x t r a n e o u s parasite
and a specific disease. T h e microSrganism exists in the infectious
and diseased o r g a n s ; it is not, as far as is known, a c o m m o n saprophyte, or associated with any other pathological condition; it iscapable o f reproducing, on inoculation, the experimental disease inmonkeys, f r o m which animals it can be recovered in pure culture.
A n d besides these classical requirements, the microSrganism withstands preservation and glycerination as does the o r d i n a r y virus o fpoliomyelitis within the nervous organs. Finally, the anaerobic
nature o f the micro5rganism interposes no obstacle to its acceptanceas the causative agent, since the living tissues are devoid o f freeo x y g e n and the virus o f poliomyelitis has not yet been detected in
the circulating blood or cerebrospinal fluid o f h u m a n beings, inwhich the o x y g e n is less firmly b o u n d ; n o r need it, even should them i c r o 5 r g a n i s m be f o u n d sometimes to survive in these fluids.
EXPLANATION OF PLATES.
PLATE 38.
FIG. I. Fluid culture showing opalescence. Natural size.
FIG. 2. Solid culture showing colonies and line of demarcation. Naturalsize.
PLATE 39.
FIG. 3. Separate globoid bodies. X I,OOO.
FIG. 4: Aggregated masses of globoid bodies. X i,ooo.
FIG. 5. Chains and pairs of globoid bodies. X I,OOO.
FIG. 6. Chains of globoid bodies compared with Stre[~tococcus pyogenes.
X I,OOO.
FIG. 7. Agar fragment showing pairs of globoid bodies compared with

Streptococcus pyogenes. )< i,ooo.
PLATE 40.
FIG. 8. Spinal cord showing degeneration of nerve cells and invasion ofleucocytes (neurophagocytosis). X I44~
Fic. 9. Perivascular and interstitial mononuclear cell infiltration of medulla.
X 98.
PLATE 4I.

FIG. IO. Interstitial infiltration and degeneration and phagocytosis of nerve
cells in intervertebral ganglion. X 16o.
FIG. II. Globoid bodies insiteof inoeulation taken up by leucocytes. X I,OOO.
FIG. 12. Film preparation from central nervous tissue of monkey inoculatedwith human virus (not culture) showing pairs of globoid bodies. X I,OOO.
Fro. 13. Section prepared from spinal cord of monkey inoculated with M Avirus (not culture) showqng intracellular pairs of glc,boid bodies. X I,OOO.

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XVIII.

F16. z.

PLATE 38.

FIG. 2.
(Flexner and Noguehi: MicroOrganism Causing Epidemic Poliomyelitis,)

T H E JOURNAL OF EXPERIMENTAL MEDICINE VOL. XVlll.

PLATE 39.

(Flexner and Noguchi: Micro6rganism Causing Epidemic PoUomyeiitis.)

T H E JOURNAL OF EXPERIMENTAL MEDICINE VOL. XVlII.

PLATE 40.

(Flex-her and Noguchi: Micro6rganiam Causing Epidemic Poliomyelitis/

T H E J O U R N A L OF E X P E R I M E N T A L

MEDICINE VOL. XVlll.

PLATE 41.

(Flexner and Noguchi: Micro6rganism Causing Epidemic Poliomyelitis.)