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Journal of Experimental Medicine

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CRISTISPIRA IN NORTH AMERICAN SHELLFISH. A NOTE
ON A SPIRILLUM FOUND IN OYSTERS.
BY HIDEYO NOGUCHI, M.D.

(From the LaboraDries of The Rockefeller Inslitute for Medival Research.)
PLATES 21 a'O 24.

(Receivedfor publication,April 1, 1921.)
In the European lamellibranchiata there have been found a large
number of comparatively coarse forms of spiral organisms, provided
with a broad, band-like membrane extending from one extremity
to the other and winding itself obliquely around the body, one edge
being attached to the body, the other free. The organism was first
discovered in oysters by Certes (1), who regarded it as akin to thetrypanosome and called it Trypanosoma balbianii. Laveran andMesnil (2) considered it a bacterium, while Perrin (3), working under
Schaudirm, thought it a protozoon. At that time Schaudinaa (4)held the view that the spirochete represented a phase of the trypanosome and that both belonged to the protozoa. The peculiar structureof the organism, as brought out by the technique employed by Perrin,
suggested the mitotic and other nuclear figures to such an extent thatthe organism was accepted as a protozoon by certain protozoologists(5-8). The investigations by Swellengrebel(9) and later by Gross (10)
led the latter to place it in the class of Cristlspira, a terminology
accepted by Dobell (11) and Zuelzer (12). The points in dispute are:(1) the interpretation of the membrane; (2) the presence or absenceof a periplast; and (3) the presence or absence of a nuclear apparatus,
especially the significance of the chambered structure of the body.
These questions, which had never been definitely settled, led thewriter to undertake examinations of American shellfish. Furthermore,
so many species have been described recently in the European shellfish
that a comparison from the standpoint of geographic distribution ofthese cristispiras seemed desirable.
295

296

CRISTISPIRA IN NORTH¢oAMERICANSHELLFISH

z~

.¢

v

m~EYo NOGUCm

297

Schellack (13), Keysselitz (5), and Gross (10, 14) created a large
number of species with only slight morphological variations. To
quote an example, Schellack set up thirteen species with certain
characteristics, as shown in Table I.
As will be pointed out later, the method of classifying these coarse
organisms by their morphological features is subject to error becauseof the great variations in the terminal portions and in the thicknessof body according to the degree of fixation, dehydration, or staining;a difference in these factors may cause the same organism to assume
a totally different aspect. Hence the writer has studied these organisms not only in stained preparations but also under the dark-fieldmicroscope.
TABLE II.

Occurrence of Cristispira in the Styles of North American Shellfish.

Shellfish.
9strea virginiana.
7enu$ tnercs~Y~a.

Modiola modiolus
Ensis americana . . . . .
Mya ar~aria . . . . . . . . . .
]f actra solidissima]lytilus edulis
Pecten irradians . . . . . . . . . . .
Fulgur canaliculatus..
Rassa obsoleta..

] No. examined.°f
specimens
298
110
97
24
24
t2 (dead?),
42
39
8
3O

Styles present.

Crislispira present.

128
99 (40 degenerated).
~
).
7O
8 (3
4
73
20
0
24 (hard).
0
1
0
34
0
34 (hard).
0
2 (~)
0
4
0

The present study deals only with salt water shellfish caught in
the neighborhood of Woods Hole during the month of August, 1916,
and includes Ostrea virginiana (oysters), Venus mercenaria (quahaugs),
Mya arenaria (long neck clams), Ensis americana (razor clams),
Mactra solidissima (large sea clams), Pecten irradians (scallops),
M ytilus edulis (mussels), Modiola modiolus (mussels), Fulgur canaliculatus (winkles, whelks), and Nassa obsoleta. The number of specimens of each species examined and the positive findings of Cristispiraand styles are shown in Table II.
As the tabulation shows, Cristisp#a was found most frequently
in the styles of oysters, next in clams, and then in modiolas. No
other species examined showed any Cristispira. In handling the298

CXISTISPIRA IN N O R ~A]~ERIC/LTq SIIELLFIStI

styles of these sheUfish it was observed that those of oysters,
although moderately solid, became liquefied into a viscid fluid upon
standing for half an hour at room temperature in a small amountof sea water after removal. The styles of modiolas are muchsmaller than those of oysters and somewhat firmer; those ofclams are much more solid and elastic than either of the others anddid not become liquefied on exposure to air and sea water at room
temperature. Scallops have large and firm styles which remain littlechanged after removal. Styles from the other varieties are verysmall and solidl The physical properties of the styles may be partly
or chiefly responsible for the presence or absence of Cristispir~ inthese shellfish; apparently the consistency of the styles of oysters
offers the optimum conditions for the habitation of the organism.
Another striking fact is that, at least in the case of the oysters,
the longer they were kept in a tank (eel pond) or the car (sea) aftercollection from their original beds, the less frequently they contained
styles. In such specimens the presence of Cristispira is also lessfrequent, and in many of them only degenerated forms may be found.
I t is rather easy to determine the freshness of oysters by examining
them for styles and the activity of the cristispira contained in them;even freshly collected oysters soon lose the styles if they are not extracted immediately. In one instance 48 oysters which had been kept
in the eel pond 24 hours after collection were opened one after anotherand left at room temperature for about 30 minutes, when they weresearched for styles, but unsuccessfully. Six more oysters from thesame lot which had not been opened all had perfect styles, and fivecontained Cristispira. These external factors must be taken into
consideration in making a survey as to the frequency of Cristispira
infectfon of oysters. This rule does not apply to those shellfish
whose styles are firm and resistant to manipulation.
The main object of the present work was to study the conditions
under which these coarse spiral organisms can be kept alive or cultivated on artificial media, in order that their biologicalandmorphological characteristics might be better studied. Experiments were also
made in which the organisms were subjected to the action of various
salts, acids, and alkalies in order to obtain an insight into the finertm:mYo ~ o o v c m

299

structure of the organism. Vital stainings with neutral red, crystal
violet, brilliant cresyl blue, methyl green, methyl orange, Bismarckbrown, and Janus green were also applied.

Dark-Field Examination of Styles.
Oysters.--The style from the oyster is a glassy, somewhat elastic,cylindrical body of about 2 to 3 cm. long and about 3 ram. in diameterat the thickest portion, tapering to a point at the end. When placedin a Petri dish it becomes a sticky viscid fluid; it dissolves in sea
water. When a drop of the liquefied style is examined under the
dark-field microscope there are observed in many instances violently
motile organisms of large size moving through the field with great
velocity. They pass out of the field so rapidly that their exact form
is not at once recognizable; they suggest butterflies constantly flapping
luminous wings; their movements are backward as well as forward.
They become sluggish within a short time and come to rest either ina tortuous form or stretched out. The body is a long cylinder withblunt ends. The end portion is slightly thinner than the rest, but
not sharply drawn. There is a thick refractive margin all along thebody, indicating the presence of definite peripheral concentration of
the protoplasma (perhaps a cell membrane). The inner part of the
body seems to present no distinct transverse partitions (so calledchambered structure), but Here are rows of refractive projections
from both sides of the wall, point to point, as if they were imperfectcross-bars; the intervals between the bars are about 3 ~. Along theentire body runs a peculiar undulatory narrow membranous structure
coiling obliquely and spirally , showing a free edge here and there(known as the crista, or ridge, of the cristispira). It follows a wavy
course, and at each wave stnnmit the light is highlyrefracted. Fromthat point innumerable fibrillar, fan-like radiations run into the bodyof the organism upon which the other edge of the membranous structure rests or is in connection (Figs. 1 to 3). The fibrils along thefree edge of the crista seem to be so arranged as to form a more compact structure than the portion which stretches between the edge andbody of the organism. Locomotion appears to be accomplished byalternate bending and relaxation of the undulatory, fibrillar, highly
elastic membrane, as is easily demonstrated in a specimen whose

300

CRISTISPIRA IN ~/ORTII AM~.RICAN S]KELL~ISII

motility is slackening because of exhaustion. In a later period of
exhaustion the organism becomes immotile, and the elastic wavy
frame of the membrane assumes its full length and shape, causing the
body to conform to its spirals (Fig. 8). Only active specimens takethe shape of a snake (Figs. 1 to 3). In a freshly extracted style whichis still unsoftened by exposure to the air we often find a nest of organisms in one field (Figs. 4 and 5), some assuming positions like coiled
snakes, and some rather regularly waved and stretched out. In a
few moments the former move rapidly from their resting places
with a violent locomotion and swim away; the latter, which represent
skeletons or elastic frames (Fig. 8), are degenerated and remain
immotile. When a liquefied style containing active specimens of theorganism is left for several hours under unfavorable conditions (room)
most of the organisms undergo degeneration and are found in various
stages from merely stretched out, regularly wavy organisms to skeletons without the body (Figs. 6, 7, and 9). The disintegration of the
body is shown by an irregular contour and various degrees of thicknessof the body substance. The skeleton often shows clearly its component elastic fibrils, some of which may take the shape of a bushy
horse tail (Fig. 6). The gradual overpowering of the body by theelastic membrane in a dying specimen is shown in Figs. 7 and 9.
Clams.--The styles of clams are thicker, firmer, and somewhat
longer than those of oysters, faintly yellowish, shiny, somewhat
opaque, and do not undergo liquefaction in sea water when removed
to a Petri dish. For dark-field examination it is necessary to macerate them in a mortar in order to obtain an emulsion. Five only, of
110 styles examined, showed the presence of active Cristispirce;
3 others contained degenerated specimens. The dark-field appearance of the cristispiras found in clams was identical with that of
the cristispiras in oysters (Fig. 10).
Modiolas.--The modiolas are small mussels, with proportionately
small vitreous styles, not longer than 1 crn.; the consistency of thestyles is softer than that of clams but firmer than that of oysters.
For dark-field examination they can be easily macerated on the slideby pressing under a cover-glass. Cristi3pir(e were present in 4 of 97
styles studied. The organism showed microscopically no distinctive
features except that of exceptional length (Fig. 11).

HmEYO ~Tootrcal

301

Examination by Means of Vital Staining.
The crisfisplras from oysters were used for this purpose.
Neutral Red.--Withln a few minutes the body showed a generalbrownish yellow coloration, with numerous paired yellowish granules
scattered with fair regularity along the entire wall. The membrane,
or crista, appeared as bushy brownish fibrils projecting in all directions. The color of the body disappeared in 20 minutes, that of the
fibrils in 6 hours. Coiled specimens did not take the stain.
Crystal Violet.--The body took a rather deep purple stain, thecrista remaining uncolored. Fine protoplasmic reticula with intersecting granules were seen. In some specimens vacuolation of thecell contents occurred, a large mass of substance occasionally protruding into the side (plasmolysis). The deep purple color was stillpresent at the end of 6 hours. There was no chambered appearanceof the body.
Brilliant Cresyl Blue.--The body took up in mottled fashion alight bluish lavender color. The cross-bars (bluish) of the body weredistinctly brought out in a few minutes; the crista remained practicallyunstained. Complete plasmolysis and decoloration occurred within
6 hours.
Methyl Green.--The body stained faint pink without showing any
structure; the crista was not stained. Decolorafion occurred in 6
hours.
Methyl Orange.--There was no coloration. The organisms remained
active.
Bismarck Brown.--The body became distinctly brownish, withmore deeply stained granules throughout the entire length. These
granules sometimes occurred in a line along one or both sides of thewall. Degenerated specimens were not well stained. The specimens
were practically decolorized in 6 hours.
Janus Green.--The body took a deep, dark bluish color, withreticular mottles, the crista a dark blue on its free margin but only
a faint blue elsewhere. In some specimens comparatively large roundgranules of varying size, stained dark bluish, were seen scatteredabout in several parts of the body. Degenerated organisms did nottake the stain. Within 6 hours the color gradually faded.

302

CRISTISPIRA IN N O R T H

AMERICANSHELLI~ISH

In general, then, the body of Crlstlspira balbianii took the vitalstaining, while the crista stained only slightly with some of the dyes
and not at all with others. The phenomenon common to staining
with neutral red, brilliant cresyl blue, Bismarck brown, and Janusgreen was the fact that within the first 5 minutes the body of theorganism took the stains in such a way as to show the presence ofchromophil granules more or less regularly and diametrically paired
on both sides of the wall. After a longer period the number ofchromophil granules became more numerous, and a sort of reticular
distribution of the same substance was brought out, giving the entire
body a mottled appearance. In the case of brilliant cresyl blue thecross-bar arrangement of the chromophil substance was distinct
but this was followed by complete plasmolysis of the organism, indicating that the chambers were the result of an unfavorable or toxic
effect of the dye. Coiled specimens usually showed no deeply
staining granules with Bismarck brown or neutral red.

Stained Preparations.
Several methods were used for the study of the morphologicalcharacteristics of the cristispiras, but the best differentiation of thevarious constituents of the organism was obtained by application of
Giemsa's stain and Heiderthain's iron-hematoxylin.
With Giemsa's stain the organisms were variously fixed. In someinstances the liquefied styles containing the cristispiras were thinly
spread over a clean slide and the moist film surface was immediately
exposed to osmic acid vapor (1 per cent) for 1 minute. 30 minutes
fixation in absolute alcohol followed. In other instances the moist
films were at once immersed in a jar containing Schandinn's sublimate
alcohol, which was maintained at a temperature of 75°C. for 5
minutes and then allowed to cool. Before applying Giemsa's
solution the films were rinsed in water, treated first with Lugol'ssolution, then with 0.5 per cent sodium thiosulfate solution, thenthoroughly washed in water. In this procedure the films were neverallowed to dry until after being stained with Giemsa's solution. In
still other instances the films were first dried in the air, then fixed
in methyl alcohol for 30 minutes, then stained with Giemsa's solution.

HmEYO NOO~CH~

303

The object of these various procedures was to study the influence offixation on the morphology of the organism.
The morphological features revealed by Giemsa's stain were very
similar, whether the films were fixed by the osmic vapor or by Schaudinn's hot sublimate alcohol method. In both preparations the
body showed a cross-bar structure consisting of deeply stained blue
bars alternating with light, almost unstained or faintly pinkish areas.
The bars, which were correspondingly wider in larger specimens,
appeared somewhat narrower toward the extremities. The relative
width of the bluish bar and of the juxtaposed light spaces was veryvariable in different specimens. In some the width was nearly thesame (Figs. 29, 30, and 40);, in others the light spaces weremuch
wider than the cross-bars, which then appeared like thin cross lines(Figs. 12, 13, and 31); in still others the reverse was true, in whichcase a deeply stained bluish body seemed to be segmented by narrow
light cross spaces (Figs. 14, 15, 17, and 32 to 34). In the specimens
fixed in sublimate alcohol there were specimens showing in some portion of the body several consecutive bars, so thinned out near thethe middle as to be almost broken (Figs. 13, 17, and 35), the appearance being not unlike that of the organism in the fresh state. The
bluish cross-bars were not of uniform width or sharply defined borders,but one end .was usually broader than the other, and sometimes a
few here and there were placed obliquely to the right angle formedby the wall and the hypothetical axis of the body. In contrast to
fresh specimens, the stained organism generally had a tapering form,
ending in sharply pointed extremities (Figs. 12 to !4, and 29 to 33).
The crista took a brilliant red hue, showing one or more heavilystained
marginal fibers, and it ended near the end of the body (Figs. 29 to33). The connection between the body and the heavy marginalfibers of the crista is so thin that the finer fibriUar structure is seldom
brought out. One may encounter in stained preparations specimens
caught in full activity (Figs. 12, 14, 13, 17, 19, 29 to 33, and 35) or in
the exhausted condition, yielding to the elastic crista, with beginningplasmolysis (Figs. 20 and 21) or with the crista skeleton (Figs. 23
and 36). Sometimes the crista alone may be found (Figs. 37 to 39).
The specimens fixed in methyl alcohol after being air-dried did notshow so sharp a structure as those just described. The organism

304

CRISTISPIRA IN N O R T H

A~RICANSHELLFISH

stained more reddish in general, and the bluish bars were not clearly
distinguished (Figs. 16, 18, and 19).
Heidenhain's iron-hematoxylin stain was applied to film preparations of the cristispira derived from clams and Mallory's to sectionsof oyster styles. In the latter the organisms were present in large
numbers, usually lying parallel with the course of protein lamell~e,
which run concentrically along the long axis of the style like a scallion.
Some were coiled, but most assumed wavy forms (Fig. 22). The crossbar structure was easily distinguished. The crista is occasionallyrevealed--it is perhaps held closely to the body. (In fresh specimens
the crista can always be seen.) Occasionally there are found specimens
cut across so that they are visible as a ring. The details of structure
are more clearly brought out in the film preparations by adequate
differentiation (Figs. 25, 27, and 44). The body is cross-barred bythin, dark grayish lines, with a more definite contour of the cell wall.
The crista is fibrillar and retains a light bluish gray color which is
deeper along the marginal fibers. The extremities of the body are
not sharply drawn out but are blunt points.
In one of the clams collected in New York Bay in September, 1916,
I encountered a rather long variety of Cristispira. It was similarin all other respects to the variety met with in the clams studied atWoods Hole. Whether this specimen of Cristispira is a new species
or merely a variety due to temporary factors has not been determined (Figs. 24, 26, 28, 45, and 46).

Effects of Chemicals.
The microchemical reactions of various components of Cr~stispCra
balbianii were studied with a view to gaining an insight into thestructure of the organism. Previous investigators have obtained
varying results in a similar study of the European specimens. The
writer's studies with the American variety were made with thefollowing substances: sodium taurocholate, sodium glycocholate,sodium oleate, saponin, cobra lecithld, acetic acid, hydrochloric acid,sulfuric acid, potassium hydroxide, and ammonia.
Method of Study.--One part of the style emulsion containing numerous active specimens of Cristispira balbianii was mixed on a coverglass with one part of the substance, dissolved in sterile sea water;

305

HIDEYO NOGUCHITABLE III.

Effect of Chemicals on Cristispira.
Date.

Chemical.

Result.

Sodium taurocholate.

Plasmolysis complete in 5 to 10 rain. Skeleton,or crista, resistant. Spirillum ostree dissolved; bacilli still motile. After 20 hrs. atroom temperature skeletons (cristas) showing their fine fibrillar composition are more
completely denuded of the cell plasma.
Practically the same as with 10 per cent.
Nearly all have undergone plasmolysis; otherwise the same as preceding experiment.

1916

Aug. 23

Sodium glycocholate.

Sodium oleate.

Similar in effect to the taurocholate solution.

This salt produced such a viscid emulsion thatno observation was possible.

Cobra lecithid.

Almost insoluble in sea water.
indecisive.

Observations

Saponin.

Complete plasmolysis in 5 to 10 mln. Cfista
not affected but assumes more regularly
waved spiral bundles of fibrils (Fig. 9), towhich irregularly protruding masses of protoplasm are seen to be attached at somepoints. Splrillum ostre~ and bacilli still
motile. After 20 hrs. at room temperature
the cristas are stiU intact. They show theirfiner structure and their relation to thebodies, which are now mere masses of broken
up protoplasm scattered along the regularly
undulated spiral cristas.
Same as with 10 per cent.
Nearly all have undergone plasmolysis.

Controls in sea
water.

Actively motile for 2 hrs. after being sealed
on slides; thereafter gradually became iramotile; structure unchanged.
After 20 hrs. no degeneration but all in "relaxed" state.

306

CRISTISPIRA I N NORTH ~M~.RICAN SHELLI~ISH

TABLE m--Concluded.
Date.

Chemical.

Result.

1916

Aug. 24 Acetic acid.

Mter 2 hrs. body smooth, showing cross-bar
structure and heavy cell wall; crista shrivelled and indistinct; Splrillura ostrece swollen and indistinct,
Same as with 50 per cent.
"save for the presence of many roundbodies suggesting extruded protoplasm of
the body of Cristist~ra balbianii.

Hydrochloric acid.

After 2 hrs. body shows more densely set
cmssobar appearance than in acetic acid,but the crlsta, though perhaps thickened
and less distinct than normally, is still attached to the body.
Same as with 50 per cent.
"except for some nodular swelling of the
body in parts.

Sulfuric acid.

Similar to results with hydrochloric acid except
for more granular appearance of the body
substance and less distinctness of the crista.

Potassium hydroxide.

Within 2 hrs. the organisms have practicallydisappeared, except for a few fragments ofthe bodies in which no structure save thecell wall can be recognized; no crista found.
Within 2 hrs. the body shows irregularly contoured thick wall with vestiges of thinned
out, broken cross-bars; crista seems to beclosely attached to the body.
Within 2 hrs. there are apparently no changes;
body shows distinct cross-bars and heavy
cell wall; crista seems to be intact.

Liquor ammoniac
fortls.

Controls in sea
water.

Within 2 hrs. no changes; immobilized.
ct

2

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ct

ct

2

tg

~t

¢g

¢g

After 2 hrs. almost all are still active.

lalDEYO NOGUC~307

the cover-glass was then sealed with vaseline on a slide and examination made under the dark-field microscope. The concentration ofeach substance and the effects of each upon the organism are briefly
summarized in Table III. The observations were made at room
temperature (23°C.).

Survival of Cristlspira balbianii under Different Conditions.
An oyster emulsion containing actively motile cristispiras was used
to study the effects of tonicity. The observations were made in hanging drop preparations after 2 hours at room temperature (22°C.).
When mixed with distilled water the organisms became immotile,then the cell body assumed a regularly spiral course in conformity
with the skeletal crista, presenting at the same time accumulationsof a highly refractive substance in round or oval masses along the
body at each turn of the spiral skeleton. The phenomenon may beinterpreted as due to plasmolysis through hypotonicity. On theother hand, in 10 per cent sodium chloride solution the organisms
remained intact, but many coiled forms (Fig. 40) were seen. The
body appeared less refractive, but there was no plasmoptysis. In
the control preparations made with sea water, most of the organisms
became immotile after 2 hours at room temperature; in those kept
at 6--8°C. all the cristispiras were still actively motile at the end of
2 hours, and one-fifth to one-tenth after 24 hours; after 40 hours only
a few were found to be active in one of the three slides. In dark-field
preparations (not hanging drop method), sealed with paraffin andkept at 6-8°C., all cristispiras were disintegrated at the end of 24hours.

Attempts at Cultivation.
Several unsuccessful attempts were made to obtain a culture of

Cristispira balbianii. The chief obstacles in the work were (1) thelack of bacteriological facilities, and (2) the impossibility of obtaining
a bacteria-free suspension of Cristispira balbianii.
The culture media employed in the first attempts were made witha filtrate of various styles (oysters and scallops), obtained by passing
the sea water solution (or emulsion) of styles (50 styles in 100 cc. ofsea water) through a Berkefeld filter V. To 3 cc. of the filtrate were308

CRISTISPIRA I N NORTH AMERICAN SH_ELLleISI=I

added, in one set, 1 cc. of 2 per cent glucose agar; in another set
1 cc. of 2 per cent plain agar; in a third set 1 cc. of ascitic fluid; inthe fourth 1 cc. of glucose broth; in the fifth 1 cc. of sterile sea water;
and in the sixth (controls) 4 cc. of sterile sea water without the stylefiltrate. All tubes were covered with a thin layer of sterile paraffin
oil to prevent evaporation of the culture media. The cultures were
set up as follows: All tubes (1 cm. in diameter and 22 cra. high) firstreceived 0.2 cc. of a rich cristispira emulsion from oysters. 3 cc. ofthe style filtrate were then added to all except the control (sixth) set.
The first and second sets were next mixed with 1 cc. of 2 per centmelted glucose agar and plain agar, respectively, the temperatureof the agar being about 42°C. at the time of mixing. The tubes werecooled quickly b y immersion in the aquarium. The glucose brothand ascitic fluid were added to the other sets and the paraffin oil was
finally added to all. Each set was made up in duplicate, one being
kept at room temperature and the other at aquarium temperature(about 7°C.).
Examination of the culture tubes was made daily during 8 days,
but no growth of cristispira was ascertained. They were brought to
The Rockefeller Institute in an ice-packed container and followed for
3 weeks longer, b u t except in one instance, in which a peculiar large
non-motile organism was seen to grow, the results were negative.
This organism, which grew in the first set of media (semisolid), bore
a close resemblance to Cristispira, except that there was no crista. I tsuggested Gross' Saprospira. Whether it was a cristispira modified
through artificial cultivation (loss of crista) or an altogether different
organism is still a question, l~iost of the tubes were more or lesscontaminated with members of the mesentericus group.
A spiral organism of oysters, Spirillum ostrece, n. sp. (Fig. 43),
likewise failed to grow. This spiral organism measures about 8 to
16 u in length and 0.5 ~ in the widest portion of the body. Bothends gradually taper off to fine filaments. The number of spirals
varies from 4 to 8 and the curves are rather shallow but very regular.
The body is elastic but not perceptibly flexible. They rotate
rapidly but seldom proceed one way or the other.
In another experiment styles were ground in a mortar with Ringer'sor Locke's solutions as well as with sea water and the whole was

n,,,Exo ~ o o u c ~309

sterilized in an autoclave. The opalescent fluids thus obtained wereused in experiments similar to those preceding with unsatisfactory
results.
The tissue culture technique was employed in order to determine
the viability of cristispiras in the presence of various extraneous
substances. By careful examination it was possible to collect a dozen
styles rich in cristispiras and apparently free from ordinary bacteria.
Spirillum ostrece was the only variety present and occurred in rather
large numbers in some styles. The styles were dissolved in 3 cc. ofsterile Ringer's solution; the work was carried out as aseptically as
circumstances permitted.
To 1 drop of the style emulsion was added 1 drop of the following
liquids respectively: (1) mixture of oyster style-Locke's solution
infusion filtrate; (2) oyster broth-Locke's solution-infusion-stylefiltrate; (3) oyster juice filtrate; (4) oyster broth; (5) Locke's solution;(6) sea water; (7) distilled water; (8) ascitic fluid; and (9) bouillon.
Eight Ringer's solution preparations were made as controls.
At the end of 24 hours at 6-8°C. many balbianii remained activein three of the eight control (Ringer's solution) hanging drop slides,
and in the slide containing ascitic fluid. In all other instances theorganisms were inactive and diminished in numbers; there was anincreasing mass of bacteria more marked in the slides containing
albuminous solutions like oyster-filtrate, Locke's solution-style infusion, and bouillon. At the end of 48 hours only a few cristispiras werestill sluggishly motile in the slides containing ascitic fluid and in oneof the Ringer's solution controls.
This observation serves to emphasize the difficulty of obtaining
a culture of this organism under the conditions of the experiments
just described.
DISCUSSION AND SUM~ARYo

Ten varieties of North American shellfish were examined for theoccurrence of Cristispira in their styles. A cristispira was found invarious numbers in Ostrea virginiana, Venus mercenaria, and Modiola
modiolus, but none in Ensis americana, Mya arenarla, Mactra solidissima, P ecten irradians, M ytilus edulis, Fulgur canaliculatus , or N assa
obsoleta. Of 298 oysters, only 128 showed the crystalline styles, in310

CRISTISPIRA IN N O R T H

A M E R I C A N SHELLFISH

which cristispiras were present in 99. Active cristispiras were foundin 59 styles only and degenerated forms in the remaining 40. In
110 clams (Venus mercenaria) 70 styles were found, and only 8 of thesecontained cristispiras; 5 yielded active and the other 3 degenerated
cristispiras. In 97 modiolas there were 73 styles, only 4 of whichcontained cristispiras.
The physical properties of the crystalline styles of these shellfish
varied considerably. The styles of the oysters were moderately soft,
and when exposed to the air or mixed with sea water they underwent
liquefaction, forming a clear, viscid material. The styles from clams
and modiolas were opaque and were more firm, not easily crushed evenin a mortar. The styles of the scallops were the most solid of all thestyles examined. It happened that the softer the styles, the more
frequent was the occurrence of the cristispira; in fact, no cristispira
was detected in styles other than those of oysters, clams, and modiolas,of which oysters had the softest styles and the largest percentage ofcristispira invasion.
The following observations were made regarding the structure of
the cristispira found in oysters. The body is a long, flexible cylinder,
with blunt extremities, towards which the diameter gradually diminishes. In motion the body rapidly stretches and contracts, formingin the contracted state several serpentine undulations. A membranous
appendage (Gross' crista) winds about the body throughout its
entire length. The inner margin is in connection with the body,the outer margin is free and is distinctly heavier. The latter isundulatory; that is, the width of the membrane, or crista, is narrower
at some points than at others. The membrane is composed of numerous fine fibrils running in a roughly parallel or slightly oblique course,
showing interwoven narrow meshes; at the outer margin there is adense smooth ridge.
The contour of the body is highly refractive, as if possessing a cell
membrane. The interior structure, as revealed by dark-field illumination, is an almost homogeneous, less refractive substance, but there
are present minute highly refractive granules more or less symmetrically arranged. There is no definite cross-bar or chambered structure.
On the other hand, when vital staining with brilliant cresyl blue isapplied, there appear numerous paired masses of lavender hue atHIDEYO NOOUCm

311

fairly regular intervals, suggesting the cross-bar aspect of a stained
specimen. In a few specimens there was seen a dim outline of crossbar effect. Neutral red, Bismarck brown, and crystal violet allbring out deeply stained granules and reticular structure but nodefinite cross-bars.
While it is difficult to recognize any definite chambered structurein the fresh state, it is very easy to bring out the characteristic banded
body by staining the organism with Giemsa's solution. The bands,
or cross-bars, which take up a deep bluish color, are of variable width,ranging from a thin line to a square or even oblong block. In somespecimens the body is almost blue with thin white cross lines, whilein others the blank body is banded with thin bluish bands at irregularintervals. The crista, or undulatory membrane, is stained bright red,particularly along the outer free" margin. The fibriUar structure of
the crista is usually recognizable. In the specimens stained with
Heidenhain's iron-hematoxylin the cross bands, which appear bluishgray, are more distinct and delicate. The bands are not, however,always thin and sharp, but in certain specimens are almost square.
The irregularity in the width of the cross bands in various parts of
one speclrnen and in different specimens suggests that the peculiar
banded appearance may be a result of fixation and not the natural
structure of the organism. At least, no such structure was recognizedunder the dark-field microscope, which would have revealed suchdefinite septa if they existed. I believe that the cross bands areformed from a homogeneous mass through sudden contraction due
to dehydration with absolute alcohol during fixation. In the prepara.
tions first dried in the air and then fixed with methyl alcohol thisbanded appearance is absent. That Cristispira has a chamberedbody, each chamber being equivalent to a single cell, is Gross' interpretation, which is not supported by the observations reported here.
Moreover, the protoplasm of the organism readily escapes from thebody in distilled water, saponin, or other chemical agents, forming
many round masses of extruded protoplasm in different parts of the
body. No septa are to be seen in these plasmolyzed or degeneratedorganisms. Acids, hydrochloric and acetic, failed to bring out any
banded structure of the body. It may be deduced that the externalsurface of the body is a highly refractive cell membrane, and that312

CRISTISPLRAIN N O R T H

AMERICAN

SI-IELLIelSII

within the cell body are homogeneous protoplasm of reticular form andnumerous highly refractive granules of varying sizes. Some of thecontents take the dark bluish color when stained with Giemsa's
solution. In a fixed specimen this particular substance becomes
contracted into a series of masses of varying dimension, leaving
between each mass a blank space of varying width, and giving acharacteristic cross-barred or banded (so called chambered) structure.
In some specimens the cross-bars are not seen, but instead a series ofbroken lines running parallel with the longitudinal axis of the body(Figs. 41 and 42).
The crista is a fibrillar structure, connected with the body at its
inner edge. The outer margin is a thickened bundle of fibrils runningan undulating course along the entire length of the crista. The crista
is elastic and when detached from degenerated organisms assumes
a rather regularly wound spiral, consisting of longitudinal bundles offibrils (Figs. 36 to 38). A fragment of two or three waves may be
encountered in a preparation containing many degenerated organisms(Fig. 39). The composition of the crlsta can best be studied indegenerated remains of the organism. During the life of Cristispira
it is stretched or relaxed according to the contraction or extension of
the body. The elasticity of the crista appears to furnish the organismwith a propelling and rotating power upon its extension after being
drawn tightly to the body by some contractile apparatus (myoneme)
present somewhere within the cell body. The crista serves as a rudder
and propeller for the swimming organism. It is interesting to compare
here the elastic and regularly waved flagella of certain bacteria andspirochetes; it is possible that the crista of Criztisp#a is a highly
modified form of flagella.
The nature of the substance which stains dark blue with Giemsa's
stain is not known, but it does not give a chromatin reaction. By
Heidenhain's iron-hematoxylin method it takes a dark grayish tint,
similar to the cell wall or crista, which are also dark gray. This
substance was regarded by Gross and Zuelzer as volutin, which is ofnutritive origin. It is probable that there are also embedded within
it minute chromidial elements. Multiplication is by transverse fission.
CrBtispira balbianii is parasitic and does not survive more thana few days in ordinary sea water emulsion, even at its optimum

am~Yo ~ o G v c m

313

temperature. In its natural habitat, or the crystalline style, it isusually pure, b u t is sometimes found in association with a t i n y spiralorganism (Spirillum ostrece). The cristispiras in the styles seem todiminish rapidly when oysters are collected from their beds andtransferred elsewhere; oysters k e p t in tanks or cars for several days
do not contain the cristispiras, and in opened oysters the styles disappear promptly at room temperature.
All efforts to cultivate this organism have failed.
BIBLIOGRAPHY.
1. Certes, A., Les parasites et les commenseaux de l'huitre, Bull. Soc. zod.
France, 1882, vii, 347; Sur le Trypanosoma balbianii, 1891, xvi, 95.
2. Laveran, A., and lVfesnil,F., Sur la nature bact~rienne du pr~tendu Trypanosome des Huitres (Tryp. Balbianii Certes), Compt. rend. Soc. biol., 1901, liii,
883.
3. Perrin, W. S., Researches upon the life history of Trypanosoma balb~anii(Certes), Arch. Protistenk., 1906, vii, 131.
4. Schaudinn, F., Generations- und Wirtswechsel bei Trypanosoma und Spiroduete, Arb. k. Gsndhtsamte, 1904, xx, 387.
5. Keysselitz, G., Beschreibung von SpCrochceta anodontee nov. spe6., Arb. k.
Gsndhtsamte, 1906, xxiii, 566.
6. Keysselitz, G., Studien fiber Protozoen, Arch. Protistenk., 1908, xi, 334.
7. HSing, A., Vergleichende Untersuchungen fiber Splrochttten und Spirillen,
Arch. Protistenk., 1911, xxiii, 101.
8. Fantham, H. B., Spirochceta (Trypanosoma) balbianii, (Certes) and Spirochceta anodontm (Keysselitz): their movements, structure, and affinities,
Quart. J. Micr. Sc., 1908, lii, 1.
9. Swellengrebel,N. H., Sur ]a cytologie compar6e des spirochetes et des spiriUes,
Ann. Inst. Pasteur, 1907, xxi, 448; Compt. rend. Soc. biol., 1907, lxii, 213.
10. Gross, J., Cristispira nov. gen., ein Beitrag zur Spiroch~etenfrage, Mitt. zool.
Station Neapel, 1910, xx, 1.
11. DobeU, C. C., On Cristispira veneris nov. spec. and the affinities and classification of spiroch~etes, O~,zrt. J. Micr. Sc., 1910-11, lvi, 507.
12. Zuelzer, M., Ltber Spirochceta plicatilis Ehrenberg und deren Verwandtschaftsbeziehungen, ArchProtistenk., 1912, xxiv, 1.
13. Schellack, C., Studien zur Morphologie und Systematik der Spiroch~etenaus Muscheln, Arb. k. Gsndhtsamte, 1909, xxx, 379.
14. Gross, J., Ueber Systemafik, Struktur und Fortpflanzung der Spironemacea,
Centr. Bakt., 1re Abt., Orig., 1912, lxv, 83.

314

CRISTISPIRA IN NORTIq" AMERICAN" SIEELLI*ISFIEXPLAN'ATION OF PLATES.
PLATE 21.

Magnification uniformly X 1,000.
FIGs. 1 to 3. Dark-fieM view of living specimens of Cristispira balblanii froman oyster. Photographed while they were temporarily at rest. The smoothwavy body with sharp contour along the wall, and a somewhat hazy alveolar
content are well shown. The undulating crista loosely attached to the body ofthe organism can be seen. What appear like pointed sticks attached to theorganism in Figs. 1 and 3 are the spirillar organisms found in fresh oysters whichwere merely entangled in the crista.
FIGS. 4 and 5. Dark-field view of Cristispira balbianii in the styles of oysters.
In the styles there are different forms undoubtedly representing various stagesof the life of the organism. In Fig. 4, there are one degenerated and two wellpreserved specimens, while in Fig. 5 there are also two intact and one long skeleton of a degenerated organism.
FIGS. 6 to 9. Different phases of degeneration of Cristlspira balblanli due tounfavorable conditions or to age. Fig. 9 shows the protoplasm bulb accumulated near the middle of the body under the influence of a concentrated solutionof saponin. Figs. 6 and 8 show the elementary fibrils which constitute the crista.
FIG. 10. Dark-field view of a cristispira from the style of a clam. I n every
respect this organism is comparable to the balbiani4 found in oysters. I t m y be
the same as that described by Dobell as Cristispira veneris.
FIG. 11. Dark-field view of a cristispira from the style of a modiola.
Generally somewhat longer than the balbianii; otherwise appears identical.
PLATe 22.
Magnification uniformly X 1,000.
FIGS. 12 to 15 and 17. Cristispira balbianii from oysters, stained with Giemsa's
solution after osmic acid or sublimate alcohol fixation. They are all wellpreserved.
Flcs. 16, 18, and 19. Cristispira balbianii stained with Giemsa's solution after
methyl alcohol fixation. All except Fig. 16 appear in good condition, although
no definite cross-bar structure can be distinguished.
Fits. 20, 21, and 23. Cristispira balbianii in process of degeneration. Fig. 20
shows the body still solid, almost intact, with a regularly wavy erista. In Fig. 21
the body is still fairly well preserved, but it follows the regular waves of thecrista, indicating the absence of any resistance to the form of the crista. Notealso the protruding mass of protoplasm near the middle of the body. In Fig. 23
there is only a shadowy trace of the body and the spiral elastic skeleton, or
crista, is distinct.

RIDEYO NOGUCSI

315

FIG. 22. A section of the oyster style fixed in sublimate alcohol and stainedwith Mallory's iron-hematoxylin. The organisms show the chambered structure,
but no crista can be distinguished.
FIGS. 25 and 27. Cristispira balbianii in films from fixation sublimate alcoholand stained with Heidenhain's iron-hematoxylin.
FIGS. 24, 26, and 28. Cristispira from a clam caught in New York Bay. The
film was fixed in sublimate alcohol and stained with Heidenhain's iron-hematoxylin.
PRATE 23.

Magnification × 1,250.
Drawings from film preparations fixed in osmic acid vapor and absolute
alcohol and stained with Giemsa's solution.
FIGs. 29 to 42. Different specimens of Cristispira balbianii showing the general chambered or cross-barred appearance of the body and undulatory membranous appendage, called the crista by Gross. The blue-stained substance whichconstitutes the cross-bars, or partitions, is seen to be regularly or sometimes irregularly distributed in amount or distance. In some, the substance may not be
attached to the wall at all, but forms longitudinal rods of varying lengths as
seen in Figs. 41 and 42. In such instances there is no suggestion of a chamberedstructure.
FIGs. 36 to 39. The final steps of degeneration of Cristispira balbianii. Fig. 36
alone still shows the degenerated body while the others show nothing but the
fibrous skeletons, or cristas, of the organism. Fig. 39 is a fragment of the crista.
FIG. 43. The spiral organism found in the crystalline styles of fresh oysters
caught in Woods Hole Bay.
PLATE 24.

Magnification X 1,250.
FIG. 44. Cristispira balbianii from an oyster. Film preparation, fixed insublimate alcohol and stained with Heidenhain's iron-hematoxylin.
FIos. 45 and 46. A cristispira (Cristlspira veneris?) from a clam collected near
New York Bay. Sublimate alcohol fixation and Heidenhain's iron-hematoxylin.

THE JOURNAL OF E X P E R I M E N T A L MEDICINE VOL. X X X I V .

PLATE 21.

(Noguchi: Crislispira in North American shellfish.)THE JOURNAL OF E X P E R I M E N T A L M E D I C I N E V O L . X X X I V .

PLATE 22.

(Noguchi: Crlstlspira in Nolth American shellfish.)THE JOURNAL OF E X P E R I M E N T A L M E D I C I N E VOL. X X X I V

PLATE 23

(Noguchi: Cristispim in North American shellfish.)THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. X X X I V .

PLATE 24.

(Noguchi: Cris!ispira in North American shellfish.)