THE FLAGELLA OF SPIRILLUM VOLUTANS

ADRIANUS PIJPER
Institute for Pathology, University of Pretoria, South Africa
Received for publication October 11, 1948
In previous publications (1946, 1947a,b, 1948a,b) the theory was proposed
that many, if not all, motile Eubacteriales move by means of spiral contortions of
their bodies, and that their "flagella" are not motor organs but twirls of mucus,
which, through the very movement of the bodies, peel off from the thin mucous
capsule. Photomicrographs and a cinemicrographic film (1947c) all made by
sunlight dark-ground microscopy were produced at the time to substantiate these
views. Most of the work was done on Salmonella typhosa as a suitable repre-
sentative of the motile bacteria concerned.
Acceptance of this new theory would affect bacterial nomenclature and classi-
fication. It was therefore suggested from several sides that further light might
suitably be thrown on the question by studying the larger spirilla with the same
technique that had been employed with Salmonella typhosa. Such cultures
proved very hard to get. Eventually Professor Pringsheim of Cambridge Uni-
versity kindly supplied a culture of Spirillum volutans. It was not a pure cul-
ture, but the contaminating smaller microbes did not hinder the investigations.
The flagella of Spirillum volutans are much more easily visible than those of
Salmonella typhosa and similar bacteria. It has even been claimed that they are
visible in a wet preparation in bright-field microscopy. This I cannot confirm,
at least not for actively moving spirilla. In bright-field microscopy I have occa-
sionally seen flagella attached to a spirillum when it was quite motionless. With
simple dark-ground methods, however, they became readily visible, no particu-
larly bright source of light such as sunlight being needed as is the case with
Salmonella typhosa. This ready visibility is commonly ascribed to their large
size. It will be shown here that this is not the correct explanation.
The strain of Spirillum volutans received from Professor Pringsheim conformed
to the usual descriptions as given by Giesberger (1936) and in Bergey's Manual
(1948). It was about 20 microns long and about 1.5 microns wide. It grew very
well and remained very actively motile in the medium prescribed by Professor
Pringsheim. This is made by putting a wheat grain at the bottom of a test tube,
covering it with soil to a depth of 4 to 5 cm, and with water 8 to 10 cm high, and
heating in a steam chamber on 2 consecutive days for an hour.
SUNLIGHT DARK-GROUND OBSERVATIONS
Sunlight was used throughout as the source of light, with the equipment de-
scribed previously (1938, 1940). Although some of the phenomena to be de-
tailed here could be seen with ordinary dark-ground methods, sunlight proved
more effective because it is more brilliant, and it was indispensable for photo-
micrography and cinemicrography of the rapidly moving spirilla. Spirillum
111
112 ADRIANUS PIJPER [voL. 57
volutans remained alive under the microscope in sunlight dark-ground conditions
for several hours. After this its movements got slower, and then it started dying.
This was an advantage because it then produced artifacts whose development
could be followed in the wet preparation under the microscope. Such artifacts
proved helpful in understanding appearances in dried and stained preparations.
Figure 1 is a typical fast-swimming spirillum. It has the usual spiral shape,
and at each end there is a revolving flagellum. Both flagella whirl around in a
plane at right angles to the long axis of the body, with the point of attachment as
pivot. This whirling movement is so fast that the shape of the flagellum is not
evident.
Occasionally a spirillum has a flagellum at one end only. It must be empha-
sized that these terminal flagella during activity never split into the large number
of constituent thinner flagella described by previous authors. To and fro move-
ments often occur, but the speed attained and the rapidity of reversal are the
same in mono- and amphitrichate specimens. There is no question of polarity.
Resting periods occur, and are followed by sudden rapid movement, both in
mono- and amphitrichate individuals. During slowing down and resting periods
and toward the end of life, details about the flagellum can be made out (figures
2, 3). All these observations are from wet preparations. The point of attach-
ment is always at the end of the bacterial body, but not necessarily exactly at the
pole (figures 3 to 5). In figure 6 its proximal end has slipped underneath the
body, giving a false impression as to the point of attachment. The flagellum has
a definite shape. It is attached by means of a thin stem (figures 2 to 5), then
follows a thicker portion, and this with a graceful curve tapers into a fine point
(figures 2 to 5). Its length varies somewhat, in different individuals, but its
shape is always the same.
With sunlight dark-ground microscopy the flagellum is never seen to penetrate
into the cell, and there never is anything like a blepharoblast visible in the cell.
This was confirmed by artifacts arising during observation. In some specimens
the cytoplasm dissolved away, leaving an empty shell, obviously the cell wall
(figures 7 to 9). Here the flagellum is seen to be attached to the cell wall, but
does not pierce it. It appears as a continuation of the cell wall; it looks as if the
material of the cell wall had been drawn out into a fine point. Occasionally a
flagellum breaks off, and the break is always at the thin stem. The broken-off
flagellum may float away, or, as in figure 10, come to rest on the body of the
microbe.
A curious phenomenon is that when a cell, having reached the end of its life,
stops its locomotion for good, the flagellum does not always become quiet. It
may go on performing a whirling or more often a lashing movement for a time,
sometimes several minutes. There may be several causes for this, chief among
which I would place tensions in the material of the flagellum or of the cell wall;
but heat, currents, brownian movement, and electrical charges probably also
play a part. This phenomenon was also described by Johnson and Baker (1947).
At this stage the flagellum often splits into a number of thinner subflagella, shaped
1949] FLAGELLA OF SPIRILLUM VOLUTANS 113
exactly like the original one. Figures 11, 12, and 13 show how this happens;
figure 14 shows the final phase. I have never counted more than 6 subflagella

Figure 1. S. volutans, swimming fast. Note two polar flagella. X 1,200.

Figure 2. S. volutans, lying still, has one flagellum only. Note shape of flagellum.
X 3,000.
Figure 3. S. volutans, lying still. Note shape and attachment of flagella. X 2,000.
Figures 4, 5. S. volutans, dying. Note shape of flagella and that attachment is not
always at extreme polar end. X 1,700.
Figure 6. S. volutans, lying still. Flagellum has slipped underneath body. X 2,000.
Figures 7-9. S. volutans, lying still. Cell contents have dissolved away, leaving empty
shell of cell wall, with which flagellum is continuous, but it does not pierce the cell wall.
X 2,000.
Figure 10. S. volutans, lying still. Flagellum has broken off and has come to rest on top
of body. Note its shape. X 2,000.
Figure 11. S. volutans, lying still and gradually splitting its flagellum. X 1,500.
in this strain of Spirillum volutans. These subflagella may exhibit movements
similar to those of the whole flagellum, but they do not move in unison, and it
114 ADRIANUS PIJPER [VOL. 57
also happens that some stop while others still go on. It is difficult to imagine
how, from the inside of the cell, such different impulses could be communicated

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Figures 12-13. S. volutans, lying still and gradually splitting its flagellum. X 1,500.
Figure 14. S. volutans, flagellum has split. X 1,500.
Figures 15, 16. S. volutans, fixed preparation, stained with Knaysi's cell wall stain.
Note that this brings out cell wall and flagellum, which is continuous with cell wall and does
not pierce it. X 2,000.
Figure 17. S. volutans, fixed preparation, stained with Gutstein's cell wall stain. Fla-
gellum and cell wall are continuous; flagellum does not pierce cell wall. X 2,000.
Figure 18. Salmonella typhosa, one swimming fast with straight tail and one resting with
spiral tail. X 1,000.
Figure 19. S. volutans, with cell wall blown out along side. X 2,000.
Figure 20. S. volutans, with cell wall beginning to be blown out. X 700.
Figures 21-24. S. volutans, with cell wall blown out at polar end, displacing place of
attachment of flagella. X 2,000.

to a number of subflagella. The main conclusion from these dark-ground obser-

vations is that the flagellum in Spirillum volutans is a direct continuation of the
cell wall.
1949] FLAGELLA OF SPIRILLUM VOLUTANS 115
STAINED PREPARATIONS
Ordinary staining methods (methylene blue, carbol fuchsin, and Giemsa) do
not stain flagella in dried and fixed preparations of Spirillum volutan8. It is
usually assumed that so-called flagellar staining methods have to be applied for
this purpose. It occurred to me that, if the flagella of Spirillum volutans are a
continuation of the cell wall, then any method capable of staining the cell wall
should make the flagella visible.
Knaysi (1941) demonstrated that when a drop of bacterial culture is dried on a
slide, fixed by heat, and mordanted for 10 minutes with a mixture of tannic acid
and potasium alum, the addition of a drop of Ziehl-Neelsen solution under a
cover slip will stain the cell wall a different color from the cell contents. Appli-
cation of this technique to Spirillum volutans gave pictures like figures 15 and 16.
Figure 15 has been overprinted, and the difference between cell wall and contents
has disappeared, but the flagellum has become stained the same color as the cell
wall. In figure 16 the cell contents have become separated from the cell wall,
and the continuity of cell wall and flagellum is obvious. Another method was
suggested by Gutstein (1924) and recommended by Robinow (1945) and Bisset
(1948). Mordanting a dried and fixed drop of culture with 10 per cent tannic
acid changes the contents of the cell in such a way that they become unstainable
with 0.02 per cent crystal violet, while the cell wall takes the stain. Application
of this method gave pictures like figure 17, showing an empty shell of cell wall and
a flagellum continuous with it.
The results with both methods show that the flagellum, in this case, is continu-
ous with the cell wall, and that it does not pierce the cell wall.
COMPARISON OF "FiFLAGELLA" OF SALMONELLA TYPHOSA AND
SPIRILLUM VOLUTANS
From the observations above and those made previously on Salmonella typhosa
(1946, 1947a,b, 1948a,b) it is evident that there is a fundamental difference be-
tween the "flagella" of Salmonella typhosa and those of Spirillum volutans.
The "flagella" of Salmonella typhosa can be made visible in activity by means
of sunlight dark-ground microscopy only, and they need a suitable medium, of
which the pH is all-important. They appear during periods of fast swimming as
a not very clearly outlined straight tail (figure 18) of varying length, and this tail
during periods of slower speed becomes a definite spiral (figure 18). When the
bacterial body wriggles forward it looks as if there were a thin coat of mucus
around the bacterial body that through the forward movement of the body is
drawn out into a tail, and there is no question of a definite point of attachment.
In keeping with this is the frequent observation that the bacterial body can turn
half a somersault in its coat of mucus while it swims on in the same direction as
before, and the tail remains where it was. The tail can untwist into a large
number of thin wavy threads, which place themselves in irregular fashion all
around the body and then float away. These structures can only be stained by
a complicated process of so-called flagellar staining, which, however, often is a
failure. On account of these and other observations, the theory was put for-
116 ADRIANUS PIJPER [VOL. 57
ward that they are not motor organs but are produced by the spiral motion of the
bacterial body.
The supposed flagella of Spirillum volutans are readily visible, sometimes even
in bright field, and quite easily with simple dark-ground methods. The medium
plays no part. They have a definite shape, a characteristic curve, and a sharp
outline. Their configuration does not change at lower speeds. They always
appear as solid permanent preformed structures. They have a definite point of
attachment, always at or near a pole. They can split into a number of sub-
flagella, but the point of attachment remains the same. Under suitable dark-
ground conditions they can be seen to be a continuation of the cell wall. In
accordance with this they stain with methods which stain cell walls; there is no
need for flagellar staining methods.
Finally, a phase contrast microscope outfit from Cooke, Troughton, and
Simms, lent for the occasion by the local representative of this firm, readily
showed the flagella of Spirillum volutans, both in full activity and at rest. This
cannot be done with the appendages of Salmonella typhosa.
ELECTRON MICROGRAPHS OF SPIRILLA
Conn and Elrod (1947) have objected to the general applicability of my views
on bacterial motility and flagella on various grounds. I intend countering these
objections more fully on a later occasion, but some of their objections can be met
here. They have seen an electron micrograph of Vibrio metschnikovii, taken by
Miss van Iterson in Dr. Kluyver's laboratory in Delft, that appeared to show the
flagellum arising from the body of the cell. I have not seen this particular micro-
graph, but through the courtesy of Dr. Kluyver I have seen similar ones from the
same source. So far I have not had an opportunity of studying vibrios with
my sunlight dark-ground technique. In the meantime Miss van Iterson has pub-
lished her observations (1947) accompanied by numerous electron micrographs.
It appears from her work that there is similarity between vibrios and the larger
spirilla under discussion here. This would mean that in vibrios the supposed
flagellum is, as in the large spirilla, a continuation of the cell wall, and not, as in
most motile bacteria, mucous twirls derived from the capsule. And so my views
on the nature and attachment of these structures in motile bacteria cannot be
affected by observations on the nature or attachment of flagella in either vibrios
or large spirilla. The so-called flagella in most motile bacteria and the so-called
flagella in the large spirilla have now been shown by me to be quite different
things. A special investigation of vibrios by means of the sunlight dark-ground
technique is indicated. The observations made in this way on live specimens
could then serve as a basis for the interpretation of their electron micrographs.
At the moment such an interpretation is possible for the large spirilla. I wish
to exclude the smaller spirilla, such as Rhodospirillum rubrum, because I have
evidence that in these also the so-called flagella are derived from the mucous coat
or capsule.
Miss van Iterson has taken an electron micrograph of Spirillum serpens, in
which case the flagella seem to pierce the cell wall and to be connected with the
1949] FLAGELLA OF SPIRILLTM VOLUTANS 117
protoplasm of the cell (1947). One is reminded of the observations of Fuhrmann
(1910), who claimed to have seen such penetration in Spirillum volutans and
produced drawings to support this, but his photomicrographs showed nothing of
the kind and his claims were completely refuted by Meyer (1912). In Miss van
Iterson's micrograph there is an open space between cell wall and protoplasm,
and the flagella seem to go through this space. For an explanation of this open
space she falls back on the usual assumption that the protoplasm has receded
from the wall. This would imply that the protoplasm has pulled the flagella in,
through a number of preformed holes in the cell wall, which seems scarcely feas-
ible. There is another explanation, more in keeping with my observations on the
development of artifacts through dying. Spirillum volutans under such condi-
tions has a tendency to blow out its cell wall. In figures 19 and 20 this has obvi-
viously taken place, chiefly along the side wall. In figures 21 to 24 this process
has affected the polar end, and here the elongation of the cell wall has displaced
the point of attachment of the flagella. They no longer appear attached at or
near the pole, but some distance away from it. If such a spirillum were rotated
around its long axis over an angle of 90 degrees, as might happen during drying,
the flagella would come to lie on top of or under the body of the cell, and give the
appearance of penetrating the wall. It must be remembered that my observa-
tions are made on wet specimens, whereas electron microscope preparations
are dried.
ACKNOWLEDGMENTS
My thanks are due to Professor Pringsheim for kindly supplying a culture of
Spirillum volutans, and to my secretary, Mrs. A. E. Brummer, for making all the
photographic prints with great skill and patience.
SUMMARY
The flagella of Spirillum volutans were studied with the author's sunlight dark-
ground technique, supplemented by staining methods, and some observations
were made with a phase contrast microscopy outfit.
They were found to be entirely different from the "flagella" -of Salmonella
typhosa and similar motile bacteria.
Objections to the author's theory on the motility and "flagella" of Salmonella
typhosa and similar motile bacteria, based on observations on spirilla, cannot
therefore be upheld.
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