ELECTRON MICROSCOPE STUDIES OF BACTERIAL VIRUSES

      S. E. LURIA', M. DELBRUCK AND T. F. ANDERSON'
The Bacteriological Research Laboratories of the Department of Surgery, College of Phyeiciane
and Surgeons, Columbia Univeraity; The Departments of Biology and Phyeics, Vanderbilt
          Univereity; and the RCA Research Laboratories

          Received for publication February 17, 1943
Physico-chemical (Schlesinger, 1933; Northrop, 1938; Kalmanson and Bron-
fenbrenner, 1939) and biological (Ellis and Delbriick, 1939) studies of bacterial
viruses (bacteriophages) in the last few years have led to a revival of interest in
this group of viruses, particularly as a material on which one can study under very
favorable conditions properties which may be common to all viruses.  Ease and
accuracy of titration and the possibility of working under biochemically con-
trolled conditions make bacterial viruses an ideal object for such investigations.
The electron microscope, recently introduced as a tool for biological research,
has been applied to the study of animal and plant viruses, and also of bacterial
viruses (Ruska, 1940; Pfankuch and Kausche, 1940; Ruska, 1941; Luria and
Anderson, 1942). Ruska (1941) published micrographs of suspensions of bac-
terial viruses, in which "sperm-shaped" particles can be seen.  Ruska suggested
that these particles should be interpreted either as the virus itself or as bacterial
constituents.
In December 1941 and March 1942 Luria and Anderson (1942), through ar-
rangements made with the National Research Council Committee on Biological
Applications of the Electron Microscope, were enabled to study several bacterial
viruses with the RCA electron microscope.  They found sperm-shaped particles
in the virus suspensions, and identified them as virus particles on the basis of
considerations which will be more fully developed in the discussion of the present
paper.
During the summer of 1942 the present authors availed themselves of the
presence of the RCA electron microscope at the Marine Biological Labora-
tory, Woods Hole, to study the interaction of bacterial viruses with their
bacterial hosts.  Previous growth experiments (Delbriick and Luria, 1942) had
served to analyze the various stages of the interaction (specific adsorption, latent
period of virus multiplication, virus liberation and lysis of the bacterium).
This analysis formed the basis for the interpretation of the results.
The present paper describes and discusses the results of this whole series of
electron micrographic studies of bacterial viruses.

1 Aided by granta from the Research Fund of Vanderbilt University and from the Dazian
Foundation for Medical Research.
* Fellow of the Guggenheim Foundation.  Now at Indiana University, Bloomington,
Indiana.
a RCA Fellow of the National Research Council.  Now at the Johnson Foundation for
Medical Physics, University of Pennsylvania, Philadelphia, Pennsylvania.
                          67


58       6. E. LURIA, M. DELBRtiCK AND T. F. ANDERSON

MATERIAL AND METHODS

The RCA electron microscope and its mode of operation have previously been
described (Anderson, 1942).  A suspension of the material to be studied is de-
posited on a thin (10-20 ml.c) collodion membrane, which in turn is supported by
a fine wire mesh screen holder.  The suspension is left for a few minutes on the
membrane in order to permit the particles to settle out and to adhere to the
membrane.  The holder is then "washed" by dipping it a few times into distilled
water; the "washing" is necessary to remove salts, which otherwise crystallize
and spoil the preparation.

Several strains of bacterial virus were studied using 60 kV electrons.  Two of
them, a and 7, received particular attention.'  In an earlier paper, the growth
of these two viruses, which are active on the same host (Eschtichia coli strain B),
has been described in detail (Delbrtick and Luria, 1942).  Adsorption of virus CL
on a growing sensitive cell of strain B at 37'C. produces, after a latent period of
13-17 minutes, lysis of the cell with liberation of about 140 infective units of
virus.  For virus y on the same host the latent period is 21-25 minutes; the
number of infective units liberated is about 135 per cell.

Crude suspensions of the viruses of a titer between 5 X loo and 2 X lOlo
units/ml. were found to give good results in the electron microscope experiments.
Suspensions of virus -y which had been partially purified by differential ultra-
centrifugation were also investigated.5

For the study of the interaction of virus and bacteria, an excess of virus was
added to a young broth culture of bacteria under standard conditions.  Samples
were taken at various intervals with a small wire loop and deposited on specimen
holders of the electron microscope.  The specimen on the holder was incubated
for a time in a moist chamber, so that the growth of the bacteria could continue
under conditions similar to those in the broth culture.  The holder was then
washed in distilled water and allowed to dry rapidly in air; the process of washing
and drying takes less than one minute.  A specimen prepared in this manner will
show the state of affairs in the growing mixture at a definite moment, which can
be taken as that of washing.  In some experiments, bacteria from a broth culture
or from a young slant were mixed with a drop of virus suspension on the holder
and, after incubation, were washed and dried as described above.

RESULT6

I. The virus particles

Micrographs of suspensions of virus OL and of virus 7 show the presence of
particles of characteristic shape and size, specific for each strain (figs. 1, 2, 3).
Regarding the identification of the particles visible on'these pictures, with the

4 Virus a and virus y, for which the authors are indebted to Dr. J. Bronfenbrenner, were
originally designated P28 and PC.  Virus 7 = PC has been purified by Kalmanson and
Bronfenbrenner (1939). The practical reasons for the change of name were given by
Delbrilck and Luria (1942).

6 One of the authors (S. E. L.) is greatly indebted to Dr. D. H. Moore for collaborating
in the work of purification.


ELECTRON MICROSCOPE STUDIES OF VIRUSES          59

viruses rather than with inert bacterial constituents, we note: the two viruses
were grown at the expense of the same host; the same bacterium then produces
particles of type a if acted upon by virus a, and particles of type 7 if acted upon
by virus 7.

The particles of virus CY have a round "head," 45-50 rnp in diameter and
uniformly dark in the micrographs, that means, uniformly scattering for 60 kV
electrons. To this round "head" is attached a "tail," about 150 rnp long and
not more than 10-15 rnp thick. The tail appears either straight or slightly
curved.

The particles of virus 7 present a very peculiar aspect.  To an oval head,
65 X 80 rnp, a straight tail, 120 rnp long and 20 rnl.c thick, is attached at one of
the narrow poles.  The head always shows a structure consisting of light and
dark areas. The structure, although striking enough to make the particles
immediately recognizable, is quite variable.  Four frequent configurations
can be described schematically as X-shaped, Z-shaped, inverted Z-shaped, and
diplococcus-shaped (C).  These various configurations can not all be accounted
for by one three-dimensional structure seen under different angles.  The inter-
pretation of these structures will be discussed later.
The particles described above are never seen in suspensions without virus
activity; their number is in direct proportion with the activity.
In suspensions of virus y partially purified by differential centrifugation, the
same particles are visible.  However, many of them appear to be damaged; the
tail is often broken, sometimes altogether missing.  Since during the process of
purification a large part of the activity had been lost, we believe that the ab-
normal particles visible in these suspensions have been mechanically damaged
and inactivated.B

Particles of another coli virus are visible in fig. 16.  They are round, 50-60 rnp
in diameter, and no tail can be seen.  This of course does not preclude the
existence of a tail, which might be too slender to be visible in the micrographs.
Particles of a staphylococcus virus' are shown in fig. 4.  They have a head
about 100 mp in diameter, and a tail about 200 rnp long.

     1. The growth oj bacterial viruses and lysis of the host
a. Virus 7;  Figs. 5-12 are micrographs of samples taken from growing mix-
tures of virus 7 and sensitive bacteria. Under the conditions of the experiment,
infected cells yield, after a latent period of 21-25 minutes, an average number of
135 infective units of virus (Delbrtick and Luria, 1942).  Results from two such
experiments will be given.  The two experiments differed in the multiplicity of ,
infection, i.e., in the number of virus units available in the mixture for each bac-
terium, as given by plaque count assays. 8  The first experiment was one of high
multiplicity, each bacterium being infected on the average by eight virus units,

6 This suggesta the opportunity of controlling on crude virus suspensions the results of
electron micrographic studies of purified preparations of viruses in general.
7 Obtained through the courtesy of Dr. 11. Zaytreff-Jern.
8 The "multiplicity of infection" is defined as the ratio adsorbed virus/bacteria (Delbriick
and Luria, 1942).


60      8. E. LURIA, hf. DELBRiiCK AND T. F. ANDERSON

while in the second experiment the multiplicity of infection was between two
and three.

Figs. 5-9 refer to the first of these experiments, in which the multiplicity of
infection was high. Adsorption experiments (Delbrtick and Luria, 1942) had
shown that under these conditions practically all the virus is taken up by the
bacteria in less than ten minutes, and that more than 99% of the bacteria are
infected.  Fig. 5 shows one of several micrographs of a sample that was dried
after 15 minutes of contact between bacteria and virus (10 minutes in the test
tube and five minutes on the specimen holder).  At this stage, all bacteria ap-
pear normal in structure.  Some particles of virus can be seen adsorbed on the
edge of the bacterium, on or within the clear peripheral zone of the cell, which
arises when the bacterial protoplasm shrinks away from the cell-wall during
the process of drying.  The existence of this clear zone enables one to see details
on about one-third of the total surface of the bacterial cell-wall.

We obtained seven good pictures of bacteria from this sample, and on these
seven bacteria, a total of 22 adsorbed virus particles could be seen in all, an
average of about three particles per bacterium.  Keeping in mind that only
about one-third of the surface is in clear view, we can estimate that there are in
reality about nine particles adsorbed per bacterium.  Within the limit of this
rather crude evaluation, we obtain, therefore, fair agreement between the
number of particles attached to the surface of the bacterium, as revealed by the
electron microscope, and the number of virus units attached to each bacterium,
as inferred from plaque count assays.  This agreement may be taken as further
evidence for the identification of the visible particles with the virus, and as
confirming the conclusion previously reached by indirect methods only (Del-
briick and Luria, 1942), that infective titers obtained by plaque count correspond
closely to the actual number of infective particles present in a suspension.  We
also note an unexpected and important fact, namely, that the adsorbed particles
remain at the surface of the bacterium; at least this must be true for the majority
of the adsorbed particles.

No free particles were visible on these micrographs.  This is to be expected
since, as pointed out above, adsorption is practically complete in ten minutes.
Growth experiments had indicated that lysis and virus liberation occur for
virus y after a minimum latent period of 21 minutes.  Electron micrographs
taken at 15 minutes confirm this by the absence of lysed bacteria.  In contrast
to this, micrographs of samples dried at 23 or 26 minutes reveal a completely
different picture.  By singular chance, we obtained from a series of unusually
o favorable fields, pictures of a number of bacteria caught in various phases of
disintegration.

Figs. 6 and 7 show a long bacterium (not unusual in young broth cultures of
this strain), one end of which has burst open and has liberated a flood of material
in which several hundred particles of virus -y are visible.  Along with the virus
particles, a granular material has come out from the bacterium.  These granules
are of uniform size and are much smaller than the virus particles, being 10-15
rnp in diameter.  The increasing transparence of the bacterium from the normal
to the bursting end shows how the bacterial content is shed from the bursting


ELECTRON MWROSCOPE STUDIES OF VIRUSES        61

end. The ditruse mass lying across the bursting end of the b,acterium probably
contains also the remains of another lysed cell.  It is most noteworthy that the
bacterial contents show, besides the virus particles, no other particles of similar
size.  The dark spots on the cell-wall of the long bacterium are either single or
groups of virus particles.  It is impossible to say whether they are inside or out-
side of the cell-wall.

It will be seen in figs. 6 and r that those parts where the two fields overlap agree
in the finest details.  Since these two figures resulted from separate exposures,
they show that the objects imaged are not noticeably altered by the very intense
electron irradiation necessary for focusing.

The cells shown in figs. 8 and 9 appear to be in a later stage of lysis.  They are
"ghosts," empty cell-walls from which all content has been liberated, and are sur-
rounded by virus particles and protoplasmic granules.  Holes of various sizes are
visible in these bacterial cell-walls.  Virus particles in large number (80 in one
case, 150 in the other) surround the empty e&walls.  Their location in the im-
mediate vicinity of the cells suggests that lysis has taken place after the specimen
had been washed, i.e., during the brief period of drying.  Therefore, the number
of visible particles should and does correspond to the average yield of virus per
bacterium, as determined by growth experiments, namely 135.

Side by side with the lyaed bacteria we find in these specimens bacteria which
are not yet lysed.  They show on their edge many more adsorbed virus particles
than those from specimens dried at 15 minutes, when lysis had not yet started.
These virus particles must have been adsorbed after their liberation from neigh-
boring lysed bacteria.  Since practically all bacteria in the specimen had become
infected in the first minutes of the experiment, these bacteria must also be close to
lysis.  We conclude that the abiity to adsorb virus remains unimpaired until
very close to the moment of lysis.  This conclusion had been reached previously
on the basis of growth experiments (Delbrtick, 1940).  Fig. 10 shows the proto-
plasmic granular material at a higher magnification.

In the second experiment mentioned above, in which the multiplicity of infec-
tion was only two or three, micrographs of specimens dried at 5,10, and 15 min-
utes show bacteria of normal aspect.  No adsorbed virus particles are visible,
corresponding to the low multiplicity of infection.  However, the bacteria are
infected; in specimens dried at 25 minutes most bacteria are seen to be lysed, and
the remaining ones show numerous virus particles adsorbed on their surface.
These bacteria appear to be damaged and near to lysis (fig. 11).

b. Virus a.  The interaction between the same strain of bacteria and virus
CT was studied in an experiment in which the multiplicity of infection was about
five.  In specimens prepared at five or ten minutes, the bacteria appear normal,
occasionally with one or a few adsorbed virus particles visible on their edge.
At fifteen minutes (figs. 13 and 14) we witness the lysis of bacteria, as expected on
the basis of growth experiments, which give a latent period of 13-17 minutes for
the lysis produced by virus cy in this host.  Lysis is accompanied by the liberation
of particles of the type characteristic for this virus.  Cells in the process of lysis
and "ghosts" are both considerably swollen.

The material which is liberated from the lysed cell along with the virus particles


62       8. E. LURIA, M. DELBRttCK AND T. F. ANDERSON

is again granular, the granules being of the same size as in the case of lysis pro-
duced by virus y (fig. 12). This is strong support of the interpretation of these
minute particles as constituents of the bacterial protoplasm.

All bacteria which are not lysed within 15 minutes have several virus particles
adsorbed (fig. 15) ; these obviously come from the lysed cells.  The bacterium in
fig. 15 has almost completely divided, but the cell-walls of the two cells are still
connected by an X-shaped bridge.             .

c. Other viruses.  Figs. 16 and 17 illustrate the action of still another virus
which is active on a different host, a motile strain of E. wli.  This virus was
studied extensively several years ago by one o;f the authors (Delbrtick, 1940).
The bacterium in fig. 16 shows flagella and adsorbed virus particles. Fig. 17 shows
an empty cell-wall after @is'.  It is clearly visible that the flagella have remained
intact. The same can be said of the case of a virus active on Salmonella sp.
(Poona type)O (fig. 18). In these two cases at least, the flagella do not appear to
be damaged by the action of the virus.

DISCUSSION

There can hardly be any doubt that the sperm-shaped particles in suspensions
of bacterial viruses are the particles of virus. They are present in amounts
proportional to the activity and they are never present in suspensions without
virus activity. The structure of the visible particles is specific for each strain
and a bacterium liberates the particles whjch are characteristic for the virus
which has acted upon it. The use of two tierent and unrelated viruses acting
on the same host eliminates the possibility that the particles might be natural
protoplasmic components of the bacterium.

The behavior of the visible particles during the reproduction cycle of the
virus, showing their specific adsorption and their liberation in the expected
amount after the expected latent period, offers further reasons for their identi-
fication.

Size and shape of the particles deserve special attention.  The size of the
"head" is close to that which had previously been inferred for various bacterial
viruses by indirect methods. For viruses (Y and y the sensitivity to irradiation
with x-rays has been tested quantitatively (Luria and Exner, 1941).  From these
data the sensitive volume, i.e., the volume within which absorption of energy
from the x-rays leads to inactivation of the virus has been calculated.  This
sensitive volume may be smaller than the true volume.  The irradiation data
therefore give minimum values for the particle sizes.  The results were 36 ml
for virus a and 50 rnp for virus y, i.e., in both cases about 300/o smaller than the
sizes given by the electron microscope pictures.  We conclude that irradiation
experiments do give values close to the real ones, and give correct values for the
relative sizes of different viruses.

Kalmanson and Bronfenbrenner (1939) have studied the diffusion of virus 7
purified by dserential filtration.  They find that their virus suspensions contain

* Obtained through the courtesy of Dr. M. L. Rakieten.


ELECTRON MICROSCOPE STUDIES OF VIRUSES         63

two fractions with different rates of diffusion.  The larger fraction diiuses at a
rate corresponding to a particle diameter of 16-M rnp.  A very small fraction
difkes faster, corresponding to a particle size of 3-4 rnp.  These authors assume
that the small particles are usually attached to larger unspecific carriers (the
slower diffusing particles), and that they occasionally become free, still retaining
their activity.  The electron microscope pictures do not support this view, since
the particles visible on these pictures are too regular in size and structure to be
interpreted as bacterial debris to which the virus might be adsorbed.  The elec-
tron microscope could not show particles having diameters of 3-4 w.  But if
such small virus particles existed, they should show up in irradiation experiments
as a very resistant fraction.  Careful search for such an x-ray resistant fraction
of virus has given completely negative results (Luria and Exner, 1941).

Size determinations of the strain of staphylococcus virus shown in fig. 4 have
not been made by any other method.  However, the filtration and centrifugation
studies of Elford and others have shown that Werent strains of staphylococcus
virus differ little in size, in contrast to the viruses of the colidysentery group.  It
may therefore be permissible to compare our values with those obtained for other
staphylococcus viruses by other methods.  Irradiation data have given 50 rnp,
somewhat lower than the value here obtained (190 mp).  Ultrafiltration studies
gave values between 50 and 78 rnp, in fair agreement with the electron micro-
scope value.  Northrop (1938) has made extensive studies with purified prepara-
tions of a staphylococcus virus.  From the rate of sedimentation in the ultra-
centrifuge a value of 60-90 rnp (mol. weight 3 X BY) was obtained, again in good
agreement with our value. Diffusion experiments on Northrop's virus gave re2
sulks which depended on the concentration of virus.  In concentrated suspensions
the difIusion rate corresponded to a molecular weight of about 3 X W, in agree-
ment with the centrifugation value.  However, in highly diluted suspensions the
rate of difksion was found to be faster, corresponding to a particle size of about
10 rnp.  In explanation Northrop proposed a reversible equilibrium between small
particles and large partikles. If this were true, plaque count assays should give
titers corresponding to the number of smaIl virus particles, since these assays are
done at extreme dilution.  The titer of large particles which are presumed ta be
present at high concentrations should then be almost a thousand times smaller
than the plaque count titer.  In contrast with this, we find approximate quan-
titative agreement between the plaque count titer and the number of particles
visible on the electron microscope pictures.  Therefore, Northrop's idea is not
applicable to our cases.

Shape and structure of the virus particles, as revealed by the electron micro-
scope, deserve special consideration.  The pictures here reproduced give the
impression of a somewhat more complex organization than the pictures of plant
viruses (Stanley and Anderson, 1941) had indicated.  These had shown either
straight rods or round particles.  Of the animal viruses, influenza virus showed
very small, round particles (Chambers and Henle, 1941). Papilloma virus
(Sharp, Taylor, Beard, and Beard, 1942a) and equine encephalomyelitis virus
(Sharp, Taylor, Beard, and Bard, 1942b) have both round particles, but while


64       8. E. LURIA, M. DELBRihK AND T. F. ANDERSON

the particles of papilloma appear to be homogeneous, those of encephalomyelitis
virus show an internal structure.  The majority of the much larger bodies of vac-
cinia virus (Green, Anderson, and Smadel, 1942) show five granules of more scat-
tering material.

The structure in the head of virus 7 cannot be ascribed to the presence of ma-
terial of higher specific scattering power.  The most likely material could be phos-
phorus, which, per atom, should scatter about four times more strongly than
carbon, nitrogen or oxygen.  In nucleic acid, for instance, there is one phosphorus
atom to 20 of the lighter atoms.  Pure nucleic acid would therefore scatter only
about 20% more than a similar compound without phosphorus. Since we can
hardly assume that the dark regions are composed exclusively of compounds of
such relatively high phosphorus content, the maximum contrast due to differ-
ences in specific scattering power could not be more than a few percent and could
not show up in our pictures.  We believe therefore that the dark parts represent
regions of greater thickness.  It is possible that the particles in the native state
are oval, but upon drying the more aqueous parts collapse while the solid parts
retain more scattering material, which forms the dark areas of the head.  The
images obtained therefore indicate that the distribution of solid material in the
head of the particle of virus y is not uniform. A detailed analysis of the structure
visible in the heads of these particles will be given elsewhere.

A word may be added regarding the tendency to speak of viruses as molecules.
This tendency received its greatest momentum from Stanley's discovery in 1935
that paracrystals of tobacco mosaic virus could be obtained by simple methods,
and from the great number of subsequent stupies of this and of other viruses along
the lines of protein chemistry. Also the electron microscope pictures of plant
viruses, revealing simple rods and spheres, seemed to encourage such a ten-
dency. While it is true that no strain of bacterial virus has yet been crystallized,
chemical studies on purified preparations have indicated that chemically, bac-
terial and other viruses are closely related (Northrop, 1938).  It is only natural
that chemical and physical studies of viruses have led scientists to the habit of
thinking of viruses in terms of molecules.  However, one should keep in mind
that the concept "molecule" is flexible when applied to structures such aa viruses.
When we speak of a long chain compound +s a molecule, neither ite configuration
nor even its composition is to be taken as necessarily definite.  The ambiguities
will be multiplied if still more complicated structures are considered, the parts of
which are not all connected by primary valencies.  Such "molecules" will share
with living things the impossibility of delimiting unambiguously which atoms
belong to them and which do not.  While no harm is done by calling viruses
"molecules", such a terminology should not prejudice our views regarding the
biological status of the viruses, which has yet to be elucidated.

The study of the interaction of virus a and virus y with their host has con-
fumed in every detail the picture of the process which had been deduced on the
basis of growth experiments.  As d'Herelle (1926) had early suggested, and as
quantitative studies had shown (Delbrtick, 1942), the infection of a bacterium,
after a latent period characteristic of each virus, is followed by lysis of the bac-


ELECTRON MICR08COPE STUDIES OF VIRUSES         65

terium with liberation of a large number of new virus particles.  Quantitatively,
too, the agreement between electron microscopic and growth experiments is ex-
cellent, both regarding the length of the latent period and regarding the amount of
virus liberated by the bacteria.  Several points, however, receive further light
from the electron micrographs.

After lysis of a bacterium a cell-wall remains which, in contrast to those ob-
tained from intense sonic vibrations (Mudd, Polevitzky, Anderson, and Cham-
bers, 1941), is always more or less damaged and variously lacerated. The new
virus is liberated from the interior of the bacterium.  The pictures, however, give
no indication in which part of the bacterium the virus is produced, whether in the
deep interior of the cell or close to the inner surface of the cell-wall.

The pictures of lysed bacteria show, besides the particles of virus, also granular
material of very regular units, 10-15 rnp in diameter.  If these are to be inter-
preted as molecules, their size corresponds to a molecular weight of the order of
10". , These particles are liberated from the cell in great abundance, and seem to
constitute the bulk of its protoplasm.  The absence among the bacterial com-
ponents of elements of size comparable to that of the virus particles explains why
the latter can be studied so favorably in crude suspensions.  It also explains the
success of work oh the purification of bacterial viruses by means of differential
centrifugation and by filtration, and should encourage further work along these
lines.

In a series of papers Krueger (1938) has proposed the idea that the bacterial
cell contains a precursor of the virus particle, which, upon infection of the cell
with a virus particle, is promptly converted into virus.  This theory was elabor-
ated as an analogue to the well-known relations between proteolytic enzymes
and their precursors.  According to this theory an uninfected bacterium of the
strain here considered should contain on the average 140 precursor particles of
virus a and 135 precursor particles of virus y.  The pictures show clearly that
this is not the case, since bacteria lysed under the influence of virus y show no
evidence of particles resembling virus a! and vice versa.

Finally, a point may be mentioned which seems to us perhaps of the greatest
consequence.  We have seen that the new virus is liberated from within the cell.
On the other hand, the pictures of bacteria infected with virus y and taken at
fifteen minutes showed that the adsorbed virus particles, or at least most of them,
do not penetrate into the interior of the cell but remain on the outer surface of the
cell-wall. This observation creates a difhculty in interpreting virus growth.
How do the infecting particles reproduce if they remain outside while the new
virus is generated in the interior of the cell?  One might assume either that the
infecting particles act through the cell-wall, or that only one particle can enter the
cell.  The latter idea seems attractive in the light of results of growth experiments
on multiple and mixed infection.  These have shown that a bacterium always
reacts as though only one particle of virus had been effective.  The pictures here
reproduced, if interpreted on the assumption that one virus particle enters the
cell, would indicate that the entry of one virus particle bars the entry of other
virus particles by making the bacterial cell-wall impermeable to them.  The highly


66       6. E. LURL4, M. DELBRtiCK AND-T. F. ANDERSON

peculiar phenomenon of mutual exclusion between virus particles attacking a
cell would thus be explained by a mechanism alternative to that proposed in a
previous discussion (Delbrtick and Luria, 1942).  An interpretation of this kind,
for the correctness of which the experiments offer as yet hardly more than a hint,
would suggest an analogy with the fecundation of monospermic eggs, and would
lend support to those theories of the systematic position of virus which consider it
as related to the host rather than as a parasite (cf. Hadley, 1928).

SUMMARY

1. Four strains of bacterial viruses have been studied with the electron micro-
scope.  In all cases the particles of virus could be identified on the micrographs.
Three of these strains show "sperm-shaped" particles, consisting of a head and a
tail.  For the fourth strain, a tail is not visible on the micrographs.  The par-
ticles of one of these viruses show a distinct structure in the head.  The particle
sizes agree well with the sizes inferred by some of the indirect methods.

2. The interaction between the virus and its host has been studied in detail in
thecaseof twoviruses which act upon the same strain of Edmichiu wli.  The
micrographs demonstrate the adsorption of virus on the host and, after the pre-
dicted time, the lysis of the host with the liberation of virus particles of the in-
fecting type.  There is quantitative agreement between the numbers of particles
visible on the micrographs and the numbers predicted on the basis of growth ex-
periments for which plaque count assays were used. Along with the virus
particles, the lysing cells shed protoplsamic material of uniform granular strut-
ture.  The size of these granules is much smaller than that of the viruses and is
independent of the virus under whose influence the bacterium is lysed.

3. Upon lysis the virus particles are liberated from the interior of the bacterial
cell, for they are not visible on its surface up to the moment of lysis.  In cases of
multiple infection, the infectins particles of virus, or at least the majority of them
seem not to enter the cell but to remain attached to the outside of the bacterial
cell-wall.

4. The bearing of these results on the problems of the nature of viruses and of
their systematic position is discussed.

We are grateful to Miss Nina Zworykin for taking many of the electron micro-
graphs shown.

BIBLIOGRAPHY

ANDEBBON, T. F.  1942 The Study of Colloids in the Electron Microscope.  Advances in
  Colloid Science, 1, 262-220.
CEAMBEIU, L. A., AND HENLE, W.  1241  Precipitation of Active Influenca A Virus from
Extra-Embrionic Fluide by Protamine.  Proc. Sot. Exptl. Biol. Med., UI, 481-484.
DELBEUCK, M. 1940 The Growth of Bacteriophage and Lyeis of the Host. J. Gen.
Phyaiol., 22, 643-000.

DELBR~~K, M. 1942 Bacterial Viruees (Bacteriophages). Advance6 in Enzymology,
  a, l-32.

DELBBUCK, M., hi) LIJBIA, S. E. 1242 Interference between Bacterial ~Viruses. I.
Interference between Two Bacterial Virueec Acting upon the Same Host, and the
  Mechanism of Virus Growth.  Arch. B&hem., 1, 111-141.


ELECTRON MICROSCOPE STUDIES OF VIRUSES         67

ELFOED, W. J. 1933 The Sizes of Viruses and Bacteriophages, and Methods for Their
  Determination.  In Doerr and Hallauer, Handbuch der Virusforschung (Wien), Vol.
  1, 128-219.
ELLIS, E. L., AND DELBEUCK, M.  1939 The Growth of Bacteriophage.  J. Gen. Physiol.,
  22,365-384.

GEEEN, R. H., ANDEBBON, T. F., AND SMADEL, J. E.  1942 Morphological Structure of the
  Virus of Vaccinia.  J. Exptl. Med., IS, 651-656.
HADLEY, P. 1923 The Twort-d'Herelle Phenomenon. A Critical Review and Presenta-
tion of a New Conception (Homogamic Theory) of Bacteriophage Action. J. In-
fectious Diseases, 42, 263-434.

D'HEBELLE, F. 1928 The Bacteriophage and Its Behavior. Williams & Wilkins,
  Baltimore.

KALMA~SON, G. M., AND BRONFENBBENNER, J. 1939 Studies on the Purification of
  Bacteriophage. J. Gen. Physiol., 23, 293-223.
KBUEQEB, A. P., AND MUNDELL, J. H. 1933 The Demonstration of Phage Precursor in
  the Bacterial Cell. Science, 33. 550-551.
LURIA, S. E., AND ANDERSON, T. F. 1942 The Identification and Characterization of
  Bacteriophages with the Electron Microscope. Proc. Nat. Acad. Sci., 23, 127-139.
LURIA, S. E., AND EXNEB, F. M. 1941 The Inactivation of Bacteriophages by X-rays.
  Influence of the Medium. Proc. Nat. Acad. Sci., 27, 370-375.
MUDD, S., POLEVITZKY, K., ANDERSON, T. F., AND CHAMBERS, L. A.  1941  Bacterial Mor-
phology as Shown by the Electron Microscope. II. The Bacterial Cell-Wall in the
  genus Bacillw~ J. Bact., 42, 251-284.

NORTHROP, J. H. 1933 Concentration and Purification of Bacteriophage. J. Gen.
  Physiol., 21. 335-366.
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  eines Bakteriophagen. Naturwiesenechaften, 28.40.
RUBKA, H. 1949 Die Sichtbarmachung der bakteriophagen Lyse im Uebermikroskop.
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RUSKA, H.  1941  Uber ein neues bei der bakteriophagen Lyse auftretendes Formelement.
Naturwissenschaften, 29, 367-368.
SCHLESINGER, M.  1933 Reindarstellung eines Bakteriophagen in mit freiem Auge sicht-
baren Mengen. Bioch. Z., 264, 6-12.

SHABP, D. G., TAYLOR, A. R., BEAED, D., AND BEARS, J. W.  1942s  Study of the Papilloma
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of the Western Strain Equine Encephalomyelitis Virus.  Proc. Sot. Exptl. Biol. Med.,
  51, 206-297.

STANLEY, W. M. 1935 Isolation of a Crystalline Protein Possessing the Properties of
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  tron Microscope.  J. Biol. Chem., 139,325-333.


f3. E. LURIA, M. DELBRijCK AND T. F. ANDERSON

PLATE 1

FIG. 1. P&i&e of virus y. X 36,000.
FIQ. 2. Particles of virus y. x 40,000.
FIG. 3. Particles of vim a. X 47,OCO.
FIG. 4. Particle8 of staphylococcue virue.  X 20,000.
FIQ. 5. E. coZi + virue 7.  15 minutee contact. A bacterium with adsorbed particlee~of
vilue. x 20,ooo.


JOURNAL OF BACTERIOLOGY. VOL. 46

PLATE 1

(S. E. Luria, M. Delbriick and T. F. Anderson: Electron Microscope Studies of Viruses)


70      6. E. LURIA,' M. DELBRiiCK AND T. F. ANDERSON

PLATE 2

FIGS. 6 and 7. E. roli + virus y.  23 minutes contact.  A bacterium immediately after
bursting, protoplasmic granules and several hundred particles of virus.  The fields of the
t,wo pictures overlap in part. X 12,500.


JOURNAL OF BACTERIOLOGY, VOL. 48                                PLATE 2

6. E. Luria. M. Delbrtiok and T. F. Anderson; Electron Micrascope Studie, of Viruses)


72       s. E. LURIA, hf. DELBRWK AND T. F. ANDERSON

PLATE 3

FIG. 8. IS. coli + virus y. 23 minutes contact.  "Ghost" of a lysed bacterium.
x 12,500.

FIG. 0. E'. coli + virus y. 23 minutes contact. Another "ghost".  X 11,500.
Flu. 10. E. coli + virus y. 25 minutes contact.  Detail of contents shed from a lysed
cell, showing protoplasmic granules and one particle of virus y.  x 95,000.
FIG. Il. E. coli + virus y.  25 minutes contact.  A bacterium with ten adsorbed particles
of virus visible on cdgcs.  x n,ooo.
Flc. 12. h'. coli + virus a.  15 minutes contact.  Detail of contents shed from a lysed
cell, showing protoplasmic granules and three particles of virus ~1.  x 100,000.


JOURNAL OF BACTERIOLOGY. VOL. 46

PLATE 3

(S. E. Lurk, Y. Delbriick and T. F. Anderson: Electron Microscope Studies of Virusas)


74

S. E. LURIA, M. DELBRijCK AND T. F. ANDERSON

PLATE 4

FIG. 13. E. coli + virus (1.  15 minutes contact. Lysis of a bacterial cell. Protoplasmic
material and 93 particles of virus.  x 13,506.
FIG. 14. E. coli + virus LI.  15 minutes contact. "Ghost" of a lysed bacterium, several
particles of virus ~1. X 15,560.
FIG. 15. E. coli + virus a.  15 minutes contact.  Dividing bacterium with 19 adsorbed
particles of virus visible on its edge.  X 16,569.


JOURNAL OF BACTERIOLOGY. VOL. 46

PLATE 4

(S. E. Luria, hf. Delbriick and T. F. Anderson: Electron Microscow Studies of Viruses)


76

S. E. LURIA, M. DELBRiiCK AND T. F. ANDERSON

PLATE 5

FIG. 16. E. coli (motile strain) + virus Pt. 22 minutes contact. Bacterium with 12
adsorbed particles of virus. 2 free particles of virus. X 19,096.
FIG. 17. E. cc& (motile strain) + virus P,. 22 minutes contact. Lysed bacterium
with flagella. X 16,599.

FIG. 18. SalmoneZla sp. (Poona type) + virus SP.  Ccntrifugation sediment of an un-
filtered lysate. Lysed cells and two apparently normal cells. Flagella. X 12,069.


JOURNAL OF BACTERIOLOGY. VOL. 46                               PLATE 6

(S. E. Luria, M. Delbriick and T. F. Anderson: Electron Microscope Studim of Viruses)