394                        SCIENCE                VOL. 04, No. 2443

             SPECIAL ARTICLES

THE SIZE OF STREPTOCOCCUS BACTERIO-

PHAGES AS DETERMINED BY X-RAY
         INACTIVATION
INFORMATION about the particle size of bacterio-

phages has been obtained chiefly by ultrafiltration and
centrifugation methods. Data of various authors, as
oollected by Elford,l seem to indicate that the particles
of each phage are homogeneous in size; and that par-
ticle size is a oharacteristie property of each phage.
Coli-dysentery phages have particles ranging from
8-20 rnp (phage "X13") to 50-75 mlr, diameter (phage
"CM"). Staphylococcus phage "K" has a diameter
of about 50-70 mu. The largest phage at present
known has particles of 80-120 mu ( Subtilis phage) .

broth in small celluloid tubes were exposed to x-rays
(475 kv equivalent constant potential, 13 ma, .25 mm
Cu plus .45 mm Al filter, 960 roentgens/min. mea-
sured in air at 37 cm focal distance, absorption and
scattering corrections negligible). Afterward the
phage content of each sample was determined by

While the particle size values as given above are
usually accepted, it must be. mentioned that diffusion
studies*. have suggested that phage particles may be
much smaller and inhomogeneous in size.

A new method has recently been developed: which,
on the basis of radiation experiments, seems to give
useful information about the particle size of bacterio-
phages.

When suspended in a suitable medium the sensitiv-
ity to x-rays of a phage strain, measured by the
percentage of active phage which remains after a
certain amount of radiation has been given, is a
highly reproducible property of that strain, and ap-
pears to be a function only of the size of its particles.
The irradiation experiments interpreted in terms of
the "hit theory" indicate that a phage particle is
inactivated by a single ionization (or excitation)
process.  In order to be effective this process must
take place within a "sensitive volume,`, the size of
which can be calculated from the rate of inactivation.
This volume is found for each strain of phage to be
of the same order of magnitude as the volume of the
phage particle itself, as determined by ultrafiltration
and centrifugation.

We have now used this method to obtain informa-
tion about the particle sizes of three streptococcus
bacteriophages, which had not previously been mea-
sured.  These phages have been described by A. C.
Evans.'  They are distinguished by their serological
specificity, by bacterial host specificity and by the
type of plaque they produce on solid medium.
Samples of the bacteriophages suspended in peptone
1 W. J. Elford, in Doerr and Hallauer, Handbuch d.
Virusforschung, p. 126, Julius Springer, Wien, 1938.
2 J. Bronfenbrenner, Jour. Ezp. Med., 45: 873, 1927;
D. M. Hetler and J. Bronfenbrenner, Jour. Gen. Physiol.,
14: 547. 1931: J. H. Northrop, Jour. Gem. Physiol., 21:
335,19&l '           -,

s E. Wollman, F. Holweck and 8. Luria, Nature, 145 :
935,194O; D. E. Lea, Nature, 146: 137, 1940.
4 A. C. Evans, U. S. Public Health Reports 49: 1386,
1934.

plaque count. The results are shown in Fig. 1.

LO

.8


.6

I?.
!I

.i

1

b-
.        J
  q ? (i  8 10 12x10
       DOSE IN r

FIN. 1.  Inactivation curves for three streptococcus bae-

teriophages.  Abscissas: dose of x-rays in roentgens.
Ordinates: proportion of bacteriophage particles remain-
ing active after irradiation.

It is evident that in each case the experimental'
points can be f'ltted by an exponential curve (straight
line on semi-logarithmic plot) within the limits of the
experimental errors, which in plaque counts with
streptococcus phages are likely to be rather large.
Having established the exponential relation with great
precision for other phages,5 we shall assume it to
apply here.

Table I gives the diameters of the "sensitive vol-
umes,,' calculated from the curves in Fig. 1 by Lea's
method.3 Assuming that the relationship which exists
for other phages between size of sensitive volume and
particle size is also valid for the streptococcus phages
we can tentatively consider the values of Table I as
estimates of particle size.  It should be `mentioned that
the recognized uncertainties in the absolute measure-
ment of x-ray dosage at 500 kv are not large enough

s 9. E. Luria and F. M. Exner, Pro& Nat. Aoad. Soi.,
-27: 370, 1941.


OCTOBER 24, 1941                  BCIENCE


                TABLE I

395

B  ..........     563
:  ..........
  ...........   %

N

* Dose giving inactivation ratio s = l/e.

to influence the present results, since particle diame-
ters depend (through volumes) on cube roots of inacti-
vation rates.

A comparison with previously measured phage9
shows that phage D is a medium size phage ; phage C
falls among small phages (like Cl3 Burnet) ; and
phage B is still smaller.

The classifioation of these three phages as separate
entities, based up to now on their different biological
properties,* finds further justification in their differ-
ent particle sizes.

A general relationship of inverse proportionality
has been shown to exist between the size of phage par-
ticles and the size of the plaques they produce on
agar (Elford, Burnet). Such comparisons are definite
only for plaques produced in presence of the same
host strain of bacteria, which was not the case in our
experiments. Nevertheless, it is probably not mean-
ingless that the smallest phage B is a "large plaque
forming" strain, whereas the larger phage D produces
very small plaques.

We hope soon to check the particle size values as
given here by other methods (ultra-centrifugation,
electron microscope).

We are indebted to Dr. A. C. Evans for supplying
the strains of bacteriophages and of host bacteria.
Only three out of four strains were studied, owing to
difficulty in obtaining reproducible counts of the
plaques formed by phage A.
                   FRANK M. EXNER
                  S. ELURIA

DEPAETI~ENT OF `CANCER RESEAIUXI AND
THE BACTEIUOLOQICAL LABORAT~RJES OF
  THE DEPARTMENT OF SWQERY,
COLLIWE 01 PHYSICIANS AND SU~OEONS,
COLUMBIA ~NIVERSI'IT