370

BIOPHYSICS: L URXA AND EXNER      PR0C.N. A.S.

THE INACTIVATION OF BACTERIOPHAGES BY X-RAYS-
         INFLUENCE OF THE MEDIUM

BY S. E. LURIA AND FRANK M. EXNER

FROMTEEBACTERIOLOGICALRESEARCHLABORATORIESOFTHE DEPARTMENTOF
SURGERY,MDFROMTHE DEPARTMENT OFC!ANCER~SEARCH,COLLEGEOF
  P~~I~~~ANDS~RGEONS,COLUMBIAUNIVERSITY,NEWYORK,N.Y.

Communicated July 12, 1941

Several studies on the inactivation of bacteriophages by x-rays have
recently been published.'  Q ua i a ive
                    1 t t   experiments of Wollman and
Lacassagne have shown that the x-ray sensitivity of different phages sus-
pended in broth increases with their particle size as determined by ultra-
filtration and centrifugation.2  Holweck, Luria and Wollman have anal-
yzed the inactivation of Cl6 phage by x-rays and alpha-particles, and inter-
preted their results in terms of the "hit theory."  Further discussion of the
same data has been given by Lea.3

We have carried out experiments with x-rays on a series of bacteriophages
(3K, C16, C13, P28), some of which have been measured by Elford.2  In
all experiments the concentration of phage particles was determined by the
plaque count method.  Samples were irradiated in celluloid tubes with
careful attention to x-ray dosage measurement.  The same x-ray generator
was used for all exposures.  Technical details will be described in a com-
plete account to be published later.

1. When bacteriophages are irradiated in the original filtrate or in
broth dilutions the results (Figs. 1 and 2) can be summarized as follows:

(a) The curve of inactivation (percentage of remaining phage as a func-
tion of the dose of x-rays measured in r) is exponential in the first part of its
path (straight line in semi-logarithmic plot).  When the dosage becomes
very high (Fig. 2) the rate of inactivation shows an increase, which how-
ever, is so slight as to be detectable only because of the high precision ob-
tained in the phage assay.  This increase in inactivation rate with high
dosage had also been found in the experiments of Exner and Zaytz&-Tern'
and is probably due to superposition on the principal inactivation process
of some effect of a different kind.  We hope to determine the nature of this
effect.

We have followed the inactivation to a diminution in titer by a factor of
2 X 10-T and have always found consistently reproducible results.  The
inactivation is a function of the total dose of x-rays, and not of the dosage
rate (no "time factor").  The fractional inactivation produced by a given
dose is independent of the concentration of the phage suspension during
irradiation.


VOL. 27, 1941      BIOPHYSICS: LURIA AND EXNER             371

(b) For x-ray tube voltages between 200 and 1000 kv. the inactivation
rate has been found independent of the quantum energy (wave-length) of
the x-rays.  This statement refers to x-ray dosage measurements based on
a Victoreen thimble chamber (wall thickness increased for measurements
at highest voltage).  It is of interest to point out the difference between
this result and that obtained with tobacco mosaic virus by Gowen4 who
finds a definite wave-length effect.

(c) The x-ray sensitivity of each bacteriophage is a highly reproducible
property of that strain.  The inactivation curve for phage Cl6 obtained a
year ago by Holweck, Luria and Wollman in Paris coincides exactly with
the curve established by us on the same phage.  The two samples, though
of common origin, had been kept many years in different surroundings and
grown on bacterial hosts of different species (Dysentery Y6R and Coli 234).

(d) The slope of the inactivation curve is different for the different
phages (Fig. 1).  Phages of smaller particle size are more resistant.  In
table 1 are shown the dimensions of the "sensitive volumes" as calculated

TABLE1

  3K      Staphylococcus "K"      45,000      48      50-70
Cl6      Coli 234 or Dysen-
         tery Y6R           40,000      50      50-75
Cl3       Coli 234           115,000       32      2&30
P28       coli "PC"            90,000      36       . . . . .
* Dose giving inactivation ratio N/N, i= l/e.

according to Lea's method,3 and the size of the particles as given by Elford.2
It is evident that the two series of values fall close to one another, in agree-
ment with the hypothesis (Holweck, Luria ancl Wollman) that in the case of
bacteriophages the sensitive volume corresponds to the phage particle it-
self.  The small particle size of P28 phage seems to be confirmed by centrifu-
gation experiments now in progress.  The utility of the radiation method
as a means of gaining information about the size of ultramicroscopic
structures seems to be well borne out by these results.

2.  We wish now to call attention to a new aspect of the inactivation of
bacteriophages by x-rays, which becomes evident when phages are sus-
pended in different media.

If a bacteriophage filtrate is diluted in distilled water, instead of broth,
so that less than 1 per cent of the original filtrate is present, its inuctivatiort
by x-rays is found to be greatly increased.  A dose of x-rays that leaves 40 per
cent active phage in broth inactivates more than 99 per cent of phage
diluted 100~ in distilled water.  This effect and the other facts related be-
low have been found to hold for all four phages so far investigated, although


372           BIOPHYSICS: L URIA A ND EXNER   :   PROC. N. A. S.

the most thorough of our studies have been made on the 3K and Cl6
phages.  We have established the following facts:
(a) The effect of distilled water as a medium does not depend on differ-

ence of pH as compared to broth.  Broth and water suspensions with pH
adjusted to different values between 6.8 and 8.0 were irradiated. All
broth suspensions gave closely the same inactivation rate.  On the other
hand, the water suspensions all showed the higher order of sensitivity
found with distilled water.

1.0





.5
9
Nl








.I0

05,            I            I            I            I
0   DOSE IN RO;NTGENS            2 xl0



                      FIGURE 1

s'

Inactivation of four bacteriophages suspended in broth.  Abscissas: dose of x-rays.
Ordinates: proportion of phage particles remaining active after irradiation.

(b) Suspensions of phage in different saline solutions (Na, K, Ca and Mg
in various proportions) and in phosphate buffer show sensitivities of the
same order as that of water suspensions.
(c) Suspensions of phage in aqueous solutions of ordinary gelatin or of
Eastman Purified Gelatin behave like broth suspensions whenever gelatin is
present in concentrations above lo-" gm. per cm.a
(d) Protection against x-ray inactivation, similar to that exerted by
gelatin and broth, though less complete, was found when solutions of egg
albumin and serum albumin were used as media.
(e) No protection was given by either reducing or non-reducing sugars
(dextrose, sucrose).


VOL. 27, 1941     BIOPHYSICS: LURIA AND EXNER            373

Q Oxidizing (HzOZ) and reducing agents (Sodium Thioglycolate and
Cysteine Hydrochloride) added to un-irradiated phage suspensions in-
activated the phage.  Similar observations have been reported by other

  .

   .
ah' I " '
     8



        .

   .
\
        .

Cl6     3K
  1'1
   x `,!\
        \ \
          o ?  ?
    ????
           ? ?
             ? ?
            ? ?
             ? ?
        ?? ?
               ?    o ? ???
           ? ?
                        o ?  ?
                      ?  ??
        ? ???
                       ?   ? ?
               ? ?

.

  I     2    +    4    9   gx105
      DOSE IN ROE.NTGENS.
                 FIGURE 2
Extension of two of the inactivation curves shown in figure 1.

workers.6 We have found such inactivation to be much more rapid in
distilled water, saline, etc., than in those media (broth, gelatin, etc.) which
protect phages against the action of x-rays.  Concentrations of protective


374           BIOPHYSICS: LURIA A ND EXNER      PROC. N. A. S.

substance necessary to give effective protection are the same in both cases.
On the other hand concentrations of thioglycolate and cysteine low enough
to have slight or no effect on the activity of phage gave no protection
against x-ray inactivation.

(g) Broth, gelatin and albumins give complete protection against the
slow spontaneous inactivation which phages undergo when highly diluted
in distilled water or saline solution.

(h) With progressively lower dilution in distilled water, so that the con-
centration of original broth filtrate is progressively higher, the inactivation
rate of phage by x-rays diminishes.  At concentrations of broth above 1
per cent the sensitivity to x-rays becomes constant, and identical with that
of suspensions in undiluted broth.  The protection is not due to substance
of bacterial origin present in the filtrate since the addition of 1 per cent of
new broth or of as little as l/lo4 gm. per cm.3 gelatin to dilute suspensions
of phage in water is enough to protect phage in the same measure.  These
quantities of broth and gelatin represent roughly comparable amounts of
protein.

(i) No amount of gelatin or albumin added to the broth suspensions of
phage gives any additional protection.  There exists a minimum sensitivity
to x-rays for each phage, which is the sensitivity measured in broth suspen-
sions and analyzed in part 1 above.

Cj) The action of x-rays on phage suspensions in water is not, as in broth,
a function of the dose of radiation only.  A large "time factor" is found, in
the sense that a given dose is more effective the longer the time over which
it is spread (within the range tested). We have not yet been able to
establish whether inactivation continues after the end of the irradiation.
However, water irradiated alone and inoculated immediately thereafter
with phage failed to show higher inactivating power than un-irradiated
water.

(k) The inactivation by x-rays of di$erent phages suspended in water
do& not follow the same rules as the inactivation in broth.  First it should
be said that it is difficult to establish the shape of the inactivation curves
owing to the presence of spontaneous inactivation and to the "time factor."
However, the order of difference in sensitivity between small and large
phages is clearly reversed:  small phages suspended in water are inacti-
vated somewhat more easily than large ones.  In this connection it is of
interest that these small phages are more sensitive also to the action of
heat.6

Discussion.-The observations listed under parts 1 and 2 above appear as
a whole to fit the following interpretation :

The inactivation of bacteriophages by x-rays is at least a twofold effect.
First there is an "indirect" effect predominant in water or saline suspen-
sions, as described under part 2 above.  This effect is probably exerted


VOL. 27, 1941      BIOPHYSICS: LURIA AND EXNER             375

through some inactivating agent produced in the medium as a consequence
of the irradiation.  This agent has a short life, as no inactivating effect is
shown by previously irradiated water.
The mode of action of this agent on phages resembles that of oxidizing
and reducing agents in that protection is afforded in both cases by the
presence of small quantities of foreign proteins. This protection seems
likely to be due to competition between the foreign protein and phage in
reacting with the inactivating agent.
In the presence of foreign proteins bacteriophages are still inactivated by
x-rays, but with reduced sensitivity and with other relationships as listed
under part 1 above.  This action we consider to be a "direct" effect on the
phage particle itself, which can be analyzed according to the pattern of the
"hit theory."  This treatment will be developed more fully in a subsequent
paper.

The indirect effect of x-rays on bacteriophages through the medium ap-
pears similar to the effects obtained by different authors' with enzymes
and simple proteins.

A protective action of foreign proteins against x-ray inactivation of
papihoma virus suspended in saline has recently been observed.8  This is
apparently another instance of the type of protection described above for
phage.

It has been shown that with bacteriophages under suitable conditions
both a direct and an indirect effect `of radiation can be distinguished.
This should serve to emphasize the importance of considering both types of
action when analyzing the effect of radiation on a particular biological
system.

We are indebted to Dr. G. P. Berry, Dr. J. Bronfenbrenner and Dr. A. P.
Krueger for kindly supplying the strains of bacteriophages and bacteria
used in these experiments.

1 Wollman, E., and Lacassague, A., Ann. Inst. Past., 64,5 (1940).
Holweck, F., Luria, S., and Wollman, E., Comgt. rend. Acad. Sci, 210,639 (1940).
Wollman, E., Holweck, F., and Luria, S., Nature, 145,935 (1940).
Exner, F. M., and Zaytzeff-Jem, `H., J. A$& Phys., 12,338 (1941).
* Elford, W. J., in Doerr and Hallauer, Handbuch der Virusforschung, page 126, Julius
Springer, Wien, 1938.
s Lea, D. E., Nature, 146, 137 (1940).
' Goweu, J. W., Proc. Nat. Acad. SC&, 26,8 (1940), and personal communication.
6 Lominski, I., Compt. rend. Sot. Biol., 119,952 (1935), and 122, 766 (1936).
( Luria, S. E. (unpublished results).
' Fricke, H., Cold S@ing Harbor Symposia on Quantitative Biology, 2,241 (1934).
Dale, W. M., Biochem. J., 34, 1367 (1940).
8 Friedewald, W. F., and Anderson, R. S., Proc. Sot. Exptl. Biol. Med., 45, 713 (1940).