T H E IMMEDIATE AND DELAYED ACTION OF X-RAYS
UPON T H E PROTOZOAN DUNALIELLA SALINA
H. J. RALSTON, PH.D.
(From the Department of Zoology, University of California, Berkeley, California)
I t was not long after the discovery of x-rays that biologists seized
upon the new tool as an interesting means of studying the effect of unusual
environmental agents upon living organisms. Much of the earlier work,
naturally, was exploratory in nature. Quantitative studies of the effects
of x-rays on living organisms really had their beginning with the work of
Crowther (l),who was the first to set up a theoretical treatment of the
killing effects of x-rays on protozoa (in this case, Colpidium colpoda), and
to test the theory by quantitative methods. Following Crowther, other
investigators have pursued the same course, using not only protozoa but
also bacteria, protophyta, and higher types of organisms. Among the
more important studies made since Crowther’s work may be mentioned
those of Packard (2), Condon and Terrill (3), Wyckoff (4),Madame
Curie ( 5 ) , and Glocker (6).
The present paper attempts to add to the foregoing studies, and t o
criticize and correct certain analytical methods used in the past.
MATERIALSAND METHODS
The organism used in this study, Dunaliella salina Teodoresco (7, 8),
is a small, egg-shaped, biflagellate protozoan, containing a cup-shaped
chromatophore in the posterior half of the body. I t varies in length from
12 to 28 micra, and in width from 13 to 17 micra. I t is an inhabitant of
concentrated salt pools the world over, the present material being obtained from such pools a t the south end of San Francisco Bay. Dunaliella
is photosynthetic, which obviates any worry about food supply so long
as the organisms are cultured in a suitable medium, and exposed to the
proper light. The culture fluid used in these experiments was a modification of one used by Dr. C. B. van Niel of the Hopkins Marine Station,
and contained KCl 0.02 per cent, NaNOa 0.02 per cent, MgClt 0.02
per cent, Na2HP040.02 per cent, NaHCOa 0.05 per cent, and NaCl 15 per
cent .
The x-ray apparatus consisted of a Coolidge type tube with a tungsten
target, permanently connected to a high-vacuum, mercury-vapor pump.
The peak voltage across the tube, as determined by means of a sphere
gap, was maintained at 52 =t 2.5 kilovolts. The current through the
tube was kept at about 10 milliamperes. The effective wavelength of
the unfiltered radiation was determined by absorption experiments, and
found to be 0.33 Angstrom unit. The intensity of the radiation a t the
point where the protozoa were exposed was measured by a Victoreen
r-meter, and found to be 43.4 f0.8 r units per minute.
288
ACTION OF X-RAYS UPON PROTOZOAN DUNALIELLA SALINA
289
The experimental procedure was as follows: 10 C.C. of the culture
medium containing the organisms, which had been sufficiently agitated to
produce uniformity, were pipetted into each of two Syracuse dishes.
These were then covered with parchment to prevent evaporation. One
dish was used for a control and the other was exposed to the radiation.
The distance from the target to the top of the culture medium was 23 cm.
The irradiated sample was immediately examined with a microscope.
Both samples were then transferred to cotton-stoppered or cork-stoppered
Erlenmeyer flasks, depending upon the nature of the experiment, and
placed next to a window shaded with gauze. From time to time, counts
of the test and control organisms were made, in order to follow the course
of the changes produced in the populations under test. In making the
counts, the culture fluid was thoroughly agitated, in order to produce a
uniform distribution of the protozoa in the medium. A 1 C.C. sample was
then withdrawn with a pipette, to which was added a trace of 5 per cent
formalin, which immediately caused the death of the protozoa, without
materially altering their visible structure for a considerable period of time.
This solution was again agitated, and a small portion was then withdrawn
and placed in a counting chamber. This consisted of a thin brass strip,
perforated in the middle and mounted on a glass slide. The fluid was
covered with a cover-glass, on which was placed a heavy lead ring, in
order to counteract surface tension effects and thus insure that the chamber always contained the same volume of fluid. The material was then
observed with the low-power of the microscope, which was furnished with
a ruled disc in the eye-piece. The field was thus divided into rectangular
areas, and the protozoa in the field could be readily counted. Ordinarily,
from 30 to 50 such counts were made, depending upon the experiment.
EXPERIMENTAL
RESULTS
Table I summarizes the experimental effects of x-rays on Dunaliellu,
immediately after irradiation. The data are shown graphically in Fig. 1,
curve A.
TABLB
I: Effect of Irradiation on Dunaliella, as Observed Immediately after Irradiation is Cornfilleted
Time of Exposure
(Hours)
No. of Samples
1
2
5
5
3
5
4
4
5
6
7
a
5
5
3
3
S
(Average survival
percentage)
100
100
100
100
100
100
2 (estimated)
0
Table I1 summarizes the results of the same experiments when a sufficient period has elapsed after irradiation so that no further change with
time takes place. These results have been plotted in Fig. 2, where the
290
H. J. RALSTON
DOTS
FIG. 1. CURVE A: SURVIVAL CURVE IMMEDIATELY AFTER IRRADIATION,
REPRECURVE CORRESPONDING TO X EQUALS
SENTING EXPERIMENTAL POINTS. CURVE B : THEORETICAL
330, LY EQUALS 50.
experimental error is indicated by the vertical lines, and the experimental
curve as a solid line.
Table I11 shows how the survival percentage of the sample which has
been irradiated for four hours changes with the time elapsing after irradiation; i.e., this table shows the continuous transition of the four-hour
sample from the data given in Table I to the data given in Table 11.
Fig. 3 shows graphically the change of the four-hour sample from Fig. 1
to Fig. 2.
When death occurs immediately after irradiation, it is manifested by
complete cessation of movement (Durtaliella is normally a very active
swimmer), by clear signs of fragmentation, and by the accumulation of
the organisms at the bottom of the dish. In samples which were exposed
for too short a time to produce immediate killing, giant and odd-shaped
forms appeared’ after a few days. The percentage of such variants increased with exposure time.
ACTION OF X-RAYS UPON PROTOZOAN DUNXLIELLA SALINA
291
FIG.2. SOLIDLINE:FINALSURVIVAL
CURVE,DOTSREPRESENTING
EXPERIMENTAL
POINTS
AND VERTICAL
LINESTHE EXPERIMENTAL
ERROR. DOTTED
LINE:THEORETICAL.
CURVECORRESPONDING TO X EQUALS 11.41, (Y EQUALS 2.31. BROKEN
LINE:THEORETICAL
CURVE CORRESPONDING TO X EQUALS 12, a EQUALS 2.5.
TABLE
11: Results of the Experiments of Table I , after a Suficient Time Has Elapsed So That No
Further Change Occurs with Time
S
Exposure
(Hours)
Days after
Exposure
No. in
Controls
No. in
Tests
(Average survival
percentage)
1
2
3
4
5
6
37
38
44
40
28
42
16
0
2704
1218
1685
2960
5037
2445
2658
1184
1564
2046
2032
45 5
98.3 f2.0
97.2 f2.5
92.8 f2.8
69.1 f2.1
40.3 f2.1
18.6 f 2.8
7
8
-
-
0
,o
0.0
0.0
292
H. J. RALSTON
TABLE
111: Chunae ~f the Four-hour Sample with Time after Irradiation
Days after
Irradiation
S
2.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.........................................
10... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
26... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3
Y,
50
97.4h4.1
93.1 f3.7
74.1 f3.6
69.1f3.1
67.1 f2.3
-
I-
Z
Irl
u
YO
-
w
a 30
20
-
I0
-
00
FIG,3.
I
I
I
I
5
I0
15
20
CHANGE IN SURVIVAL OF THE
I
a5
FOUR-HOUR
SAMPLE
WITH
I
30
TIMEAFTER
IRRADIATION
Dots represent experimental points, vertical lines' the experimental error.
DISCUSSION
AND CONCLUSIONS
The delayed action of x-rays on organisms is a well known phenomenon.
Roentgenologists are familiar with the fact that the erythema produced
on the skin of a patient after exposure to x-rays may'not appear for as
long as ten days or two weeks. Wyckoff and Luyet (9) studied the effect
of x-rays upon yeast, and found that injury was followed by the development of extraordinarily large numbers of two-celled colonies, which died
without further budding on prolonged incubation. The reproductive
ACTION OF X-RAYS UPON PROTOZOAN DUNALIELLA SALINA
293
function was thus destroyed, although the yeast cells were not immediately killed. Brown, Luck, et a1 (10) found that x-rays in sublethal
doses produced an inhibition of reproduction in Euplotes taylori which was
not manifested for some time after irradiation. Scott (11) x-rayed the
eggs of Cullifihora and showed that although 20 per cent of the eggs
continue to develop after irradiation with 300 r units, only about 1.5 per
cent actually hatch. Vintemberger, quoted by Packard (12), exposed one
of the blastomeres of the frog embryo to x-rays, and found that at gastrulation the irradiated half ceased to function. Langendorff and Langendorff (13) exposed Axolotl eggs in the meta-telophase of the first segmentation division, and found that with 200 r units, death resulted after
twelve days. Enzmann and Haskins (14) studied the delayed killing of
Drosophila after irradiation of young larvae, and considered the cause to
be mitotic derangement. Many other studies, in which not only x-rays
b u t also ultra-violet rays and radium were used, could be quoted, in which
the delayed effect was exhibited.
This delayed action effect plays an important r61e in the present study,
but before discussing further this phase of the work, it will be necessary
to consider the theoretical treatment of killing curves first promulgated
by Crowther (1).
Assume that an individual organism will be killed if it absorb one
quantum of x-ray energy. Let No be the original number of organisms,
and nl the number hit once. Then
where a is a constant. This on integration yields
NO- nl
is the fraction surviving. Now assume that two quantum
No
hits are necessary to kill. Then
where
where n2 is the number hit twice. I t should be noticed that
to be identical in the two cases.
Substituting the value for nl from (2) :
(Y
is assumed
or
This yields, on integration :
NO- n2
No
=
+ at).
fF(1
(5)
294
H. J. RALSTON
Similarly, it can be shown that for x hits required to kill, the fraction
surviving S after time t is given by
The most convenient way of determining a and x from a known S will
depend largely upon the magnitude of both a and x. Thus Claus (15)
found by a method of trial and error that no value of a existed for x equals
one which adequately represented his data, but that for x equals two, he
was able to find an a which, when substituted in equation (6), agreed
with his experimental results. At this point it might be said that Claus
gave no indication of his experimental error, and so it is impossible to say
whether or not his choice of x and a was unique. Claus worked with
Bacillus coli.
For larger values of x,such a method of trial and error becomes very
cumbersome and time-consuming. However, Crowther (1) put equation
(6) into the form:
where r&) is the incomplete gamma-function of x, and I‘(%) is the complete gamma-function of x. Pearson’s Tables of the Incomplete Gamma-
rat(%)
Function evaluate the ratio -up to x equals 51, at equals 98. Using
(x)
these tables, Crowther, again by a trial and error method, was able to
determine an x and an a which would adequately represent his data for
immediate killing.
How a relatively large experimental error may lead to ambiguity in
fitting theoretical curves to experimental data is shown in Fig. 4. In this
figure, the crosses represent Crowther’s experimental points for the survival one hour after irradiation, and the vertical lines indicate the stated
experimental error (7 per cent). Crowther believed that the theoretical
curve for x equals 42, a equals 15, adequately fitted his data. We have
calculated this curve, shown in Fig. 4,as a broken line. We have also
calculated the theoretical curve for x equals 46, CY equals 16, shown in the
same figure as a solid line. I t is evident that Crowther’s curve not only
is not unique, but that our curve actually forms a better fit. More than
that, it is also evident that several curves could be drawn which would
fall within the experimental error range. Crowther was unable to find
any x for the given value of a, 15, for the survival curve for two hours
after irradiation, which fact he attributed to the observation that “the
Colpidia make a very marked recovery in an interval of two hours.”
Since Crowther worked with the protozoan Colpidium colpoda, his work
parallels the present study.
For values of x and a beyond those given in the tables, the method of
trial and error becomes practically impossible, and so the following method
was used. Differentiating equation (6) we get
ACTION OF X-RAYS UPON PROTOZOAN DUNALIELLA SALINA
I
I
I
I
295
I
FIG. 4. CROSSES:
CROWTHER’S EXPERIMENTAL
POINTSFOR SURVIVAL
OF COLPIDIUM
ONE
HOURAFTER IRRADIATION(VERTICAL LINES REPRESENT THE EXPERIMENTAL ERROR). BROKEN
LINE:THEORETICAL
CURVECORRESPONDING
TO x EQUALS 42, OL EQUALS 15. SOLID
LINE:THEORETICAL CURVECORRESPONDING
TO x EQUALS 46, (Y 16.
This we can set equal to the measured value of the slope. I t will be convenient to take such a value of t t h a t the slope is a maximum, i.e., at the
point of inflection. Differentiating again we get:
and this is p ut equaI to zero (see Madame Curie’s analysis, 5 ) . These
two equations may easily be solved for x and a,as follows:
By Stirling’s theorem :
(x - 1) s
5
zi271-(x - l>(x
- l)Z-1e-(z-1).
Substituting in equation (7), we get:
log
( - $)
=
x log a
+ (x - 1) log t + 0.434(x - 1 - at)
- [$ log 27r(x - 1) + (x - 1) log (x - l)].
(9)
296
H. J. RALSTON
Putting equation (8) equal to zero, it follows that a t the point of inflection:
x-1
a=-
ti
'
where ti is the time corresponding to the point of inflection.
in equation (9), we get:
log ( x
- 1) = 2 log
Substituting
( - y)i+ 2 log + log 27r.
ti
($)$is the slope of the curve a t t,, and both of these values are approximately determined directly from the curve. Having found x, a can be
determined from equation (10).
With the known values of x and a!, the slopes a t any time can be calculated from equation (9), and the final curve built up from the values
of the slopes at the various points. Fig. 1, curve B, shows a curve built
up in this fashion, using x equals 330, a! 50, which may be compared with
the immediate killing curve. Since no experimental data are provided
for times intermediate between six and seven hours, no attempt was made
to get a closer fit. It is obvious, however, that the calculated curve agrees
rather well with the experimental curve. I t should be emphasized that
this process is in no sense a curve-fitting, but is merely a method for
building up a curve corresponding to any particular x and a.
Turning once more to the final survival curve, Fig. 2, solid line, we
can determine an x and an a by the above described method. Due to
the fact that the experimental points are an hour apart in time, the measured values of the slope and the point of inflection are again subject to
some uncertainty. With our first choice for these quantities, x was calculated to be 11.41, and a! 2.31. The theoretical curve corresponding to
these values is represented in Fig. 2, dotted line. Since this curve even
for t as low as four hours deviated more than the experimental error, i t
shows that the choice of slope and point of inflection was somewhat in
error. Furthermore, if x is to have the physical interpretation ascribed
to it, it must be an integer. Choosing the values x equals 12, a! equals
2.5, we get the broken curve in Fig. 2, which adequately represents all
the data up to and including t equals 6. We wish to note, at this point,
that this curve is not unique, but it does form the best fit. In order to
find an unique curve, it would be necessary to narrow down considerably
the experimental error, but it is not likely that the value of a! would be
materially changed.
I t is impossible to choose any pair of values for x and a which will
represent all of the experimental data. I t is obvious, as'crowther pointed
out, that if a represents the probability that a quantum of x-ray energy
be absorbed in a particular part of the cell, it cannot change with time.
We therefore suggest that the final survival curve represents the sum of
two separate processes: the first, which is a lethal effect observable immediately after x-raying, and the second, which permits an organism to live
but inhibits reproduction, and so represents killing after some time has
ACTION OF X-RAYS UPON PROTOZOAN DUNALIELLA SALINA
297
If the absorption coefficient for x-rays be regarded as a constant
aimmediate
= -50
- - 20 must
for the various parts of the cell, then the ratio
elapsed.
afinrtl
2.5
be equal to the ratio of some two volumes in the cell. By actual measurement of stained cells, it was found that the ratio of the average volume
occupied by the cytoplasm to that occupied by the nucleus was 23.4,
which closely corresponds to the ratio a-immediate to a-final. I t therefore appears that immediate killing is due to a general cell effect, while
the delayed effect is a consequence of nuclear damage, probably a disturbance of the mitotic mechanism.
SUMMARY
1. The survival curves of Dunaliella populations were determined after
x-irradiation.
2. I t is shown that the experimental curve changes in form with time
after irradiation.
3. The suggestion is advanced, and quantitatively supported, that
two separate processes are involved, one causing immediate death and
affecting the cytoplasm of the cell, and the other acting indirectly by way
of the nucleus, inhibiting reproduction and thus representing death after
some time has elapsed.
4. The analysis of survival curves as exemplified by the work of previous investigators is discussed and criticized.
AcknowZedgment: The author wishes t o express his deepest thanks to Prof. A. R.
Olson, of the Department of Chemistry, University of California, for his invaluable
assistance and advice during the course of experimentation and preparation of this
p,aper. Any errors or defects, however, are the author’s sole responsibility.
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