Rad~t. Phys. Chem. Vol.15, pp.83-89.

Pergamon Press Ltd. |980. Printed in Great Britai~

THE SPECTRUM OF MICROBIAL RADIATION SENSITIVITY

A. Tallentire

Department of Pharmacy, University of Manchester, Manchester, U.K.

ABSTRACT

The innate sensitivity of micro-organisms towards high energy radiation varies widely;
different types, species and strains exhibit greatly differing sensitivities. Certain
environmental factors are also able to influence the response of a given type of micro-
organism to radiation, e.g., the presence of oxygen, water or adventitious water-soluble
organic material. Such environmental influences hold equally for all types of micro-
organisms, irrespective of the innate sensitivities which they possess.

INTRODUCTION

The foregoing papers in the Seminar have been focussed on defining the effects of radiation
in relation to the plastics or polymers that comprise the items destined for radiation
sterilization. It is true to say that, in general, the preference is for the radiation to be
without effect in this regard. Usually the action of the radiation on plastics is
destructive so that, in practice, the principal concern is to prevent or suppress the
radiation action with the objective of conserving the original properties of the polymer.
The present paper considers not the action of radiation on the polymer but that on its
likely microbial contaminants; this latter action is again a destructive one, but in this
instance there is advantage in sustaining or enhancing the action rather than preventing it.
This point should be borne in mind, just as it should be remembered that the real objective
behind the radiation sterilization process is to free items of viable mlcro-organlsms. In
other words, radiation treatment, in the context of the present Seminar, is concerned with
inactivating micro-organlsms. Frequently this primary purpose is forgotten. To underscore
this thought, it is worthwhile considering limiting circumstances; if items intended for use
in a sterile condition could be produced at an equal acceptable level of sterility assurance
with or without a radiation treatment then, provided that equal economic considerations obtain,
irradiation would be omitted.

One question that will undoubtedly be raised in subsequent papers is "what dose of radiation
is needed to achieve freedom of items from viable micro-organlsms?" A rational and
considered answer to this question must be based on a knowledge of how micro-organisms
respond to irradiation, and the present paper is aimed at providing, in general terms,
background knowledge of this kind.

DOSE/RESPONSE RELATIONSHIP

Consider first the form of the response of micro-organisms to high energy radiation. This
can be found from a relatively simple kind of experiment. A population of micro-organisms
of a given type is grown in the laboratory and exposed to increasing graded doses of
radiation. After each radiation dose, the number of micro-organlsms surviving is scored
in order to provide a dose/response relationship. When the number of survivors is plotted
on a logarithmic scale on the 'Y'-axis against radiation dose on the 'X'-axis, this
relationshlp can be represented by a curve such as that shown in Fig. i. As depicted, the

83
84 A. Tallentire

form taken by the curve generally approximates to a straight line, so that overall the
response may be described by the slope of the curve which, of course, is constant. The

O
z

0=
IO
uO

OO=

=E
U.
O

=E
.~ ~ADIATION DOSE (Oy) \ =P
z
O
q
16

Fig. i. Typical representation of a dose/response relationship

significance of this finding is that radiation inactivation of a microbial population

follows an exponential or a near exponential course (in chemical terms this would be thought
of as a process following first order kinetics), meaning that, regardless of the extent of
dose applied, there is a finite probability of a micro-organism surviving.

For a curve with a constant slope, the response can also be represented by the d e c a l
red~t~on dose or DIO. This is the dose needed to reduce the number of surviving cells
by a factor of 10 (or put another way, inactivate 90% of the population). The relationship
between the slope of the dose/response curve and the decimal reduction dose (DIo value), is
a simple one in that the D~O value is the reciprocal of the slope. As the Seminar progresses
we will hear more about thls characteristic of radiation response, since it is those micro-
organisms, encountered in practice, which respond least to radiation (i.e. exhibit the
least slope for the dose/survival curve and, therefore, the highest DIO value) that set the
dose of radiation to be used in sterilization processing.

INNATE RADIATION SENSITIVITY

Implicit in the above is the notion that populations of different types of micro-organisms
respond differently to irradiation and, of course, this is so. An illustration of this
difference is given in Fig. 2. Five types of micro-organisms were produced in our laboratory
under similar conditions, irradiated under conditions that were identical and scored for
survival using methods and techniques closely resembling one another. The observed
responses differ appreciably from one another (23Ox maximally) implying that the different
types of micro-organisms possess different i~ate sensitivities to high energy radiation.

What is it that determines the relative sensitivities of the microbial types to radiation?

Traditionally, differences in sensitivity have been attributed to differences in the size

of targets that exist in cells, the target being defined as a volume in which specific
radiation-induced damage must occur for the cell to be inactivated (Ref. i). Although
this idea appears to hold for cells drawn from different major taxonomic groups (Ref. 2),
available data suggest that it does not apply to microbial cells considered alone (Ref. 3).
On the other hand, there is some evidence to suggest that subtle differences in the
 Spectrum of Microbial Radiation Sensitivity 85

composition of the target may contribute to the determination of innate sensitivity (Ref. 4).
However, to account for the wide spectrum of sensitivities to radiation seen with microbial
cells (Fig. 2) alternative explanations must be sought.

phaericus C1A
ores
kGy
n,
u~
~, 1~2
_/
bJ
(.) Ps. ~8. pumilus
aeruginosa
~) 16~ ~spores
z
o
~0= 20Gy

5t(~oh. aureus Strep.

~ l.TkGy

h
D,0 = 100Gy faecium

I00 2~0
; '~ ~ , ~ , ,
DOSE, Gy DOSE, kGy

Fig. 2. Responses to Co60 y-rays of five different types

of micro-organisms suspended in aqueous aerated buffer or
water

Generally the different types of microbial cells are in the same size range (around IO-12g)
and they largely consist of the same constituents (proteins, carbohydrates, nucleic acids,
lipids etc). It seems reasonable to suppose then that within fairly narrow limits the same
amount of damage, and generally the same kind of damage, is induced per unit of radiation
dose in radiation sensitive and radiation resistant types of micro-organisms. In these
circumstances, the observed differences in response might well be reflections of the relative
abilities of different types of micro-organlsms to cope with or handle the induced damage.
The clearest demonstrations of determination of radiation response by this means reside in
the field of low energy radiation biology (or photobiology) where ultra-violet light (UV)
is the radiation to which cells are exposed. In contrast to gamma or electron irradiation,
ultra-violet radiation is relatively specific in its action; it is mainly absorbed in the
cells' DNA where one of the products, known to be lethal to the cell on replication, is a
dimer formed from two adjacent pyrimidine nucleotides. These dimers can be measured
quantitatively and it has been found that they are produced in near equal amounts per unit
of ultra-violet radiation dose in the DHA of UV sensitive and UV resistant cells (Ref. 5).
However, the UV resistant cells are able to remove the dlmers before they exert their lethal
effect, whereas the UV sensitive cells cannot (Ref. 6). Repair of ultra-violet induced
damage of this kind can occur by a number of different pathways (Ref. 7). Often the repair
pathway is made up of a sequence of enzyme mediated steps so that repair of radiation damage
is under genetic control. Thus, sensitivity to ultra-vlolet light is principally an innate
character which can be transmitted within a microbial species from one generation to another.

The genetic control of sensitivity to high energy radiation, such as gamma-rays or electrons,
is not nearly as fully understood as that for ultra-violet radiation and, in the high energy
field, much remains to be unravelled. However, repair processes for gamma- or X-ray
damage induced in the main chain of the D N A o f vegetative bacteria certainly exist (Ref. 8),
as also do spore specific repair mechanisms (Ref. 9). Currently much investigational effort
is being directed towards identifying and characterizing these repair processes and other
processes associated with different forms of damage induced by ionizing radiations, such as
damage occurring in DNA bases and in membranes. Accordingly, we can look forward within
the next few years to significant advances in knowledge about the determinants of the
innate radiation sensitivities of micro-organlsms.
86 A. Tallentire

EFFECTS OF ENVIRONMENT ON RADIATION RESPONSE

The spectrum of innate radiation sensitivities seen with different types of micro-organisms
is obviously an important consideration in radiation sterilization processing. At the moment
it is something over which we have little or no control (very likely the frequency of
occurrence of micro-organisms of low sensitivity on items destined for sterilization is
determined largely by chance and, as such, it has to be accepted). However, modification of
radiation response of a given type of mlcro-organism is possible and, in contrast to
innate sensitivity, this kind of variation, dependent upon the cells' immediate environment,
is amenable to control. One example of modification of response is given in Fig. 3. This
shows the existence of the so-called oxygen effect to an almost equal degree in two types
of organisms drawn from the extremes of the spectrum of innate radiation sensitivity.

\ Wet Ps. oeruginOs~ ournilus spores

(.D
Z
c,lrs

2 equilibrated
~ 1c;:

/2"
LU
C)
h
O
1(3'
Z
O
I--
C)
~ 0 =20Gy~

i 1001 I ~0 I
(Gy) RADIATION DOSE (kGy)

Fig. 3. The enhancing action of oxygen in two types of micro-

organisms of high (LHS) and low (RHS) innate radiation
sensitivities

Where tests have been done, similar findings have been made for types of micro-organisms taken
from the whole range of sensitivities, lending support to the belief that manipulations of
response can be achieved, within limits, in all micro-organisms.

The water content of the microbial cell at the instant of irradiation is also known to affect
greatly its radiation response (Ref. i0 and ii). Contaminating micro-organisms on plastic
items undergoing radiation treatment are usually in a 'dry' state and so it is reasonable to
ask how this condition influences their response to radiation. Figure 4 depicts, in a
general way, alterations in response of bacterial spores present in N 2 or 02 brought about
by changing water content over a wide range bounded by the 'wet' and 'dry' states (spores
suspended in water and equilibrated to 2.3 x 10-2 N/m2 H20 vapour respectively). For spores
in N2, progression from the 'wet' to the 'dry' state causes a lessening in response to
radiation, whereas for oxygenated spores, a similar progression results in an increase in
response. Similar overall water effects have been recognised in vegetative bacteria (Ref. 13)
and mould spores (Ref. 14).

The water effects represented in Fig. 4 were seen with bacterial spores mounted on the surface
of cellulose acetate membrane filters; the spores were discrete and isolated in the absence
of a soluble carrier and therefore changes in response to radiation seen with changes in water
content were exclusively water effects. In practlce, not all cells that are adventitious
contaminants occur in an isolated state. Some can be expected to be either in contact with
or intimately associated with dust, skin scales, dried human secretions, etc. in which
 Spectrum of Microbial R a d i a t i o n S e n s i t i v i t y 87

c a s e t h e y w i l l r e s i d e on o r i n a n o r g a n i c s u b s t r a t e o f one f o r m o r a n o t h e r . All available

e v i d e n c e i n d i c a t e s t h a t , i n t h i s k i n d o f e n v i r o n m e n t , m i c r o b i a l c e l l s e x h i b i t a low r e s p o n s e
to radiation (e.g. Ref. 15-17).

2"©
8. megoterium spores
N2

v 1'0

02

'DRY' 'WET'
WATER CONTENT

Fig. 4. A representation of the way in which cellular water content

affects the radiation response of anoxic or oxygenated spores /curves
constructed from the data of Tallentire and Powers (Ref. 12)]

In the context of irradiation of plastic materials, gaseous environment, water content, and
the presence of organic material are vitally important determinants of cellular radiation
response. Clearly such environmental influencies can be superimposed on an innate
radiation sensitivity falling within widely spaced limits, similar to those illustrated in
Fig. 2. It need hardly be said that with radiation processing, as with other methods of
sterilization, the highest assurance of achieving freedom from viable micro-organisms is
desired, and therefore the aim must be to shift the spectrum of response as far as possible
to the left. To achieve this, the microbial contaminants must be bathed on ozygenj 'dry'
and cleGn.

EFFECTS OF MODE OF RADIATION DELIVERY ON RESPONSE

So far consideration of microbial response to radiation has been restricted to two

determinants: the micro-organisms themselves and their environment. A third possible
determinant, worthy of some thought, is the incident radiation. It must be asked "can the
form of the radiation or the way in which it is delivered to the micro-organisms influence
their response?"

For the range of energy levels used in sterilization processing with the two usual radiation
types (electrons and ga~na-rays), it is well established that variations in electron or
quantum energy do not influence microbicidal efficiency. Furthermore, for continuously
delivered radiation given to 'dry' items, such as applies with g--me-ray processing of
plastic materials, variations in the rate at which dose is delivered also show no effect
(Ref. 18). There is too the possibility of the processing dose being given in fractions;
fractionation may be intended as a design feature of the sterilizing plant (e.g. the
sterilizing dose ~s given as two equal fractions with a substantial fixed time interval
between them) or it may be enforced, arising out of maintenance demands or mechanical break-
down (here a variable time interval may elapse between doses). For microbial contaminants
occurring in a 'dry' condition, as on plastic materials, fraetionated radiation doses give
the same response to radiation as those delivered continuously (Ref. 19). On the other hand,
for certain micro-organisms, present in a liquid substrate under appropriate conditions,
fractionated radiation doses can bring about a reduced response (Ref. 20); apparently, repair
of radiation damage occurs during the interval between dose fractions. However, the
88 A. Tallentire

occurrence, in practice, of conditions needed to achieve repair of this kind is so

unlikely with plastic items destined for radiation sterilization that it seems reasonable to
discount this laboratory observed effect.

Recently, we have noted another way in which microbial response to radiation may be
changed as a result of changing the mode of delivery of dose. This has been revealed while
undertaking a fundamental study in radiation biology aimed at investigating the time-scale
of processes responsible for cell death using, as the radiation source, a beam of electrons
delivered from a linear accelerator as pulses. The functional characteristics of the
accelerator are such that, when operated in the repetitive mode, it can deliver a train of
electron pulses of energy 8-10 MeV, at repetition rates variable over the range i to 50
pulses per second, for pulse lengths from 5 ns to 5 ~s. With a fixed beam current and a
given pulse length, varying the distance between the micro-organisms under irradiation and
the exit window of the accelerator gives wide variations in mean pulse dose, and for a given
integrated radiation dose, varying pulse repetition rate or pulse length changes the
integral dose-rate.

Up to the present time, our studies have mainly been concerned with varying the magnitude
of the mean absorbed dose associated with the pulses of electrons, a number of which make
up the integrated radiation dose. For anoxic bacterial spores suspended in water, we find
that with relatively low pulse doses (< i0 Gy) the response to radiation is equal to that
seen with gamma-radiation, but on increasing the pulse dose from I0 Gy to I kGy the response
increases almost two fold (Ref. 21).

Radiation sterilization became a commercial reality in the middle 1950's when it was
established that all types of micro-organisms were inactivated by exposure to high energy
radiation, provided that a sufficient dose was applied. An electron accelerator was the
first commercial radiation sterilization source and when it was commissioned there was little
concern, in relation to effectiveness, about whether the beam of electrons was continuous
or pulsed, and if the latter, about the repetition rate, pulse length, pulse dose, etc.;
effectiveness was simply measured against likely microbial contaminants. In the
intervening years there has been a wealth of investigation in the field of radiation
microbiology, both of a fundamental and applied nature, so that we are more knowledgeable
about radiation inactivation than any other form of cell inactivation. However, as
indicated above, new phenomena still appear. Clearly the message is that any departure
from a known, effective method of radiation processing which could lead to changes in
types of microbial contaminants, in the environment of these organisms or even in the
radiation itself should not be taken without proper and exhaustive experimental
justification.

REFERENCES

(I) D.E. Lea, Actions of Radiations on Living Cells, University Press, Cambridge, 1955.

(2) A.H. Sparrow, A.G. Underbrink and R.C. Sparrow, Chromosomes and cellular radiosensi-
tivity. I. The relationship of D O to chromosome volume and complexity in
seventy-nine different organisms, Radiat. Res. 32, 915 (1967).

(3) A.G. Underbrink, A.H. Sparrow and V. Pond, Chromosomes and cellular radiosensitivity.
II. Use of interrelationships among chromosome volume, nucleotide content and
DO of 120 diverse organisms in predicting radiosensitivity, Radiat. Bot. 8,
205 (1968).

(4) H.S. Kaplan and R. Zavarine, Correlation of bacterial radiosensitivity and DNA base
composition, Biochem. Bioph~s. Res. Commun. 8, 432 (1962).

(5) R.B. Setlow and W.L. Carrier, The disappearance of thymine dimers from DNA: an error-
correcting mechanism, Proc. Nat. Acad. Sci. (U.S.) 51, 226 (1964).

(6) R.P. Boyce and P. Howard-Flanders, Release of ultraviolet light-induced thymine dimers
from DNA in E. coli K-12, Proc. Nat. Acad. Sci. (U.S.) 51, 293 (1964).
 Spectrum of Microbial Radiation Sensitivity 89

(7) K.C. Smith, Multiple pathways of DNA repair in bacteria and their roles in mutagenesis,
Photochem. P h o t o b i o l . 28, 121 (1978).

(8) R.A. McGrath and R.W. Williams, Reconstruction in vivo of irradiated Escherlchia coli
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(9) N. Munakata and Y. Ikeda, A mutant of Bacillus subtilis producing ultraviolet-sensitive

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(IO) E.L. Powers and A. Tallentire, The roles of water in the cellular effects of ionizing
radiations, pp. 3-67. In Actions Chimiques et Biolo~iques des Radiations Vol.
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(II) E.L. Powers, Water as a modulator of radiation damage to micro-organisms, pp. 97-118.
In Sterilization by Ipnizln~ Radiation Vol. II, Multiscience, Montreal, 1978.

(12) A. Tallentire and E.L. Powers, Modification of sensitivity to X-irradlation by water

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(15) E.A. Christensen and K. Sehested, Radiation resistance of StreptOcoccus faecf~m and
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(16) C. Emborg, The influence of preparation technique, humidity and irradiation conditions
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(17) A. Tallentire, Aspects of microbiological control of radiation sterilization, Int. J.

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(18) F.J. Ley, The influence of dose rate on the inactivation of micro-organisms, Int. J.
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(20) M. Fox and J. Hopkins, Post-irradiation DNA degradation and response to fractionated
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