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RADIATION BIOLOGY 
VOLUME II 



COLLABORATING EDITORS 

Farrington Danikls, Professor of Physical C'hemistry, University of 
Wisconsin, Madison 

Alexander Hollaender, Director, Biology Division, Oak Ridge 
National Laboratory, Oak Ridge 

John R. Loofbourow, Professor and Executive Officer, Biology Depart- 
ment, Massachusetts Institute of Technology, Cambridge 

Arthur W. Pollister, Professor of Zoology, Columbia University, 
New York 

Lewis J. Stabler, Professor of Field Crops, University of Missouri, and 
Agent, U.S. Department of Agriculture, Columbia 



^ / 



RADIATION BIOLOGY 



Volume II: Ultraviolet and Related Radiations 



Edited by 
ALEXANDER HOLLAENDER 

Director of Biology Division 
Oak Ridge National Laboratory 

With the cooperation of 

Farrington Daniels Arthur W. Pollister 
John R. Loofbourow Lewis J. Stadler 



Prepared under the Auspices of the Committee on 

Radiation Biology, Division of Biology and Agriculture 

National Research Council 

National Academy of Sciences 

Washington, D.C. 



New York Toronto London 
McGRAW-HILL BOOK COMPANY, INC. 

1955 



RADIATION BIOLOGY, VOLUME II 

Copyright, 1955, by the McGraw-Hill Book Company, Inc. Printed in the 
I'nited States of America. All rights reserved. This book, or parts thereof, 
may not be reproduced in any form without permission of the publishers. 

Library of Congress Catalog Card Number 5;i-()042 



TlIK .\1.\1'LE PRESS COMl'A.N'Y, YORK, FA. 




This volume is dedicated to the memory of 
two members of the Volume II Editorial Committee 

Dr. Lewis J. Stadler 

Professor of Field Crops, University of Missouri 
Agent, U.S. Department of Agriculture, Columbia, Missouri 

and 
Dr. John R. Loofbourow 

Professor and Executive Officer, Biology Department 
Massachusetts Institute of Technology 



PREFACE 

This second volume of "Radiation Biology" covers the 
field of ultraviolet radiation. It also contains considerable 
material on high-energy radiation. Since both parts of the 
spectrum induce similar biological effects, no clear line can be 
drawn between the two. Microbiology, for example, is dis- 
cussed in both volumes but occupies a more prominent place 
in the second. Actually, Volume I and Volume II compose a 
unit and should be read as such. 

Alexandek Hollaender 




vu 



14 71 



CONTENTS 



Preface vii 

1. Photochemistry ] 

Robert Livingston, Professor of Physical Chemistry, University of Minne- 
sota, Minneapolis 

2. Practical Applications and Sources of Ultraviolet Energy ... 41 

L. ./. Bvitolph, Engineer, General Electric Company, Lamp Division, 
Cleveland, Ohio 

3. Sunlight as a Source of Radiation 95 

./. .4. Sanderson, Superintendent Optics Division, Xaval Research Labor- 
atory, Washington, D.C. 

Edward O. Hulhurt, Director of Research, Xaval Research Laboratory, 
Washington, D.C. 

4. Technique of Study of Biological Effects of Ultraviolet Radiation 119 

Jesse F. Scott, Associate in Biophysics, Massachusetts General Hospital, 
Boston; and Research Associate, Department of Biology, Massachusetts 
Institute of Technology, Cambridge 

Robert L. Sinsheimer, Associate Professor of Biophysics, Physics Depart- 
ment, Iowa State College, Ames 

5. Ultraviolet Absorption Spectra 165 

Robert L. Sinsheimer (see Chap. 4) 

6. A Critique of Cytochemical Methods 203 

A. W. Pollister, Professor of Zoology, Columbia ITniversity, New York 
City 

7. The Effect of Ultraviolet Radiation on the Genes and Chromo- 
somes OF Higher Organisms 249 

C. P. Swanson, Professor of Botany, Johns Hopkins University, Balti- 
more, Maryland 

L. J. Stadler, Professor of Field Crops, University of Missouri, and Agent, 
U.S. Department of Agriculture, Columbia 

8. The Effects of Radiation on Protozoa and the Eggs of Inverte- 
brates Other than Insects 285 

Richard F. Kimball, Senior Biologist, Biology Division, Oak Ridge 
National Laboratory, Oak Ridge 

9. Radiation and Viruses 333 

S. E. Luria, Professor of Bacteriology, University of Illinois, Urbana 



IX 



69919 



X CONTKNTS 

10. Effects of Radiation on Bactkria 365 

M . h'. Zilh . I'rolrssor of liactoriology, Cornell University, Ithaca, N.Y. 
Alexander HoUnnuler. Miophysieist, BioloRy Division, Oak Ridge National 
Laboratory, Oak Hidni' 

11. R.\i)iATioN Stiidies ON Fungi l-^l 

Seymour I'omper, Hioloni-^t. Riolop;y Division, ();ik Hidge National 
Laboratory. Oak Ridge 

Kinihiill ('. Alirood, Senior Biologist, Biology Division. Oak Hidge Na- 
tional Laboratory, Oak Ridge 

12. I'lIOTOREACTrVATION 456 

Renato Dulbecco. Associate Professor of Biology, California Institute of 
Technology. Pasadena 

13. Sunburn 487 

Harold F. Blum, Physiologist, National Cancer Institute, and \'isiting 
Lecturer, Princeton University, Princeton, N..J. 

14. Ultraviolet Radiation and Cancer 529 

Harold F. Blum (see Chap. 13) 

Name I.ndex 561 

SiTBJECT Index 577 



RADIATION BIOLOGY 
Volume I J 




CHAPTER 1 

Photochemistry 

Robert Livingston 
School of Chemistry, University of Minnesota, Minneapolis 

Introduction. Primary steps: Absorption — Franck-Condon principle — Direct optical 
dissociation — Half life of the excited state — Predissociation — Internal conversion — Phos- 
phorescence and long-lived fluorescence — Long-lived energetic states — Quenching of excited 
states — Transfer of excitation — Solvent effects — Cage effect — Photochemical transfer of 
electrons or protons to the solvent. Secondary steps: Bimolecular steps — Unimolecular 
steps — Tennolecular steps — Diffusion-controlled processes. Mechanism of complex 
reactions: General problem — Steady-state approximation. Examples of the principal 
types of photochemical reactions: Decomposition reactions — Reactions of molecular 
oxygen — Polymerization and dimerization — Intramolecular changes — Sensitized reac- 
tions. References. 

INTRODUCTION 

The development of photochemistry as a quantitative science was made 
possible by the discovery of the Einstein photochemical equivalence law 
and by the accumulation of spectroscopic knowledge. The equivalence 
law can be stated as follows: a photon can induce a photochemical reac- 
tion only by being absorbed and, on being absorbed, will activate one and 
only one molecule. The spectroscopic behavior of practically all (stable) 
diatomic molecules is well understood. In studying the optical proper- 
ties of complex molecules, it is usually necessary to be guided by general 
principles and qualitative analogies. In principle, there is little difference 
between photochemical reactions utilizing visible light and those produced 
by ultraviolet radiation. 

The observed change in a photochemical reaction, as in a thermal reac- 
tion, is the result of the concurrence of a number of simple reaction steps. 
A set of reaction steps, which is consistent with all available information 
about a reaction, is called the "mechanism of the reaction." It is con- 
venient to divide the steps which constitute the mechanism into two 
groups, called "primary" and "secondary" steps. The primary steps 
are those chemical or physical processes which are the direct consequence 
of the absorption of the photon and which involve only the absorbing 
species (and possibly the solvent). Reactive molecules, radicals, or 
atoms are produced by the primary steps. These reactive entities can 

1 



2 RADIATION HIOLOGY 

(hen uiulergc) (or initiate) a series of siinplc thermal reactions, the second- 
ary steps. Secondary reaction steps arc imiinolecular, himolecular, oi- 
termolecuhir reactions. ( )1 these, himolecuhir reactions, which involve 
a radical or atom and a stable molecule, are probably the most important. 
Bimolecular reactions also occur between two radicals or atoms. In the 
gas phase, recombination of two atoms always occurs as the result of a 
three-body collision. This is very probably the only important type of 
termolecuhir reaction step. Unimolecular reaction steps are limited 
to the "spontaneous" decomposition or rearrangement of comple.x 
molecules. 

PRIMARY STEPS 

ABSORPTION 

Each photon is absorbed by a single molecule,' and, under all ordinary 
conditions, multiple e.xcitation of a molecule by successive capture of two 
or more photons is of negligible importance. By capture of a photon the 
molecule is raised from the ground state to a higher electronic state. 
With few exceptions (notably molecular oxygen and "odd" molecules; 
Herzberg, 1950), the ground state of a stable molecule is a singlet one, 
and the molecule will be excited preferentially to a higher singlet level. 
The selection rule, which "forbids" transitions betw^een states of different 
multiplicities, is called the "intercombination rule." For ordinary mole- 
cules the probability of a transition between energy levels of unlike multi- 
plicity can be 10^-fold less than that for an otherwise similar transition 
which does not violate the rule. This is one of the few selection rules 
which apply to complex molecules as well as to atoms and diatomic mole- 
cules. For certain cases, such as those involving heavy atoms, e.g., mer- 
cury, the rule applies less strictly, and the probability of transition is 
reduced by a factor of only about 10'*. 

FRANCK-CONDON PRINCIPLE 

The Franck-Condon principle is probably of greater importance than 
the selection rules in determining the photochemical behavior of mole- 
cules. This principle states that electronic transitions which involve 
appreciable changes in the positions or momenta of the constituent 
nuclei have relatively low probabilities.- At ordinary temperatures, 
molecules which exist in thermal e(|uilibrium with their surroundings are 
in their lowest oscillational states. Accordingly, the Franck-Condon 
principle and the potential-energy curves determine the oscillational 

' In this disrussion, certain typos of crystals and crystalline micelles are to be 
regarded as single molecules. 

^ This discussion of the Franck-Condon principle is based on its original classical 
formulation. For a discussion of the modifications introduced by quantum theory see 
Herzberg (1950). 



PHOTOCHEMISTRY 



U 




A^B* 



A+B 



r 

[a) 



states of the excited electroiiic state that can be reached directly by the 
absorption of a photon. This is illustrated for a diatomic molecule in Fig. 
1-1, in which the ordinate r represents the distance between the nuclei and 
U is the potential energy of the molecule. In Fig. 1-la, the eciuilibrium 
separation for the nuclei is about the same in the excited state as it is in 
the ground state. The probable transitions from the ground state can be 
represented by vertical arrows lying 
between a and 6 since they corre- 
spond to small changes of position or 
momentum. In this way, a simple 
application of the Franck-Condon 
principle demonstrates that direct 
optical dissociation is improbable for 
a molecule whose potential-energy 
curves are of the type illustrated in 
Fig. 1-la. In the gas phase at low 
pressure, where the life expectancy 
of an excited molecule is short com- 
pared to the time interval between 
collisions, such a molecule would 
emit one quantum for each one ab- 
sorbed. As is suggested by the vari- 
ations in the length of the downward 
arrows, the fluorescence or emission 
spectrum is much more complex than 
the corresponding absorption spec- 
trum. In a condensed phase or in a 
gas at higher pressure, there is a high 
probability of the excited molecule 
losing its excess oscillational energy 
by collisions of the second kind 
(Franck, 1922) during the lifetime of 
the excited state. As a result, prac- 
tically all the transitions, corresponding to the fluorescence, will start 
from the lowest vibrational level of the excited state. Stokes's law 
holds under these conditions, and the fluorescence spectrum is shifted 
to the red side of the absorption spectrum. Freciuently the fluorescence 
spectrum is approximately a mirror image of the absorption spectrum 
(Lewschin, 1931). 



u 




A+B 



'B 



r 

(b) 



Fig. 1-1. Potential-energy diagrams for 
a diatomic molecule. 



DIRECT OPTICAL DISSOCIATION 

In the case illustrated by Fig. 1-1 b, the molecule is nuich less stable in 
its excited than in its ground state and, correspondingly, the equilibrium 
separation of the nuclei is increased. It follows, from the Franck-Condon 



\ RADIATION BIOLOGY 

priiuiplt', I hut the range of probable transitions (starting from the ground 
state) Hes between the vertical arrows a and c. Transitions whose arrows 
lie between arrows b and c are similar to those discussed in connecti(jn with 
Fig. 1-1 a. Transitions arising from points to the left of arrow 6 result in 
the formation of an excited molecule whose vibrational energy is greater 
than that required to dissociate it into atoms, one normal and one excited. 
Accordingly, direct optical dissociation is a probable event. If the gas is 
irradiated with photons of energy greater than that indicated by the 
length of arrow h and less than that of arrow a, the radiation will be 
strongly absorl)ed. For each such absorption act, a molecule will be dis- 
sociated (within the half period of a single vibration), and there will be no 
fluorescent emission. Since the end state is not quantized, the absorption 
spectrum will be contiiuious in this region. 

HALP^ LIFE OF THJ: KXCITED STATE 

If no chemical process, such as optical dissociation, can take place, the 
half life (ti,,,) of the excited state of a molecule is greater than 10~^ sec, and 
the molecule will lose all or part of its energy of excitation by emitting a 
photon. Since the restrictions which limit the probability of a transition 
apply equally to absorption and emission, a high absorption coefficient 
corresponds to a short half life of the excited state. For example, the 
direct optical excitation of a normal molecule from its ground singlet state 
to an excited triplet state (with a half life of lO"'' sec) would be so improb- 
able that it would not be observed in the absorption spectrum under ordi- 
nary conditions. These conditions were put in quantitative form by Ein- 
stein in 1917 (Herzberg, 1950, pp. 20, 381). If the effect of possible 
degeneracy of the levels is neglected, the Einstein relation can be put in 
the following form: 

NaC In 2 _ 1 



aT\., — 



Stt X 10» v^ 



X 4 (1-1) 



where Na is Avogadro's number, c is the velocity of light, and v is the 
freciuency of the radiation absorbed or emitted. The Beer's law extinc- 
tion coefficient, off is defined by the following equation, in which in is the 
concentration of the absorbent in moles per liter, / is the length of the 
light path in centimeters and /n and Iir are the intensities, respectively, of 
the incident and transmitted light: 

tr — InC 

It should be noted that the product ary, is inversely proportional to the 
square of the fre(}uency. While Eq. (1-1) applies only to monochromatic 
light (and therefore approximately to atomic spectra), it can be modified 
to apply to the broad-band absorption and emission of a molecule. For 
the latter application, a must be known as an empirical function of u, and 



hi — ^jiNn, 




Ifi _ kfi _ 


T 


labs kfi + kc 


To 



PHOTOCHEMISTRY 5 

the expression must be integrated over all frequencies which correspond 
to the electronic transition under consideration. 

Equation (1-1) is based on the assumption that an excited molecule can 
lose its energy of excitation only by emitting a photon. If the energy can 
be lost in any other way, either spontaneous or induced, the lifetime of the 
excited state will be correspondingly reduced. When the system is illu- 
minated with light of constant intensity, a steady-state condition will pre- 
vail, and the concentration A^'.v of molecules in the excited state will 
be constant. If "intensities" are expressed in photons absorbed per 
cubic centimeter per second and the rate Vc of the nonradiative disap- 
pearance of excited molecules in corresponding units, then 

labs = Ifi + ?'c = {kfi + k,)NN. 

The coefficient k^ can be a function of added substances, but, for any given 
solution, kfi -\- k^ is a constant and is equal to the reciprocal of the mean 
life r of the excited state under these special conditions. Since 

it follows that 

(1-2) 

where ro is the natural mean life or the life which the excited state would 
have if the emission of fluorescent light was the only possible degradative 
process. The ratio of Ifi/ labs is called thfe fluorescence yield. 

PREDI8S0CIATI0N 

The fluorescence yield, and correspondingly the mean life, of an excited 
molecule may be reduced by a process known as "predissociation." 
This process is possible when a stable vibrational level of an electronically 
excited state overlaps a dissociation region of another state. For a dia- 
tomic molecule this corresponds to the crossing of the potential-energy 
curves of two excited states. Under these conditions, when both the 
energy and the nuclear configuration are the same in the two states, 
the molecules can cross over from the stable, quantized state into the 
unstable, nonquantized state. While the energy of the primary excited 
state must be greater than the thermochemical energy of dissociation, it 
may be much less than that required for direct optical dissociation. The 
probability of such a transition may have any value from unity to prac- 
tically zero. It is determined by the Franck-Condon principle and by 
certain selection rules. The chemical detection of the resultant atoms or 
radicals is the most sensitive test for the occurrence of predissociation. 
The weakening of the fluorescence intensity is a direct measure of the 
probability of predissociation. The disappearance of the rotational 
structure of an absorption band indicates that the probability of the cross- 



fi KADIATION HIOLOGY 

oNtT Iroiu OIK' state to llic otlicr lias hccoMK- so j;rcat that the mean life of 
the excited molecule has been reduced to a value comparable to its period 
of rotation. 

Some niolcculcs which cxhil)it a fluorescence yield of unity at low pres- 
sures dissociate when they are illuminated at high pressures. A collision 
of the excited molecule with a normal molecule of its own kind or of an 
added gas induces its dissociation. A process of this type, which is called 
"induced predissociation." was first observed for I2 (lierzberg, 1950). 

INTERNAL CONVERSION 

In addition to fluorescence, direct optical dissociation, and predissocia- 
tion, excited complex molecules can undergo a process called "internal 
conversion" (Franck and Sponer, 1949). This process consists in a radia- 
tionless transition from a low oscillational level of a higher electronic state 
to a high oscillational level of a lower electronic state. The difference in 
energy between the two electronic states appears as an increase in the 
oscillational energy of the molecule after the transition. In internal con- 
version, both the initial and final states are (juantized; in this respect 
internal conversion differs from predissociation. Like predissociation, it 
can occur only when the molecule is in a specific nuclear configuration for 
which the total energy and the nuclear configuration of the molecule are 
the same in the two electronic states. Since a complex molecule has 
many generalized oscillational degrees of freedom, the time required for 
the molecule to reach the required configuration may be relatively long. 
Under experimentally realizable conditions, the time between collisions 
appears to be much less than the average time required for the molecule in 
the lower electronic state to return to the crossing point and thus to have 
a chance of coming back to the original state. Collisions between sur- 
rounding molecules and the vibrationally excited molecule quickly reduce 
the vibrational energy of the latter and so make the reverse transition 
impossible. In this way, internal conversion followed l)y a number of col- 
lisions of the second kind can lead to the complete degradation of the 
energy of excitation into thermal energy of the system. This is very 
probably the explanation of why many molecules which absorb strongly 
in the visible or near ultraviolet are nonfluorescent and do not react 
photochemically. 

Immediately after the act of internal conversion, the molecule has a 
large amount of energy in its oscillational degrees of freedom; in other 
words, it is a " hot " molecule. As such it can undergo pyrolytic reactions 
such as decarboxylation or the elimination of a hydrogen molecule. It is 
diflRcult to conceive of any other simple explanation of the direct photo- 
chemical dissociation of a complex molecule into two stable molecules, a 
j)rocess which requires the simultaneous breaking of several bonds and the 
formation of new bonds. Depending on the nature of the molecule and 



PHOTOCHEMISTRY 7 

the amount of energy available, internal convn'rsion may lead to the dis- 
sociation of a molecule either into two stable molecules or into radicals. 

PHOSPHORESCENCE AND LONG-LIVED FLUORESCENCE 

Metastable states appear to play an important part in the photo- 
chemistry of complex molecules. The existence of these states has been 
demonstrated indirectly by the analysis of photochemical data (Shpol'skii 
and Sheremet'ev, 1936) and directly by a study of the "phosphorescence" 
and "long-lived fluorescence" of these molecules (Pringsheim, 1949; 
Forster, 1951). Practically all complex molecules (at least those which 
contain a double bond) are either fluorescent or phosphorescent (or both) 
when they are dissolved in glassy media or adsorbed on suitable solids. 
One of the first examples to be studied quantitatively was the dye trypa- 
flavin adsorbed on siUca gel (Pringsheim and Vogels, 1936). At ordinary 
temperatures this system emits a strong fluorescent green band and a 
separate weak orange band. The green band is made up of ordinary 
short-lived fluorescence and a relatively long-lived emission, having the 
same wave-length distribution. The half life Ty^ corresponding to the 
latter process is an exponential function, ry, = ke^'^'^, of temperature. 
There is no short-lived fluorescence corresponding to the orange band. 
The half life corresponding to this latter transition is independent of tem- 
perature and equal to 1.2 sec. As the temperature is reduced, the life 
corresponding to the green phosphorescence eventually becomes longer 
than that pertaining to the orange, long-lived fluorescence, and the slow 
emission becomes predominantly orange. This general behavior is 
exhibited by a wide variet}^ of substances (Kasha, 1947). Many measure- 
ments of this type (Lewis and Kasha, 1944, 1945) have been made with 
absorbing substance dissolved in a solvent, such as a mixture of ether, 
pentane, and alcohol, which is fluid at ordinary temperatures and becomes 
rigid at low temperatures. Under these conditions, only ordinary 
fluorescence is observed in the fluid solvent, the temperature-dependent 
phosphorescence appears when the solvent becomes very viscous, and the 
temperature-independent, long-lived fluorescence becomes noticeable at 
still lower temperatures. 

A reasonable explanation of these phenomena, which was first proposed 
by Jablonski (1935), is illustrated by the simplified energy diagram of Fig. 
1-2. The several electronic-energy levels are represented by horizontal 
lines, capped by a bundle of horizontal lines which indicate the overlap- 
ping generalized oscillational levels. For an ordinary stable molecule, the 
ground level N and the two excited levels F and F' are singlet states. The 
metastable level M is, presumably, a triplet level. The transitions which 
correspond to arrows 1 and 2 represent the absorption of photons, which 
raise the molecule into its first or second excited (singlet) state. In its 
initial state the molecule will be in thermal equilibrium with its surround- 



8 RADIATION HIOLOGY 

ings, and its oscillational enerf^y will Ix' at or near its zero-point value. As 
determined l»\ the Franck-Condon principle, the electronically excited 
molecuh^ will usually have an excess of oscillational eiierj^y. In a con- 
densed medium or in a gas at moderate pressure, the molecule will (|uickly 
lose this excess of vibrational energy by successive collisions of the second 
kind. As a result the fluorescent light, transition 3, will usiuilly have a 
larger wave length than the corresponding absorption. In addition to 
these permitted transitions, radiationless transitions, 4, 5, and (>, are pos- 
sible. Each of these acts corresponds to a process of internal conversion. 
The occurrence of step 4 is proved by the fact that fluorescence cor- 
responding to transitions from /'" to A'^ or from F' to /'' is never observed 




Fk',. 1-2. Schematic potential-energy diagram for a complex molecule. 

with complex molecules such as dyes. Illumination with light of shorter 
wave length, which raises the molecule to the second (or a higher excited) 
level F', results only in the long-w'ave-length fluorescence (transition 3). 
Phosphorescence and long-lived fluorescence involve the metastable state 
M . In order to reach this state the molecule must undergo an act of 
internal conversion, step 5. Once in state M , the molecule can return to 
state F, by way of step 5, only if it receives thermal energy e(|ual to or 
greater than e, the difference between the energies of levels M and F. 
This sequence of events corresponds to the temperature-dependent phos- 
phorescence. Step 8 is forbidden, by the intercombination rule, and will 
occur only if there is no other probable mode of escape from M. The 
relativ^e importance of this emission, the long-lived fluorescence, increases 
as the temperature is lowered. Internal-conversion steps, 6 or 7, con- 
tribute to the inefficiency of photochemical and fluorescence processes, 
(^ne offect of adsorbing the molecule or dissol\iiig it in a rigid medium is to 



PHOTOCHEMISTRY 9 

reduce its number of oscillational degrees of freedom and thereby to lower 
the probability of internal conversion. It seems reasonable to assume 
that this effect of binding the molecule to its surroundings has a relatively 
small effect on the initial transition from F to M (or F' toF), since the 
molecule and its surroundings momentarily have available an energy 
excess equal to the difference between the energy of the photon and the 
energy difference betw'een levels F and N. 

If the preceding simple explanation is correct, the difference between 
the energ}^ levels F and .1/ can be determined in two independent ways, 
which should yield identical results. The difference in potential energy 
between levels F and .V is measured approximately by the quantity hp.i, 
corresponding to the long-wave-length limit of the normal fluorescence. 
Similarly, the energy difference between M and N is approximately equal 
to hvs for the long-wave-length limit of the long-lived fluorescence. The 
energy difference between F and AI is therefore equal to hv^ — hva. This 
same energy difference should be obtainable from measurements, at two 
or more temperatures, of the half life of the phosphorescence. In terms 
of the simple Jablonski mechanism, e in the following equation should be 
equal to Aj^s — hv^: 

For all cases for which the experimental evidence is available, this is 
approximately true. Although there are minor and understandable dis- 
crepancies (Pringsheim, 1949, p. 441), the available data support this 
interpretation. The measured values (Kasha, 1947) of the M-F energy 
difference lie in the range 5-40 kcal/mole. The lower values belong to 
dyes, and the higher ones to simpler molecules, such as the aromatic 
hydrocarbons. Since e~'^*^ will be a very small fraction for the higher 
values of e, phosphorescence will be very inefficient and probably unde- 
tectable in these cases. 

LONG-LIVED ENERGETIC STATES 

It Avas formerly maintained by many photochemists that fluorescence 
and photochemical action are strictly complementary actions. How- 
ever, a large amount of information, chiefly qualitative but in part quan- 
titative, which is definitely incompatible with this belief, has accumulated. 
Efficient photochemical reactions are known which involve compounds 
whose fluorescence yield is small, even in dilute solutions in inert solvents. 
Particularly in some dye-sensitized photooxidations involving weakly 
fluorescent sensitizers (Shpol'skii and Sheremet'ev, 1936; Franck and 
Livingston, 1941), the absorbed photon has too small an energy to dis- 
sociate the absorbing molecule. At least in these cases a long-lived 
excited state must be an intermediate in the photochemical reaction. 

To demonstrate this, let us consider a particular example: the auto- 



10 RADIATION BIOLOGY 

oxidutioii of allyltliiouicii pliotosensitizod \>y cliloiopliyll ((lulTroii, 1927). 
It seems safe to assume that the reaction involves an interaction between 
an energy-rich chlorophyll molecule and either an oxygon or an allyl- 
thiourea molecule. It iiotinal chloropiiyll is represented by G, its fluo- 
rescent state by G*, some long-lived excited state by G', and the reacting 
molecule, either oxygen or the reducing agent. I)y B, a generalized (and 
simplified) mechanism for the reaction may be written as follows: 

(1) G -\- hu ^ G* ' (absorption), 

(2) G* -^G -\- hvf (fluorescence), 

(3) G* —> G' (internal conversion), 

(4) G'-^G (degradation), 

(5) B + G* — >-^ G + products (chemical reactions). 

Expressed in appropriate units (einsteins per liter per second), the rate of 
step (1) is i'l = lab.- The rates of the four subsequent steps are, respec- 
tively, V, = /.-,[. I *],/•:, - A-4.l*],r4 = k,[A'], and V, = k,[B][G*]. As long 
as the measurements of the fluorescence and of the chemical reaction are 
made under the same conditions, it is immaterial whether ks is a function 
of the nature of the solvent, i.e., whether the solvent quenches the fluo- 
rescence. Under steady-state conditions, 

djA*) „ , d{A') „ 

-^^0 and ^^-0, 

and thus 

Vi = i>2 + Vs + /'o, and v^ = Vi 

may be written. Substituting for the several Vi, the following values for 
the fluorescence yield <pfi and for the photochemical yield are obtained: 

^ la ^ ^2 

^'' Us k2 + k, + kABy 

V, k,[B] 



f = 



labx ki + A:3 + k^{B] 



In a dilute solution and in the absence of quenchers the fluorescence yield 
iPfi for chlorophyll (Prins, 1 934) is approximately 0. 1. It is not detectal)ly 
quenched by allylthiourea, and it recjuires about 2 X 10^- mole of oxygen 
to reduce the fluorescence to half its maximum value. A quantum yield 
ip of practically unity was obtained in a solution containing about 0.1 mole 
of allylthiourea and 0.002 mole of oxygen. The natural mean life of the 
excited state must be of the order of 10~^ sec; therefore k-i =10^ sec~'. 
Since in the absence of quenchers the fluorescence yield is approximately 
0.1, 



^h = 0.1 = f:^; 



»o 



PHOTOniKMlSTRY H 

Therefore ks is about 10* sec~'. Approximately, 

The addition of 0.1 mole of allylthioiirea to a chlorophyll solution reduces 
its fluorescence by less than 5 per cent. Accordingly, if a direct reaction 
of this substance with the (singlet) excited chlorophyll is responsible for 
the sensitized reaction, then k^lB] < 0.05 X 10^ or kf, < 5 X 10^ liters 
raole~^ sec"'. However, the quantum yield for the reaction is 

k,[B] 



f 



10^ + k,[B] 



In the presence of 0.1 mole of allylthiourea the quantum yield is certainly 
greater than 0.9, which reciuires that kf, be greater than 10^". Since the 
difference between these two estimates is 200-fold or more, the postulate 
that the reducing agent reacts directly with the singlet excited state must 
be rejected. If it is assumed that a collision between an oxygen molecule 
and an excited chlorophyll molecule is responsible for both the reaction 
and the fluorescence quenching, the computed values of k^ are 10^" and 
5 X 10" sec-i (moles/liter) -\ respectively. Not only is this fiftyfold 
discrepancy too great to be explained in terms of experimental uncer- 
tainties, but also the value (5 X 10") required by the photochemical data 
is unreasonably large. The frequency factor for a bimolecular reaction 
between molecules of ordinary dimensions and mass at room temperature 
is given by the simple collision theory (Moelwyn-Hughes, 1947) as about 
10" sec-i (liter/mole). Although the size of the chlorophyll molecule 
might possibly increase this number fivefold, 5 X 10" is surely an upper 
limit. In order for the rate constant to be as large as the frequency fac- 
tor, the steric factor must equal unity and the energy of activation must 
be zero. Moreover, the rate of such a reaction, occurring in solution, will 
be determined by the frequency with which the reaction partners can 
diffuse together (Fowler and Slater, 1938) and not by the total number of 
collisions between them. In a condensed phase, collisions occur in 
bursts of (probably) 100 or 1000, and the number of encounters is corre- 
spondingly reduced. It must be concluded that this reaction cannot be 
induced by a collision of the directly excited chlorophyll molecule with 
either of the reaction partners. Therefore the sensitizer molecule must 
be capable of existing in some long-lived energy-rich form. The same 
conclusion may be reached from the results of analysis of kinetic data for 
other sensitized reactions (Franck and Livingston, 1941). 

More direct evidence is offered by the experimental investigation 
(Gaffron, 1937) of the photochemical autooxidation of the aromatic 
hydrocarbon, rubrene. Since the quantum yield for this reaction remains 
high even when the oxygen concentration is relatively low, it can be shown 



12 RADIATION BIOLOGY 

that tlie half Hfe of the excitod ruhrene molerule is very long (possibly 1 
sec or more). This is very mwh {greater than the maximum half life of 
the directly excited, sinj^let state. 1 ii this case, as in those sensitized reac- 
tions previously referred to, a long-lived excited state must be an inter- 
mediate in the photochemical reaction. 

At the present time, there appear to be no data availal)l(' from which it 
can be concluded that the long-lived state recpiired by photochemical evi- 
dence is identical with the phosphorescent state, usually studied at low 
temperatures and in rigid media. However, in the absence of information 
to the contrary, this identification is commonly made as a simplifying 
hypothesis. In one case, that of fluorescein in boric acid glass, direct 
measurements of the paramagnetism of the excited substance (Lewis et al., 
1949) have demonstrated that the phosphorescent state is a triplet one. 
Comparison (McClure, 1949) of the measured half lives of the long-lived 
fluorescence of a large number of aromatic compounds and of a few ali- 
phatic ketones makes it appear probable that the phosphorescent states 
of these compounds are likewise triplet states. 

It should not be assumed that all photochemical reactions involve either 
dissociation or a long-lived excited state. Benzene and some of its methyl 
derivatives react photochemical ly with molecular oxygen to form perox- 
ides. Their limiting fluorescence yields are high, and their fluorescence 
and photochemical reactions are complementary functions of oxygen con- 
centration (Bowen and Williams, 1939). It is probable, therefore, that 
the photochemical reaction in these cases goes by way of a direct inter- 
action between an oxygen molecule and the singlet excited, i.e., fluores- 
cence, level of the hydrocarbon molecule (Kasha and Xauman, 1949). 

QUENCHING OF EXCITED STATES 

A low quantum yield for a photochemical reaction may, under some con- 
ditions, be the result of the quenching of the excited molecule by impuri- 
ties, reaction products, the solvent, or even one of the reactants. Such 
quenching brings about a decrease in the fluorescence yield, which is not 
accompanied by an ol^servable chemical reaction. The mechanism of the 
process has been the subject of mu(;h speculation (Pringsheim, 1949, p. 
335), and it appears probable that no one explanation is consistent with 
all the experimental facts. In some cases the quenching appears to be 
the result of the reversible formation of nonfluorescent complexes, 
whereas in others it may be due to collision (or the close approach) of a 
quencher and excited molecule. It is possible (Rollefson and Stoughton, 
1941; Livingston and Ke, 1950) that the quencher acts by inducing a 
transition of the potentially fluorescent molecule from its excited singlet 
state to its lowest triplet state. Fluorescence (juenching of this type 
v/ould not necessarily be accompanied by a corresponding reduction in 
the quantum yield of a photochemical reaction. 



PHOTOCHEMISTRY 13 

At moderately high concentrations (10~* M or greater), the fluorescence 
of most compounds decreases as the concentration is increased. In some 
cases this self-quenching is due to the reversible formation of nonfluores- 
cent dimers (Rabinowitch and Epstein, 1941; Lewschin, 1935). This is 
by no means invariably true. More generally, self-quenching appears to 
be related to the "migration of excitation energy" (Vavilov, 1943; For- 
ster, 1948, 1950; Franck and Livingston, 1949). As a consequence of this 
effect, photochemical reactions which do not involve direct optical dis- 
sociation or predissociation should be expected to become corresponding!}- 
inefficient at high concentrations of the light absorber (cf. Gaffron, 1927). 

TRANSFER OF EXCITATION 

Transfer of energy of excitation between like or unlike molecules may 
well play an important role in photochemistry, particularly in heterogene- 
ous sj^stems and in solutions in which the concentration of the nonabsorb- 
ing reactant is relatively small. In crystals such a transfer of excitation 
may be caused by migration of electrons in a conductivity band of the 
crystal (Franck, 1948), by "exciton migration" (Frenkel, 1931), or by 
a radiationless transfer which may be called "classical resonance" 
(Franck and Livingston, 1949). In a solution such a transfer of excita- 
tion can occur only on collision or as the result of classical resonance. 
This latter possibility has been carefully analyzed by Forster (1948), who 
has shown that for certain dyes and pigments such a transfer can occur 
efficiently at distances as great as 50 or 100 A. Sensitized fluorescence, 
in which the photon is absorbed by one molecule and the energy trans- 
mitted to an unlike molecule which emits its characteristic fluorescence, is 
one consequence of classical resonance. It is a well-established phenom- 
enon in gases at low pressure, such as mixtures of mercury and thallium 
(Carlo and Franck, 1923), and has been reported for at least one case in 
liquid solutions (Watson and Livingston, 1950). Transfer of excitation 
between like molecules can be most readily detected by the depolarization 
(Vavilov, 1943; Forster, 1948) of fluorescence which is excited with plane 
polarized light. In some cases, self-quenching (Watson and Livingston, 
1950) and quenching by added substances (Forster, 1950) appear to be the 
consequence of classical resonance. 

Energy of excitation may be exchanged between different groups within 
a complex molecule by a similar mechanism (Franck and Livingston, 
1949). Weissmann (1942) made the interesting observation that light 
which is absorbed by the organic part of the europium salicylaldehyde 
molecule excites fluorescence characteristic of the europium ion ; this effect 
is very probably the result of such a radiationless transition between the 
separate parts of the molecule. When the carbon monoxide-myoglobin 
complex is illuminated with light of either 5400 or 2800 A, carbon monox- 
ide is split off, and the quantum yield of the process is about unity 



14 RADIATION BIOLOGY 

(HiicluT and Kaspcr.s, 1947). Myoglobin is iin enzyme, consistinf^ of two 
heniin groups attached to a protein having a molecular weight of about 
32,000. The absorption at o4()0 A is due almost entirely to the hemin 
group, and the corresponding photochemical dissociation is presumably 
either predissociation or direct optical dissociation. At 2800 A, much of 
the absorption (about 40 per cent) is due to the tyrosine and tryptophane 
groups which are presumably distributed throughout the protein mole- 
cule. It is very probal)le that the energy is transferred from these pri- 
marily excited groups to the hemin-carbon monoxide complex by classical 
resonance. The elTect is almost certainly not caused by a general degra- 
dation of the protein, since the quantum efficiencies of such reactions are 
smaller by several orders of magnitude (Finkelstein and McLaren, 1941)). 

SOLVENT EFFECTS 

The presence of a solvent may affect the primary process in several 
different ways; namely, it may decrease, leave unaltered, or even increase 
the quantum yield of the photochemical reaction. The solvent may also 
bring about a change in the reaction products. What the effect will be in 
a specific instance cannot, in general, be predicted. However, a knowl- 
edge of the possible influences of a solvent on the primary step is of great 
value in interpreting experimental results. 

In one sense the simplest, and perhaps the most important, effect of the 
solvent on the primary process is its influence on the normal ecjuilibrium 
state of the reactant (i.e., absorbent) molecules. Acidic or basic media 
often determine the charge on solute molecules and thus alter their 
absorption spectra and their relative probabilities of fluorescing, dissociat- 
ing, undergoing internal changes, or degrading their energy of excitation. 
Changes in the molecular state, such as dimerization or the formation of 
molecular complexes with the solvent, are of even more common occur- 
rence and are likewise effective in changing the photochemical properties 
of the solute. Usually such changes can be detected by measurements of 
nonkinetic properties, such as absorption spectra, conductivity, or trans- 
ference, and the coUigative properties of the solution. 

Since solute molecules are continuously in a state of multiple impact 
with the surrounding molecules, any oscillational energy which the solute 
molecule may possess, in excess of the thermal eciuilibrium amount, is 
quickly drained off by successive collisions of the second kind. This loss 
of oscillational energy may stabilize an electronically excited molecule by 
reducing its probability of predissociating or of undergoing internal con- 
version. Conversely, the solvent may "induce predissociation" of a 
molecule by light of wave lengths too long to bring about dissociation of 
the isolated molecule (Herzberg, 1950). After an act of internal conver- 
sioji, a complex molecule has momentarily a large fraction of its energy of 
excitation in its generalized oscillational degrees of freedom. If all its 



PHOTOCHEMISTRY 15 

excitation energy is present as oscillational energy (i.e., if the energy-rich 
molecule is in its electronic ground state), the solvent will increase the 
probability of this energy being lost to the surroundings as heat and so 
reduce the quantum yield. ^Nlany dyes and other complex molecules go 
(by internal conversion) from their excited singlet states to a relatively 
long-lived (triplet) energetic state, which is chemically an activated state. 
Under these latter conditions, deactivation by collisions of the second kind 
has little, if any, effect on the probability of reaction. 

CAGE EFFECT 

When a dissolved molecule dissociates, its fragments (atoms or radicals) 
are surrounded by a barrier of solvent molecules. Held in this cage, they 
will make a number of collisions with one another before they can move 
out of the cage. This increases the probability that they will recombine 
and so reduces the efficiency of the photochemical reaction. There are 
several factors which influence the importance of this "cage effect." 
When the absorbed photon has greater energy than is required to dis- 
sociate the molecule, the excess kinetic energy of the resulting atoms will 
increase their chance of escaping from the cage. This chance of escape is 
greater for small atoms (especially hydrogen atoms) than it is for larger 
radicals. In the case of large radicals, there is a compensating factor. 
The recombination of large radicals requires some (small) energy of acti- 
vation and demands that very strict conditions of relative orientation be 
fulfilled. Both these requirements greatly reduce the probability of 
recombination at a collision and correspondingly decrease the importance 
of the cage effect. Experimental investigation (Rollefson and Burton, 
1939, Chap. XIV) of the cage effect demonstrates that it is real and can be 
of importance in photochemical reactions. However, it appears to be 
unexpectedly specific. 

A solvent may change the nature of the reaction products either by 
reacting (in one or more secondary steps) with the primary products or by 
altering the relative probabilities of alternative primary reactions. An 
excited complex molecule can dissociate either into two radicals or into 
two stable molecules. It has been reasonably substantiated (Bamford 
and Norrish, 1938) that certain ketones undergo both types of dissociation 
to a comparable extent in the gas phase but, in solution, dissociate only 
into stable molecules. This difference in products has been attributed to 
the action of the solvent, which cages in the free radicals and so brings 
about their recombination. 

PHOTOCHEMICAL TRANSFER OF ELECTRONS OR PROTONS TO THE 

SOLVENT 

In condensed systems the absorption of a photon of ultraviolet or even 
visible light can lead directly to the formation of an ion by the ejection of 



16 RADIATION niOLOGY 

oitluM- an electron or a proton. An important special case of this type is 
the photocondnctivity of ionic crystals (AFott and CJnrney, 1940). 
Althouj^h this latter phenomenon is of vital importance in determininji; the 
photochemical and optical properties of crystal phosphors, it appears to be 
too specialized for this discussion. 

When certain compounds dissolved in glassy media at low temperatures 
are illuminated with ultraviolet light, semi(iuinones are formed hy the 
ejection of an electron from the absorbing mcjlecule (Lewis and Bigeleisen, 
1943). In these low-temperature rigid solutions the return of the elec- 
tron is greatly retarded, and the spectrum of the resulting semi(iuinone 
can be directly measured. In some instances the semi(iuinone is stabi- 
lized by the thermal loss of a proton to the surroundings. A number of 
organic compounds (all of which contain basic nitrogen, sulfur, or o.xygen) 
exhibit this property. There can be no reasonable doubt ihat a similar 
photoionization can occur in aqueous solutions. The near-ultraviolet 
absorption of ferrous ion is very probably due to the transfer of an elec- 
tron from the central ion to the surrounding shell of water molecules 
(Zimmerman, 1949). Unless this excited system is stabilized by a 
secondary reaction, such as the elimination of elementary hydrogen or the 
reduction of an oxidizing agent, it will return after a short time to its 
original state. 

The transfer of a proton from an absorbent molecule to a solvent mole- 
cule or between the two components of a molecular complex has been 
demonstrated by three independent methods. If aqueous solutions of 
organic acids or bases are exposed to ultraviolet radiation which does not 
decompose them, it has been reported (Terent'ev, 1949) that the pH of 
the solution changes reversibly. In the majority of the cases studied, 
the pH increased upon illumination by a few tenths of a unit. This sug- 
gests that the excited molecules are weaker acids than the corresponding 
normal molecules. 

More detailed and systematic measurements were made by Forster 
(1950) on solutions of hydroxy and amino derivatives of sulfonated 
pyrenes. These compounds display either one or the other of two differ- 
ent absorption spectra and of two different fluorescence spectra, depending 
on the pH of the solution. The shifts in the emission and absorption 
spectra do not occur in the same pH range. From an analysis of the.s(^ 
spectral shifts the ionization constants of the normal and excited mole- 
cules can be determined. The changes in the ionization constants, due to 
electronic excitation, are surprisingly great. For example, the ionization 
constant of hydroxy trisulfonated pyrene is increased by a factor of 10" 
upon excitation. Since the acid-base equilibrium is realized during the 
lifetime of the excited (fluorescent) state, this photochemically induced 
ionization must be classed as a primary step. It does not involve internal 
conversion. 



PHOTOCHEMISTRY 17 

Tereniii and Kariakin (1947) prepared thin films by subliming simul- 
taneously acridine and an organic acid (e.g., succinic acid) onto a plate 
kept at — 180°C. When irradiated with wave length 3660 A, the newly 
prepared films exhibited the violet fluorescence which is characteristic of 
neutral acridine. When the material had stood for some time at — 180°C, 
or for a much shorter time at room temperature, this fluorescence was 
replaced by the green emission which is given by acridinium salts. A 
short irradiation with wave length 2537 A restored the film to its violet 
fluorescent state. On standing in the dark the fluorescence again became 
green. These changes could be repeated indefinitely. The effect of 
irradiation with actinic light must be the transfer of a proton across the 
hydrogen bond from the amino group to the carboxyl ion. This reaction 
is essentially similar to those which were studied in aqueous solution. 
They all involve the transfer of photons across hydrogen bonds. Terenin 
(1947) has suggested that processes of this type may play an important 
role in the photochemistry of biological systems. 

SECONDARY STEPS 

The photochemical secondary steps, those simple reactions which trans- 
form the primary products into the stoichiometric reaction products, are 
identical with the reaction steps which are responsible for the observed 
kinetics of thermal (dark) reactions (Laidler, 1950, Chap. 7). The indi- 
vidual steps are kinetically simple — commonly unimolecular, bimolecular, 
or termolecular chemical reactions. Under some conditions the kinetics 
of the over-all reaction are influenced by the rates of the diffusion, adsorp- 
tion, or desorption processes. 

BIMOLECULAR STEPS 

Bimolecular reactions involving a reactive entity and a normal molecule 
are of special importance in the mechanisms of photochemical reactions. 
Reactions between stable molecules and atoms have been extensively 
investigated. The following reactions are typical examples whose exist- 
ence has been reasonably well established (Steacie, 1946; Polanyi, 1932; 
Laidler, 1950): 

Na -\- Clo -^ NaCl + CI, 

CI + H2 ^ HCl + H, 

H + CH4 -^ H2 + CH,3, 

OH + H2O2 -^ H2O + HO... 

Similar reaction steps such as 

H + CH3 -^ CHo + Ho, 

which invohc a radical in place of the stable molecule, appear to occur 
with equal facility. Reactions of this general type, which do not change 



18 HAUIATION UIOLOGV 

the total number of part ides involved, are truly bimolecuhir and can occur 
in dilute f>;as as readily as in a condensed system. When such reactions 
are exothermic, their heats of acti\ation are usually low, ratifying from 
to 10 kcal. It does not follow that any such reaction which can be pos- 
tulateil will occur in practice. For example, the reaction 

D + C'M4-^CH3l) + H 

apparently does not take place under ordinary conditions, and theoretical 
calculations indicate that its heat of activation may be as high as 40 kcal. 
The reaction between atomic sodium and cyaiingen, 

Na + C2N2 -^ NaC^X + CN, 

is slow o\>ing to the snialiness of its probability factor, although its energy 
of activ'ation is approximately zero. 

When sufficient energy is available an atom may react with a molecule 
to form two radicals. A step of this kind (Lewis and von Elbe, 1938), 

H + U^^ OH + H. 

undoubtedly plays an important role in the explosive combination of 
hydrogen and oxygen. 

Combination of two atoms results in the formation of a molecule pcjs- 
sessing more oscillational energy than is necessary to dissociate it. As a 
result, the life of the quasi molecule is equal to the time of a single oscilla- 
tion (about 10~^'^ sec), and at ordinary pressures it dissociates before it 
has a chance to make a stabilizing collision with a normal molecule. In 
other words, combination of atoms in the gas phase as a bimolecular reac- 
tion cannot occur. However, two radicals, or an atom and a moderately 
large radical, can combine to form a molecule whose energy can be dis- 
tributed among several degrees of freedom and whose mean life will there- 
fore be comparable to the time between collisions in an ordinary gas. 
Examples of this type are 

H + C2H4 -> C2H5 
and 

2C2H.^ — > ( '4H11). 

There is, of course, no restriction on the combination of atoms in a con- 
densed system, since the colliding atoms will be continuously in collision 
with the surrounding solvent molecules. 

Disproportionation and probably metathelical reactions occur and 
influence the mechanisms of reactions. The reaction 

2C2H:,-. C2H4 + r>\u 

is detectable under suitable conditions but is apparently less probable 
than the simph? combination of the ra(li<'als to form l)utane. 



PHOTOCHEMISTRY 19 

Secondary steps are, of course, not restricted to reactions involving 
radicals; the reactive species may be a relatively unstable molecule or an 
excited atom or molecule. Tn some cases the excited molecule in a sing- 
let, fluorescent state may enter into the reaction, as is illustrated by 

Hg*(6^Pi) + H2^HgH-^H, 
"" ^'cH^ + 02^ peroxide. 

Most photochemical reactions of complex molecules, such as the autooxi- 
dation of rubrene, which take place by way of an excited state appear to 
involve a long-lived (possibly triplet) excited state. 

UNIMOLECULAR STEPS 

True unimolecular reaction steps are limited to complex radicals or 
molecules. They can result in the formation of (1) two radicals, (2) two 
stable molecules, or (3) a stable molecule and a radical: 

(1) Hg(C2H5)2^HgC2H5 + C.>H,, 

(2) CH3OH -^ HCHO + H2, 

(3) CH.CO -> CH3 + CO. 

Internal rearrangements of complex molecules can be the result of uni- 
molecular reactions. Examples of this type, which have been studied, 
include cis-trans isomerizations, racemizations (probably), as well as reac- 
tions of the following type: 

CHo 



CH2 CHo — > CH:{ — CH = CH2. 

Many first-order reactions which occur in solution involve a molecule of 
the solvent and are therefore bimolecular rather than unimolecular 
reactions. 

TERMOLECUI.AR STEPS 

With few possible exceptions, termolecular gas-phase reactions are 
recombinations of atoms (or radicals), occurring as three-body collisions 
iiu'olving some other molecule or radical, i.e., 

2Br + No^ Br2 + N2. 

In solution, termolecular reactions involving one or more molecules of the 
solvent are probably of much more frecjuent occurrence. 

DIFFUSION-CONTROLLED PROCESSES 

The rates of some reaction steps which take place in condensed systems 
or in gases at moderate or high pressures are controlled by diffusion proc- 
esses. Most of these reactions involve heterogeneous or microhetero- 
geneous media. There is an important class of such reactions which occur 



20 U\ni\TI<>\ lUOLOGY 

ill homogeneous, coruhMised phases. Steps of this type have very low 
heats of activation and steric faetors of the ordcf of magnitude of unity; 
accordingly, practically every collision results in reaction. Examples of 
this type are the combination of atoms or small radicals, the quenching of 
the fluorescem-e of dyes by efficient (juenchers, and enzymatic reactions 
at low concentrations of the substrate. These reactions occur at every 
encounter of the reactant molecules. The frequency of encounters, 
unlike that of collisions, is determined by the rate of diffusion, which in 
turn is dependent on the viscosity of the solution. 

Under the normal conditions in which gas-phase reactions are com- 
monly studied, recombination of atoms frequently is a wall-catalyzed 
process, whose rate is fixed by the rate of diffusion of the atoms to the wall 
of the vessel. The rate of atomic recombination 2 A = A2, occurring 
both by three-body collisions and by wall catalysis, can be represented by 
an equation of the following form (Kassel, 1932, pp. 170-180): 

^ = kPcW + ^^ [A]. 

The factors Pc and P« are linear functions of the partial pressures of the 
components of the gas. The coefficient k' is influenced by the geometry 
of the vessel, becoming greater as the surface-to-volume ratio of the 
vessel increases. In a photochemical steady state the homogeneous 
recombination (whose rate is proportional to the square of the concentra- 
tion of atoms) is favored by an increase in the intensity of the absorbed 
light. 

If atoms or radicals are formed in a liquid-phase photochemical reac- 
tion, the observed quantum yield is likely to be influenced by the rate of 
stirring of the solution since, in an unstirred solution, the steady-state con- 
centration of atoms will, in general, be spatially nonuniform. This effect 
is especially important if a large fraction of the actinic light is absorbed 
in a thin film near the window through which the light enters. 

MECHANISM OF COMPLEX REACTION 

GENERAL PROBLEM 

Few, if any, chemical reactions are kinetically simple in the sense that 
they involve only one reaction step which is of simple order and which 
is identical with the stoichiometric reaction. The observable course of a 
photochemical reaction is the result of the simultaneous occurrence of a 
number of reaction steps. A set of reaction steps, which is consistent 
with the stoichiometry and kinetics of a reaction, constitutes the mecha- 
nism of the reaction. The kinetic equation for the over-all reaction can be 
obtained by combining the rate equations for the several steps in such a 
way that the concentrations of the reaction intermediates are eliminated. 



PHOTOCHEMISTRY 21 

For relatively simple reactions (Kassel, 1932; Laidler, 1950) this can be 
done by solving simultaneously the differential rate equations for the 
individual steps. In general, the resulting solution cannot be obtained in 
terms of simple functions, and the analysis demands a mathematical 
skill which the average photochemist does not possess. As a result 
it has become conventional among students of kinetics to use approx- 
imate methods to deduce the over-all rate equation from a postulated 
mechanism. 

STEADY-STATE APPROXIMATION 

The most generally applicable of these simple methods is the so-called 
"steady-state approximation." A steady state may be defined as a con- 
dition in which the rates of change of the concentrations of the several 
intermediates are very small compared to the rates of change of the con- 
centrations of the reactants and products. This condition is realizable 
whenever the ratio of the concentrations of the intermediates to the con- 
centrations of the reactants is very much less than unity. When this 
condition is not attained, the method is not applicable; however, it should 
not then be necessary since the (larger) concentrations of the intermedi- 
ates could be measured by experimental means. In no reaction is the 
steady state attained instantaneously. However, the time required for 
its attainment is usually a negligibly small fraction of the half time of the 
reaction. The steady-state approximation consists in setting the rates 
of change of each of the intermediates equal to zero and in solving simul- 
taneously the resulting algebraic equations. This process can be 
explained most easily by outlining the details of two well-known examples. 

Examples. The decomposition of hydrogen iodide is a classic example 
(Warburg, 1916; Bonhoeffer and Farkas, 1928) of a carefully studied and 
thoroughly understood photochemical reaction. Although this reaction 
is so simple that it is not necessary to use the steady-state approximation 
method in its analysis, it will serve to introduce the fundamentals of this 
procedure. Gaseous hydrogen iodide strongly absorbs light of wave 
length 3000 A or shorter. It is nonfluorescent, and its absorption spec- 
trum is continuous, showing no vibrational or rotational structure. It 
follows from these observations that the primary act is one of direct 
optical dissociation, 

HI 4- /iv ^ H + I. 

The secondary steps must be reactions of hydrogen and of iodine atoms. 
They may combine to form hydrogen iodide or molecular hydrogen and 
iodine, or more probably they may react with hydrogen iodide: 

I + HI^l2+ H, 
H 4- HI-^ H2 + I. 

The first of these tw^o reactions can be ruled out since it is strongly endo- 



22 RADIATION HIOLOGY 

thermic (A// = 34 kcal). Tlu* .socoikI, l)oiiit>; an cxijlhc'iiuic reaction 
hetwocn an atom and a diatomic molecule, .sliould have a .small heal of 
activation and a steric factor not dirt'eriiig {greatly from unity. The two 
possible alternative reactions that a hydrogen atom can undergo, 

M + H + I-^ III + M 

and 

M + 11 + II -^ U, + M, 

can occur only by three-body collisions with any molecule, M, or by 
diffusion to the wall. The relativ^e importance of these atomic combina- 
tions is further reduced by the fact that the steady-state concentrations of 
the atoms must be much smaller than the concentration of the reactant, 
hydrogen iodide. Since the hydrogen atom concentration is kept very 
small by its efficient reaction with hydrogen iodide, the only reaction 
which iodine atoms can enter into, appreciably, is association to form 
molecular iodine. Accordinglj^, the mechanism for the reaction may be 
written as follows: 



(1) HI + /;i^-^ H + T* (primary step), 

(2) H + HI ^ Ho + I I 

(3) M + 21 -^ lo + M ) 



(secondary steps). 



Expressing the "intensit.y " lahs of the absorbed light in the photochemical 
units of einsteins per liter per second and the rates of the chemical reac- 
tions in moles per liter per second, the rate equations for the three steps of 
the mechanism may be written 

Ih = labs, 

Vo = k.j[Hl][Ul 

ih = k,Pc[l]-. 

The rate of decomposition of hydrogen iodide is the sum of the rates of 
steps (1) and (2), 

- '-^ =^ V, + Vo = La,. + A-.[HIlfin. 
Introducing the steady-state assumption 

then 



dt -^' 



lab. = A-2[HI][H] 
may be written. Therefore 

dt ~ *'"''• 



PHOTOCHEMISTRY 23 

The (luaiitum yield ^ expressed in terms of molecules of hydrogen iodide 
decomposed is 

d[lil]/d( , 
(f = J = 2. 

^ abs 

This is in excellent agreement with the experimentally determined quan- 
tum yield of 2.00, the value which has been observed over a wide range of 
conditions, including pressures down to 0.008 mm Hg. There can be no 
reasonable doubt of the correctness of this mechanism. 

The thermal formation of hydrogen bromide was perhaps the first reac- 
tion to which the steady-state approximation method was applied (Chris- 
tiansen, 1919; Herzfeld, 1919; Polanyi, 1920). At temperatures in the 
range from 150° to 200°C, a photochemical formation of hydrogen bro- 
mide can be observed which is relatively free from disturbance by the ther- 
mal reaction. The quantum yield of this reaction is the following func- 
tion of concentrations and intensity (Bodenstein and Lutkemeyer, 1924): 

dlHBiydt Av[H2] 



I'^s rH K I [HBr] 

Absorption of blue or near-ultraviolet light leads to optical dissociation of 
bromine. The primary step of this reaction is therefore 

(1) Br. -\- hu^ 2Br. 

The bromine atoms might be expected to react with either molecular 
hydrogen or hydrogen bromide: 

(2) Br + H. ^ HBr + H {AH = 16.7 kcal), 
(2') Br + HBr ^ Br + H {AH ^ 40.6 kcal). 

Both these reactions are endothermic, with the heats of reaction indicated. 
Since the steric factors for these two reactions are very probably of the 
same order of magnitude, the rate of the second reaction should be smaller 
than that of the first bv a factor of about 



g— 40,600/2r 
g— 16,200/2r 



g— 24,400/2r 



In the temperature range under consideration this is a very small number, 
and the second reaction may be justifiably dropped from consideration. 
The hydrogen atoms, formed in step (2), can undergo similar reactions, 

(3) H + Ih:, -^ HBr + Br 
and 

(4) 11 + HHr^ U,-\- Br, 



12 1 RADIATION HIOLOGY 

l)()th of which are exothermic. Xoiie of these reactions reduce the total 
immhcr of atoms present. Therefore the formation of atoms in step (1) 
must be balanced by their disappearance in atomic combination reactions: 

(5) M+2H-^Br2 + M, 

(6) M + H + Br -^ HBr + M, 

(7) M + 2H -^ H2 + M. 

Since no hydroj^eii atoms arc formed directly and since, furthermore, steps 
(3) and (4) are much more efficient than the endothermic step (2), the 
concentration of hydrogen atoms must be much less than that of bromine 
atoms. Accordingly, the rates of step (6) and (7) must be small com- 
pared to the rate of step (5). As a working hypothesis, let us assume that 
only steps (1) through (5) affect the course of the reaction. The corre- 
sponding mechanism may be written as follows, where the expression for 
each rate follows its chemical equation: 

(1) Br, + /)i'-^2Br Vi = Us, 

(2) Br + H.2 -^ HBr + H v^ ^ /,-,fH,][Br]. 

(3) H + Br., -^ HBr + Br v^ = A-4Br,][H], 

(4) H + HBr^ H., + Br v^ - A-4[HBr][H], 

(5) M + 2Br-^ Br,, -f- M v, = k,Pc[Br]- = /.{[Br]-. 

Introducing the steady-state assumptions 

^[Br] _ m] _ 

dt ~^ ^'^"^ dt ""' 

then 

2^1 + v-.i + ''4 = ''2 -f 2?'-, 
and 

V-2 = Vs + ?'4 

may be written. Therefore 

Vl ^ Vr, 



or 



Similarly. 
and 



[Br] = (J^'- 

/;,[H.,][Br] = {A-.[Br,l + ^•4[HBr]![H] 



k,[BT,] + A-4[HBr] \ /,-.; / 

In terms of the mechanism the rate of formation of hydrogen bromide is 
given by 

d[HBr] 

-r. — = V2 + V:i — Vi 

dt 

= A-2fH.,l[Br] -f lUBv,] - A^4[HBrl}[H]. 



PHOTOCHEMISTRY 25 

Introducing the values for the concentrations of the intermediates and 
simplifying, 

d( ~ A-4[HBr] ■ 



A-.[Br2] 
The corresponding equation for the quantum yield, 

?t^Hl 

f/[HBr]A// A-f ^ '^ 

<P = - 



labs ji/, I /i:4[HBr: 

^abs I 1 I" 



A::i[Br2] 



is identical in form with the empirical equation. The empirical constant 
k' corresponding to A-3/A-4 of the theoretical equation is independent of 
temperature over a wide range. This is consistent with the mechanism 
since steps (3) and (4) are exothermic reactions of an atom with a dia- 
tomic molecule and should have small heats of activation. The observed 
heat of activation for A'^ is 17.6 kcal. This is only slightly greater than 
the (endothermic) thermochemical heat of step (2). The mechanism has 
been further tested by investigating the effect of such substances as 
inert gases on the photochemical rate and by comparing the photo- 
chemical to the thermal rate. The results of all these tests are consistent 
with the proposed mechanism. It may be concluded, therefore, that it is 
very probably correct. 

Limitations of the Method. In the two examples just considered the 
determination of the mechanism was simplified by rejecting possible reac- 
tion steps on the basis of thermochemical data. Frequently the necessary 
thermal data are not available, and there is no a priori reason for rejecting 
any of the chemically possible steps. Under these conditions the kinet- 
icist endeavors to find a mechanism consisting of a minimum number of 
reaction steps which is consistent with the stoichiometry and the kinetics 
of the reaction. Whenever possible, the rate constants for the individual 
steps are evaluated in terms of the empirical rate equation. Unless these 
several constants fall within the (frequently rather wide) range of values 
permitted by rate theory, the mechanism must be rejected. If the mech- 
anisms of diff"erent reactions have steps in common, the rate constants 
for these steps should have the same values regardless of what mechanism 
they occur in. 

There is no general method by which the mechanism of a reaction may 
be derived from empirical rate data. The process of devising a mecha- 
nism is essentially a " cut-and-try " procedure, in some respects similar to 
the methods used in solving differential equations. As in the latter case, 



2(» 1{ \I)I AlION lUOLOGY 

certain gciuMal rules iiiid aiialojiiics are helpful in fi;ui(linjj; the inluition of 
the kineti('i.sl. It is rarely true that a reaetion mechanism c-an ho 
re^rarded as true or even as very pr()l)ahly true. Usually all that can be 
claimed for a mechanism is that it is consistent with all pertinent infor- 
mation. This does not pieclude the possibility that some other mecha- 
nism, or even many other mechanisms, may likewise be compatible with 
the data. A mimber of reaction mechanisms which were at one time 
tentatively accepted had to be discarded later when more information 
became available. In spite of these iinsatisfactor\' characteristics of the 
mechanisms of complex reactions, there ajjpears to be no way of obtaining 
information about complex reactions other than by postulating and test- 
ing mechanisms. 

EXAMPLES OF THE PRINCIPAL TYPES OF PHOTOCHEMICAL REACTIONS 

The types of photochemical reactions which have been studied most 
extensively are decompositions, oxidations, polymerizations, hydrolyses, 
and internal rearrangements. A great part of the early photochemical 
literature is devoted to oxidations, especiall}^ chlorinations. Much of the 
classical information about chain reactions was derived from these latter 
measurements. From a biological point of view the details of these inves- 
tigations are of little immediate interest, and for this reason they are 
omitted from the following discussion. 

DECOMPOSITION REACTIONS 

The photochemical decomposition of a wide \'ariety of compounds, 
ranging in complexity from hydrogen iodide to complex azo dyes, has been 
investigated, in many cases with considerable care. The decomposition 
of hydrogen sulfide is a good example of a carefully studied reaction of a 
simple molecule. The al)sorption of hydrogen sulfide is continuous, 
becoming appreciable at about 2800 A and reaching a maximum near 
1900 A. The corresponding primary act is presumably one of direct 
ojitical dissociation, yielding a hydrogen atom and a hydrosulfide radical. 
Photochemical measurements (Forbes et al., 1938) made with radiation 
wave length of 2080 A demonstrate that one molecule of hydrogen sulfide 
is decomposed for each photon absorbed, over a wide range of pressures 
and of light intensities. The following mechanism is consistent with all 
available reliable information and, for this simple system, is probably 
correct : 



H,S + /^j' ^ IIS + H (primary step), 

H -h HoS -> H, + HS j 
2HS -> HoS + S 



(secondarj^ steps) . 



The photolysis of a variety of aldehydes and ketones has been investi- 
gated extensively and in detail (Xoyes and Leighton, 1941). The results 



PHOTOCHEMISTRY 



27 



of these studies are far from simple. It appears to be fairly definite that 
two different kinds of primary acts can occur. The excited molecule can 
dissociate either into radicals or into two such stable molecules as a hydro- 
carbon and carbon monoxide. The formation of the radicals is probably 
a process of predissociation, and the production of molecules, the result 
of internal conversion. Croton aldehyde, at temperatures below 150°C, 
is photochemically stable in spite of the facts that it is nonfluorescent and 
that its absorption is continuous. Although this failure to react is con- 
ceivably the result of rapid recombination of radicals formed in the pri- 
mary act, it seems much more likely that the energy of excitation is lost 
by way of an act of internal conversion, followed by degradative colli- 
sions, i.e., collisions of the second kind, with surrounding molecules. 

The photochemical decomposition of formaldehyde is probably as sim- 
ple a reaction of this type as has been studied. Although the results of 
the several investigations of this reaction (Steacie, 1946) do not agree in 
all particulars, the broad outline of the mechanism appears to have been 
reasonably well established. The products of the reaction are carbon 
monoxide and molecular hydrogen. At 110°C the ciuantum yield is 
approximately unity for wave lengths from 2600 to 3500 A. At the 
longer wave lengths the absorption spectrum shows fine structure but 
corresponds to a region of predissociation in the shorter wave-length 
range. The yield increases with increasing temperature, reaching a 
value of about 100 at 350°C. Hydrogen atoms can be detected under all 
experimental conditions, but there is some evidence that there is an appre- 
ciable direct formation of molecular hydrogen when the gas is illuminated 
with light in the longer wave-length region. The following mechanism 
seems to be compatible with the published results: 

(1) HCHO + hv— CHO + H (chief primary step), 

(2) HCHO + hv -^ [HCHO] -^ CO + H, (primary step at long 

wave lengths), 

(3) HCO ^ H + CO (chain-carrying secondary 

step), 

(4) H + HCHO ^ H2 + CHO (chain-carrying secondary 

step), 

(5) M + 2H -^ H2 + AI (chain-breaking secondary 

step) . 

Steps (3) and (5) can occur either in the gas phase or by diffusion to the 
wall. For the gas-phase reaction the heat of activation of step (3) is 
about 13 kcal. There are, of course, a number of other possible steps, but 
these five are sufficient to explain the available data. 

REACTIONS OF MOLECULAR OXYGEN 

In biological systems those photooxidatix'e reactions which involve 
molecular oxygen are by far the commonest. The primary step of such a 



28 RADIATION BIOLOGY 

reaction may be either the optical dissociation of the oxygen molecule or 
the photoexcitation or dissociation of a molecule of the reducing agent. 
Since oxygen absorbs chiefly at wave lengths less than 1800 A, reactions 
of the first type are limited to relatively transparent substrates such as 
hydrogen or carbon monoxide (Noyes and Leighton, 1941, pp. 246-254). 
In those cases where the reducing agent absorbs the light, the initially 
reactive species may be an excited singlet (i.e., fluorescent) state, a long- 
lived (triplet) excited state, or a pair of radicals, produced by some type of 
photodissociation. 

Solutions of aryl hydrocarbons in hexane or similar solvents are fluores- 
cent. In the presence of o.xygen, their fluorescence is quenched and 
peroxides are formed (Bowen and Williams, 1939). With few exceptions 
the sum of the fluorescent yield and the peroxide quantum yield is dis- 
tinctly less than unity, in some cases being as small as 0.1. None of the 
data for the 14 hydrocarbons investigated by Bowen and Williams are 
consistent with the view that the only effect of o.xygen is to quench the 
fluorescence by reacting with the excited molecule (in its singlet, fluores- 
cent state) to form a peroxide. Apparently oxygen can quench the 
fluorescence of these molecules without forming any detectable product. 
In five cases (benzene, w-xylene, fluorene, acenaphthene, and triphenyl- 
methane) the evidence is compatible with the postulate that only the 
singlet, fluorescent state is involved in the peroxide formation. For the 
others (especially hexamethyl benzene, anthracene, naphthacene, toluene, 
and p-.xylene) the experimental results strongly indicate that some or all 
of the peroxide is formed by a reaction between an oxygen molecule and 
an energy-rich nonfluorescent (triplet?) state of the hydrocarbon. This 
is particularly obvious for hexamethylbenzene, where a quantum yield 
of peroxide formation almost fivefold greater than the maximum fluores- 
cent yield was observed. The preceding conclusions are based on the 
assumption that only the 10 following reaction steps occur. In these 
ecjuations, A* stands for the singlet, excited state and A' for the triplet 
state of the hydrocarbon molecule, A. 



(1) 


\ + hu-^ A*, 


(6) (), + A* ^ AO2, 


(2) 


A*^ A + ItPf, 


(7) 0, + A' ^ AO2, 


(3) 


A* -^ A, 


(8) 0-2 + A* ^ A + O2, 


(4) 


A* -^ A', 


(9) O2 + A* -^ A' + (),, 


(5) 


A' -^ A, 


(10) ().. -f A'^ A + ()... 



Oxidations which are initiated by the photochemical dissociation of the 
reductant frequently exhibit the characteristics of chain reactions. Their 
quantum yields are functions of the temperature, of the concentrations of 
reactants and products, and sometimes of the intensity of the absorbed 
light. Often the products are complex, and the relative amounts of the 
several compounds formed vary with the conditions. The detailed 



PHOTOCHEMISTRY 29 

mechanism of the reaction for such a case has not been estabhshed with 
reasonable certainty. 

The photochemical oxidation of formaldehyde (Style and Summers, 
1946) is a good example of this type of reaction. It has been studied in 
the temperature range of 100° to about 275°C and at a variety of pressures 
and compositions. Its principal products are CO, CO2, HCOOH, Ho, 
and H2O. Their several yields vary from values of less than 1 to 30 or 40, 
depending in a coniplex way on the conditions. It is well established that 
H, CHO, and HO2 are reaction intermediates. The (chief) primary 
process is 

HCHO + hp^U + CHO. 

Although the detailed mechanism is not known, the experimental evidence 
is compatible with the postulate that the following reactions serve as 
(some of) the secondary steps of the reaction: 

H + HCHO ^ H. + CHO, 
CHO + HCHO -^ Ho + CHO + HCHO, 
2CH0 ^ CO + HCHO, 
M + H + O2 ^ H62 + CO, 
CHO + 02^ H62 + CO, 
HO2 + HCHO ^^ [CO + CO2 + HCOOH + H], 
2HO2 -^-^ H2O + 3.2O2. 

Organic peroxides are the principal products of some reactions of this 
general type. Peroxides may also serve as photochemical sensitizers. 
For example, the chief product of the photochemical oxidation of cyclo- 
hexene is the corresponding peroxide. As the concentration of the perox- 
ide builds up in an illuminated solution containing oxygen and cyclo- 
hexene, the peroxide absorbs an increasing amount of the incident light, 
and the reaction is accelerated (Bateman and Gee, 1948). These 
observations are consistent with the postulate that the primary process 
in the absence of the peroxide is 

RH (cyclohexene) + /( j^ -^ R + H, 

and in the presence of the peroxide is predominantly 

ROOH + hv^ ROO + H. 

At temperatures at which the thermal reaction can be neglected, the over- 
all process is a short chain reaction. It seems very probable that R, ROO, 
H, and HO2 are important intermediates in this process. Since the 
quantum yield of the reaction is inversely proportional to the square root 
of the intensity of the absorbed light, the chain-breaking step must be a 
bimolecular reaction between chain carriers (i.e., intermediates) leading 
to the production of stable molecules. 



30 KADIAIION lUoLOfJV 

POLYiMKHIZATION AM) 1)1 MKRIZATION 

There are two general types of polymerization: .simj)l(' reactions leading 
to the formation of definite molecules, such as dimers or t rimers, and 
chain reactions, whose products are macromoleciilcs of indefinite molecu- 
lar weight. One of the first polymerizations to be studied with reason- 
able care (Luther and Weigert, 1905) is the dimerization of anthracene. 
At moderately elevated temperatures (80°-200°C') the dimer reaches a 
measurable sleady-state concentration in a dilute solution of anthracene 
illuminated with ultraviolet light (wave length 3GG0 or 3130 A). Over 
the range of intensities and concentrations studied, the steady-state con- 
centration is directly proportional to the intensity of the absorbed radia- 
tion. The quantum yield of dimerization increases with increasing 
anthracene concentration, approaching a limiting yield of about 0.5 
(Weigert, 1927). The available measurements are insufficient to deter- 
mine the mechanism of the process. It appears very probable that the 
first five steps which were proposed in the discussion of the oxidation of 
aromatic hydrocarbons occur in the polymerization reaction. However, 
any of the three following alternative reactions may be responsible for the 
formation of the dimer: 

A* + A-> Ao, 

A' + A -^ Ao, 

2A' -^ A,. 

The last of these is consistent with the observed limiting yield of 3-^. 
This interesting reaction is certainly worthy of further study. 

The gas-phase polymerization of cyanogen (Hogness and Ts'ai, 1932) 
is at least superficially simple. The absorption of this compound is 
fairly strong in the wave-length region 2150-2250 A. The absorption 
bands are diffuse, and the gas is nonfluorescent. Under the conditions of 
measurement the ciuantum yield is 3.0. The product is a brownish solid. 
The authors propose the following mechanism: 

C2N2 -\- hv —* 2CN (primary process), 

CN + C2N2^ (CN)3 (secondary process). 

This is probably an oversimplification, since it is difficult to understand 
why the (CN):i molecules would react with themselves to form para- 
cyanogen but would not r&act with the remaining cyanogen. 

The formation of large polymer molecules (Mark and Raff, 1941 ; Bawn, 
1948) ma}^ occur either by successive condensation or by addition of sim- 
ple molecules. Addition polymerization is essentially a chain reaction 
and, as such, can be studied effectively by photochemical methods. 
Determinations of the chain lengths of thermal reactions can be made 
only indirectly, usually by the use of inhibitors (Alyea and Biickstrom, 
1929). In a photochemical reaction the ratio of the over-all (juantum 



PHOTOCHEMISTRY 31 

yield to the yield of the primary process (which is commonly close to 
unity) is a direct measure of the average chain length. Furthermore, 
knowledge of the nature of the primary product is frecjuently very helpful 
in the prediction of the secondary steps. 

The primar}^ act in association polymerization is the formation of two 
radicals or a diradical. Each radical or diradical can then add to a 
monomer molecule, forming a new radical of greater molecular weight. 
Large polymers are built up by the successive addition of monomer mole- 
cules to the growing radical. In most cases studied, the addition of 
monomer to the radical rec^uires a heat of activation of a few kilocalories. 
The specific rate of addition is only slightly influenced by the size of the 
radical. In the absence of inhibitors the chain is, in the great majority 
of cases, terminated by a reaction between two radicals. This chain- 
stopping step eliminates two radicals either by their disproportionation or 
by their addition. 

Free radicals may be formed by the photochemical dissociation of the 
monomer or of an added sensitizer such as acetone (Jones and Melville, 
1946). Since the chains are broken by bimolecular reactions between 
growing radicals, the rate of polymerization is proportional to the square 
root of the intensity of the absorbed light. 

A determination of the ratio of the rate constants for the chain-propa- 
gating and chain-terminating steps may be made by analyzing the 
kinetics of a polymerization reaction. This analysis is made by the usual 
steady-state approximate method. Special methods are required to 
evaluate either of these individual constants. Melville (1947) has shown 
that these individual constants can be obtained if the polymerization 
occurs under intermittent illumination. This technic^ue, which has 
proved very useful in the study of polymerization kinetics, is a relatively 
old one in photochemistry, having been used by Berthoud and Bellenot in 
1924 and subjected to a thorough theoretical analysis by Dickinson 
(Noyes and Leighton, 1941, pp. 202-209). 

INTRAMOLECULAR CHANGES 

Relatively few photochemical isomerizations have been studied ciuanti- 
tatively. One group of reactions which has received some attention is 
the cis-trans isomerizations. For reasons of experimental convenience, 
most of the kinetic measurements have been made with substituted 
ethylenes. However, knowledge of their spectroscopic properties is 
limited to the simpler compounds. Figure 1-3, which is taken from the 
work of Mulliken (1942), is a schematic representation of the electronic 
energy levels of ethylene. In addition to the ground level A'^, two excited 
singlet levels, V and R, are shown. Absorption bands, corresponding to 
transition from N to either V or R, are strong. According to the Franck- 
Condon prijiciple, the angle between the hydrogens cannot change appre- 



32 



RADIATION HIOI.OGY 



V 
R 



\ 





c'iably tluriiifi the electron triinsitioii, and therefore tlie energies corre- 
sponding to transitions N —^ R and N —* V will overlap. Transitions 
from iV to R result in sharp bands, and from A'' to F in diffuse general 
absorption. 

The optical and photochemical properties of cis- and trans-atiWyvAiv. 
were carefully investigated by Lewis ct at. (1940). 7Vo/is-stilbene is 
fluorescent (wave length 3300 to 4400 A), and its absorption spectrum 
(wave length 2000 to 3400 A) shows distinct "oscillational" structure; 
as-stilbene is nonfluorescent and its absorption spectrum is apparently 
structureless. Irradiation of either pure compound with radiation of 

wave length 2537 A produces partial 
stereoisomerization. Since cz's-stilbene 
undergoes a photochemical side reac- 
tion to an unknown product, quantum 
yields had to be based on measurements 
of the initial rates. Starting with the 
pure fzs-compound, the quantum yield 
of /raws-stilbene formation is 0.26 and 
of the side reaction is 0.10. The cor- 
responding quantum yield for the forma- 
tion of cis- from ^rans-stilbene is 0.35. 
The interpretation of these facts is ren- 
dered uncertain by the lack of knowl- 
edge of the potential-energy diagram for stilbene. The steric interference 
between the phenyl groups, which is responsible for the relative instabil- 
ity of the cts-form, undoubtedly renders the potential-energy curve for 
the ground state unsymmetrical and probably has a similar effect on the 
curves for the excited states. Conjugation between the benzene rings 
and the ethylenic link must also affect the energy levels. For lack of 
other information, let us assume that the potential-energy curves for 
stilbene, although asymmetric, are otherwise essentially similar to those 
for ethylene. A molecule excited to the state V will quickly lose its extra 
energy of oscillation by successive impacts with solvent molecules and 
will end up in the (approximately) 90° trough of the electronic state. 
The sul)se(iuent transitions of the molecule are, of course, independent 
of whether it was originally a normal cis- or /raw. s-stil bene molecule. 
Since transitions between states A'' and V are permitted, it might be 
expected that the excited mole('ule could emit a (luantum and return to 
the ground state of either the cis- or trans-iorm, depending on the relative 
asymmetries of states A'^ and V. If this were the mechanism of the 
process, the quantum yields of fluorescence and isomerization would not 
be complementary, and the limit of the sum of these yields would be 2 
rather than 1. That this mechanism does not apply to this case is shown 
by the nonfluorescence of cis-stilbene. The strong fluorescence of the 



AT 

0° 90° 180 

Fig. 1-3. Schematic potential-energy 
diagram for ethylene. 



PHOTOCHEMISTRY 33 

trans compound demonstrates that some (or all) of these excited mole- 
cules are in a state which is peculiar to the /rans-configuration, possibly- 
state R. If czs-stilbene is excited to state R it must go, by internal con- 
version, to state 1' (or possibly to the ground state with a high excess of 
oscillational energy) in a time much less than the natural half life of the 
excited state. The fluorescent yield of /rans-stilbene was not measured. 
Lewis and his coworkers (1940) assumed that the ratio of the nonradia- 
tive return to the cis- and /ra/ts-forms was independent of whether the 
excited state was formed by the irradiation of normal cis- or trans-siW- 
bene. This assumption leads to a value of about 0.5 for the fluorescent 
yields. Although this latter assumption is consistent with the available 
data, it is by no means the only reasonable interpretation. Olson's con- 
clusion (1931) that the compound formed from the excited state will be 
predominantly the isomer of lower stability should not be expected to 
apply to a reaction which takes place by way of internal conversion. 
The probability of such a process will depend on the relative forms of the 
several potential-energy surfaces and on their points of intersection and 
not merely on relative times spent in the two rotational configurations. 
Photochemical reactions of this type deserve much more attention than 
they have received. They are intrinsically interesting, and an under- 
standing of them should prove helpful in the interpretation of more com- 
plex photochemical processes (Pinckard et al., 1948; Stearns, 1942). 

The photoisomerization of o-nitrobenzaldehyde to o-nitrosobenzoic 
acid involves the breaking of two bonds and the formation of two new 
ones, but the reaction appears to be strictly an intramolecular process. 
The course of the reaction is independent of whether the compound is 
present as crystals, is in solution in a solvent, e.g., acetone, or is in the 
vapor phase. No detectable oxygen is liberated. In the condensed 
systems (Leighton and Lucy, 1934) at room temperature the quantum 
yield is 0.50 ± 0.03. In the vapor phase at 90°C, where the vapor pres- 
sure is about 4 mm of Hg, the quantum yield is 0.70 + 0.05, but the 
yield is reduced by the addition of molecular nitrogen, reaching a value 
of about 0.5 at a nitrogen pressure of 700 mm of Hg (Ktichler and Patat, 
1936). The yield in solution for the corresponding reaction of 2,4-dini- 
trobenzaldehyde is also about 0.5 but is approximately 0.7 for 2,4,G-tri- 
nitrobenzaldehyde. It is plausible that the reaction involves the hydro- 
gen-bonded quasi six-membered ring and that it takes place by way of an 
act of internal conversion. Why the yield reaches a limiting value of 0.5 
in condensed systems or in the presence of an inert gas is not obvious. 
The theoretical predictions of Leighton and Lucy are incompatible with 
the subsecjuent experiments of Ktichler and Patat, and therefore this 
detailed theory apparently must be rejected. 

The photochemical denaturation of proteins and inactivation of 
enzymes can be classed, somewhat arbitrarily, as rearrangements of the 



34 RADIATION BIOLOGY 

hydrated protein molecules. These reatrtions have been studied exten- 
sively in recent years (McLaren, 1!)M)), and some empirical generaliza- 
tions can lie deduced from the results of these studies. The (juantum 
yields of the reactions an^ in the range from 1()~- to 10~'. Radiation of 
wave lengths shorter than 3100 A is reciuircd to produce the reactions. 
Photochemical denaturation is irreversible. The primary jjliotochemical 
product remains in solution at low temperatures (e.g., 4°Cj, but a precipi- 
tate forms rapidly when the previously irradiated solution is heated to 
40°C. The (juantum yield of the primary process is practically inde- 
pendent of temperature over the narrow range availal)le. Photochemical 
denaturation results from the irradiation of "dry" proteins as well as 
proteins in dilute aqueous solution. The (juantum yield is not a function 
of the intensity of the absorbed light. 

If the quantum yield is strictly independent of the intensity of the light 
absorbed by the native protein, the primary act involves the interaction of 
one photon with each molecule; i.e., it is a "single-hit" process, in which 
there is no cooperative action between two or more photons either suc- 
cessively or simultaneously. It should be realized, however, that no 
simple mechanism predicts that the yield is independent of the product 
of incident intensity and time of irradiation since the (dissolved) dena- 
tured protein must act as an efficient internal filter. The "one-hit" 
kinetics have been interpreted in terms of a primary act in which one 
peptide linkage is broken by a single absorl)ed photon. The low (juantum 
yields indicate that this primary process is very inefficient; most of the 
absorbed quanta are degraded to heat. One plausible explanation for 
the observed inefficiency is that the light, which is absorbed by aromatic 
nuclei in the molecule, becomes available for chemical action by an act 
of internal conversion. A few facts which support this tentative explana- 
tion are (1) the yield increases with increasing frequency (i.e., energy) of 
the photon, (2) the yield decreases with increasing size of the molecule, 
and (3) the yield is greater for adsorbed films of proteins than it is for 
solutions. 

SENSITIZED REACTIONS 

In sensitized reactions the substance which absorbs the light does not 
undergo any permanent chemical change. This absorbing substance, 
called the "sensitizer," catalyzes the photochemical reaction. The 
simplest known example of this type is the xenon-sensitized photochemical 
dissociation of hydrogen (Calvert, 1932). The resonance radiation of 
xenon has a wave length of 1409 A, which corresponds to an energy of 
193 kcal/mole. Molecular hydrogen, whose dissociation energy is 103 
kcal/mole, does not absorb radiation of wa\'e length longer than 849 A. 
If a mixture of xenon and hydrogen is illuminated with a xenon arc, hydro- 
gen atoms are formed as was demonstrated by their color reaction with 



PHOTOCHEMISTRY 35 

solid tiHigstic oxide. If pure hydrogen is substituted for the mixture of 
gases, there is no reaction. The reaction steps are as follows: 



Xe + hv, - 


->Xe* 


(absorption), 


Xe*- 


-^ Xe + hv. 


(fluorescence). 


H., + Xe* - 


^ Xe + 2H 


(collision of the second kind) 



Mel-cury vapor is a l)etter-kno\vn sensitizer for the dissociation of hydro- 
gen (Noyes and Leighton, 1941). The first resonance radiation of mer- 
cury, wave length 2537 A, corresponds to an energy of 112 kcal/einstein, 
which is only slightly more than is necessary to dissociate molecular 
hydrogen. It should be expected that the reaction 

H., + Hg*(()^Pi) -^ Hg((3'Po) + 2H 

should be very efficient. Although the interaction between an excited 
mercury atom and a hydrogen molecule is indeed very probable, HgH 
appears to be one of the products: 

H2 + Hg* ^ HgH + H. 

If other reactant gases, such as carbon monoxide or ethylene, are present, 
the hydrogen atoms initiate a series of reaction steps leading to a variety 
of products. Many such mercury-sensitized reactions have been 
studied. 

Photochemical cis-trans isomerizations are sensitized by iodine (Ber- 
thoud and Urech, 1930; Dickinson et al, 1949). The sensitized reaction 
is a short chain process with an appreciable heat of activation. The pri- 
mary act is the photodissociation of molecular iodine. Iodine atoms can 
add, with an appreciable heat of activation, to carbon atoms adjacent to 
the double bond. This opens the double bond, permitting rotation of the 
groups. Subsecjuently the iodine atom can split off. This mechanism is 
summarized in the following equations, in which C and T stand, respec- 
tively, for the CIS and trans form of the molecule: 

I2 + hv-^2\, 

i+ T^fi, 

i + c ^ CI, 

CI ^ fi, 

2\ -^ I2. 

In some systems an absorbing compound, undergoing a permanent 
photochemical reaction with a yield of about 1, simultaneously induces a 
chain reaction between other reactants. The chain reaction so over- 
shadows the inducing reaction, that the whole process may be thought of, 
loosely, as a sensitized photochemical reaction. Examples of this type 
are the oxidation of hydrogen (Farkas et al, 1930) and the polymerization 



36 KADIATIOX BIOLOGY 

of elliyk'iu' (^'I'uylor and Enioliius, li)31j induced by tlie prcdisfjuciatiou 
of ammonia: 

NHs + hv^ XH2 + ft. 

When a mixture of hydrogen, oxygen, and ammonia, at a moderate!}' 
elevated temperature, is illuminated with light of wave length 2200 A or 
shorter, ammonia is decomposed and water is formed. The cjuan^um 
yield for the formation of water increases from about 25 at 290°C to 
approximateh^ 380 at 405°C. At 420°C, irradiation of the system results 
in an explosion. The kinetics are complex (Lewis and von Elbe, 1938) 
and probably involve the amide radical as well as the hydrogen atom. 

If a mixture of carbon monoxide and chlorine is illuminated with light 
which is absorbed by the chlorine, a chain reaction ensues, the product 
of which is phosgene. The kinetics of the reaction are complex, but the 
primary act is certainly the dissociation of chlorine and the radicals CI 
and COCl are involved in the secondary reactions. If an excess of oxy- 
gen is added to the system, the formation of phosgene is suppressed, and 
the predominant process becomes the sensitized formation of carbon 
dioxide (Rollefson and Burton, 1939, pp. 313-319). The quantum yield 
of carbon dioxide formation is large and is a complex function of tem- 
perature and the partial pressures of the reactants. A numl)cr of reac- 
tions of this general type have been studied, but the mechanism of none 
of them is completely understood. 

The photolysis of ethyl iodide is sensitized (West and Miller, 1940; 
West, 1941) by naphthalene and a number of its derivatives. The direct 
photolysis of ethyl iodide occurs both in the gas phase and in solution. 
In hexane solutions the quantum yields corresponding to wave lengths 
3130 and 2537 A are about 0.30 and 0.40, respectively. The quantum 
yield of the naphthalene-sensitized process is about 0.30 for cither wave 
length. The maximum fluorescence efficiency of naphthalene in hexane 
solutions is approximately 0.15. As was clearly stated by West (1941), 
this demonstrates that ethyl iodide can interact with a nonfluorescent 
excited state as well as with the fluorescent excited state of the naphtha- 
lene molecule. The yield of the sensitized reaction is independent of 
the naphthalene concentration but falls off to small values when the e{\\y\ 
iodide concentration is decreased much below 10~'- M. These results, 
as well as observations on the effect of changing the \iscosity of the 
solvent, show that the sensitization is a coUisional process, that the 
efficiency of such collisions in producing the reaction is high (probably 
greater than 0.1), and that the collisions in the condensed system occur 
in bursts (p. 15). All these data are consistent with the following 
mechanism, which is strikingly similar to the mechanism here offered 
as an explanation (p. 28) for the photoautooxidation of aromatic hydro- 
carbons (Howen and Williams, 1939): 



PHOTOCHEMISTRY 37 

A* -^ A + hvf, 

A* -^ A', 

A' -> A, 
A* + Co^sl -^ A + C2H5 + I, 
A' + C2H5I -^ A + C2H5 + I. 

Photoaiitooxidations of reactive reducing agents are sensitized by a 
wide variety of dyes and pigments (Hurd and Livingston, 1940). These 
reactions occur in aqueous solutions or in organic solvents, such as 
methanol or acetone. In cases where the kinetics have been studied in 
detail, they appear to be complex, and in no case has a completely satis- 
factory mechanism been proposed. These reactions are produced by 
either visible light or ultraviolet radiation, depending chiefly on the 
absorption spectra of the sensitizers. 

Sensitized photochemical redox reactions are of great importance in 
biology, the outstanding example being photosynthesis by green plants 
(Rabinowitch, 1945). Photodynamic action and certain pathological 
skin reactions (Blum, 1941) are also of interest. The oxidative inactiva- 
tion of enzymes is sensitized by riboflavin (Galston and Baker, 1949) as 
well as by certain dyes. 

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1:21-34. 



38 RADIATION BIOLOGY 

Calvert, H. R. (1932) Die Zorlogung von WasserstofT Molokiilon durch Stosse mit 

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Forbes, G. A., J. Fl Cline, and B. C. Bradshaw (1938) The photolysis of gaseous 

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F^olgerungen i'lber Fluoreszenz, photochemische Prozesse und die Elektronen 

Emission gliihender Korper. Z. Physik, 9: 259-266. 

(1948) Bemerkungen fiber Lumineszenz von lonenkristallen. Ann. Physik, 

3: 62-68. 

Franck, J., and II. Livingston (1941) Remarks on the fluorescence, phosphorescence 
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Mod. Phys., 21: 505-509. 

Franck, J., and H. Sponer (1949) Comparison between predissociation and internal 
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Gaffron, H. (1927) Sauerstoff-tjbertragung durch Chlorophyll und das photo- 
chemische Xquivalent-Gesetz. Ber. deut. chem. Ges., B60: 755-766. 

(1937) Die lange Lebensdauer angeregter organischer Molekiil(> erlautert 

am Beispiel der Rubrenoxydation. Z. physik. Chem., B37: 437-46!. 

Galston, A. W., and R. S. Baker (1949) Inactivation of enzymes by visible light in 
the presence of riboflavin. Science, 109: 485-486. 

Herzberg, G. (1950) Spectra of diatomic molecules. 2d ed., D. Van Nostrand Com- 
pany, Inc., New York. 

Herzfeld, K. F. (1919) Zur Theorie der Reaktionsgeschwindigkeiten in Gasen. Z. 
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Hogness, T. R., and L.-S. Ts'ai (1932) The iihotochemical polymerization of 
cyanogen. J. Am. Chem. Soc, 54: 123-129. 

Hurd, F., and R. Livingston (1940) The quantum yields of some dye-sensitized 
photooxidations. J. Phys. Chem., 44: 865-873. 

Jablonski, A. (1935) Weitere Versuche i'lber die negative Polarization der Phos- 
phoreszenz. Acta Phys. Polon., 4: 311-324. 



PHOTOCHEMISTRY 3<) 

Jones, T. T., and H. W. Melville (1946) The free radical polymerization of the 

vapours of certain vinyl derivatives, l^roc. Roy. Soc. London, A187: 37-53. 
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singlet level of naphthalene. J. Chem. Phys., 17: 516-520. 
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York. 
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Lewis, B., and G. von Elbe (1951) Combustion, flames and explosions of gases. 

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solutions. J. Am. Chem. Soc, 72: 909-915. 
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McCIure, D. S. (1949) Triplet-singlet transitions in organic molecules. Lifetime 

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Noyes, W. A., and P. A. Leighton (1941) The photochemistry of gases. Reinhold 

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Trans. Faraday Soc, 27: 69-76. 



40 RADIATION lUOI.OOY 

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University of Chicago. 

Manuscript received by the editor Mar. 19, 1951 



CHAPTER 2 

Practical Applications and Sources 
of Ultraviolet Energy 

L. J. BUTTOLPH 

General Electric Company, Lamp Division, Cleveland, Ohio 

Inlroduclion. Germicidal-aclion curves: Action curve tentative at shorter wave lengths- 
Action curve approximate at longer wave lengths—The unique 2537 .4 mercury line 
Susceptibility to ultraviolet: Injury, mutation, and kill—Comparison of susceptibility to 
ultraviolet and to other lethal agents-Logarithmic nature of kill— Unit kill— Reactivation 
by heat and light— Germicidal action of ultraviolet of wave lengths greater than 2800 A— 
Composite of killing factors. Killing exposures: Reciprocity of time and intensity. 
Erythemal action of wave lengths 2537 and 2967 A: American Medical Association 
tolerance— Face and eye protection and treatment. Commercial sources of ultraviolet- 
High- and low-pressure mercury arcs— Ultraviolet of wave length 2537 A— Conversion 
factors— Intensity— Ozone formation— Photochemical effects of 2537 and 1849 A energy- 
Temperature and ventilation— Depreciation. Ultraviolet disinfection: Air disinfection— 
f luid disinfection- Disinfection of surfaces of granular materials. Ultraviolet-induced 
m utantsfor new fungi. Protection and processing of products: Mold, antibiotics, and par- 
enteral fluids-Blood plasma— Syrup, fruit-juice, and wine storage— Meat storage 
Higher pressure mercury sources of ultraviolet: Intensity and variations with distance- 
Individual line intensities-Starting and restarting times-Life and depreciation- 
Research determination of output and intensity— Mercury-amalgam and other metal 
arcs — S unlamps. References. 

INTRODUCTION 

Innumerable applications of ultraviolet energy are suggested in a volu- 
minous amount of old literature, in which there is little of practical value 
because of the failure to specify the ultraviolet wave lengths, the inten- 
sities, and the exposure times used. This is equally true of the many 
chemical, the indefinite therapeutic, and the few biological effects of the 
ultraviolet. Ellis et al. (1941) have comprehensively reviewed the 
chemical and biological applications of the ultraviolet; Laurens (1933) 
has done the same for the physiological effects. Meyer and Seitz (1949) 
and Roller (1952) have excellently reviewed the sources, measurement, 
and various applications of the ultraviolet. Lea (1946) has contrasted 
the excitation effects of ultraviolet with the ionization effects of X ray and 
shorter wave length radiations in a practical discussion of the theoretical 
bjises of both effects, 

41 



42 



RADIATION HIOLOGY 



The practical biological applications of ultraviolet are those utilizing its 
erythenial effects, its ergoslerol activation, and its inactivating and 
mutational effects on bacteria, fungi, and viruses. The action spectra 
describing all these effects as functions of wave length are subjects of 
other chapters; this chapter is concerned with one outstandingly practical 
biological application, the germicidal effect. There arc included dis- 
cussions of commercially available sources of ultraviolet for this effect and 
for research on this and other effects. 



GERMICIDAL-ACTION CURVES 



If bacteria are irradiated with ultraviolet of various wave lengths and 
with an identical exposure (intensity times time) for each wave length, 






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Fig. 2-1. Bactericidal- and orytluMiial-action curves. 
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3000 



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3400 



{International Commission on 



which is sufficient to give a convenient unit of killing, oO, 63.2 (lethe; see 
p. 49), or 90 per cent, at the optimum wave length, the data may be 
plotted as a germicidal-action curve. Such a curve has not been stand- 
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Gates (1929-30), Hollaender et al. (1940), Jones ct al. (1940), and others 
have studied the action of specific wave lengths on specific organisms. 
Caspersson (1931, 1937) associates the germicidal-action curve with the 
ultraviolet-absorption curve of the nuclear protein. For most bacteria 
and fungi and for some viruses, the optimum killing w^ave length is at 
^2650 A. The relative effects at longer and shorter wave lengths are so 
similar that a single tentative action curve for the average germicidal 
effect on various bacteria and fungi and on many viruses is shown in Fig. 
2-1 along with an erythemal-action curve standardized by the Inter- 



APPLICATIONS AND SOURCES OF ULTRAVIOLET 



43 



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iniX 39WJ.N30a3d 



44 



RADIATION BIOLOGY 



national Commission on lllnmination (ICI), Berlin (11)35). Both curves 
are plotted with the relative effect at the optimum wave length as 100 per 
cent. The relative 100 per cent bactericidal elTcctiveness of 2V)'){) A and 
the 85 per cent relative effectiveness of 2537 A energy shown in Fig. 2-1 
should not be confused with the possibility of a 100 per cent absolute kill 
by 2537 A energy shown in Figs. 2-2 and 3. The ICI factors for the 
erythemal-action curve and tentative factors for the bactericidal-action 
curve proposed in Fig. 2-1 are shown for indixidual mercury lines in Table 
2-1. These factors are useful for calculating the relative effectiveness and 



Table 2-1. I^kytiikmal- and Bactericidal-action Factors 



Mercury 


ICI 


Tentative 


spectrum 


erythemal 


bactericidal 


lines, A 


factors 


factors 


2353 


0.55 


0.35 


2446 


0.57 


0.58 


2482 


0.57 


0.70 


2537 


0.55 


0.85 


2576 


0.49 


0.94 


(2650) 




1.00 


2654 


0.25 


0.99 


2675 


0.20 


0.98 


2700 


0.14 


0.95 


2753 


0.07 


0.81 


2804 


06 


0.68 


2857 


0.10 


. 55 


2894 


0.25 


. 46 


2925 


0.70 


0.38 


2967 


1.00 


0.27 


3022 


0.55 


0.13 


3130 


0.03 


0.01 



efficiency of mercury sources whose relative line intensities are known 
(see Tables 2-3 and G). 

ACTION CURVE TENTATIVE AT SHORTIHl WAVE LENGTHS 



The germicidal-actioii i-nvvc for wave lengths less than 2500 A is still 
tentative since theory and some research suggest that the action continues 
to increase at shorter wave lengths (greater frequencies and greater energy 
content of the quanta). A rather rapid drop in the curve at wave lengths 
less than 2500 A is, on the other hand, characteristic of the absorption 
curve of nuclear protein and very representative of practical germicidal 
effects in which nearly all the liquid and gaseous elements in the environ- 
ment of an organism absorb the ultraviolet of shorter wave lengths and 



APPLICATIONS AND SOURCES OF ULTRAVIOLET 



45 



thus protect the organism itself. Perhaps the decreasing germicidal 
action at wave lengths less than 2500 A found by some workers may 
represent the absorption curves of the culture media rather than the 
absolute susceptibility of naked bacteria to ultraviolet killing. 



8 



' o'i^^gth 



'^ J^ 9 !^Q 



A 
o 

~ £\j O 







Fig. 2-3. Composite of Figs. 2-1 and 2. 



ACTION CURVE APPROXIMATE AT LONGER WAVE LENGTHS 



The germicidal action of radiant energy extends even into the visible 
spectrum, the action decreasing rapidly with increasing wave length 
(decreasing frecjuency and energy content of quanta). The action is of 
the order of magnitude in the near ultraviolet and visible estimated by 
Luckiesh and Taylor (1946; see also Hollaender and Claus, 1935-36) in 
Fig. 2-4 where a logarithmic ordinate is used to extend the killing action 
to lower effectiveness levels. The reactivating effects of wave lengths 
3600-4400 A reported by Kelner (1949) suggest that, for all practical pur- 
poses, the curve of Fig. 2-4 might well end at 3600 A. In any case, since 



•46 



RADIATION BIOLOGY 




^ 0.00002 



^ 00001 



7000 



"2000 3000 4000 5000 6000 

WAVE LENGTH, A 

Fig. 2-4. Relative bactericidal action extended to the near-ultraviolet and visible 
regions for E. colt on agar. (Luckiesh et al., 1947 ; Hollacndcr and Claus, 1935-36.) 

no effects on fungi are reported, the curve probably applies only to the 
more susceptible organisms, at wave lengths greater than 3600 A. 

THE UNIQUE 2537 A MERCURY LINE 

Sixty per cent of the electrical input to a low-pressure mercury arc is 

converted directly into radiation of wave length 2537 A. Wave length 

2537 A produces 85 per cent of the maximum germicidal effect on most 

bacteria, fungi, and viruses which is possil)le at ^2650 A. This efficient 

production of ultraviolet of nearly optimum germicidal wave length is 

one of the more unusual coincidences in biophysics. The shape ot the 

germicidal-action curve is su(^h that the effectiveness of 2537 A ultraviolet 

is only 10-20 per cent less than the maximum effectiveness possible at 

2650 A, an uncertainty well within the variations in the action curves for 

various organisms and within the experimental errors inherent in the 

determination of the curves. For these reasons the low-pressure mercury 

arc has been selected as the practical source of ultraviolet for germicidal 

effects. About the only practical interest in the germicidal-action curve 

is to appraise the relative inefficiency of high-pressure mercury arcs in 

fused (luartz glass, the only artificial sources of ultraviolet that are at all 

comparable with the low-pressure arcs. In Fig. 2-5 the action curves 

are superposed on block diagrams of the relative line intensities of typical 

high- and low-pressure mercury arcs. The block diagrams are calculated 

on the basis of etjual amounts of power (in watts) into th(> two types of 

arcs in order to show graphically their relative ultraviolet efficiencies as 

well as their germicidal and erythemal effectiveness. 



AMPLICATIONS AND SOURCES OF ULTRAVIOLET 



47 



1 




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6200 


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Fig. 2-5. Relative energy distribution of (a) high- and (6) low-pressure mercury arcs, 
transmission of arc tube glasses, and action curves. 



SUSCEPTIBILITY TO ULTRAVIOLET 
INJURY, MUTATION, AND KILL 

An understanding of the biophysical nature of cell injury, mutation, 
and kill is not essential for the practical applications of the ultraviolet! 
In this discussion, for practical purposes, an organism is considered dead 
when it is unable to reproduce. The possibility of this being a condition 
far short of complete destruction, and the logarithmic nature of ultra- 
violet killing, leave considerable uncertainty as to just when all the organ- 
isms in one group are dead. In most of the practical ultraviolet applica- 
tions a complete kill is not necessary, but whenever it is necessary it can be 
pro\'ided by adequate factors of certainty in exposure. 

There is ample evidence from practical experience that the growth of 
fuiigi can be prevented by exposures of the order of those effective for bac- 
teria killing and by exposure of only a fraction ( a tenth to a hundredth) 
of those required for the killing of bacterial spores. The wilting and 
death of common plants such as ivy and tomato under such exposures sug- 



48 KADI ATION lUOLOr.Y 

gest suppression of tlio mycdia without serious damage to the spores, buf 
the mechanism should tx' ;iii interesting subject for research. 

COMPARISON OF SUSCKl'l I HlLl'l^ TO TLTRAVIOLET AK\) TO OTHIOR 

ij/niAi, a(;kxts 

The intrinsic susceptil)ility of \arious species of bacteria, fungi, and 
viruses to killing by ultraviolet of 2537 A, or any other wave length, varies 
over an exposure range of as much as 1-3. The extrinsic susceptibility, 
determined by acquired tolerance and age, may also \'ary over a range of 
1-3. In contrast with liacteria and viruses, for any arbitrary percentage 
kill of various mold spores, the exposure may be 15-300 times that 
required for the same kill of dry air-borne Escherichia coli, with a difTer- 
ence as great as 1 to 1000 between the most susceptible bacteria and the 
most resistant mold spores. The exposure necessary to kill any one kind 
of organism may vary considerably, depending on its environment, tem- 
perature, illumination, and physical condition, illustrated in Pig. 2-2 by 
humid water-borne E. coli which requires four times the exposures of dry 
air-borne E. coli for comparable killings. 

Although the effects of radiation of shorter wave lengths on various 
microorganisms seem to be very similar to those of the ultraviolet, there 
is little similarity between the effects of ultraviolet and of other lethal 
agents such as heat, dryness, or chemicals except in the logarithmic nature 
of the kill. Markedly thermoduric organisms, for example, are readily 
killed by ultraviolet energy. 

LOGARITHMIC NATURE OF KILL 

The practical ultraviolet killing of nearly all microorganisms is more 
or less logarithmic in nature. Wyckoff (1932) and Rahn (1932, 1945) 
have discussed this rule and its exceptions in detail. The logarithmic 
nature of kill is in accord with the general exponential attenuation law, 

,V = AV-", (2-1) 

where 

iVo = the initial concentration of organisms, 

,V = concentration of organisms after an exposure I'or tinu- / to an ultra- 
violet intensity, 
/ = the ultraviolet intensity, and 
e = the base of the natural logarithms (about 2.718). 
For a unit exposure, 7/ = 1, the concentration of survivors .V becomes 

N = Noe-' = 0.3(58 No, (2-2) 

and the kill, .Vo — N, becomes 

No- N = 0.632 .Vn. (2-3) 



I 



Al'l'LICATIONS AND SOURCES OF ULTRAVIOLET 



49 



The same attenuation law also covers the dilution of fluid-borne con- 
tamination by the admixture of a neutral and sterile fluid when the unit 
volume of diluent is substituted for / in the exponent. 



UNIT KILL 

This relation suggests 63.2 per cent as a basic unit of sanitation for 
which the term "lethe" has been suggested. With air sanitation in mind, 
Wells (1940) has also used the term for a unit ultraviolet exposure pro- 
ducing a 63.2 per cent kill of a standard organism (E. coli) under elabo- 
rately specified conditions. A lethal exposure then becomes equivalent 



/xw/cm^ TO KILL IN I min 




2 3 4 5 6 7 8 9 10 11 12 13 14 15 
MINUTES TO KILL WITH I //watt/cm^ 

Fig. 2-6. Typical data from Fig. 2-2 shown on a logarithmic scale of percentage of 
survivors and uniform scale of exposures. 

in effect to the air change of mechanical ventilation, as discussed later. 
The lethe unit of kill is indicated in Figs. 2-2 and 6. 

The exponential form of the curve of killing as a function of exposure 
has also led to the suggestion of a unit kill of 50 per cent, by analogy with 
the half-life rating of radioactive materials. 

The logarithmic nature of the ultraviolet germicidal efi^ect is illustrated 
in Fig. 2-6 where the data on dry and wet E. coli are plotted on a loga- 
rithmic scale of survivors and on an arithmetic scale of exposures. Most 
of the published data plot as straight lines on such scales within the 
experimental errors of the measurements [see Lea (1946) for a discussion 
of methods of plotting such data]. An arithmetic or linear scale of kill is 
used in Fig. 2-2, along with a logarithmic scale to e.xpand the range of 



.-,0 RADIATION BIOLOGY 

lesser exposures and yet cover a thousandfold exposure range. The sig- 
moid form of the curves is inherent in the scales used. 

REACTIVATION BY HEAT AM) I.KIIIT 

The j)ractical signilicaiu'e of the reactivating effect of time, heat, and 
light on ultraviolet-injured bacteria and molds was overemphasized by 
the manner in which the experimental data were first presented by Kelner 
(1949). For example, a definite 20 per cent revival of the total initial 
number of irradiated bacteria per experimental unit volume was presented 
as a 3000 per cent increase in the number of viable organisms from the 
indefinite few left after a theoretical killing of 99.99994 per cent. By this 
method of presentation, if the "killing" had been complete, the per- 
centage increase in viable organisms would have been infinite regardless 
of the actual revival. 

The 20-25 per cent revivals of bacteria resulted from light exposures of 
the order of 5-8000 ft-c-hr, exposures provided only by 2 3 days of the 
highest levels of practical indoor illumination. The eciuivalent of a 2- to 
3-hr exposure to 100 ft-c provided less than a 0. 1 per cent revival, and few 
practical germicidal applications involve exposures of bacteria to as many 
foot-candle-hours. This suggests that the reactivating effect of light is 
of little or no significance indoors but may somewhat reduce the apparent 
susceptibility of organisms to the ultraviolet of the sun. 

GERMICIDAL ACTION OF ULTRAVIOLET OF WAVE LENGTHS GREATER 

THAN 2800 A 

Buchbinder ct al. (1941) have shown that sunlight, direct and through 
window glass, as well as the ultraviolet from common artificial light 
sources, has measurable germicidal effects on bacteria exposed to common 
illumination intensities for a day or two (Fig. 2-4). The daylight inten- 
sities and exposure times may have been somewhat comparable with 
those used in the reactivation experiments, in which case the killing must 
have been the difference between the germicidal action of wave lengths 
greater than 3000-3200 A and the reviving or protective action of wave 
lengths greater than 3600 A. 

COMPOSITE OF KILLING FACTORS 

Only a three-dimensional model would completely represent the rela- 
tions of wave length and exposure to killing. However, the outstanding 
practicality of wave length 2537 A suggested making its plane representa- 
tive of the wave length and plotting on it a fourth indeterminate variable, 
the .susceptibility of organi.sms to killing by that wave length, as typical 
of the killing by other wave lengths. The result was a consolidation of 
Figs. 2-1 and 2 as Fig. 2-3. 



■ APPLICATIONS AND SOURCES OF ULTRAVIOLET 51 

KILLING EXPOSURES 
RECIPROCITY OF TIME AND INTENSITY 
Like the photographic effects of Hght, the germicidal effect of ultra- 
violet results from an exposure (intensity times time). The basic factors 
in an exposure are the incident power, the time, and the irradiated area. 
The erg, often used in biophysical work, is a unit of energy only and must 
be referred to time in order to define power. Ergs per second become 
units of power, and ergs per second per square centimeter become units of 
uitensity. Ergs per second per square centimeter-second, or, more 
usually, ergs per square centimeter, become units of dose or exposure. 
In practical work there are advantages in basing the intensity unit 
directly on the watt since it is also used to define the power output of 
ultraviolet sources. The microwatt, equal to 10 ergs/sec, becomes a con- 
venient unit of power, and the microwatt per square centimeter becomes 
a convenient unit of intensity. For practical purposes the microwatts 
per square centimeter equal the milliwatts per square foot, the multiply- 
ing factor being 0.9290. The microwatt per square centimeter-minute, 
often written as microwatt-minute per square centimeter, becomes a prac- 
tical unit of exposure equal to 600 ergs/cml The microwatts per square 
centimeter-minute emphasizes the reciprocity of exposure intensities and 
times which may be adjusted over a very wide range to obtain a specified 
exposure under various conditions. 

Theoretically, an e.xposure of 25 MW-min/cm-' or 1500 ergs/cm^, for 
example, may be obtained either in a long time (1 day) with a low inten- 
sity (0.018 ultraviolet /xw/cm^) or in a short time (0.001 min) with a 
high intensity (25,000 ultraviolet /xw/cm^). In practice, the exposure 
time IS usually determined by the nature of the job to be done and ranges 
from a fraction of a second for the disinfection of rapidly moving air or 
products to 1-10 min for air disinfection in relatively quiet places. The 
exposure intensity must then be adjusted to obtain an adequate exposure. ' 
Such intensities may range from a few ultraviolet microwatts per square 
centimeter for bacterial air disinfection to several ultraviolet milliwatts 
per square centimeter for product disinfection from molds, as suggested 
by Fig. 2-7a,b and in greater detail for the short exposures in air ducts 
by Fig. 2-8. 

The reciprocity of time and intensity is also illustrated by the upper and 
lower legends on the otherwise identical scales of exposure of Fig. 2-2. 

ERYTHEMAL ACTION OF WAVE LENGTHS 2537 AND 2967 A 

As shown graphically in Figs. 2-1 and 5, the germicidal ultraviolet is 
also erythemal in action, the effect at 2537 A being about half as great as 
at the optimum wave length of 2967 A. As indicated in Figs. 2-1 and 7a 



52 



RADIATION lfI()L(JGY 




0.000 001 



0.001 0.01 0.1 I 10 100 1,000 

EXPOSURE TIME, min 



100,000 




I U II 



1,000 



7 hr 



I hr 

EXPOSURE TIME, min 
Fifi. 2-7. Reciprocity of tiinc and intensity for various exposures and the kill of typical 
inioroorganisius. 



APPLICATIONS AND SOURCES OF ULTRAVIOLET 



53 



erythemal exposures are about 10 times the germicidal and are comparable 
with the fungicidal exposures, so that the time element becomes important 
in practical applications. Intensities, which are germicidal within the 
few seconds and minutes reciuired, in many cases become erythemal with 
10-15 times longer exposures of the face and eyes, thus making some form 
of protection usually necessary. As suggested in Fig. 2-76, an exposure to 



1,0 00 500 

80 



250 



LINEAR AIR SPEED, ft/mm 
100 50 25 to 5 



10,000 
8,000 

6,000 
5,000 
4,000 
3,000 

2,000 




80,000 

60,000 
50,000 
40,000 

30.000 
20.000 



q: 

UJ 

< 

O 
< 
O 



o 

a: 
o 



t,000 
800 



< 

u. 

CO 



o 



o 
S 



o 
> 

< 



0.001 0.0O2 0.005 OOI 002 0.05 01 0.2 

EXPOSURE TIME IN AIR DUCTS, min/ft 

Fig. 2-8. Reciprocity of time and intensity for short-time exposures in air ducts. 

2537 A energy of 450,000 ergs or 750 MW-min/cm- will produce a minimum 
perceptible erythema of many skins (see Chap. 13, this volume). 

AMERICAN MEDICAL ASSOCIATION TOLERANCE 

The American Medical Association (1948) specifies 0.5 M^/cm"^ as the 
maximum permissible ultraviolet intensity for 7-hr day exposures, an 
exposure of 126,000 ergs, or 210 MW-min/cm-, but they reduce this to 144 
MW-min/cmVday (an intensity of 0.1 fiw/cm^) for contimious exposure 
and acknowledge that the specification is based on experience with wave 
lengths 2800-3200 A rather than wave length 2537 A. 



54 



RADIATION BIOLOGY 



Altlit)ut»;h this exposure toleranee mi{i;ht seem to supply an unnecessary 
factor of safety, it provides for some unusual skin sensiti\ities bordering 
on the pathologic. Experience in hospitals and in inchistria' applications 
indicates, for example, that some adult face skins are more sensitive to 
ultraviolet irritation than the face skin of the average infant . The Amer- 
ican Medical Association has not had occasion to specify a corresponding 
sui^erythemal exposure for '29()7 A ultraviolet, but its unit exposure for 
a minimum perceptible erythema on average untanned skin is 300 nw- 
min/cm-, as indicated in Fig. 2-76. 

It should be noted here that there is no theory or evidence that the 
erj'thema produced by 2537 A energy differs at all from that produced by 
29G7 A except that the former is more superficial and transient. Expo- 
sures to comparable erythemas which result in skin peeling after 2957 A 
produce only scaling after 2537 A. This difference in effect, shown in the 
erythemal-action curve of Fig. 2-1, may be due entirely to the slightly 
deeper penetration of 29G7 A into the skin (see Chap. 13, this volume). 

Useful germicidal intensities of 2537 A ultraviolet range from 5 to 6000 
Mw/cm-, and the corresponding suberythemal exposure times are from 
less than 40 min to 2 sec. The intensity of ultraviolet reflected from sur- 
faces and walls of a minimum reflectance of 5-10 per cent may range from 
5 /iw downward. From this it is obvious that there is always a problem 
of face and eye protection in practical applications of ultraviolet energy. 

Table 2-2 extrapolates the American INIedical Association's permissible 
exposure through practical ranges of time and intensity. 

Table 2-2. M.\ximum Permissible Daily F^cposures 



Exposure time 


Intensity on 


Exposure time 


Intensitj- on 


per 24 hr, hr 


faces, /xw/cm^ 


per 24 hr 


faces, Mw/cm* 


24 


0.1" 


2hr 


1.8 


18 


0.2" 


1 hr'' 


3.6 


12 


0.3 


30 min 


7.2- 


9 


0.4 


10 min 


21.6' 


6 


0.6 


1 min** 


216.0 


4 


0.9 


30 sec 


432.0 


3 


1.2 


5 sec 


2()00.0 



"Permissible intensity in hospital infant wanis; onc-liftieth or one one-hundredth 
that recommended for hospital air disinfection. 

* Exposures (time times intensity) of 3.6 juw-hr/cm'. 

'Intensity recommended for hospital upper-air disinfection; tolerated only 10-30 
min if on the faces of personnel. 

'' Exposures (time times intensity) of 216 /j\v-min/cm^. 

FACE AND EYE PROTECTION AND TREATMENT 

Commercially available sun glasses and face shields designed to cover 
the eyes from the sides and the ears completely provide adequate protec- 
tion. Hands and arms may be protected by plastic or rubber or very 



APPLICATIONS AND SOURCES OF ULTRAVIOLKT 55 

closely woven textile gloves, but these gloves should be tested before long- 
time use. 

The discomfort from ultraviolet-irritated eyes may be relieved by 
exposing them for 15-20 min to as high an intensity of heat as can com- 
fortably be borne from a heat lamp or from an ordinary 50- to 60- watt 
incandescent lamp held close to the eyes; the treatment is effective 
through closed eyelids (author's personal experience). In extreme cases, 
a doctor should be consulted, but when this is impracticable, the usual 
first-aid treatment is the application of ice packs. In any case, the irri- 
tation produced by the ultraviolet may disappear within a day or two and 
much more quickly than a corresponding degree of irritation from a longer 
wave ultraviolet source. A severe conjunctivitis may, however, make 
the eyes susceptible to secondary infection until the lesions are healed. 

COMMERCIAL SOURCES OF ULTRAVIOLET 

Mercury-vapor sources of ultraviolet for practical and experimental 
uses may be grouped as (1) commercially available low-pressure (0.004- 
0.02 mm of Hg) germicidal lamps, (2) high-pressure (400-60,000 mm of 
Hg or 0.5-75 atm) photochemical, therapeutic, and filtered sunlamps, and 
(3) special experimental lamps of limited availability. The character- 
istics of all but the low-pressure lamps are discussed later in this chapter. 

Arc lamps with rare-earth cored carbons provide powerful sources of 
energy for many photochemical, photographic, and photocopying appli- 
cations of the ultraviolet. For various reasons their biological applica- 
tions have been rather limited and are not discussed in this chapter. 

HIGH- AND LOW-PRESSURE MERCURY ARCS 

As indicated graphically in Fig. 2-5, low-pressure mercury arcs are 5- 
10 times more efficient in germicidal action than high-pressure arcs in 
envelopes of the same transmission. It should be noted that high-pres- 
sure quartz-mercury arcs may be of practical use, regardless of efficiency, 
in places where it is impossible to provide the essential ultraviolet inten- 
.sities from the much more bulky low-pressure lamps. For example, the 
germicidal effect per unit of total volume of a 360-watt high-pressure arc 
in quartz is 5-10 times that from low-pressure arcs, but the germicidal 
efficiency of the high-pressure arc is one-fifth to one-tenth that of the low. 
For another example, the germicidal effectiveness of the radiating part of 
the UA-3 and UA-11 high-pressure mercury arcs of Table 2-6 can be 
duplicated only by 8-12 times the radiating length of the more efficient 
low-pressure lamps. 

ULTRAVIOLET OF WAVE LENGTH 2537 A 
INHERENTLY LOW EMISSION INTENSITY OF SOURCES OF 2537 A 

The possibility of a source of 2537 A ultraviolet with the high power 
output per unit of source area, or radiant-flux density, of the high-pressure 



56 



RADIATION niOLOGY 



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APPLICATION'S AND SOURCES OF ULTRA VK^LET 



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58 RADIATION BIOLOGY 

mercury arcs lias long taiitali/od exporimoutcrs. From the well-known 
absorption of the 25.37 A resonance line by mercury vapor and from an 
erroneous association of efficient 2r)37 A production solely with low 
mercury-vapor pressure, the experimenters have inferred that water or air 
cooling of a lamp should permit a great increase in the electric power 
input and the 2537 A emission. In the search for the optimum con- 
ditions for 2537 A production, rather definite optima of vapor pressure 
and power input, corresponding to lamp-tube temperatures of 40°-60°C, 
have been found. Radical decreases in mercury pressure by cooling or 
increases in power input, either separately or concurrently, produce rela- 
tively small changes in 2537 A output but produce radical changes in 
efficiency. The output ratings of commercial sources are the maxima 
consistent with good efficiency and life. Users of 2537 A sources who 
may be willing to sacrifice both life and efficiency for higher output power 
density must now be reconciled to a maximum emission of the order of 
30-50 ultraviolet mw/cm- of source surface provided by about 0.1-0.15 
watt of electrical power input per square centimeter of tube surface. 
This is about twice the output of commercial sources. The difference 
between power input and emission (3 to 1) results from the inefficiency 
of the conversion of electrical power to radiant power in the lamps and a 
subsequent absorption of about 20 per cent in the glass tube. Tenfold 
increases in power input, which are possible by water or air cooling, pro- 
vide but slight increases in the 2537 A output per unit of source area. 
These generalizations have little or no bearing on the radiation character- 
istics of higher pressure mercury arcs discussed later. 

SOURCES OF 2537 A ULTRAVIOLET 

Table 2-3, based partially on the lES Lighting Handbook, 2d ed. 
(1952), presents the physical, electrical, and radiation characteristics 
of most of the commercially available sources of 2537 A energy. In each 
ease the amount of electrical input (in watts) to the arc, the length of 
the radiating source, and the total radiated 2537 A energy in watts, here- 
after called "ultraviolet watts," and the ultraviolet watts per square 
centimeter at a distance of 1 meter are closely associated. This permits 
calculation of the efficiency of the sources, of their input and output per 
unit of source length and area, and of the ultraviolet intensity provided 
by them at various distances. Division of the intensity in ultraviolet 
microwatts per sc^uare centimeter at 1 meter by 10,000 provides a useful 
practical rating in ultraviolet watts per square foot at 10 ft. Multipli- 
cation of ultraviolet microwatts per square centimeter at 0.9290 converts 
to ultraviolet milliwatts per square foot, but for practical purposes they 
are equivalent. Multiplication of the 10-ft rating by 100 and division 
by the distance squared provides intensity in the same units for other 
distances greater than the length of the radiating source. 



APPLICATIONS AND SOURCES OF ULTRAVIOLET 



59 



CONVERSION FACTORS 
POWER, INTENSITY AND DISTANCE, AND WORK AND ENERGY 

The use of metric and U.S. units, separately and together, named units, 
and various time units, with little standardization of practice, requires 
frequent use of conversion factors. In Table 2-4 are listed various con- 

Table 2-4. Conversion Factors 



From 



Multiply by 



To 



Power Output and Intensity 



UV watts output, total 

UV watts output, total 

UV pw/cm^ 

UV output /steradian 

UV output/steradian 

UV output/steradian 

UV intensity, unit area, 1 meter , 
UV intensity /cm^ at 1 meter. . . . 

ergs /sec 

joules /sec 



10.0" 
0.001 
. 929 

10.0" 
0.01 
0.0001 
0.176 

10 X 103 
0.1 
1.0 



UV Mw/cm^ at 1 meter 
UV juw/sq ft at 10 ft 
UV mw/sq ft 
UV output total 
UV output/cm^ at 10 cm 
UV output /cm 2 at 1 meter 
UV intensity, unit area, 10 ft 
UV intensity /sq ft at 10 ft 

UV MW 

UV watts 



Work and Energy* 



joules 

joules 

ergs 

ergs 

g-cal 

Btu 

r (roentgens) in air 

Photons or quanta at 2536 A. 



1.0 

0.01665 X 10« 

0.1 

0.011665 
69.77 X 10' 
17.4 X 106 

0.00018315 

0.0131 X 10 '2 



UV watt-sec 
UV /iw-min 
UV MW-sec 
UV MW-min 
UV /Ltw-miu 
UV /iw-min 
UV MW-min 
UV MW-min 



Exposures 



joules /cm 2. 
ergs /cm 2. . 
g-cal /cm 2 . 
r/cm^ 



0.01665 X 106 
0.001665 
69.77 X 103 
0.00018315 



UV MW-min/cm^ 
UV nw-min/cm^ 
UV MW-min/cm^ 
UV MW-min/cm^ 



" Approximate. 

^ When on equal areas — exposures. 

versions to the centimeter-microwatt-minute units used in this chapter. 
The relation between ultraviolet output and maximum intensity refers 
only to essentially linear sources, to distances greater than the length of 
the source, and to directions of maximum intensity perpendicular to the 
center of the source. Energy per steradian refers only to the steradian in 
that maximum intensity direction and, literally, only to a very small part 
of the solid angle represented by the steradian. For this reason, energy 



60 HADIATION HIOI-OC.Y 

per steradiiui is uii unsatisfactory description of the output of linear 
sources. 

2050 A i:cn IVALKVCE 

Since 25;i7 A (Micrj>;y has only about 85 per cent of the hactcricidal 
action of 2050 A energy (Table 2-1), the ultraviolet watts output and 
ultraviolet microwatts of Table 2-3 must be multiplied by 0.85 for the 
2050 A bactericidally equivalent ultraviolet watts of Table 2-0. 

INTENSITY 

RELATIVE ENERGY DISTRIBUTION 

The inherent spectra of all low-pressure mercury arcs are dominated 
by the 2537 and 1849 A lines. Other lines are so relatively weak and 
from such low-intensity sources that they are of little practical value 
(Table 2-5 and Fig. 2-56). The output of the 1849 A line is determined 

Table 2-5. Relative Energy in Various Spectral Lines or Groups of 

Typical Low-pressure Lamps 

Wave Length, A Relative luiergy, % 

2537 100 

2652 0.14 

2753-2893 0.12 

2967 . 37 

3022 0.17 

3126-3132 1.43 

3650-3()63 1 . 30 

3906-4077 1 . 60 

4339-4358 3 . 40 

5461 2.25 

5770-5791 0.60 

over a wide range by the fused cjuartz and the special glasses, in various 
thicknesses, whose transmissions are shown in Fig. 2-5a. Since the 
1849 A energy is rapidly absorbed by air (about 50 per cent in 1 in.) and 
since it penetrates licjuids and cellular proteins much less effectively than 
the 2537 A, little practical bactericidal application has been found for 
energy of this wave length. Since 1849 A energy is only slightl}^ absorbed 
by nitrogen but is readily absorbed by oxygen, it provides ozone in air, 
relatively uncontaminated by oxides of nitrogen, and some practical 
application of this energy for this purpose is being made. Commercially 
available sources in thin glass provide 1849 A energy to an extent 1-2 per 
cent that of 2537 A energy. Greater 1849 A energy output is possible 
through thin fused-fjuartz glass. 

RESEARCH SPECIFICATIONS OF ULTRAVIOLET INTENSITY 

The ultraviolet power output and intensity ratings of Table 2-3 are 
average values for new sources. The variation and service depreciation 



i 



APPLICATIONS AND SOURCES OF ULTRAVIOLET Gl 

of all commercial sources are such that the output ratings should be used 
or approxmiations only. A description of a lamp type and of its elec- 
trical characteristics is an essential guide to the mechanics of a laboratory 
research but is. of little value as a means of specifying the radiation 
nitensities provided. The effective intensities obtained fn any „h 
should be measured at the irradiated surface or throughout the irradiated 
volume and should be specified in general terms entirely independent of 
the source, the usual laboratory unit being the microwatt per square 
centimeter, and the corresponding engineering unit, the milliwatt per 
square foot. ^ 

INTENSITY VARIATIONS WITH DISTANCE 

For distances greater than the length of the radiating source the 

intensity varies inversely as the square of the distance. For distances 

ess than about one-third the radiating length of these linear type sources 

he intensity varies inversely as the distance. The variation at the 

transition distances can be measured directly or estimated from the 

ac-tual mtensities produced by such typical sources as are shown in 

HIGH INTENSITIES AT CLOSE RANGE 

The maximum hitensity prcn-ided by a single tubular source is at its 
own surfac-e. This is a useful point at which to start a study of the 
variation of intensity at short distances from the tube. Distances are 
measured from the center of the tube although it radiates ultraviolet 

r' r^ 1 T r^ ""^ ^^' *"^' '''^'^ *^^ «^^^^^- The effective emitting 

ng h of the l-in.G30T8 tube of Fig. 2-9 is 32 in., and its circumferenc^ 

•s 314 m. From this surface area of about 100 sq in., 7 ultraviolet watts 

^00 ' "">' T""'T '""^'''"'^^ "^ ^-^^ ultraviolet watt/sq in. or 

il^800 Mw/cm-. A surface or a material in contact with the tube would 
therefore be irradiated at that intensity. 

Similarly, a cylindrical surface 2 in. in diameter would have double the 
tube area^ J* .would intercept practically all the radiated 7 ultraviolet 
watts with half the intensity. Similarly, the intensity on a 3-in cylin- 
drical surface would be one-third as great, and on a 4-in. cvlinder one- 
ourth as o^reat On cylinders of these diameters, small compared with 
heir eng h, the ultraviolet intensity is uniformly distributed, except 
tor a length at each end equal to about the radius of the tube Th,> 
intensity on the surfaces of such irradiated cylinders varies with their 
diameters or inversely as the distance from the tube axis to the irradiated 
suitace. This relation is true out to distances of the order of one-third 
the effective length of the source, as shown in Fig. 2-9. 



62 



RADIATION mOLOGY 



ti31N30 3901 kNOtiJ 93HDNI 

_ _ o o o 




y31N3D 3901 WOaJ 133J 



APPLICATIONS AND SOUKCES OF ULTRAVIOLET 63 

AVERAGE INTENSITY IX SPACE 

In the disinfection of fluids, the average ultraviolet intensity, or average 
radiant-energy density, throughout the concentric cylindrical space sur- 
rounding the tubular sources becomes a basic factor. It is especially 
basic in the case of air where there is no absorption to modif}^ the linear 
decrease in intensity inversely with the distance from the lamp tube. 
It can be the basic intensity factor in the disinfecting exposure of air 
where it is possible to provide enough turbulence of flow to expose the 
air to the full range of intensities, and so to an average intensity, during 
its travel through an irradiated zone. Since the intensity at less than 
source-length distances from linear sources varies inversely as the distance 
and the volumes of successive increments of annular space increase 
directly as the distance, the products of annular volume increments and 
their energy density become constant. Thus, within source-length dis- 
tances, the average intensity occurs at the average distance of one-half 
the radius of the irradiated zone and is twice the intensity at the outer 
limits of the zone, as pointed out by Luckiesh and HoUaday (19-l:2a,b). 

At distances greater than source length, as in directly irradiated rooms 
or very large plenum chambers of ventilating systems, the intensity 
throughout the spherical space surrounding a central ultraviolet source 
varies inversely as the stjuare of the distance, and the volumes of suc- 
cessive increment shells of space increase directly as the square of the 
distance, so that the products of successive volume increments and their 
energy density become constant. Here again the average intensity 
would occur at the average distance of l/\/3 or 0.577 the radius of the 
irradiated spherical volume and would be three times the intensity at 
the outer limits of the volume, as developed by Wells (1940), if it were 
not for the toroidal rather than spherical spatial distribution of the 
energy about a linear source. Also, since irradiated rooms are cubical 
rather than spherical in form, the average intensity occurs at more 
nearly half the average radial distances to the walls and is again about 
half the intensity at the outer limits of the spherical or cubical zone. 

INCREASE OF INTENSITY AND UTILIZATION BY' REFLECTORS 

Efficient sources of the germicidal ultraviolet are inherently low in 
intensity compared with high-pressure sources designed for photo- 
chemical and therapeutic use. A maximum intensity of 10-20 ultra- 
violet watts/sq ft (10,000-20,000 ultraviolet-/iw/cm'") can be available 
at a tube surface for experimental work, but, at practical working dis- 
tances, only about one-fourth this intensity can be obtained as irradiation 
over an extended area (see Fig. 2-106). 

The ionized mercury vapor in germicidal tubes almost completely 
absorbs any 2537 A energy which might otherwise pass through the glass 
tube itself from an outside source. For this reason, only the thin layer 



64 KADIATION HIOLOGY 

ot incri'ury xapor prtictii-uUy in roiitacl with the luinp tuhe i.s aiiolTcctix-e 
source of 2537 A energy. Thus, when several such tubes are placed in 
contact, side by side to form a grid, only about one-third their total 
ultraviolet output reaches a parallel irradiated surface of an area about 
one-third the total surface areas of the tubes (Fig. 2-1 Oaj. It is of 
interest to note that, with such a rectangular or sciuare grid source 
formed by such an as.sembly, the intensity on the irradiated surface 
remains constant for distances out to about half the width of the assem- 
bly. As the distances are increased to the length of the rectangle, there is 
a transition to a variation inversely with the square of the distance. 
Single tubes are, in effect, rectangles of so small a width as to have the 
characteristics of theoretical line sources (Fig. 2-9). 




(a) 




(6) 
Fig. 2-10. Effect of bactericidal-tube spacing on utilization of total ultraviolet output. 
(a) Close spacing, (b) Open spacing under reflectors. 

The high intensities possible with germicidal tubes in contact in a grid 
pattern can be produced more economically with about one-third as 
many tubes fitted with reflectors. In a tube-and-reflector system the 
tubes should be spaced on centers three or four times their diameter 
(Fig. 2-10^). 

All reflectors for practical uses with germicidal lamps should be of 
speciallj^ processed, polished aluminum (60-70 per cent reflectance) or 
polished chromium plate (40-50 per cent reflectance). Luckiesh and 
Taylor (1946) have shown that no other reflecting materials are of 
practical value. Special aluminum paint may be used in some places, 
such as in air ducts, if the service or maintenance is such as to make 
occasional repainting practical. Such a paint is made of pure aluminum 
flakes in a vehicle of plastic lacquer of high ultraviolet transmission. 

Specular aluminum reflectors, designed to intercept about two-thirds 
of the tube energy, redirect about (55 per cent of the energy to the irradi- 
ated surface. The efficiency is therefore 65 per cent of two-thirds or 43 
per cent plus the 33 per cent directly from the tube or a theoretical total 
of ^^75 per cent. In practice, commercial etiuipment is only capable of 
doubling the effective radiation from an equivalent grid of bare tubes. 



APPLICATIONS AND SOURCES OF ULTRAVIOLKT ()0 

The mercury column in the tube absorbs almost all the energj^ redirected 
to it by the reflector and prevents higher utilization. 

OZONE FORMATION 

One-tenth to 5 per cent of the mercury line 1849 A energy is trans- 
mitted by the glasses used for the tubes of germicidal lamps as indicated 
in Fig. 2-oa. Energy of this wave length, transmitted only a few inches 
through air, easily breaks the weak bonds of the oxygen molecule to 
permit the formation of ozone near the ultraviolet source. Roller 
(1946) found such ozone to have a half life of 15 hr, from concentrations of 
several hundred parts per million, in the dry glass containers in which it 
had been formed. Ewell (1942) had found, however under more prac- 
tical conditions, that humidity, light, 2537 A ultraviolet, and surface 
absorptions greatly catalyzed the reversion to oxygen. Under such 
conditions he found ozone to have a half life of 2-3 min when irradiated 
with 2537 A ultraviolet and of 6-7 min when not irradiated, in both cases 
from concentrations of 3-4 ppm. 

Since this ozone diffuses throughout an irradiated space, its inherent 
instability is considerably increased by 2537 A energy, and short-lived 
atomic oxygen occurs in a uniciue manner. Commercial sources permit 
air disinfection with eciuilibrium ozone concentrations less than the 1 
part per 10 million considered permissible by the American Medical 
Association (1948). Other sources provide the somewhat higher concen- 
trations traditionally used in certain food-storage applications, where the 
odor-masking effects of ozone and its concentration by absorption on 
moist surfaces may be of some value. 

PHOTOCHEMICAL EFFECTS OF 2537 AND 1849 A ENERGY 

The photochemical actions of the 2537 and the 1849 A energy are out- 
side the scope of this chapter except in so far as they are incidental to some 
of the practical applications in other fields. The 2537 A energy con- 
siderably increases the normal oxidizing action of oxygen without its 
obvious ionization. This is greatly increased wherever there is ioniza- 
tion and ozone formation by 1849 A energy. In all germicidal applica- 
tions of the ultraviolet the possibility of objectionable chemical changes 
should be investigated, e.g., formation of phosgene and hydrogen chloride 
in poorly ventilated dry cleaning rooms where carbon tetrachloride may 
be used, formation of hydrogen sulfide and mercaptans in egg-drying 
plants where egg powder may be in the air, and modification of the flavor 
of irradiated foods such as meat, milk, cheese, and butter. 

TEMPERATURE AND VENTILATION 

Like fluorescent lamps, commercial germicidal lamps are designed to 
operate under average conditions of room temperature and ventilation. 



66 



RADIATION BIOLOGY 



Unusual enclosure or extremes of air temperature, such as in refrigerators, 
ovens, and air ducts, will reduce the ultraviolet (jutput of the germicidal 
tube to the same extent as the light output of a similar fluorescent lamp. 
The reduction is about 10 per cent at 50° and 100°F, 20 per cent at 40° 
and 1 10°F, and 30 per cent at 35° and 120°F. 

DEPRECIATION 

In common with fluorescent lamps, bactericidal tubes depreciate 
rapidly during the first 100 hr of operation. This is considered a part of 
the manufacturing process, and commercial lamps are given an initial 
rating as if at \0d hr of normal operation. In Fig. 2-11 the approximate 



120 



1000 



2000 



LIFE, hr 

3000 4000 



1 — I — I — I — I — I — I — I — I — I — I — I — I — \ — I — I — I — I — I — I — I — I — I — r 



5000 6000 7000 8000 
120 




10 20 30 40 50 60 70 80 90 100 
PERCENTAGE KILL BY UNIFORM EXPOSURES 



Fig. 2-11. Depreciation, life, and bactericidal-effectiveness curves of typical low- 
pressure ultraviolet sources. 



depreciation of low-pressure sources is shown. Those in high-transmis- 
sion glasses start from 20 to 25 per cent above the 100-hr rating, whereas 
those in f used-quartz glass or in similar Vycor glass start from only 
slightly above the 100-hr rating, considered as 100 per cent in Fig. 2-11. 

The depreciation rate, and therefore the effective life, is affected by the 
length of the operating periods; the shorter the average operation per 
start, the faster the depreciation and the shorter the effective life. In 
general, such lamps fail to start and operate normally at about the end of 
their effective life, i.e., at about 60 per cent of their 100-hr rating. Lamps 
with so-called "cold" electrodes provide some exception to these rules, 
but they still have the basic depreciation limitations of constant opera- 
tion. They may continue to start and operate normally after their out- 
put has dropped to ineffective levels. 

It should be noted that ultraviolet killing is an exponential rather than 
linear function of ultraviolet intensity. The curve at the right of Fig. 
2-1 1 provides a typical illustration of the relatively small amount of 
change in ultraviolet killing that may result from a large depreciation. 



APPLICATIONS AND SOURCES OF ULTRAVIOLET 67 

ULTRAVIOLET DISINFECTION 
Practical uses of the lethal action of ultraviolet energy are limited only 
by the practicality, in each case, of providing an adequate exposure 
(nitensity times time) on microorganisms. Exposure time is usually 
defined by mechanical conditions leaving little chance for radical change, 
and the exposure intensity must be adjusted for an adequate exposure 
product. Provision of an adequate ultraviolet intensity is conditioned 
on two basic and independent factors: (1) the variation with distance 
from the source or sources and (2) the absorption of the ultraviolet by 
intervening media. The variation with distance has been represented in 
general terms as insignificant within a few inches from a relatively large 
assembly of sources and reflectors, as inverse with the distance for a few 
inches from single tubular sources and reflectors and a few feet from large- 
area sources, and as inverse with the square of the distance at greater than 
source-length distances from single tubular sources and reflectors, as well 
as at considerably greater distances from large areas. The absorption 
of germicidal ultraviolet by air is entirely negligible even for the irradia- 
tion distances of large auditoriums. 

AIR DISINFECTION 

Ultraviolet air disinfection is commonly accomplished by placing germi- 
cidal lamps in the rooms or in the air ducts serving such rooms. The two 
methods well illustrate the definition of exposure times by the mechanics 
of the problems, with adequate exposure intensities to be provided if pos- 
sible. As detailed later, an effective exposure for air disinfection is 
15,000 ergs/cm^ or 25 ultraviolet /xw-min/cml In the upper air of 
occupied rooms, practical exposure times may be 1-5 min, and eff'ective 
intensities may be 25-50 ultraviolet Mw/cml In air ducts the exposure 
times may be H-}i sec, and the corresponding intensities may be 10,000- 
25,000 ultraviolet /xw/cm^. 

DUCT AIR DISINFECTION 

The disinfecting exposure of duct air is defined by its transit time 
through an ultraviolet-filled zone of definite length and by the average 
ultraviolet intensity during the transit time throughout that zone. In 
Fig. 2-2, 15 ultraviolet MW-min/cm^ is suggested as a disinfecting exposure 
for most air-borne microorganisms except fungi. In an air duct with a 
cross section of 2 sq ft and a rating of 1200 cu ft/min, the linear flow of 
600 ft/min through an ultraviolet-filled zone 3 ft long provides an expo- 
sure time of 0.005 min. The average ultraviolet intensity throughout 
the zone must then be 3000 ultraviolet yuw/cm^, or about 1800 ultraviolet 
mw/sq ft for an exposure of 15 ultraviolet ^w-min/cm-. The average 
intensity throughout a cylindrical zone radially irradiated by a linear 



68 RADIATION BIOLOGY 

source on its axis of a length greater than the diameter of the zone has 
been shown to be that on a concentric cylindrical surface of one-half the 
radius. It follows then that the ultraviolet intensity at any given dis- 
tance from a germicidal lamp, as indicated in Fig. 2-9, will be the average 
intensity throughout a cylindrical zone of a radius twice that distance. 
Thus the GSGTO lamp of Fig. 2-9 provides an intensity of 2800 mw/sq ft 
at a distance of 2'^^ in. and so an average of that intensity throughout 
a cylindrical space of about the length of the ultraviolet source and a 
radius of 53^ 2 ii'^-i or a cross-section of 0.95 sq ft. Although two such tubes 
would provide an effective initial average intensity in the cross section 
of the duct, three would be specified to increase the minimum intensity 
at remote parts of the duct and for an effective intensity at the end of tube 
life. Luckiesh and HoUaday (1942a) have developed the theory of 
ultraviolet duct-air disinfection in minute detail, and Buttolph (1945, 
1951) has given it practical application. 

Turbulent Flow for Average Exposure. In small ducts that require only 
one or two germicidal tubes which are of necessity placed parallel with the 
direction of air flow, there may be a 10-to-l variation in the ultraviolet 
intensity at distances 1-10 in. from the tube. To ensure that all the air 
receives an average intensity exposure in its travel through the irradiated 
zone, either the streamlined flow of the air must be broken into turbulent 
flow by baffles or more germicidal tubes must be used. In the latter case, 
as in all cases where many tubes are used, they may be spaced to provide 
a sufficiently uniform ultraviolet intensity to take care of the streamlined 
air flow. 

Increase of Average Intensity and Uniformity by Reflective Duct Walls. 
Duct walls of pure aluminum of 65-75 per cent reflectance for 2537 A 
will nearly double the effectiveness of the germicidal tubes by at once 
nearly doubling the average intensity and by greatly increasing the uni- 
formity of distribution by multiple reflection. 

Disinfected Duct Air as Alternative to Make-up Air in Sanitary Ventila- 
tion. The most that ultraviolet disinfection can do is to make all the air 
handled by a duct bacteriologically equivalent to make-up air. When- 
ever the use of enough ultraviolet to provide a theoretical 99 per cent 
disinfection of the duct air may be impractical, it should be noted that 
one-half as much ultraviolet will still prox'ide 90 per cent disinfection, and 
one-fourth as much will provide about 70 per cent disinfection. In such 
cases, disinfection provides the equivalent of 90 and 70 per cent make-up 
air in contrast with the 10-20 per cent usually believed to be economically 
practical in the winter. 

Outdoor air is usually considered satisfactory for the sanitary ventila- 
tion of living and assembly (juarters, and its usefulness is limited only by 
the considerable cost of heating and circulating it in adeciuate quantities. 
In food and pharmaceutical plants it may, however, carry enough mold 



APPLICATIONS AND SOURCES OF ULTRAVIOLET 69 

and bacterial contaminants to be a continuous hazard to the products. In 
these instances, ultraviolet air disinfection can perform a job which is not 
yet possible by available methods of air washing and filtration. 

ROOM AIR DISINFECTION 

Ultraviolet disinfection of air is accomplished in occupied rooms by 
germicidal tubes in cylindrical parabolic refiectors which are designed 
to project the energy for maximum distances through the air of the room 
above the head level of the occupants. It is accomplished in vacated 
rooms or where protection of the occupants can be provided by bare 
germicidal tubes centrally placed in the rooms or on the ceilings. Because 
of the greater distances the variations in ultraviolet intensities are much 
greater through irradiated rooms than in irradiated air ducts. Fortu- 
nately the convective circulation and the relatively low-intensity long- 
time exposures practical in irradiated rooms provide an average intensity 
exposure such as is obtained in air ducts only by induced turbulent flow 
or by the use of many separate sources of ultraviolet. 

Unoccupied Rooms or Rooms with Occupants Protected. In the rela- 
tively simple case of the vacated room or where occupants may be ade- 
quately protected, effective ultraviolet intensities may be provided by 
centrally placed bare ultraviolet sources. The effective intensity is then 
determined entirely by the time available to disinfect the air. Assuming 
this time to be 5 min, the intensity for an exposure of ultraviolet ^w- 
min/cm- needs to be only 5 ultraviolet /xw/cm'. This intensity which 
can be provided by the G36T6 tube of Fig. 2-9 at a distance of 12 ft will 
disinfect not only the air but also the walls. Bacteria which might be 
deposited on the walls from the air are thus subjected to only the mini- 
mum intensity present in a 20- by 20-ft room, such as a hospital room 
between occupancies or a room in a pharmaceutical factory. In rooms 
where only air-borne bacteria are a problem the average rather than the 
minimum intensity becomes the basis of installation, and the same bare, 
centrally placed tube will provide an average intensity of 5 mw^sq ft 
throughout a 40- by 40-ft room to disinfect the air in only 5 min. 

Occupied Rooms. Occupants in a room add three serious complications 
to ultraviolet air disinfection that compel entirely different approaches 
to the theory and the practice. In the occupied room the problem is not 
the simple one of cleaning up the residual contamination but the dynamic 
one of killing or removing air-borne microorganisms as rapidly as they 
appear from the noses, throats, and clothing of the occupants. The kill- 
ing or removal must be in such a way as to reduce to a minimum their air- 
borne life under an equilibrium condition of origin, of necessity several 
feet from the place of their killing or removal. In the occupied room the 
maximum intensities tolerated on more sensitive faces range from 0.1 
ultraviolet mw/sq ft for continuous exposure to 0.5 ultraviolet mw/sq ft 



70 RADIATION BIOLOGY 

for 7 lir of exposure per day. Altliough ideally placed, these intensities 
are not high enough for rapid disinfection. In occupied rooms the value 
of air disinfection is primarily that of the health value in removal of 
microorganisms. In the absence of any criteria of the health value or 
hazard of air-borne organisms, the natural ventilation believed of value 
becomes a secondary criterion. 

Conrcrtirc Circulation and Upper-air Irradiation. The (;onvective cir- 
culation of air, by which the heating of a room from a few localized 
sources of heat is possible, involves the use of vertical components which 
provide an interchange of air between the upper and lower parts of a room 
equivalent to from several air changes per hour to several per minute. 1 n 
occupied rooms the basic convective ciicul.itioii is increased by the body 
heat, the breathing, and the movement of the occupants. These factors 
increase the circulation in proportion to the crowding, the contamination, 
and the need for air disinfection. Ultraviolet disinfection of the upper 
third or fourth of a room can provide in these portions of the room a 
reservoir of air for the dilution of the lower air at rates equivalent to 
unusual natural or mechanical ventilation. Lacking more direct criteria 
of value, it becomes convenient to consider ultraviolet air disinfection as 
equivalent to and a substitute for outdoor air for sanitary ventilation 
purposes. 

Upper-air Method of Disinfection. Luckiesh and Holladay (1942b) 
treat the upper part of a room as a duct containing air in random circula- 
tion at a velocity (5-10 ft/min) equivalent to about one one-hundredth 
the linear velocity in wall ducts and room units and irradiated with an 
average ultraviolet intensity (0.025 ultraviolet watt/sq ft) which is about 
one one-hundredth that provided in air ducts. The upper part of the 
room is then treated as a duct serving the lower part. There is, however, 
no such definite separation between the two parts of the room as this 
oversimplification suggests, and the following analysis (Buttolph, 1951) 
is believed to be more realistic. 

An ultraviolet (2537 A) intensity of 5 ultraviolet mw/sq ft, effective 
throughout a cubic foot, will kill respiratory and E. coli test organisms 
at the same rate as they might otherwise be washed or diluted out of the 
same cubic foot of air by one air change per minute. This is the theo- 
retical reduction of 62.3 per cent of Fig. 2-2. An additional air change or 
an additional 5 ultraviolet mw/scj ft can dispose of 62.3 per cent of the 
remaining 37.7 for a theoretical reduction of 82.5 per cent. The effect 
is the same whether by 5 mw for 2 min or 10 mw for 1 min, two air 
changes in successive miimtes or two air changes in 1 min. These rela- 
tions are plotted on linear and on semilogarithmic scales for comparison 
in Fig. 2-12. 

An average ultraviolet intensity of 5 mw/ s(i ft throughout the entire 
cubage of a room would theoretically provide the disinfection equivalent 



APPLICATIONS AND SOURCES OF ULTRAVIOLET 



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in the room of one air change per minute, such as is thought to be desir- 
able in very crowded rooms. Only one-tenth that intensity (0.5 ultra- 
violet mw/sq ft) is, however, tolerated on sensitive faces for 7 hr per day 
without objectionable "sunburning." So only about one-tenth of an air 
change eciuivalent per minute is practical by this method. This is, 
however, equivalent to the six air changes per hour, or 30 cu ft of out- 
door air per child per minute sometimes specified for school rooms. 
An average intensity of 5 ultraviolet mw/sq ft throughout a whole room, 
with only 0.5 permitted in the lower 
part of the room, can be obtained 
b}^ an average of 14 mw/sq ft in an 
upper third of the room or 18.5 in 
an upper fourth. Since the ultra- 
\'iolet in the lower part of the room 
is due mostly to the diffuse reflec- 
tion from the upper side walls and 
ceiling, its intensity may vary little 
throughout the entire area, especi- 
all}^ under 10- to 12-ft ceilings. 
However, since the ultraviolet in 
the upper part of the room is pro- 
jected through it from a few sources 
(usually in cylindrical parabolic re- 
flectors on the walls), the intensity 
may vary from 2 or 3 ultraviolet 
mw/sq ft to 2 or 3 ultraviolet 
watts/sq ft (a thousandfold varia- 
tion). Such an uneven distribu- 
tion of the energy in the room is 
effective only because the convective circulation exposes nearly all the 
air to nearly the entire range of intensities to provide an integrated lethal 
exposure, as with turbulent flow in an air duct. 

Hospital Room Disinfection. The first uses of ultraviolet for air dis- 
infection in hospitals were to provide the equivalent of local curtains or 
barriers between the surgeon and his operation (Hart, 1936; Overholt and 
and Betts, 1940) and across the front of infant cubicles (Sauer et al., 
1942; Del Mundo and McKhann, 1941; Robertson et al., 1939, 1943). 
Ultraviolet intensities ranging from 20 mw at the floor to 200 mw at 
head level can readily be provided. The 20-mw intensity becomes as 
effective as the 200-mw intensity because the width of the di\'ergent beam 
and the distance of the bacterial travel through the beam, and thus the 
average exposure time, are ten times as great at the floor. These inten- 
sities may produce erythema at head level in 1 min and at floor level in 
10 min, so that such installations are limited to pharmaceutical plants 



f r I — \ — 1 — 




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20 




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10 20 30 40 50 

mwatt-min/sq ft OR mwatt-min/cu ft 
2-12. Comparison of linear 
logarithmic plotting of percentage sur- 
vival of air-borne bacteria as a function 
of dilution by air or by equivalent ultra- 
violet irradiation. 



72 



KADI \TI()N MIOLOGY 



and infant wards of hospitals where essential discipline of personnel is 
possible. Sut'h an nltraviolet l)arrior obviously also provides general air 
disinfection b}' virtue of the circulation of room air through it. Because 
of its p()siti\-e functioning, the ultraviolet l^arrier has somewhat the same 
psychologic as well as engineering appeal as ultraviolet air disinfection in 



110° 100° 90° 80° 70° 60° 50° 



120° 




300 400 500 600 

PER CENT OF BARE LAMP INTENSITY 
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1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 
DISTANCE FROM FIXTURE, ft 

(6) 
Fig. 2-13. (a) Spatial distribution of ultraviolet from typical bactericidal tubes and 
open reflectors, (b) Isointensity lines in milliwatts per square foot in a plane perpen- 
dicular to the center of the G30T8 tulie of Fig. 2-9. 

air ducts; the door opening becomes a duct from one room to another in 
spite of its unconventional cross section compared with its length. 

The variation in ceiling height and the difference in exposures per 
day in patient and service rooms of hospitals have led to two distinct 
types of commercial equipment, an open type for use under high ceilings 
and where there may be personnel exposure of about 8 hr per day (Fig. 
2- 13a) and a louvered tj^pe for use under low ceilings and where there may 
be continuous exposure (Fig. 2-14a). Figures 2-13a and 14?> suggest a 
possible way to provide energy intensities of 15-20 m\v/sq ft in the upper 
third or fourth of a room. 



APPLICATIONS AND SOURCES OF ULTRAVIOLET 

IIO" 10 0° 90° 80° 70° 60° 50° 40" 30° 20° 



73 




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1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 
DISTANCE FROM FIXTURE, ft 

Fk;. 2-14. (a) Spatial distribution of ultraviolet from typical bactericidal tubes and 
louvered reflectors. (6) Isointensity lines in milliwatts per square foot in a plane 
perpendicular to the center of the G30T8 tube of Fig. 2-9. 

Barrier-type units have usually been custom made, but a typical 
combination unit is described by Fig. 2- 15a and h. 

HEALTH VALUE OF AIR DISINFECTION 

Ultraviolet air disinfection is but one of the factors in a complete air 
sanitation. It is comparable with the removal of dust and noxious 
vapors. Although air disinfection has been used in industrial applications 
as such a general sanitary measure, it early came to be thought of as 
having more specific possible value in preventive medical and public- 
health applications. Buttolph (1951) has proposed a tentative standard 
of air sanitation relating ventilation and disinfection to room occupancy. 
It calls for one air change per miimte where there may be as little as 300 
cu ft of room volume per occupant. From that it assumes the need for 
dilution with fresh air or eciuivalent ultraviolet air disinfection to be 
proportional to the crowding, to vary dii-ectly with the iuiml)er of occu- 
pants in a room and inversely with its volume. 



74 



RADIATION BIOLOGY 



Hospitals. When new low-pressure sources of 2537 A ultraviolet, with 
a germicidal ellicieucy fivefold greater than that of previously available 
sources revived the interest in practical applications, they were first 
found by Hart (193G) and Overholt and Betts (1940) to improve the air 



(10" 100° 90° 80° 70° 60 




4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 
DISTANCE FROM FIXTURE, ft 

(6) 

Fig 2-15 (a) Spatial distribution of ultraviolet from typical bactericidal tubes and 
louvered reflectors with a downward barrier reflector, (h) Isointen.s.ty lines ni milli- 
watts per square foot in a plane perpendicular to the center of the G30T8 tube ot 
Fig. 2-9. 

sanitation in operating rooms. This success led to trials by Sauer et al. 
(1942), Del Mundo and McKhann (1941), and Robertson et al. (1939; 
1943) in infant nurseries where an even more definite improvement was 
found by air sampling as w(^ll as by records of the spread of contagion 
among infants. On the basis of these tests the use of germicidal lamps 



APPLICATIONS AND SOURCES OF ULTRAVIOLET 75 

is approved for general hospital use by the American Medical Association 
(1948). 

Lurie (194G) and Vandivicre ct at. (1949j ha\e shown that tubercle 
bacilli, either in sputum or air-borne, are readily killed by practical 
exposures to ultraviolet. Wells and Ratcliffe (1945; Wells et al., 1948) 
have shown that, in experimental animals, tuberculosis is spread mostly 
l)y air-borne organisms so small that they remain suspended in air for long 
periods. These studies suggest a unique value for ultraviolet air sani- 
tation in tuberculosis hospitals and perhaps even in some homes. 

Schools. The promising results in hospitals led at once to trials in 
public schools. Wells et al. (1942) and Wells (1945) in Swarthmore 
and Wells and Holla (1950) in Pleasantville studied the spread of measles 
and chicken pox as typical of respiratory diseases in general to find that 
ultraviolet air disinfection suppressed the epidemic occurrence of these 
diseases in the sense that their incidence was spread out over longer- 
than-usual time intervals. This modification of the pattern of epidemic 
spread was thought worth while in spite of some uncertainty as to a 
significant long-run reduction in the total cases. 

Only measles and chicken pox were studied as respiratory diseases, 
typical in their air-borne manner of spread but atypical in the individual 
immunity they impart, with the preconception that influenza and the 
common cold could not be studied directly because of their indefinite 
diagnosis, their spread in every environment outside the schools, and the 
almost universal susceptibility to them. As anticipated, air disinfection 
provided no measurable effect on the incidence of colds and influenza 
among the school children, and Downs (1950) reported no effect in a 
surrounding community. The studies by Wells and Holla (1950) showed 
that measles and chicken pox are too completel}^ typical of respiratory 
diseases to simplify greatly the study of their epidemiology in schools 
since two-thirds of their spread occurred outside the school coverage of 
the ultraviolet installation. 

In large consolidated schools served by busses from small towns and 
the surrounding country, Perkins et al. (1947) hoped to study also measles 
and chicken pox with a minimum spread outside the school environment. 
There was early indication of some effect on the epidemic spread of the 
diseases in the schools, but there was also subsequent evidence of their 
spread in the busses, perhaps enough to blanket the marginal effect of the 
air disinfection in the school buildings. 

Ultraviolet air disinfection is justified in school rooms as a supplement 
to air sanitation by ventilation, especially in northern latitudes. It has a 
place as a general sanitary measure along with dust suppression, wash- 
room sanitation, and habits of personal cleanliness which may be taught 
and practiced in schools regardless of their effectiveness outside the 
school. 



76 RADIATION BIOLOGY 

Institutions. Many institutions, through their isolation from sur- 
rounding communities, have provided an opportunity to study the 
possil)le vakie of a unixcrsal practice of air sanitation in large com- 
munities. Schneiter et al. (1944) early reported a study in a training 
school for delin(iuent boys started with such e(|uipment as was then 
available. DuHuy ct al. (1948) have since reported no effect on the 
incidence of disease among the boys, and that air sampling showed little 
air disinfection. If there was no disinfection, no effect should have l)een 
expected, but the more probable explanation is that the air-sampling 
method did not properly detect the presence or absence of respiratory- 
disease organisms, that the obsolete equipment did not pro\'ide an 
effective use of the ultraviolet energy even though it was supplied in 
excess, and that any possible effect on the spread of respiratory diseases 
in the sleeping rooms was nullified by the lack of separation of the boys 
in the irradiated dormitory from those in the control dormitory during 
their class, intimate play, and eating periods. 

Navy Barracks. In contrast with these results, studies in Navy 
barracks consistently indicated disinfection of the air and a significant 
reduction in the spread of general respirator}^ diseases. Wheeler et al. 
(1945) reported a 25 per cent reduction of respiratory illness and a 50 
per cent reduction of the relatively highly resistant saprophytic organisms 
dominating the air contamination. Miller et al. (1948) reported, in a 
similar but theoretically more effective installation, a 19.2 per cent over- 
all reduction in total respiratory disease and a 24 per cent reduction in the 
unusually high streptococcus-disease rates. Willmon et al. (1948), 
reviewing four years of Navy barracks study, are less certain of the 
amount of reduction in disease, and Jarrett et al. (1948) reported about 
50 per cent reductions in bacteria count but were dissatisfied with 
the open-plate method which overemphasizes heavier dust-borne 
contaminants. 

Conservatism as to Value. The universal appeal of air disinfection as a 
general sanitary measure and the limited evidence of its specific health 
value have led committees of the American Public Health Association 
(1947) and of the National Research Council (1947) to issue warning 
statements about air disinfection in general and about the ultraviolet 
method in particular. Both committees point out that, at best, air dis- 
infection can reduce only that limited part of the spread of respiratory 
disease which may be air-borne, and they emphasize the difficulties in 
obtaining effective ultraviolet air disinfection without face and skin 
irritation of room occupants. 

There is need for further study of the extent to which air disinfection 
might supplement the use of face masks, the smothering of the cough and 
sneeze, and the physical isolation of patients suffering from respiratory 
disease. 



APPLICATIONS AND SOURCES OF ULTRAVIOLET 77 

Air disinfection would seem to have most of the possibilities and 
limitations in health value of unusual amounts of ventilation with outdoor 
air, amounts ordinarily impractical in the cost of moving and heating. 
Ultraviolet energy in germicidal barriers across openings would seem to 
provide a bacterial isolation of rooms and people where doors or glass 
partitions may be impractical, as in some hospital infant wards. Ultra- 
violet barriers in air ducts can supplement and in some cases serve the 
purpose of excessive filtration of duct air, as in pharmaceutical factories. 

FLUID DISINFECTION 

When intervening media are gases of negligible absorption and licjuids 
of so great an absorption that the effective penetration distance is 
negligible, the intensity at the irradiated surface is obviously dominated 
entirely by the distance through the gas. When, however, there is 
irradiation through a gas such as air and into the mass of a licjuid of low 
or intermediate absorption, the intensity at any given point in the licjuid 
is determined primarily by the total distance from the source to the 
point, and secondarily by the absorption from the surface of the liciuid 
to the same point. This absorption can vary over a 10,000-fold range, 
from water of low iron content which can be disinfected in a duct in much 
the same way as air, when due allowances are made for its greater absorp- 
tion and the increased ultraviolet tolerance of wet bacteria, to milk and 
serum which must be processed in films of thicknesses less than a few 
thousandths of an inch. 

WATER DISINFECTION 

Water was the first liciuid to be disinfected by ultraviolet and with 
commercial equipment using high-pressure mercury arcs. The method 
could not compare with chlorine disinfection, economically, and it did not 
provide any evidence of effective use as does "residual chlorine." 
Although low-pressure sources of germicidal ultraviolet have greatly 
reduced the cost, ultraviolet is now used only in instances where chemical 
methods or boiling cannot be tolerated and where there may be routine 
bacteriological control, such as in beverage, food, and pharmaceutical 
processes. 

Water-borne E. coli recjuire an 8-10 times greater ultraviolet exposure 
for a given kill than when air-borne, 150-200 ultraviolet /iw-min/cm- for 
a theoretical 99 per cent kill in distilled water (Luckiesh and Holladay, 
1944). The ultraviolet absorption of all water is much greater than that 
of air and of itself varies more than tenfold from a 90 per cent absorption 
in 5 in. to the same absorption in 50 in. (Fig. 2-16, from data by Luckiesh 
('/ a/., 1947, 1949). This variation is apparentl}^ almost entirely due to 
dissolved iron salts. Since water readily acciuires sufficient iron for such 
\ariations in absorption from contact with iron pipes and storage tanks, 



78 



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APPLICATIONS AND SOURCES OF ULTRAVIOLET 



79 



regardless of its previous condition, water-disinfecting equipment must 
he designed to ensure adequate treatment of the most absorptive water, 
with some provision for effective use also with water of considerably less 
absorption. Because of these factors, water disinfection must be based 
on exposures of the order of 700-1000 ultraviolet /xw-min/cm^. 

Water-disinfecting Devices. Ultraviolet water-disinfecting devices fall 
into three general classes: (1) devices in which water under pressure 
is disinfected by lamps either immersed in the water or separated from it 
by a concentric jacket, (2) devices in which the lamps are isolated in an 
air-pressure chamber over the surface of the water, and (3) devices which 




IP OP G 

Fig. 2-17. Basic types of ultraviolet water-disinfecting devices. I. P., source immersed 
in water under pressure. O.P., source in air over water, subject to air pressure and 
gravity flow. O.G., open gravity type. 

provide for disinfection of free-flowing water in a trough over which ultra- 
violet sources may be suspended. The open gravity type is used in fac- 
tory processes involving small quantities of water and in isolated camp 
and farm installations where there may already have been a routine bac- 
teriological control. A variety of pressure-type units have been devel- 
oped commercially, usually with considerable difficulty, where the ultra- 
violet source has been entirely surrounded by water but isolated from it 
by a concentric ultraviolet-transmitting jacket because of ultraviolet- 
absorbing deposits on the lamp or its surrounding jacket. Complete 
immersion of the ultraviolet source is usually impractical also because of 
the excessive cooling of the mercury arc. The more complicated but 
more promising method isolates the ultraviolet source under air pressure 
and over the water in the remaining half or two-thirds of the tank. 
Examples of these typical devices are shown schematically in Fig. 2-17. 

ABSORPTIVE LICjUIDS 

Since water was first disinfected by ultraviolet with commercial equip- 
ment, reported by Recklinghausen (1914) and the entire subject reviewed 
in a U.S. Public Health Service report (1920), attempts have been made 
to disinfect more absorptive liciuids such as wine, beer, and milk. The 



80 UADIATION BIOLOGY 

method has soomed even more attractive for pharmaceutical liquids such 
as vaccines, serums, l)lood, and blood plasma, to which pasteurizing tem- 
peratures may he destructive. Such licjuids are much more al)sorpti\'e 
of ultraviolet than is water. 

Kjjvdivv Depth of Penetration. Absorption of ultraviolet by these 
li(iuids is expressed in Fig. 2-lG, in practical terms of the depth or film 
thickness through which there is a 90 per cent absorption of 2537 A ultra- 
violet. This "effective depth of penetration" can, however, be made 
fully effective only in film-spreading devices which ensure enough tur- 
l)ulence in the film to expose all particles to the full range of ultraviolet 
intensities through the depth of the film during the exposure time. 

Film Spreaders. Liciuids with effective depths of penetration less than 
0.1 in. must be irradiated in special ecjuipment which will expose those 
liquids in moving layers of less than this depth. With the more absorp- 
tive or more viscous liquids, films of controlled thickness cannot be pro- 
duced by gravity, and it is necessary to resort to centrifugal methods or 
to the wetting of moving surfaces from which the liquids are removed 
after irradiation. Some of the many possible thin-film irradiators are 
suggested schematically in Fig. 2-18. The film thickness and rate of 
flow (exposure time) is by gravity and limited to it. In type C, both 
thickness and flow are by centrifugal force and thus are subject to con- 
siderable control. In type D, the film flow is by gravity and the slope 
of the cylinder, \vhile the film is spread by centrifugal force, the latter 
to some extent decreasing the gravity flow. Type E is almost entirely 
dependent on adhesion and viscosity for the film formation and is inher- 
ently limited as to the thinness of film formed. Type C provides for 
short-time irradiation of thinner films, but with difficulty in providing the 
required high ultraviolet intensit.y. Types D and E provide for longer 
time, lower intensity irradiation, but with diflRculty in providing suffi- 
ciently thin films of the more absorptive liquids. In types B, C, and D, 
the irradiated liquid may be protected from contact wdth air and ozone by 
filling the irradiators with a neutral gas such as nitrogen or carbon diox- 
ide. In types C and E, the thickness of the irradiated film can be con- 
trolled, regardless of viscosity, by the use of mechanical film spreaders. 
Type F defines the film thickness between flat plates of fused-quartz 
glass and the exposure time by the rate of flow. All but type F have the 
time-proved feature of irradiation of and through the free-flowing and 
continually renewed surface of the irradiated liquid. Type F has the 
important feature of being inherently a closed system adaptable to con- 
tinuous pressure operation. The slower moving liquid surface in con- 
tact with the device is exposed for a longer time to compensate partially 
for the reduction of the ultraviolet intensity by the absorption of the 
liquid. Deposits of the nature of polymerization products, which usually 
form on the film contact surfaces of these devices, are less objectionable 



APPLICATIONS AND SOURCES OF ULTRAVIOLET 



81 



in all types other than F. Types C, D, E, and F have recently l)eeii 
made commercially available, primarily for the inactivation of serums and 
vaccines. Types A and B have been adapted to milk disinfection. 

Laboratory Methods. Useful laboratory methods for the ultraviolet 
disinfection of absorptive liquids have been reported. Hollaender and 
Oliphant (1946) have used a spherical fused-quartz glass flask, inclined 
about 45° from the vertical and about one-third full of liquid, rotated 
slowly on its axis. The film of li(iuid which was dragged up and over on 




MECHANICAL 
AND GRAVITY 



D E F 

Fig. 2-18. Basic types of thin-film ultraviolet irradiators for relatively opaque liquids. 



the inner side of the lower two-thirds of the flask was irradiated through 
the upper third by ultraviolet sources surrounding the flask; this pro- 
vided a closed system but only for intermittent operation. Hollaender 
has also suggested an inclined fused-ciuartz tube containing indentations 
along its lower third and paralleled above by three or more tulnilar ultra- 
violet sources which irradiate a turbulent stream of liquid through the 
uncoated upper two-thirds of the tube. This is a closed system without 
trouble from deposits and is adaptable to continuous pressure operation. 
It would seem to have practical possibilities in spite of its ineflRcient use of 
the ultraviolet sources. 



82 RADIATION lUOLOGY 

Effective Exposures. Even with absorption minimized by the thinnest 
fihn.s provided in centrifugal devices, exposures at least as great as for 
\vater-l>orne organisms would be re(iuired: 200 300 ultraviolet ^iw-min/ 
cm-. The minimum exposure for the thicker films pro\'ided by gravity 
devices might i)e increased se\-eralfold to 700-1000 ultraviolet /iw-min 
cm- such as is retiuired in the disinfection of absorptive water through 
several inches of depth. Since it is difficult to provide ultraviolet inten- 
sities greater than 10,000 ultraviolet ^w/cm- (10 ultraviolet watts/scj ft), 
the exposure times in such devices should not be less than 1-2 sec for the 
thiimest films to 4-6 sec for the thickest. 

Although the speed of 500-1000 rpm of the centrifugal devices provides 
linear film speeds of 200-400 ft/min, this is in a helical, nearly circular 
path. Only the relatively slow, forward component contributes to the 
exposure time. The centrifugal force provides this forward component 
directly only in rotors essentially conical in shape (Fig. 2-1 8C). In a 
cylindrical rotor the centrifugal force spreads the film in both directions 
perpendicular to the helical path, l)ut there is effective movement of the 
film as a whole only in so far as the gravity-pressure equilibrium is dis- 
turbed by the delivery of liquid from the film and only in so far as the 
rotor is so inclined as to permit gravity flow. Note also in this connec- 
tion that the rate of liquid flow through these devices defines the exposure 
time only through the amount actually and momentarily being processed 
in the film, an amount usually very difficult to measure accurately. 

Operating Controls. Where there may be variations in ultraviolet 
absorption or penetration into an irradiated film such as are shown in Fig. 
2-16, there must be provision in commercial devices by which the film 
thickness and the ultraviolet exposure are completely controlled bj' the 
licjuid absorption. The ideal control would provide an automatic adjust- 
ment of the exposure to the absorption with the film thickness held con- 
stant mechanically. The exposure should, in turn, be based directly on 
the ultraviolet intensity at the film surface, rather than on the electrical 
characteristics of the sources, to compensate for their output depreciation. 
Fast-acting relays and valves should stop the delivery of material 
instantly upon the shortest power failure and should provide for rejection 
or reprocessing of material remaining in the device. 

DISINFECTION OF SURFACES OF GRANULAR MATERIALS 

There is practically no penetration or reflection of ultraviolet energy 
in the irradiation of granulated or powdered materials. Only the upper 
fourth or fifth of the surface of individual particles can be irradiated at 
any given instant of time. However actively stirred or agitated, the sur- 
face of such particles is therefore efTectively irradiated only one-fourth 
to one-fifth of the time. The particles shade each other as soon as there 
is a layer more than one particle thick, so that there is a "coefficient of 



APPLICATIONS AND SOURCES OF ULTRAVIOLET 83 

.shading" similar to the coefficient of absorption of lifiuid.s which defines 
the "effective depth of agitation" for granular materials, analogous to 
the effective depth of penetration of water. Experience indicates that the 
effective depth of agitation is of the order of ten times the diameter of 
granulated products. 

The theory of treating granulated and powdered solids is practically 
the same as for highly absorptive liquids, with effective depths of agita- 
tion of the order of the effective depth of penetration into sugar syrups. 
Although the bacteria on the surface of dry sugar crystals, for example, 
may be about ten times as susceptible to ultraviolet killing as are those 
in water, this is offset by the presence of the individual sugar crystals 
on which the bacteria ride at the surface of the layer for only about a 
tenth of the time. When the crystals are in the irradiated layer, bac- 
teria on them are irradiated only about a fifth of the time on the exposed 
sides of the moving crystals. The result is that granular particles can 
be disinfected with only about one-fifth the efficiency of water disinfec- 
tion and that the method is not practical on powdery materials. The 
disinfection of the surfaces of granular materials is well illustrated by the 
commercial method used on canner's sugar. 

SUGAR 

Thermoduric bacteria survive the vacuum evaporator temperatures of 
sugar-syrup concentration and, rejected by the sugar crystals during 
formation, remain in the final film of dilute syrup left on the crystal sur- 
faces. Ordinarily harmless, they may cause serious spoilage in canned 
foods and beverages. 

Such sugar, preferably in coarse crystals in a layer of about 3>-^ in. at 
rest, is continually vibrated, stirred, or cascaded on a conveyor under 
closely spaced germicidal lamps (Fig. 2-10) providing of the order of 
23,000 ultraviolet mw/scj ft of conveyor surface. The length and speed 
of the conveyor may be such as to provide a total exposure time of 15-5 
sec for an exposure of the order of 500 mw-min/sq ft. 

GRAINS AND SMOOTH-SKIN FRUIT 

An ultraviolet method has been reported by Ewest and Leicher (1939) 
to be effective in reducing the superficial mold contamination of hard 
grains such as that which develops after storage in the tropics. A similar 
but simpler method is reported by Matelsky (1950) as effective on 
smooth-skinned fruit such as cherries. 



ULTRAVIOLET-INDUCED MUTANTS FOR NEW FUNGI 

The use of ultraviolet to produce mutants of fungi in a search for new 
or better commercial characteristics deserves mention because of its 



84 KADIATION MIOLOGY 

iiovoUy rather than its commercial importance. Emmons and FIol- 
laender (1939) showed that the curve which represents the efficiency of 
different wa\(> lenf!;ths of ultraviolet producing mutations in fungi par- 
allels closely the germicidal-action curve. Hollaendcr and l^mmons 
(1941, 194()) correlated mutants of fungi produced by ultraviolet irradia- 
tion with the naturally occurring species probably produced by sunlight. 
From this, techni(iues have been developed for obtaining mutants, for 
example, for better yields of citric and itaconic acids and of penicillin 
and the similar antibiotics (see chapters on bacteria and fungi). 



PROTECTION AND PROCESSING OF PRODUCTS 

Because of its high germicidal effectiveness compared with its other 
photochemical, erythemal, and thermal effects, ultraviolet energy, espe- 
cially of wave length 2537 A, has been used for the protection and disin- 
fection of many products of so unstable a composition as to prohibit the 
use of more conventional methods. Such applications have developed 
in food, pharmaceutical, and beverage processing and storage places. 

MOLD, ANTIBIOTICS, AND PARENTERAL FLUIDS 

The mold-derived antibiotics and many parenteral li(}uids are very 
susceptible to contamination by the normal mold and bacterial content of 
air. This contamination is often of a chemical nature precluding terminal 
sterilization by heat. Some serums and antitoxins are developed by the 
growth of bacteria and viruses which must be finally inactivated by 
methods which will not at the same time destroy the desirable properties 
of the preparation. Ultraviolet has served to protect such materials 
during processing and to provide a final inactivation where controlled 
bacterial growth has been a part of the process. The germicidal lamps 
are used in ducts and hoods, over work tables, for upper-air disinfection, 
and also for thin-film irradiation (Fig. 2-18) l)y the methods previously' 
discussed. 

BLOOD PLASMA 

One of the more unusual applications of ultraviolet is for the dis- 
infection of blood plasma of the hepatitis virus. Several commercial 
devices have been developed. All provide for irradiation in films of the 
order of }/ioi)o in- in thickness or for the violent agitation of somewhat 
thicker films. It is important to remember that, because of the low 
penetration of the ultraviolet, changes in film thickness, even of micro- 
scopic dimensions, may seriously interfere with any of these methods. 
These methods and devices have already been discussed generally for 
absorptive liquids (Figs. 2-16 and 18). Preliminary to the use of any 



APPLICATIONS AND SOURCES OF ULTRAVIOLET 85 

of those methods, there must be a complete removal by centrifugiiig or 
filtration of all clumps down to an empirically determined and specified 
size. A machine or process should produce a specified degree of sterility 
in test runs of plasma contaminated with a test organism of an ultra- 
violet-exposure tolerance comparable with that of the hepatitis virus. 
Sarcina Infca, whose packet growth habit may simulate the minute 
clumps remaining in plasma after clarification, is suggested. 

In operation there should be a continuous record of plasma flow and 
ultraviolet intensity, similar to the controls used in continuous methods of 
milk pasteurization. A cadmium photocell (Figs. 2-1 and 5b), described 
by Taylor and Haynes (1947), and a recording microammeter are suitable 
for the ultraviolet control. The rate of flow may be controlled by a pump 
with variable-speed drive. Rapidly operating relays and electric valves 
should stop or divert the delivery of plasma from an intermediate storage 
reservoir of a capacity much greater than that of the irradiating device. 

SYRUP, FRUIT-JUICE, AND WINE STORAGE 

The sugar content of sugar and fruit syrups is usually such as to 
prevent fermentation even though a mold scum ma}^ form on exposed sur- 
faces. Whenever there is condensation of moisture on the sides and tops 
of syrup-storage tanks, it may dilute the surface layer of the stored syrup 
enough to permit destructive and otherwise very objectionable fermen- 
tation in addition to the usual mold formation. Germicidal tubes are 
being used to prevent such fermentation and mold on the surfaces of 
tank-stored sugar and fruit-juice syrups used for soft drinks and con- 
fectionery. Continuous irradiation with an intensity of at least 5 ultra- 
violet mw/sq ft is required. 

MEAT STORAGE 

The most extensive single industrial use of germicidal lamps is to 
reduce the growth of bacteria and molds on the surface of meat and on 
shelves, walls, and floors of retail-meat-storage refrigerators operated 
at 35°-45°F. There is little need for such provisions in cold-storage 
rooms operated below zero, nor has there been any such need in the larger 
meat-processing factories where exact control of temperature, humidity, 
and air movement produces similar results. Proper use of the ultraviolet 
does not take the place of established periodic sanitary maintenance but 
does supplement it by a continuous suppression of spoilage and odors. 
The ultraviolet intensities required are of the order of only the 5 mwsci ft 
effective for mold suppression in other applications. 

Ultraviolet is effectively used to suppress surface slime molds on meat 
stored for 3 days at a temperature of about 60°F for rapid aging or 
tenderization. Contrary to the impression of some, the ultraviolet has 
no direct effect on the enzymatic tenderizing. 



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APPLICATIONS AND SOUHCES OF ULTKAVIOLET 87 

HIGHER PRESSURE MERCURY SOURCES OF ULTRAVIOLET 

Electric-discharge mercury lamps in tubes of high ultrav^iolet trans- 
mission develop relatively full-line spectra when operated at higher 
(over 400 mm or 0.5 atm) pressure. The total ultraviolet per watt of 
electrical input drops to about one-half that of a low-pressure lamp in 
tubes of the same transmission. The energy is distributed variably 
among about 20 lines instead of being almost entirely concentrated at 
2537 A, as shown graphically in Fig. 2-5. Lines other than 2537 A of 
good energy content become very useful for studies of biological effects 
as functions of wave length. The high intrinsic output of the higher 
pressure sources becomes essential for effects requiring ultraviolet inten- 
sities unobtainable even at the surface of the low-pressure sources. 
High-pressure lamps in the lower wattage sizes are essential to any 
research involving isolation of spectral lines or bands by optical methods. 
Their adaptability can be inferred from the source dimensions given in 
Tables 2-3 and 6, based partially on the lES Lighting Handbook, 2d ed 
(1952). 

INTENSITY AND VARIATIONS WITH DISTANCE 

The radiant-energy intensity at a distance of 1 meter, in microwatts 
per square centimeter, can be approximated from the total watts output 
rating of Table 2-6 by multiplying by a factor of 10. Conversion to 
other units and distances can be made by the methods outlined for low- 
pressure sources. 

INDIVIDUAL LINE INTENSITIES 

Persons who need a more detailed analysis of the line spectra of the 
sources listed in Tables 2-3 and 6 but who do not have facilities for making 
line-intensity measurements under the actual conditions of their experi- 
mentation should correspond directly with manufacturers of the source 
being used. The relative energy distribution among the lines of high- 
pressure mercury lamps varies between individuals and between groups 
in such a complicated fashion that no general rules can be given and 
detailed listings here become impractical. A fairly representative high- 
pressure spectrum is shown graphically in Fig. 2-5 and is typical of such 
lamps as the UA-3 and UA-1 1 of Table 2-6. 

STARTING AND RESTARTING TLMES 

With few exceptions, the higher pressure lamps start with low-pressure 
characteristics and require 3-5 min to reach normal operating temper- 
ature, pressure, and radiation output. If momentarily extinguished, they 
also require a cooling and restarting period equal to or in some cases 
one-half longer than their warm-up time. 



88 RADIATION HIOLOGY 

LIP'E AM) l)i;i'Hi:('IATION 

Life ratings of all electrit'-dist'harge lamps involve complicated factors 
of economy as \vi>ll as physical mortality. The hurn-out life is usually 
that at which one-half the lamps of a test fiiroup reach a physical or 
economic end of life. The latter is very dependent on variable factors of 
lamp and electric costs and the nature of the ai^plication. For oj)erating 
intervals of less than 5 hr the life decreases rapidly with the length of the 
operating interval. For longer operating intervals the total life may 
increase to several times the 5-hr interval rating. The general form of 
the depreciation curve is that of Fig. 2-11. 

An important variable in the depreciation of all higher pressure mercury 
arcs is the "solarization" effect of the ultraviolet and heat on the trans- 
mission of the fused (juartz generally used. The effect increases as the 
wave length decreases and may reduce the output of 2200-2500 A energy 
to one-third the initial and of 2500-2800 A to one-half the initial in a 
few hours of operation. This accounts for some of the discrepancies in 
published data on the shorter ultraviolet output of commercial sources. 
The effect is relatively small at wave lengths longer than 2800 A. 

RESEARCH DETERMINATION OF OUTPUT AND INTENSITY 

As was emphasized for low-pressure sources, although their commercial 
radiation ratings should be fully specified in all research reports, these 
ratings can be considered only a first approximation to the energy output 
and intensity actually effective in any specific research. Whenever 
direct measurements of energy at the point of application are impractical, 
calibrated lamps should be used. The intensities that they provide can 
be calculated for various distances by the methods outlined for low- 
pressure sources as long as there are no intervening optical systems or 
filters. 

In work at wave lengths less than 2800 A, and especially with the higher 
pressure sources, the source itself should be frequently calibrated for out- 
put where direct measurements of the irradiated surfaces of material are 
in practice. 

MERCURY-AMALGAM AND OTHER MET.\L ARCS 

Laboratory workers generally fabricate their own electric-discharge 
metal- vapor arcs, other than mercury, although such lamps have been 
occasionally imported for sale in the United States. Various commercial 
types availal)l(' in the i)ast are described by Meyer and Seitz (1949). 
Their availability is too uncertain and thcii- radiation characteristics are 
too unstandardized for inclusion in this chapter. It should be noted 
here, however, that cadmium-amalgam lamps proxiding low-intensity 
cadmium lines of wa\-e lengths greater than .SOOO A arc commercially 



APPLICATIONS AND SOURCES OF ULTRAVIOLET 



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90 



KADIATION HIOLOCJY 



;i\:iihil)l(' as arc also similar ijotassiuin and sodium lamps. Xoiic ol llicm 

arc, howcx'cr, sources of ult i'a\'iolct. ol the \va\'c lcnti;llis jicncially desired 

in biological work. 

SUN I,. \ MI'S 

Commercial sunlamps have been used for some biological research. 
They provide energy down to 2800 A from sources of sufficiently high 
intrinsic intensity to adapt them to optical methods of isolating narrow 
spectral regions. They have the general characteristics of high-pressure 
quartz-mercury arcs with the radiation limited to about 2800 A by the 
enclosing bulbs or tubes. Laboratory housings for the S-4 lamp have 
becMi comnKM-cially available. Lamp ('II-3 of Table 2-6 is proving more 




2000 2400 2800 3200 3600 4000 4400 4800 5200 5600 6000 6400 

WAVE LENGTH, A 

Fig. 2-19. Relative energy distril)uti()ii of fluorescent and S-4 type .sunlamps. 

useful in the same devices because it provides essentially the full (|uartz- 
arc spectrum. Table 2-7 permits comparison with sources of Tables 2-3 
and 6. Figure 2-19, which shows the relative energy distribution of sun- 
lamps, is comparable with Fig. 2-5. The S-1 lamp spectrum is similar 
to that of the S-4 but with addition of a continuous visible and infrared 
spectrum from a tungsten filament in multiple with the mercury arc and 
operating at a temperature of about 2000° K. The RS spectrum has 
similar visible and infrared components from a tungsten filament in series 
with the arc. 

As indicated in Fig. 2-19, tluorescent sunlamps provide a continuous 
ultraviolet spectrum with a peak at about 3100 A. Mercury-arc lines 
transmitted by the phosphor make an insignificant addition to it. The 
generated energy in these lines is comparable with that in the lines of the 
germicidal lamp and is transmitted to an extent of 60-70 per cent by the 
phosphor. Although the low emission intensity of these lamps makes 
them unsuitable for use with optical isolating systems, they are compara- 
ble with the high-pre.ssure mercury arcs for low-intensity general irradia- 
tion with energy of wave lengths in the 3000-3200 A range. They are 



APPLICATIONS AND SOURCES OF ULTRAVIOLET 91 

unique sources of energy in the 3200-3400 A region of the ultraviolet 

spectrum. 

REFERENCES 

Ainerican ^lodical Association, Council on Physical Medicine (1948) Acceptance 
of ultraviolet lamps for disinfecting purposes. J. Am. Med. Assoc, 137: 
1600-1603. 

American Public Health Association, Subcommittee for Evaluation of Methods to 
Control Air-borne Infections (1947) The present status of the control of air- 
borne infections. Am. J. Pub. Health, 37: 13-22. 

Buchbinder, L., M. Solowey, and E. B. Phelps (1941) Studies on microorganisms in 
the simulated room environments. III. The survival rates of streptococci in the 
presence of natural, daylight and sunlight, and artificial illumination. J. 
BacterioL, 42: 353-366. 

Huttolph, L. J. (1945) Principles of ultraviolet disinfection of enclosed spaces. 
Heating, Piping Air Conditioning, 17: 282 290. 

(1951) Ultraviolet air disinfection in room air conditioners. Refrig. Eng., 

59: 54-57, 73. 

Caspersson, T. (1931) Uber den chemischen Aufbau der Strukturen des Zellkernes. 
Skand. Arch. Physiol., Suppl. 8 {also Referat, Protoplasma, 27: 463-467, 1937). 

Del Mundo, F., and C. F. McKhann (1941) Effect of ultraviolet irradiation of air on 
incidence of infections in an infants' hospital. Am. J. Diseases Children, 61: 
213-225. 

Downs, J. (1950) Control of acute respiratory illness by ultraviolet lights. .\m. J. 
Pub. Health, 40: 1512-1520. 

DuBuy, H. G., J. E. Dunn, F. S. Brackett, W. C. Dreesen, P. A. Neal, and I. Posner 
(1948) .\n evahiation of ultraviolet radiation of sleeping quarters as supplement 
of accepted methods of disea.se control. Am. J. Hyg., 48: 207-226. 

Ellis, C, A. A. Wells, and F. F. Heyroth (1941) The chemical action of ultraviolet 
rays. 2d ed., Reinhold Publishing Corporation, New York. 

Emmons, C. W., and A. HoUaender (1939) The action of ultraviolet radiation on 
dermatophytes. II. Mutations induced in cultures of dermatophytes b^- expo- 
sure of spores to monochromatic ultraviolet radiation. Am. J. Botany, 26: 
467-475. 

Ewell, A. W. (19-t2) Production, concentration, and decomposition of ozone by 
ultraviolet lamps. J. Appl. Phys., 13: 759-767. 

Ewest, H., and A. Leicher (1939) Getreideentmuffung durch ultraviolette Strahlen. 
Miihle, 76: 569-570. 

Gates, F. L. (1929-30) .\ study of the bactericidal action of ultraviolet light. J. 
Gen. Physiol., 13: 231-260. 

Hart, D. (1936) Sterilization of air in the operating room by special bactericidal 
radiant energy — results in thoracoplasties. J. Thoracic Surg., 9: 520. 

Holiaender, .\., and W. D. Claus (1935-36) The bactericidal effect of ultraviolet 
radiation on Escherichia coli in liquid suspensions. J. Gen. Physiol., 19: 753-765. 

Holiaender, A., and C. W. Emmons (1941) Wavelength dependence of mutation 
production in the ultraviolet with special emphasis on fungi. Cold Spring 
Harbor Symposia Quant. Biol., 9: 179-186. 

(1946) Induced mutations and speciation in fungi. Cold Spring Harbor 

Symposia Quant. Biol. 11: 78-84. 

Holiaender, A., M. F. Jones, and L. Jacobs (1940) The effects of monochromatic 
ultra\'iolct radiation on eggs of the nenuitode, Enlirohiux venniciilnris. I. Quan- 
titative response. J. Parasitol., 26: 421-432. 

Holiaender, A., and J. W. Oliphant (1946) Experimental inactivation of etiologic 



92 RADIATION BIOLOGY 

agt'iit ill scruiii hy ultraviolet irradiation. U.S. Pub. Health Service, I'ul). 

Health Kept. tJl: oUS (■>()2. 
Illuininatiiifi; iMinineeriiif!; Soeiety (l'J52) IKS liniitiiiK handbook. 2d ed., lUuiiii- 

natiiin iMi^ijiiieerinf!; Soeiety, New York, 
luteniatioiud (^oniniission on Illuinination, Berlin (UKiS) Compt. rend., •.»: 596-625. 
Jarrett, K. T., M. l^ Zelle, and .\. HoUaeiuhT (1<)J8) Studies of the control of aeute 

respiratory disea.se among \aval reeruits. .Vni. J. Hyg., 48: 2'.i'.i-2'.i\) . 
Jones, M. F., L. Jacobs, and A. Hollaender (1940) The effects of monoclnoin.itic 

ultraviolet radiation on eggs of the nematode, Enterobius vermicularis. II. Sub- 
lethal effects. J. Parasitol., 26: 4:^5 445. 
Kelner, A. (1949) Effect of visit)le liglit on the recovery of Slreptomyces griseua 

conidia from ultraviolet irradiation injury. Froc. Natl. Acad. Sci. U.S., 35: 73-79. 
KoUer, L. R. (1946) Ozone production by low-pressure mercury arcs. Gen. Elec. 

Rev., 49: 50-53. 

(1952) lUtraviolet radiation. Jolm Wiley & Sons, Inc., New York. 

Langnniir, A. I)., Iv T. Jarrett, and A. Hollaender (1948) Studies of the control of 

acute respiratory diseases among Xaval recruits. Am. J. Hyg., 48: 240 250. 
Laurens, H. (1933) The physiological effects of radiant energy. Chemical Catalog 

Company, Inc., New York. 
Lea, D. E. (1946) Actions of radiations on living cells. Cambridge University 

Press, Cambridge, England (also The Macmillan Company, New York, 1947). 
Luckiesh, M. (1946) Applications of germicidal, erythemal and infrared energy. 

D. Van Nostrand Company, Inc., New York. Pp. 110-117. 
Luckiesh, M., and L. L. Holladay (1942a) Designing installations of germicidal 

lamps for occupied rooms. Gen. Elec. Rev., 45: 343-349. 

(1942b) Tests and data on disinfection of air with germicidal lamps. Gen. 

Elec. Rev., 45: 223-231. 

(1944) Disinfecting water by means of germicidal lamps. Gen. Elec. Rev., 



47: 45-50. 
Luckiesh, M., and A. H. Taylor (1946) Transmittance and reflectance of germicidal 

(X2537) energy. J. Opt. Soc. Amer., 36: 227-234. 
Luckiesh, M., A. H. Taylor, and G. P. Kerr (1944) Germicidal energy — a practical 

method of measuring transmission and absorption of germicidal energy' by water. 

Gen. l']lec. Rev., 47: 7-9. 
Luckiesh, M., A. H. Taylor, T. Knowles, and E. T. Leppelmeier (1947) Killing air- 
borne respiratory microorganisms with germicidal energy. J. Franklin Inst., 

244: 267-290. 
(1949) Inactivation of molds by germicidal ultraviolet energy. J. Franklin 

Inst., 248: 311-325. 
Lurie, M. B. (1946) Control of air-borne contagion of tul)erculosis. Am. J. Nursing, 

46: 808-810. 
Matelsky, I. (1950) Rays curb bacteria, boost fruit quality. Food Ind., 22: 1722- 

1723. 
Meyer, A. E. H., and E. O. Seitz (1949) Ultraviolette Strahlen. 2d ed., Walter de 

Gruyter & Co., Berlin. 
Miller, W. R., E. T. Jarrett, T. L. Willmon, A. Hollaender, K. W. Brown, T. Lewan- 

dowski, and R. S. Stone (1948) Evaluation of ultraviolet radiation and dust 

control measures in control of respiratory di.sease at a Na\al training center. 

J. Infectious Diseases, 82: 86-100. 
National Research Council, Committee on Sanitary lOtigineering (1947) Recent 

studies on disinfection of air in military estabiisiiinents. Am. .1. P<ib. Health, 

37: 189-198. 
Overholt, R. IL, and R. H. Betts (1940) Comparative report on infection of thoraco- 



APPLICATIONS AND SOURCES OF ULTRAVIOLET 93 

plasty wounds; experiences with ultraviolet irradiation of operating room air. 

J. Thoracic Surg., !): 520-529. 
Perkins, J. E., A. M. Bahlke, and H. F. Silverman (1947) Effect of ultraviolet 

irradiation of classrooms on spread of measles in larsc rural central schools. 

Am. J. Pub. Health, 37: 529-537. 
Rahn, O. (1932) Physiologj^ of bacteria. Blakiston Company, Philadelphia. 
(1945) Physical methods of sterilization of microorganisms. Bacteriol. 

Revs., 9: 1-47. 
Recklinghausen, AI. v. (1914) Sterilization of water by ultraviolet rays of the mer- 
cury-vapor quartz lamp. Proc. Am. Inst. Elec. Engrs., 33: 1217-1242. 
Robertson, E. C, M. E. Doyle, and F. F. Tisdall (1943) Use of ultraviolet radiation 

in reduction of respiratory cross infections in a children's hospital — final report. 

J. Am. Med. Assoc, 121: 908-914. 
Robertson, E. C, M. E. Doyle, F. F. Tisdall, L. R. Koller, and F. S. Ward (1939) 

Air contamination and air sterilization. Am. J. Diseases Children, 58: 1023- 

1037. 
Sauer, L. W., L. D. Minsk, and I. Rosenstern (1942) Control of cross infections of the 

respiratory tract in a nursery for young infants: a preliminary report. J. Am. 

Med. Assoc, 118: 1271-1274. 
Schneiter, R., A. Hollaender, B. H. Caminita, R. W. Kolb, H. T. Eraser, H. G. DuBuy, 

P. A. Neal, and M. B. Rosenblum (1944) Effectiveness of ultraviolet irradia- 
tion of upper air for the control of bacterial air contamination in sleeping quarters 

— preliminary report. Am. J. Hj^g., 40: 136-153. 
Taylor, A. H., and H. Haynes (1947) New meters for germicidal energy. Gen. 

Elec. Rev., 50: 27-29. 
U.S. Public Health Service (1920) Ultraviolet rays in water purification. U.S. 

Pub. Health Service, Pub. Health Kept., 34: 2831-2834. 
Vandiviere, H. M., C. E. Smith, and E. J. Sunkes (1949) Unpublished report at the 

American Public Health Association meeting. New York, Oct. 28, 1949. 
Wells, M. W. (1945) Ventilation in the spread of chickenpox and measles within 

school rooms. J. Am. Med. Assoc, 129: 197 200. 
Wells, M. W., and W. A. Holla (1950) Ventilation in the flow of measles and chicken- 
pox through a community. J. Am. Med. Assoc, 142: 1337-1344. 
Wells, W. F. (1940) Bactericidal irradiation of air, physical factors. J. Franklin 

Inst., 229: 347-372. 
Wells, W. F., and H. L. Ratcliffe (1945) The behavior of inhaled particles in different 

states of aerosol suspension as indicated by pulmonary tuberculosis in rabbits. 

Am. J. Med. Sci., 209: 412-413. 
Wells, W. F., H. L. Ratcliffe, and C. Crumb (1948) Mechanics of droplet nuclei 

infection. II. Quantitative experimental air-borne tuberculosis in rabbits. 

Am. J. Hyg., 47: 11-28. 
Wells, W. F., M. W. Wells, and T. S. Wilder (1942) Environmental control of 

epidemic contagion, an epidemiologic study of radiant disinfection in day schools. 

Am. J. Hyg., 35: 97-121. 
Wheeler, S. M., H. S. Ingraham, A. Hollaender, N. D. Lill, J. Gershon-Cohen, and 

E. W. Brown (1945) Ultraviolet light control of air-l^orue infections in a Naval 

training center. Am. J. Pub. Health, 35: 457-468. 
Willmon, T. L., A. Hollaender, and A. D. Langmuir (1948) Studies of the control of 

acute respiratory diseases among Naval recruits. Am. J. Hyg., 48: 227-232. 
Wyckoff, R. W. G. (1932) The killing of colon bacilli by ultraviolet light. J. Gen. 

Physiol., 15: 351-361. 

Manuscript received by the editor Aug. 4, 1952 



CHAPTER 3 

Sunlight as a Source of Radiation 

J. A. Sanderson and E. O. Hulburt 

Naval Research Laboratory 
Washington, D.C. 

The sun as o radiator. Sunlight on top of the atmosphere. Solar ultraviolet radiation 
at the earth's surface. Atmospheric ozone. Calculated ultraviolet intensity at the earth's 
surface. Observed ultraviolet intensity at the earth's surface. Solar infrared spectrum 
at the earth's surface. References. 

THE SUN AS A RADIATOR 

As a star, the sun is quite ordinary ; it is placed by astronomers in spec- 
tral class Go of yellow stars which, on the average, are about one one- 
hundredth as bright as average blue stars and a hundred times brighter 
than average red stars. It is located near the middle of the main sequence 
into which stars fall when absolute magnitudes are plotted against spec- 
tral class. As the source of a spectrum of radiations extending from 
X-ray to radio wave lengths which fall on the earth, conveniently situated 
to utilize them in sustenance of life, it is a fascinating object of never- 
ending study. The story of the sun has been well written by Abetti 
(1938), by Menzel (1949), and by Hoyle (1949). Hoyle devoted his 
effort to specialized problems of solar physics, while Abetti and Menzel 
gave more general discussions of knowledge of the sun. The radiations 
of the sun probably derive their energy from nuclear reactions deep in 
the gaseous interior, where the temperature is about 40 million degrees 
and the density is 76 compared with a density of 1.4 for the entire sun. 
These radiations finally emerge from the outer layers, mainly at tempera- 
tures of 4000°-G000°K, and then travel unmodified for 8 brief minutes 
until they reach the svn-face of the earth, at which point they are the 
principal interest of this chapter. 

Since the sun is gaseous throughout, transitions in temperature, the 
states of matter, and the character of the radiation emitted are gradual 
along the radius. Yet several marked distinctions in these properties 
exist, which permit the division of the outer reaches of the incandescent 
mass of gas into several regions, each characterized l\v its radiative prop- 
erties. The photosphere, or light sphere, is the innermost and most 

95 



96 RADIATION" HlOI.onv 

sharply deliiiccl of these t raiisitioiuil regions. When viewed in visible 
light the photosphere appears to ))e a sharply defined disk, 1.39 X 10^ km 
in diameter, considerably brighter at the center than at the edge, or limb. 
It is the source of the continuous spectrum of the sun, or rather, the re}>;i()n 
in the sun in which predominantly contiiuious emission changes rather 
abruptly into emission of spectral lines. This transition, owing to a 
rapid decrease in opacity of the solar gases in the region of the photo- 
sphere, takes place in a few hundred kilometers in a level where the pres- 
sure is about 10~' atmosphere. 

The transmission coefficient tx of a homogeneous layer of absorbing gas 
for light of wave length X is given by 

h = r-'-x" (3-1) 

Avhere «x = the absorption coetficient for wave length X and h = the 
thickness of the absor))ing layer. The sharpness of the plnjtosphere 
depends on the circumstance that, for visible light, the values of a\h are 
sufficiently high that a relatively thin layer h of the gases is opaque; that 
is, the i)roduct a^h is very great. Relative to any comparison wave 
length, say, in the visible region, radiation emerging from the sun at other 
wave lengths where ax is greater will come from higher and therefore 
cooler layers, and the intensity will be lower. At wave lengths where ax 
is smaller, a thicker layer of the sun is required to be opaque, and the 
radiations which escape come from a region of higher temperature and 
are, accordingly, relatively more intense. Thus a part of the visible and 
near-infrared continuum of the photosphere matches the spectrum of a 
()000°K black body rather well, whereas the blue and ultraviolet intensi- 
ties lie considerably below a (i()00°K source, and there is evidence that the 
infrared spectrum in the 8- to 13-ju region is fitted better by a 7000° K 
intensity curve. 

Although the photosphere is sharp in visible light, it is not uniformly 
l)right along the diameter, the center of the disk being considerably 
brighter than the edge. This phenomenon, readily observable with the 
eye or in photographs of the sun, was examined by Abbott et al. (1922) at 
seven wave lengths between 3737 and 10.080 A. Considering the inten- 
sity at the center of the disk to be unity for each wave length, it was 
found that the intensity decreased toward the edge of the sun for all 
wave lengths and that the diminution in intensity was more pronounced 
for short than for long wave lengths. For example, at a distance from 
the center equal to 0.95 of the photospheric radius, the intensity of wave 
length 3737 A was 0.4319 of the central intensity, whereas at 10,080 A it 
was 0.7331 of the central intensity. Thus the limb of the sun is not only 
less bright but is also redder than the center. The effect is explained by 
the considerations in the foregoing discussion and is probably due both 
to absorption and scattering by overlying layers of gases and to the fact 



SUNLIGHT AS A SOURCE OF UADIATIOX 97 

that radiations Avhirh emerge from the edge of the spherical sun come 
from higher and cooler levels than the radiations from the center of the 
disk which must pass through minimal thickness of overlying gases in 
escaping. Since the attenuation by scattering and absorption is greater 
for short than for long wave lengths, the effect is more pronounced in the 
blue than in the red end of the spectrum. 

Although the sun is entirely gaseous, the sharpness of the photosphere, 
in comparison with more nebulous layers above it, has led to the custom 
of referring to these latter regions as the atmosphere of the sun. There 
are three regions of the solar atmosphere distinguishable by the states in 




Fig. 3-1. Ultraviolet solar spectruiu ol)tained in 1942 at Arosa, Switzerland. {Gotz 
and Caspar is, 1942.) 

which matter exists in them and by the radiations which they emit — the 
reversing layer, the chromosphere, and the corona. The reversing layer 
is the innermost of these regions and lies just above the photosphere. 
It extends to a height of 1500 km above the photosphere and is the region 
in which the transition from continuous to line emission occurs. The 
temperature is about 4830°K, and the pressure is probably 10"^ to 10~^ 
atm. In the reversing layer the dark Fraunhofer lines of the solar spec- 
trum are formed by atomic absorption at discrete wave lengths of the 
continuous radiation from the underlying hotter photosphere. The ultra- 
violet spectra of Figs. 3-1 and 2 show many of the large number of 
Fraunhofer lines in the biologically effective region of the solar spectrum. 
Babcock et al. (1948) have investigated the ultraviolet solar spectrum 
between 2935 and 3050 A with a 21 -ft grating spectrograph and have 
listed 665 absorption lines in this erythemal region. 

It must not be supposed that the dark lines represent points of zero 
intensity in the solar spectrum; they appear so in Figs. 3-1 and 2 because 



98 UADIATION lUOLOCiY 

of liij^h phototiiiapliic contrast. Duiiiiji a tcjtal oclipso of tlic .sun when 
the moon ohscurots the pliotosphcrc, llie reversing layer and regions 
al)ove it are .seen to emit l)riliiant spectral lines, the so-called "flash 
spectrum." Although the dark lines reduce the intensity of higher tem- 
perature radiation from the photosphere, they are (^nly relatively dark 
and radiate toward the earth with inttMisities appropriate to a lower tem- 
perature source at about 4800° K. 

The Fraunhofer absorption is stronger in the ultraviolet than in the 
visible solar spectrum. Pettit (1940) mapped the spectral energy 
between the Fraunhofer lines, using a high-dispersion spectrograph and 



2900 2800 2700 2600 2500 2400 2300 2200 2100 

WAVE LENGTH, A 
Fig. 3-2. Ultraviolet solar spectrum obtained during a rocket flight of June 14, 1949, 
by Johnson, Purcell, and Tousey (1952). 

sensitive photocells, and, from his measurements, estimated the attenua- 
tion of radiation from the photosphere by the overlying layers of the sun. 
Comparison of his results for integrated radiation from the sun and for 
radiation between the Fraunhofer lines indicates that the total intensity 
between 3200 and 4000 A is about 70 per cent of the radiation which 
would be emitted by the unobscured photosphere, whereas between 4000 
and 7000 A the ratio is about 91 per cent. 

The chromosphere is a region consisting principally of hydrogen, helium, 
and calcium, located immediately above the reversing layer; it extends 
from the top of the reversing layer at about 1500 km above the photo- 
sphere to a height of 12,000 km which is the greatest height at which H„, 
the first Balmer line (6563 A), occurs. Other lines of the Balmer series 
of hydrogen and line spectra of other un-ionized atoms fade out at lower 
heights. The fla.sh spectrum of the chromosphere as seen during a solar 
eclip.se contains emission lines of helium and ionized helium together with 
lines of ionized metals. Menzel points out the existence of ionized helium 
in the chromosphere and Hoyle gives results obtained from studies of line 
widths in the flash spectrum as evidence that the temperatures in this 
region are 20,000-30, 000°K. Prominences originating in the chromo- 
sphere and sometimes rising several hundred thousand miles also contain 
matter at these temperatures. The chromosphere also emits continuous 
radiation, but none of the.se emissions — the line spectra, the spectra of 



SUNLIGHT AS A SOUKCi: OF KADIATION 99 

prominences, or the continuum — are strong. The quantity a\h in Eq. 
(3-1) is small throughout the chromosphere, and light from the underlying 
layers is readily transmitted. Since the chromosphere is a weak absorber, 
it is a weak emitter. 

The corona is the outermost observable region of the sun, being observa- 
ble only during a solar eclipse or by use of the coronagraph. The corona 
begins in the region where the total continuous radiation from the solar 
atmosphere is about equal in intensity to the total line emission, and this 
region lies about 12,000 km above the photosphere. The corona extends 
outward for very great distances. S. P. Langley is said to have observed 
a coronal streamer extending to 12 solar diameters during the eclipse of 
1878. Photographs usually show the corona extending to about 1 solar 
diameter because of the rapid decrease in its brightness with increasing 
height. 

The visible light from the corona consists principally of light from the 
photosphere scattered by electrons, but emission lines of highly ionized 
calcium, iron, nickel, and argon are also present, the most intense being 
the green line at 5303 A due to Fe(XIV), that is, iron with half its 26 elec- 
trons removed. Temperatures of about one million degrees are required 
to produce the states of ionization and other effects observed in the 
corona. Nevertheless, the entire visible light from the corona is about 
half that of the full moon, or about one one-millionth that of the sun, and 
its contribution to the visible light and ultraviolet radiations which reach 
the surface of the earth is inconsequential. The corona and upper 
chromosphere are, however, of interest as the source of radio waves and of 
X rays emitted by the sun. Although these emissions are probably too 
weak to be of biological importance, they deserve a brief description. 

Radio emissions originating outside the earth, presumably from inter- 
stellar space, were discovered in 1932 by Jansky (1933) in experiments 
with 30-meter waves, but emissions from the sun were not known until 
1945 when the improved sensitivity of receiving techniques brought 
about their detection (Hagen, 1951). It was found that the solar radio 
waves originate in the upper chromosphere and corona in thick regions 
of the solar atmosphere which center at heights above the photosphere of 
approximately 8000, 10,000, 13,000, and 18,000 km for wave lengths 0.8, 
3, 10, and 50 cm, respectivel3^ For example, during optical totality of 
the solar eclipse of May 20, 1946, the solar radiations in the respecti^'e 
wave lengths were reduced to 1, 6, 19, and 33 per cent of their values for 
the uneclipsed sun. To account for the observed radio-wave energy the 
temperatures of the regions which were emitting the wave lengths 0.8, 3, 
10, and 50 cm were calculated to be 7000, 10,000, 2(),000, and212,000°K. 

In contrast with the limb darkening of the sun for visible light, when 
observed with radio waves, the sun brightens at the limb, the l)rightening 
increasing with the wave length. The solar radio emission is not constant 



100 UADIATION Hlol-OGY 

in intcihsity but varies in an iin'{;iiiai- and unpredictable manner. At the 
shortest wave length the intensity is most constant. The correlatioii 
with sunspot area is poor at the shortest wave length and improves at a 
wave length of about 10 cm. At longer wave lengths the fluctuations in 
the intensity become more erratic and abrupt. \'ig(>i-()us investigation of 
all these phenomena is in progress. 

X rays and short ultraviolet radiations from the sun which had never 
been detected at sea level were measured by narrow-band photon counters 
carried aloft in a rocket (Friedman et al., 1951) on Sept. 29, 1949. Ultra- 
violet radiation in the wave-length band 1150-1350 A was observed 
above 05 km altitude, and in the band 1425-1700 A above 100 km. 
Solar X-ray emission was first recorded at 85 km with a counter sensi- 
tive from to 10 A, which indicated, because of the known absorption 
of the atmosphere, that the solar emission became undetectable below 
1 A. The measured intensities recjuired effective temperatures of the 
emitting regions, again probably the upper chromosphere and corona, of 
4500°, 5000°, and 10«°K for the bands 1425-1700, 1150-1350, and 7-10 A, 
respectively. It seems nearly certain that X rays longer and softer than 
10 A are emitted by the sun, and further rocket experiments have been 
planned to investigate the subject. 

SUNLIGHT ON TOP OF THE ATMOSPHERE 

Two methods have been used to determine the solar spectral energy on 
top of the atmosphere: (1) by measuring the spectral intensity of the 
sunlight reaching the surface of the earth and correcting for the trans- 
mission of the terrestrial atmosphere, and (2) by sending apparatus on 
rockets to altitudes above most of the atmosphere. Both methods are 
difficult; the first has been in use for many years but can obviousl}^ give 
information about only those wave lengths which are detected at the 
earth's surface; the second is relatively recent and has yielded important 
new results in the ultraviolet portion of the spectrum. In the first 
method, unfocused radiation from the entire sun is allowed to fall on the 
slit of a double monochromator, that is, two monochromators in series in 
order to reduce contamination, by radiation scattered by the optical 
parts of the instrument, of the spectrum falling on the exit slit. Back of 
the exit slit, in a position to intercept the portion of the spectrum emerg- 
ing from the monochromator is mounted a bolometer, a thermocouple, or 
a calibrated photoelectric cell with which the intensity may be measured 
point by point throughout the spectrum or in selected portions of it. 
Glass or quartz prisms and lenses in the monochromator are used to 
measure the major portion of sunlight reaching the surface of the earth, 
which lies between 2900 A and 2.5 ju- Rock-salt pi-isms and diffraction 
gratings are used to observe the infrared solar spectrum from 2.5 to 25 n. 
This portion of the spectrum has been of little direct interest in bio- 



SUNLIGHT AS A SOURCE OF RADIATION 101 

physics, although it is of impcjrtaiR-e in the physical state of the atmos- 
phere and in meteorology. 

The measurement of the spectral distribution of intensity in sunlight 
and its correction for atmospheric attenuation has been a major function 
of the Astrophysical Observatory of the Smithsonian Institution, begin- 
ning in 1892 under the direction of S. P. Langley who invented the bolom- 
eter and first measured the spectral distribution of energy in the solar 
spectrum, and continuing under Abbott and others. Their measure- 
ments have been made at Mt. Wilson, Mt. Whitney, and Washington, 
D.C. A convenient summary of their work is to be found in the Smith- 
sonian Physical Tables (Fowle, 1934b). Many details of method are 
described in a later publication by Abbott et al. (1942). Independent 
and, in some spectral regions, improved measurements of solar spectral 
intensity have been made by other investigators. In nearly every case, 
however, the measurements were scaled to fit the Smithsonian curves 
which therefore remain the standards over most of the spectrum. 

In the ultraviolet and visible portions of the spectrum it is observed 
that the atmospheric absorption follows an exponential law. Hence 

i = iV-^T^"'^, (3-2) 

where i and /n = the intensities of a beam of sunlight at the bottom and 
top of the atmosphere, respectively, and a, the atmospheric attenuation 
coefficient, refer to a wave-length interval from X to X + (fX; Z = the 
zenith angle of the sun; and 7 = a factor which accounts for the curva- 
ture of the earth, ^^alues for 7 are listed in standard tables; 7 is very 
close to unity for Z < 80°. In the infrared for certain bands of water 
vapor and other gases, Eq. (3-2) does not agree with the observed absorp- 
tions. However, Eq. (3-2) is not wTong; the discrepancy is due to the 
use of insufficient dispersion to resolve the narrow and complex structure 
of many of the bands. 

The air mass M is defined by 

M = 7 sec Z. (3-3) 

From Eq. (3-3), .1/ - 1 for Z = and 7 = 1, and therefore .1/ is the 
amount of atmosphere from the surface to space in a vertical direction. 
Then 

(3-4) 



I = loC 



-aM 



To determine io on top of the atmosphere, i is measured for several 
values of M and is plotted for each wave length on a logarithmic scale 
against ]\I . The straight line thus obtained is extrapolated to zero air 
mass, which gives lo when proper account is taken of the transmission of 
the spectrograph and the spectral response of the accompanying bolome- 



I ()-i 



RADIATION mOLOGY 



Icr or photoelectric cell. The determination reciuire.s that the atmos- 
pheric attenuation a remain unchanged for several hours as Z changes. 
Several sets of data obtained hy this method are plotted in Fig. 3-3. 
They include the data of the Smithsonian Institution (Ahl)ott et al., 
1923; Fowle, 1934h) of Pettit (1940) at Mt. Wilson, and of Gotz and 
Schonmann (1948) at Arosa. The curve of Moon (1940) is an average 
of all data existing in 1940. The data at an altitude of 55 km were 
obtained in 1947 i)y Durand ci al. (1949) of the Naval Research Labora- 
tory with a spectrograph on a rocket; at 55 km the pressure was 2 X 10~' 
too 



90 



80 - 



70 - 



t 60 

z 

H 

? 50 



UJ 

> 



40 



30 



20 



10 



r 

/ 1 
/ / 


^ 




£i 


SMITHSONIAN PHYSICAL TABLES (FOWLE, 1934b) 


/ I 
J' 




o 


SMITHSONIAN INST, 1920-1922 ( ABBOTT e/ O/, 1922) 


/ r' 




v\ 


• , 


PETTIT, 1940 


- hi 

1 1 , 




^\ 


A 


NRL, 65 KM, 1947 (HULBURT, 1947) 




v\ 





GOTZ AND SCHONMANN, 1948. 


h 
- 1 







MOON, AVERAGE TO 1940 
6000°K RADIATION 




q \ 


r\ 






A 




"M 






^ 


• 


r i 






\ 




/ • 






■^ 




/*? 









^ 


-/Al 








>v 


h> 








%D 


/ -J 








^S„^A 


- A 








^^^.^o o 


tf' 








^~— ~.^___o_ 


A 










U 1 






1 1 


1 1 1 1 1 1 1 



0.2 



0.4 



0.6 



0.8 



1.0 



2.0 



2.2 



2.4 



2.6 



1.2 1.4 1.6 1.8 

WAVE LENGTH, /Z 

Fig. 3-3. Solar-spectrum curves on top of tlio atniosphore. 

atm, and the spectrograph had risen through 4999 5000 of tlie atmosphere 
and had only 1/5000 al)o\^e it. The solid-line curve of Fig. 3-3 is the 
spectral intensity of a l)lack body at 6000° K. All the curves of Fig. 3-3 
were arbitrarily adjusted to have their maxima at 100. It is seen that 
the ()000°K curve lies abov^e the solar values in the ultraviolet and also in 
the red and infrared to 1.4 m- 

In Figs. 3-1 and 2 are reproduced perhaps the best photographs which 
have been made of the ultraviolet portions of the solar spectrum. The 
spectrum of Fig. 3-1 was taken by Giitz and Casparis (1942) at Arosa, 
Switzerland, using all po.ssible care to reduce the scattered light which 
always veils the short-wave-length limit of the solar spectrum l)elow the 
ozone layer. The spectrum of Fig. 3-2 is a composite made from four of 
a series of spectra obtained by F. S. Johnson, .1. 1). Purcell, and W. 



SUNLIGHT AS A SOURCE OF HADLATION 



103 



Toiiscy (1952) of the Naval Research Laboratory with a sportrosraph 
on a rocket flying above the ozone region from al)()ut 60 to 110 km on 
June 14, 1949. The spectra of Figs. 3-1 and 2 overlap at the absorption 
band 2882 A. 

The curves of Pettit and the NRL (Durand and coworkers, unpub- 
lished; cited in Hulburt, 1947) shown in Fig. 3-3 in the ultraviolet, are 
replotted in Fig. 3-4 with the ordinate on an absolute scale and with the 



120 



no - 





PETTIT, 1940 

NAVAL RESEARCH 
LABORATORY, 
55KM, 1947 

STAIR, 1951- 

6000°K 
RADIATION 



J L 



2500 



3500 



3000 
WAVE LENGTH, A 

Fig. 3-4. Ultraviolet portion of the solar spectrum on top of the atinospliere. 

scale of the abscissa expanded over that of Fig. 3-3 in order to bring out 
detail. The data of Stair (1951) of October 1950 are added. The fluc- 
tuations in the curves indicate certain major absorptions in the solar 
spectrum but are not fine-grained enough to bring out all the Fraun- 
hofer lines. Pettit's value of u at 3400 A is taken as standard, and the 
other two curves are adjusted to it. The ()000°K black-body curve of 
Fig. 3-3 is continued in Fig. 3-4 to emphasize the fact that the solar 
intensity continues to fall below it as the wave length is decreased to 
2200 A. 

In the infrared beyond 2.6 n, io decreases rapidly with increasing wave 
length approximately as for a black body at 7000°K. For example, rela- 



101 



HADIATION HIOLOGY 



tive to the inaximuin intensity at KiOO A the intensities at 5, 8, and 14 /x 
are l.i) X 10 •', 3 X 10-', and 3.7 X l()-'\ respectively, hut the idea that 
io is approximate to or is less than tiic intensity of a hlack body at ()()00°K 
must not !)(' pushed too far because, as brought out in the preceding sec- 
tion, both in the region of X rays and of radio waves the solar energy is 
such as to indicate the existence of emissive regions of the sun, probably 
in the corona, which are at temperatures much greater than ()000°K. 

In Table 3-1 is given the energy in several portions of the solar spec- 
trum on top of the atmosphere calculated from the areas under the aver- 
age of the curves of Figs. 3-3 and 4. In obtaining the values in column 



Table 3-1. Spectral Distribution of Soi.ar Radiation 



\Vavo-leii}j;th 


K 


•action 


of 


Flux doiLsity 


interval 


total radiation 


cal/cin^ mill 


2200-3150 A 




0.014 




027 


3150-4000 A 




0.079 




0.153 


4000-7000 A 




0.403 




0.78 


7000 A-2.6m 




474 




0.92 


2.6-14 M 




0.03 




0.06 



3 of the table the solar constant, which is the flux density of total solar 
radiation on top of the atmosphere at the earth's mean distance from the 
sun, was taken to be 1.94 cal/cm- sec. It is seen from Table 3-1 that 
about half the total solar radiation lies in the visible and ultraviolet 
regions below 7000 A and about half in the infrared. 



SOLAR ULTRAVIOLET RADIATION AT THE EARTH'S SURFACE 

The intensity of the solar ultraviolet radiation that reaches a particu- 
lar point on the surface of the earth depends on the amount of ozone, air 
haze, and clouds between that point and the sun. Of these materials, 
ozone is important because of the absorption of its great ultraviolet band 
which begins at about 3400 A and increases rapidly for shorter wave 
lengths. Air, haze, and clouds atteiuiate the rays of the sun mainly by 
scattering with little true absorption; the scattering, of course, causes the 
sky. For ultraviolet radiation, true absorption of the oxygen of the air 
does not set in appreciably until the wave length becomes less than about 
2700 A. 

The absorption coefficient a of ozone, observed by Tsi-Ze and Shin- 
Piaw (1932, 1933) is plotted in curve I, Fig. 3-5, against the wave length; 
about one-fifth the observations are shown by the dots. The absorption 
coefficient a is defined by 

i = z>-«, (3-5) 



SUNLIGHT AS A SOURCE OF RADIATION 



10.3 



where io and i are the intensities of a collimatod beam of lij2;ht in the 
wave-length region from X to X + dX, entering and emerging from a 
layer of ozone x cm in thickness at normal temperature and pressure 
(NTP), i.e., at 0°C and a pressure of 1 atm. The long-wave-length region 
of the ozone absorption from about 3400 to 3100 A has a band structure 
known as the Huggins bands; the smooth absorption below 3100 A is 



lOOOrr 




2200 2400 2600 2800 3000 

WAVE LENGTH, A 



J200 



3400 



''Fig. 3-5. Ultraviolet absorption coefficients of some atmospheric gases. 

known as the Hartley continuum. The absorption in the Huggins bands 
varies with the temperature in a complicated way, which is of no interest 
here; roughly, a decreases between 10 and 30 per cent when the tempera- 
ture falls from 18° to -50°C (Vigroux, 1950). It is seen from the ozone 
curve of Fig. 3-5 that, below 3100 A, a rises to very high values; therefore 
it is not surprising that even the few millimeters of ozone which e.xists 
in the upper atmosphere is sufficient to prevent the detection of ultra- 
violet wave lengths from 2915 to 2150 A at the surface of the earth. 
In the standard atmosphere the pressure is 1.0132 X lO*' dynes /cm-, 
and the total air in a vertical column of the standard atmosphere from 
sea level to space amounts to 8 km of air at XTP; 20.75 per cent of this is 
oxygen. In Fig. 3-5, curve II is the attenuation coefficient a per atnios- 



ion 



RADIATION BIOLOGY 



phore of puii' air calculated from the Rayleigh scattering theory with 
polarization dofoct. and curve III is a per atmosphere observed by Vassy 
(1941) for what may be termed a "fairly clear atmosphere at sea level." 
The observed (Buisson el al., 1930, 1932) absorption coefficient a of the 
molecular oxygen gas contained in 1 atm, from which the Rayleigh term 
has been subtracted, is plotted in cur\e IV, Fig. 3-5. There are no avail- 
able data on the attenuation of ultraviolet radiation passing down through 
a hazy or cloudy atmosphere. 

ATMOSPHERIC OZONE 

Ozone exists in the upper atmosphere of the earth for the most part 
in the region between 15 and 35 km above sea level; it extends in rapidly 



T3 

Uj" 
O 

3 



o 

I 

0. 

< 

O 

UJ 

o 




Fig 
NTP 



JAN FEB MAR APR MAY JUNE JULY AUG SEPT OCT NOV DEC 

3-6. Average contours of equal thickness of ozone. Unit, I0~' cm of ozone at 
(Gotz, 1944.) 



decreasing concentration as high as 50 km and down to sea Jexel whcMv 
there are often traces amounting to as much as 0.05 mm of ozone at NTP 
per kilometer of air (or 5 X 10^'' by volume of air). The total thickness 
of ozone in a vertical column of air from sea level to space varies from 
about 1.5 to 5 mm at NTP depending on the latitude and the season. 
This is brought out in P'ig. 3-6 which gi\-es an axcrage world-wide distribu- 
tion of ozone derived by Gotz (1944) from a summary of nearly all avail- 
able observations. Figure 3-(i shows the increase of ozone thickness with 
increasing latitude up to about (50° foi- all seasons of the year and the 
spring maximum and autumn minimunv for latitudes greater than 20°. 
Whether there is a diurnal variation in the ozone thickness above any 
station is not known with certainty; a few data mentioned by Dobson 



SUNLIGHT AS A SOURCE OF KADIATION 107 

(1930) indicate that a diurnal variation, if it exists, is small. Regarding 
the change of ozone thickness with sunspots, there are few long-contiiuied 
series of ozone data. Fowle (1934a, 1935) obtained yearly a\'erage values 
at north latitude 34° in Arizona and California. Ilis values from 1921 to 
1928 varied with the sunspots, but the correspondence was not main- 
tained from 1928 to 1934. A coamection between ozone and sea-level 
barometer changes is complicated and has not been clearly established. 
From the data of six ozone stations in Europe, Dobson concluded that the 
smallest amount of ozone occurred to the southwest of a barometric high 
and that the passage of a barometric low over a station was accompanied 
by an increase in ozone content. In general, since most of the ozone lies 
between 15 and 35 km, its variations would be expected to be correlated 
with changes of pressure and winds in the stratosphere rather than in the 
troposphere, and at this time the relation between stratospheric and 
tropospheric weather cannot be said to be completely known and 
understood. 



CALCULATED ULTRAVIOLET INTENSITY AT THE EARTH'S SURFACE 

A complete calculation of the solar ultraviolet radiation falling on the 
surface of the earth from the sun and sky is very complicated and will not 
be attempted. It is, however, instructive to calculate the ultraviolet 
intensity of the direct rays of the sun at the surface of the earth for vari- 
ous zenith angles of the sun. Let in and i be the intensities, respectively, 
of the rays of the sun outside the atmosphere and at the surface of the 
earth. Then 

where a = the absorption coefficient of ozone per centimeter, 
X = the thickness of the ozone in centimeters at NTP, 
a„ = the attenuation per atmosphere below the ozone region due 

to pure air and haze, and 
Z = the zenith angle of the sun. 
The symbols i, in, a, and a^ refer to the wave-length region from X to 
X + r/X. In Fig. 3-7 the curve labeled to is the Naval Research Labora- 
tory curve of Fig,. 3-4 and is plotted in arbitrary units against the wave 
length for the ultraviolet region of the solar spectrum where the absorp- 
tion of ozone becomes important. The values of i for ozone of thickness 
4 mm, for no atmospheric attenuation, and for various zenith angles were 
calculated from Eq. (3-6) with .r = 0.4 cm, «„ = 0, and values of a 
obtained by drawing a smooth curve through the fluctuations of curve I, 
Fig. 3-5. The i vs. X curves are plotted in Fig. 3-7 for six values of Z. 
The total ultraviolet energy E in wave lengths less than 3200 A was 



108 



RADIATION HIOLOGY 



calculated I'loni \\\v integral 



/^' - fr 



i(i\ 



(3-7) 



and the / \'s. X cur\"c.s lor the se\eral xaiues of Z. The resulting values 
of E are plotted against Z in the 4-mm curve of Fig. 3-8. Families of 
i vs. X curves similai' to those of Fig. 3-7 were calculated for other ozone 
thicknesses, and from these curves and Ei\. (3-7) the E vs. / curx'es of 





1 1 


- 


SOLAR ENERGY //^\\ 




10 


- 


ABOVE ATMOSPHERE ./ //V\^ 




9 


- 


'°//^/r^ 


V) 

3 


8 


- 


/ //// 


a> 

> 

o 
<u 


7 
6 




/ lili/ 


o 


5 




nil 1 ^ '""^ 


C 

o 


4 




/III/ ■* '^^ ^3 


(>1 






z ////// 




3 
2 
1 


- 


/ 'o'o / / / 

/ / f ^0 

/ J 1 ^ ^ 1 




'0 


1 1 1 1 1 1 1 1 ■-'- 


<\i^^r^i^\-i^-J^\ >< 1 1 1 1 1 t L . 1 1 1 [ 1 1 1 1 1 1 1 L I r ■ 1 



2900 3000 3100 3200 3300 3400 
WAVE LENGTH, A 

Fig. 3-7. Observed solar-spectral-energy curve ?n above atmosphere and calculated 
curves of i after passing through 4 cm of ozone at various zenith angles. 

Fig. 3-8 for 1, 2, and 3 mm of ozone were calculated. The curves illus- 
trate the manner in which E decreases with increasing ozone thickness 
and increasing zenith angle. Curves for integrals to wave lengths less 
than 3200 A were similar to those of Fig. 3-7 but descended more rapidly 
to low values. 

The effect of the atmosphere, in addition to the ozone, is shown by the 
dotted curve of Fig. 3-8, which refers to a "fairly clear" atmosphere with 
1 mm of ozone. It was calculated from Eqs. (3-6) and (3-7) with «„ from 
curve III, Fig. 3-5. The dotted curve of Fig. 3-8 brings out the almost 
obvious fact that a relatively small amount of atmospheric haze is more 
effective in reducing the ultraviolet energy of the direct rays of the sun 
than, for example, doubling the ozone thickness of the upper atmosphere. 
However, we must hasten to remark that haze reduces the total ultravio- 



SUNLIGHT AS A SOURCE OF RADIATION 



109 



let radiation falling on the earth's surface far less than it reduces the 
effectiveness of the direct rays of the sun. The reason is that the light 
scattered out of the direct solar 



mm Oi 



rays by haze is not entirely lost, 
since haze exerts little absorption, 
but reappears as sk}^ light, which 
for thin haze is mainly directed 
downward. 

A fraction of the sky light is 
scattered outward to space and is 
lost to the earth; the fraction in- 
creases with increasing haze and 
clouds. As has been mentioned, an 
exact calculation of all this is com- 
plicated and would require a com- 
plete theory of sky brightness and 
polarization for ultraviolet wave 
lengths in terms of the ultraviolet 
optical constants of the atmosphere 
in all stages of haziness. Such a 
theory has not been formulated, and 
such constants have not been determined; therefore only a survey of the 
observational material is presented in the following section. 



o 
o 



V 



^ 




I mm O3 ^^ 

+ "FAIRLY CLEAR" ^ 



J I I I 1 L 



30 60 

Z, deqrees 
Fig. 3-8. Calculated solar ultraviolet 
energy E for wave length.s loss than 
3200 A for several cases. 



OBSERVED ULTRAVIOLET INTENSITY AT THE EARTH'S SURFACE 

Many measurements of ultraviolet radiation from the sun and sky were 
carried out at Washington, D.C., by Coblentz and Stair (1943). The 
first series of measurements were made with photocells arranged to record 
En, the radiation received on a plane normal to the rays of the sun, from 
the sun and a circular region of sky around the sun as a center 22° in 
diameter. Data were taken during the clearest days over the years 
1936 to 1941; for illustration the values of En for 1937 are shown in Fig. 
3-9. Two types of photocells were used, one sensitive to wave lengths 
less than 3200 A and the other sensitive to wave lengths less than 3132 A; 
the spectral-sensitivity curves of the photocells were not reported. In 
Fig. 3-9 the abscissas are the air mass M and the zenith angle of the sun 
Z, and the two scales of ordinates are the En in microwatts per square 
centimeter for the respective types of photocells. It is seen that they 
were approximately proportional and that the ultraviolet intensities of 
wave lengths below 3200 A were roughly 2.5 times the intensities below 
3132 A. The data of the other years from 193() 1941 were similar to 
those of Fig. 3-9 for 1937. For any zenith angle the variation of the 
ultraviolet intensitv of a factor of about 2, shown in Fig. 3-7, was proba- 



uo 



RADIATION TUOI.or.Y 



bly due largely to haze variations within the (luulitative specification of 
a "clear" day and, to a lesser extent, to ozone variations. No ultra- 
violet variation with sunspots appeared, and if any existed, it was 
obscured l)y the haze variations. The sunspol numbers for the years 
H)8() 1941 were 80, 114, 110, 89, ()8, and 48, respectively. There was an 
ill-defined seasonal variation in K„, which is not brought out in Fig. 3-9, 
partially attributable to the spring-to-fall decrease in ozone (see Fig. 3-6), 
in that the ultraviolet intensity was often greater in the autumn than in 
the spring from equal solar zenith angles. Ho\vever, high En values fre- 
(jucntly occurred in late winter and early spring which may have been 



150 - 



5 



J- 100 



o 
o 




V 



^ 



50 60 

AIR MASS M 

Z, degrees 
Fu;. 3-9. Ultraviolet intensity E,, during clear days in \\ asliiiigtoii. 1937. 
and Stair, 1943.) 



{Coblentz 



due to unusually clear skies or to local ozone variations attributable to 
stratospheric weather. 

After 1941 Coblentz and Stair (1944) changed their plan of observation 
and measured E/,, the ultraviolet energy from the sun and the entire 
hemisphere of the sky, falling on a horizontal plane, instead of PJ„, the 
ultraviolet energy from the sun and 22° of the sky, falling on a plane 
normal to the sunbeams. Values of Eh obtained for clear days in Wash- 
ington are plotted in Fig. 3-10 as dots, circles, crosses, and triangles for 
June 4, Sept. 18, and Dec. 21, 1943, and Oct. 17, 1944, respectively. In 
Fig. 3-10 the two dotted curves outline the spread of value of E„ for clear 
days in Washington during 193(5 and 1941. Since the points fell in the 
region between the dotted cur\'es. it was concluded that Eh and A'„were 
approximately the same; in general, of course, Eh and En cannot be 
expected to be equal. "N^nlncs of Ei, for .some clear days in high latitudes, 
obtained (Coblentz el <d., 1912) on a trip to Greenland in 1941 are shown 
by the solid lines of Fig. 3-10. The increase in Eh with latitude may have 
been due to less liaze and Ics.s ozone at the higher latitudes, but these 
features were not measuriMl. 

A series of measurements of Eh for ultra\iolet \\a\e lengths less than 



SUNLIGHT AS A SOURCE OF RADIATION 



111 



3022 A was made in Cleveland in i<j;3() and 1937 by Luekiesh d <il. (1937, 
1939). The results exhil)ited about the same variation with Z and the 
same spread as the Washington data of Fig. 3-9 and therefore are not 
plotted in Fig. 3-10. For wave lengths less than 3022 A, Eh was about 
one-fourth the Eh for wave lengths less than 3200 A. 

For ultraviolet wave lengths less than 3130 A, Eh was observed during 
1932 and 1933 by Ives and Gill (1937) in 14 cities scattered over the 
United States. In Fig. 3-11 their results are given for two groups, where 
group 1 refers to the most smoky localities and group III refers to the 

200 



50 



t<4 



E 

4; 




- 


s 


i 




_ 




\ 


150 


- 


« 


^ 




- 




< 








o 








o 




- 




CM 








ro 




- 




V 


100 


_ 


~>^ 


r< 




, 




l_ 








o 
















c; 








Em 




- 





60.8° 




30 60 90 

AIR MASS Af 

Z, degrees 
Fig. 3-10. Values of Eh and E,, for wave lengths less than .3200 A. The symbols 
indicate Ei, for some clear days in Washington during 1942 and 1943. The solid-line 
curve is for Eh for higher latitudes in 1941. The £„ for clear days in Washington 
1936-1941 was within the dotted curves. (Coblentz and Stair, 1944; (Coblentz el al., 
1942.) 

least smoky localities; the data for clear and cloudy skies were plotted 
separately. It is seen in Fig. 3-11 that the curves for the more smoky 
localities lie below those for the less smoky both for clear and cloudy skies 
and also that the curves for cloudy skies lie below those for clear skies. 
Therefore both smoke and clouds decreased the amount of ultraviolet 
radiation that reached the surface of the earth. Comparison of Fig. 
3-10 with the "clear-sky" data of Fig. 3-11 shows good agreement, when 
it is remembered that Eh for wave lengths less than 3200 A is about 2.5 
times Eh for wave lengths less than 3130 A. 

The conclusion is therefore that, from Fig. 3-10, a rough estimate may 
be made, correct perhaps within a factor of 2, of Eh in clear weather for all 
seasons of the year and all times of the day. If a more e.xact value of 
Eh is required, provision must be made to measure it. To make the 
rough estimate, an average curve was drawn through the data of Fig. 



112 



RAnr\TI<)\ ItlOLOGY 



3-10 which ti;ave Eh in terms of the zenith nnp}o of the sun. From this, 
I'Jh was calculated throuj^ihout the day for various latitudes and seasons. 
The results are plotted in Fijj;. 3-12 for (he twenty-second day of March, 
June, September, and December for north latitudes 0°, 20°, 40°, ()0°, and 
80°. The curves for March and September are the same at all latitudes 
becaus(» the zenith ;inu;lo of the sini is the same at these epochs; at the 
— I 1 1 1 1 — 




1 


1 


1 IT 


1 ■■■■ 


i 




GROUP m 




\ 




CLEAR SKY 


*" 


\ 






- 


\ 


i 




1^ 


- 


\ 




- 


- 


\ 




- 


- 


\ 




- 




1 


?\^^ 


1 



N* 




3 4 1 2 3 4 

M M 

Fig. 3-11. Values of Eh for wave lengths less than 3130 A. Group I. for most smoky 
loealities; Group III. for least smoky localities. {Ives and Gill. 1937.) 

equator the curves for .June and December are the same for the .same 
reason. For December at 60° north latitude and for March, September, 
and December at 80°, the sun does not rise above 10° above the horizon, 
and Kh i« zero throughout. At 80° the value of Eh in June remains above 
zero all night, being 3 at midnight because the midnight sun is 14° above 
the horizon. In the curves of Fig. 3-12, no seasonal adjustments have 
been made for the fact that the sun is about 3 per cent nearer the earth 
on December 22 than it is on June 22. Since the values of Eh of Fig. 
3-12 are based on the data of Fig. 3-10 obtained on clear days in the 
United States, they may be expected to be correct for localities outside 
the United States only if the localities have the same ozone and the same 



SUNLIGHT AS A SOURCE OF RADIATION 



113 



clearness of atmosphere and sky as those that occurred in the United 
States. 

A few measurements have been made of the ratio of the ultraviolet 
radiation on a horizontal plane from the total hemisphere of the sky 



200 



E 
u 



o 
o 

CM 
rO 

V 

u 
o 
<t- 

r< 



100 




200 



E 
u 



o 

o 

V 



^ 



100 




8 9 10 II 12 I 2 3 4 5 6 
AM NOON PM 

Fig. 3-12. Calculated values of Eh for wave lengths less than 3200 A through the day 
for various seasons and latitudes. 

to that from the sun, i.e., Eh (sky)/A\ (sun). For clear days in Cleve- 
land in 1936 and 1937 for wave lengths less than 3022 A the ratio was 
1.0, 1.1, 1.5, 2.2, 4, and 10 for zenith angles 30°, 40°, 50°, 60°, and 
70°, respectively. During the mid-day hours of June 13, 1928, a very 
clear day, for zenith angles about 20°, Pettit (1932) found that, for wave 
lengths less than 3200 A, the ratio was 1, 0, 0.55, and 0.43 at altitudes 
above sea level in the vicinity of Pasadena, Calif., of 845, 3400, and 5700 
ft, respectively. In general, these ratios are greater than the correspond- 
ing values for visible light; the ratio for visible light is about 0.2 for a 



II I 



RADIATION HIOLOGY 



dear sky at sea level and Z = 30°. The ratio, either for ultraviolet 
radiation or for \isil)lc light, increases with increasing haze, and in thick 
haze or cloud oxcrcast, when the direct rays of the sun are reduced to 
zero, the ratio becomes infinite because there is still light from the sky. 
The distribution of ultrax'iolet radiation for \va\-c lengths less than 
3200 .\. o\'er the sky was measured by Pet tit (1932) duiing the spring of 
1928. His average values of the ultraxiolet sky brightness at Pasadena 



I 

o 
m 

10 



- 


---; 












- 


n / 










/ 
/ 


;/ 


>/i 


\\ 

Nx 


^^ 








'*>.^ 




' -7\ 


- 


1 1 1 1 


1 1 1 1 


1 1. 


1 


1 1 


1111 



90° 



60° 



30° 



30° 



60° 



90° 



HORIZON ZENITH HORIZON 

Fig. 3-13. Sky brightness for ultraviolet radiation and visible light. Curve I. for 
ultraviolet wave lengths less than .■?200 A in California. Curves II and III. for 
visible light in Brazil and Switzerland. 

on a meridian through the sun with the sun at an altitude of 50° are 
shown by the solid-line curves of Fig. 3-13. The values varied con- 
siderably from day to day because of changes in haze. For comparison 
two curves of sky brightness for visible light are plotted in Fig. 3-13, one 
observed in Brazil (Richardson and Hulburt, 1949) and one in Switzer- 
land (Dorno, 1919, Table Illd). The curves refer to a clear sky, to a 
meridian through the sun, and to the sun at an altitude of 50° ; the three 
curves are adjusted to pass through the same point at the zenith. The 
difference in the two visible-light curves was probably due to the haze 
conditions in the respective atmospheres. The curves show the well- 
known lirightness of the sky near the sun and indicate that the sky near 
the horizon was relatively darker for ultra\iolet radiation than for \'isiblc 
light. 



SOLAR INFRARED SPECTRUM AT THE EARTH'S SURFACE 

In Fig. 3-14 is gix-en the solar spectral-intensity curve /H on top of the 
atmosphere and the tiansmission cur\'es of th<' important terrestrial 
al)sorber, water, in liciuid and \apor form as ol)ser\ ed with spectrometers 
of low resolving power. The transmission curves for litiuid water refer 



SUNLIGHT AS A SOITRCE OF RADIATION 



to tliicknossos of 1 and 10 mm of distilled water a1 20°C calculated from 
the accurate ah.s()ri)tioii coefficients of ('urcio and Petty (1951). The 
water-vapor curve represents the transmission through 1.85 km of atmos- 
phere along a horizontal path containing some haze and a total of 17 
mm of precipitable water. That is, the water vapor in a column 1 cm 
square and 1.85 km long would, if condensed to the liquid phase, form a 
column of licjuid water 1 cm square and 17 mm long. The portion of the 
water-vapor cur\'e for wave lengths longer than 0.9 /x was measured in 



1.00 



mm WATER 



17 mm WATER 
VAPOR 




0.8 



1.0 



2.0 



22 



2.4 



2.6 



1.2 1.4 1.6 1.8 

WAVE LENGTH, /.^ 

Fig. 3-14. Solar inten.sity 'o and transmis.sion of water and water vapor. 

1949 by Gebbie et at. (1950). The short-wave-length portion of the 
curve below 0.9 yu was from miscellaneous older measurements along 
horizontal paths in the real atmosphere; the attenuation below 0.6 /x was 
largely due to haze because water vapor is very transparent in this region. 
The curves of Fig. 3-14 bring out the well-known differences in the 
absorption of water in the liquid and vapor phases. For example, 
beyond 1.4 /x the strength of absorption by liquid water is much stronger 
than by water vapor. Thus, 10 mm of liquid water and 17 mm of water 
vapor are opaque at 1.4 m, but, although the transmission of water vapor 
rises at 1.65 /x to a high value, liquid water remains completely opaciue at 
longer wave lengths. Also, it is seen that the absorption coefficients of 
1 7 mm of water vapor and of 1 mm of liquid water are comparable on the 
short-wave-length side of the 1.9-m water-vapor band, but, although 
water vapor regains its transparency at 2 /x, 1 mm of liquid water does 
not again become transparent. Furthermore, the wave lengths of maxi- 



10 



RADIATION' laoLOGY 



imim alisorption do not coiiicidc for the two phases. Approximately the 
same iiumher of al)sorption hands exist at wave lengths helow 1.9 fi, hut 
the \apor hands app(Mir at somewhat shoi-ter \va\'e lenf^ths than the liquid 
bands. 

Therefore sunlight that reaches the earth through several centimeters of 
precipitahle water vapor is not entirely' depleted of energy capable of 
being absorbed !)>■ liiiuid w atcr in organisms or in the seas, and, in particu- 
lar, this is true in the so-called "atmospheric windows" at 1.65 and 2.2 n, 



I 17mm WATER VAPOR 




17mm WATER VAPOR 




0.05 



0.04 



0.03 



0.02 



o 



0.01 



7 8 9 

WAVE LENGTH,/^ 

Fig. 3-15. Solar intensity I'o in tho infrared and transmission of water vapor, ozone, and 
carbon dioxide. 



where atmospheric water vapor transmits copiously and relatively small 
thicknesses of liquid water absorb strongly. The effect of absorption 
in the infrared is to heat the absorber. In the 2.2-/i region and at longer 
wave lengths where liquid water is highly absorbing, heating is produced 
principally at the surface. On the other hand, for shorter wave lengths 
below 1 /i, the radiation peiietrates to a greater depth before being com- 
pletely absorbed and produces warming in depth. 

Figure 3-15 is an extension of the solar-intensity and the atmospheric- 
transmission curves of Fig. 3-14 to 14 n to show other atmospheric infra- 
red absorption bands. The absorption at 2.7 m is due to both carbon 
dioxide and water vapor. The strong absorption at 4.2 fx is due to carbon 
dioxide, and the great band from 5.2 to 7.5 /x is due to water vapor. 
Beyond this band to about 14 ^l the lower atmosphere is relatively trans- 



SUNLIGHT AS A SOURCE OF llADIATION 117 

parent except for the complicated but slight absorption of water vapor. 
The 9.()-M infrared band of ozone of the upper atmosphere is shown. At 
14 n, a strong band of carbon dioxide sets in, and thereafter, for longer 
wave lengths up to about 400 m, or 0.4 mm, water vapor is a strong 
absorber with the exception of a narrow crevice of transmission at 22 ju. 
In conclusion, it is apparent that the curves of Figs. 3-14 and 15 can be 
used to make rough estimates, perhaps correct within a factor of 3, of 
the solar energy in the infrared at the surface of the earth if the amount 
of water vapor in the overhead atmosphere is known, but, if greater pre- 
cision is required, provision must be made to measure the radiation 
directly. 

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Abbott, C. G., F. E. Fowle, and L. B. .AJdrich (1922) Annals of the Astrophysical 
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Abetti, G. (1938) The sun. Cro.sby Lockwood and Son, Ltd., London. 

Adel, A. (1939) Atmospheric absorption of infrared solar radiation at the Lowell 
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Babcock, H. D., C. E. Moore, and M. F. Coffeen (1948) The ultraviolet solar spec- 
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Buis.son, H., G. Jausseran, and P. Rouard (1930) Sur la transparence de la basse 
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Dorno, C. (1919) Himmelshelligkeit, Himmelsstrahlung und Sonnenintensitat. 
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1 18 ItADl \'I'I()\ lilOLOOY 

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ilagen, J. P. (1951) A study of the radio-frequency radiation from the sun. Astro- 

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Luckiesh, M., A. H. Taylor, and G. P. Kerr (1937) Ultraviolet energy in daylight — 

a two year record. J. Franklin Inst., 223: 099-714. 

(1939) A four year record of ultraviolet energy in daylight. J. Franklin 

Inst., 228: 425-431. 

Menzel, D. H. (1949) Our sun. The lilakiston Company, Philadelphia. 

Moon, P. (1940) Propo-sed standard solar-radiation curves for engineering use. 

J. Franklin Inst., 2.30: 583-017. 
Pettit, E. (1932) Measurements of idtraviolet solar radiation. Astrophys. J., 75: 

185-221. 

(1940) Spectral energy curve of the .sun in the ultraviolet. Astrophys. J., 

91: 159-185. 

Richardson, R. A., and E. O. Hulburt (1949) Sky brightness numsurements near 

Bocaiuva, Brazil. J. Geophys. Research, 54: 215-227. 
Stair, R. (1951) Ultraviolet spectral distribution of radiant energy from the sun. 

J. Research Natl. Bur. Standards, 40: 353-357. 
Tousey, R., and E. O. Hulburt (1947) Brightness and polarization of the daylight 

sky at various altitudes above sea level. J. Opt. Soc. Amer., 37: 78-82. 
Tsi-Ze, X., and C. Shin-Piaw (1932) L'absorption de la lumiere par I'ozone entre 

3050 et 3400 A. Compt. rend., 195: 309-311. 
(1933) L'absorption de la lumiere par I'ozone entre 3050 et 2150 .\. Compt. 

rend., 190: 910-918. 
Va.ssy, A., (1941) Sur l'absorption atmosphcrique dans I'ultraviolet. Thesis, Uni- 
versity of Paris. 
Vigroux, M. E. (1950) L'absorption de I'ozone dans la region des bandes Huggins. 

L'influence de la temperature. Compt. rend., 230: 2170 2172. 

Manuscript received btj the editor Mar. 12, 1951 



CHAPTER 4 

Technique of Study of Biological Effects 
of Ultraviolet Radiation 

Jesse F. Scott* 

Department of Biologij, Massachusetts Institute of Technology 

Cambridge, Massachusetts 

and 

Massachusetts General Hospital 

Boston, Massachusetts 

Robert L. Sinsheimer 

Department of Physics, Iowa State College 
Ames, Iowa 

Introduction. Sources: Classification of light sources — Physical parameters of sources 
— Choice of a source — Practical aspects. Detectors of ultraviolet radiations: Fluorescent 
screens — Thermal detectors — Photochemical detectors — Photographic detectors — Photoelec- 
tric detectors. Methods of spectral isolation: Filters — Dispersing systems. References. 

INTRODUCTION 

The technique of the study of the effects of any radiation on Hving 
systems is divisible, on an operational basis, into (1) the means of produc- 
ing the radiation, (2) the means of manipulating and estimating the vari- 
ous parameters of the radiation, and (3) the means of demonstrating and 
analyzing the effects of the radiation on the biological system under 
study. This operational outline will be adhered to in a discussion of 
sources of ultraviolet radiation, detectors of ultraviolet radiation, means 
of spectral isolation, and accessor}^ optical components. The various 
means of demonstrating and analyzing the effect of ultraviolet radiation 
on the biological systems are considered in great detail elsewhere in this 
volume and will not be taken up here. It must be emphasized that this 
chapter will not deal with the detailed technique of any particular study 
but will be concerned with materials for such an investigation. This 
approach is dictated by the great variety of problems in this field. For 
example, one investigator may be interested in the abiotic activity of the 

* A Scholar in Cancer Research of the American Cancer Society. 

1H» 



1_>() RADIATION lUOLOGY 

.sunlight as a function of season, altitude, or some other parameter. A 
second investigator, using the same biological test object, might wish to 
determine the action sp(M'trum for the lethal effect of ultraviolet radiation 
in groat detail over a wide range of wave lengths. Each of these workers 
would draw from the same reservoir of available tools but would combine 
them in a different fashion for his own particular problem. The function 
of this chapter is to serve as a guide. 

SOURCES 

The source of radiation is of great importance among materials for a 
study of effects of ultraviolet radiation on biological systems. .V number 
of excellent chapters have been written containing detailed considera- 
tions of light sources in general and of tlu; ultraviolet light sources in par- 
ticular (see, e.g., Ellis d at., 1941; Forsythe, 1937; Harrison etal, 1948; 
Sawyer and Vincent, 1939; and Roller, 1952). No effort will be made 
to reproduce these detailed discussions in this chapter but rather to dis- 
cuss types of light sources and their characteristics, factors to be con- 
sidered in the choice of a light source, and finally to present a tabular 
compilation of noncommercial and commercial laboratory light sources 
which have characteristics making them particularly useful in a study 
of radiation effects. 

CLASSIFICATION OF LIGHT SOURCES 

Sources of radiant energy have been classified in a number of ways 
among which are: (1) the spectral range of radiation of useful intensity, 
(2) the method used for exciting the radiation, and (3) the distribution of 
energy within the spectral range. The ultraviolet spectrum, which is 
discussed in this volume, covers the range 4000-10 A. This broad range 
has been subdivided primarily on technical grounds into the near, the far, 
and the extreme ultraviolet. Sources differ considerably in the fraction 
of the total energy emitted in each of these ranges. The near ultraviolet 
extends to 3000 A which is near the short wave-length limit for the sunlight 
at the earth's surface. The far ultraviolet in biological work extends to 
about 1900 A. In this vicinity quartz begins to absorb strongly as does 
atmospheric oxygen (Schneider, 1940; Ladenburg et al., 1932). Because 
of this increasing atmospheric absorption below 1900 A, the extreme 
ultraviolet is also known as the vacuum ultraviolet. The lower limit 
of the extreme ultraviolet is arbitrary. There is considerable overlap in 
this region between the longer wave lengths of radiation produced by 
the techniciues employed in the excitation of X radiation and those found 
by excitation of ultraviolet radiation. 

Method of Exciting the Radiation. Ultraviolet radiation of any portion 
of the ultraviolet spectrum may be produced by any one or more of the 
following means of excitation: (1) incandescent or thermal, (2) spark, 



ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 121 

(3) arc, or (4) discharge. The modern tungsten lamp is an excellent 
example of a source of the first category. The radiation takes place as a 
result of heating of the surface of the radiator by some means; in this case 
it is by the passage of an electric current. For fundamental reasons 
sources of this category have limited utility, and that only in the near 
ultraviolet. In most cases the intensity of the emitted radiation falls 
rapidly between 4000 and 3000 A, approaching zero at the latter figure. 

Spark sources emit radiation excited by the passage of a high-voltage 
discharge between electrodes. The material of the electrodes enters the 
spark stream, contributing the major fraction of the radiation through its 
excitation by the electrical energy. In the arc also the electrode material 
evaporates into the arc stream to produce a large portion of the emitting 
ions in this stream. Arcs are generall}^ low-voltage, high current dis- 
charges. Radiation is produced in the discharge tube by excitation and 
ionization of the gas contained at reduced pressure. The radiation is 
excited by a relatively high potential between electrodes which, them- 
selves, do not contribute significantly to the ion stream. The distinction 
between these methods of excitation is not sharp and the reader will find 
that the foregoing system of classification is not rigidly adhered to in the 
literature. This is understandable when it is noted that discharge tubes 
operated at very high current densities may show evidence of evaporation 
of the electrode material into the ion stream by the appearance of radia- 
tion characteristic of the electrode material. The heating of the elec- 
trodes of an arc by ion bombardment ma^^ be sufficient to make the ther- 
mal radiation from the electrode a significant contribution to the total 
radiation from the source. A spark operated in air produces radiation 
which is characteristic of the electrode material, but if operated under 
water, the radiation produced bears no relation to the electrode material. 

Spectral Distribution. Of somewhat more practical importance is the 
classification of light sources with respect to the distribution of the spectral 
energy emitted. Sources are classified as continuous, line, or band. 
Continuous spectra generally arise from thermal emittors or from non- 
quantized energy transitions; line spectra arise from quantized atomic- 
energy transitions; and band spectra arise from molecular-energy transi- 
tions or from atomic-energy transitions occurring at high temperatures 
and pressures. On the basis of the method of production of these various 
types of spectra it would be expected that many sources would exhibit 
other than the nominal type of spectrum. Thus, when the hydrogen 
discharge tube is operated at extremely high current densities in an effort 
to achieve high brilliance, line spectra are frequently found superimposed 
upon the typical continuous ultraviolet spectrum of hydrogen. These 
lines arise from the evaporation and subsequent excitation of electrode 
material in the ion stream. Mercury discharge tubes, which at low pres- 
sures and current densities show well-defined line spectra, show increasing 



122 J{\1)I ATION 1!1()L(K;Y 

broadoiiinji; of the lines and the development of iippreciahle contirniou.s 
l)a('kt>;roun<l ;is the ciincnt density and vapor |)ressure are inereased. 

PHYSICAL I'AHA.MKTKUS OK SOl'llCKS 

Two of the physical parameters which may influence tlu; ciioice of a 
particular lif>;ht source were mentioned in the precedinj^ discussion of pos- 
sible classification of sources, namely, the useful spectral range eovercjd 
by the emitted radiation and the distribution of energy within tliat range 
(i.e., continuous or discontinuous). A third factor of importance is the 
amount of radiation emitted at a particular wave length or over a certain 
band of wave lengths. The amount of radiation may be considered in 
two ways: first, the total amount of radiation emitted from the whole 
of the luminous body of the source. Second, the amount of radiation 
may be considered to be that quantity emitted by a unit area or volume 
of the source into a unit solid angle. The significance of these two modes 
of expressing the intensity parameter of a light source will be discussed. 
Following is a list of the terms which are used to describe the intensity 
parameter: 

1. Radiant Flux: Radiant flux is the rate of flow of radiant energy with 
respect to time. The quantity is also called "radiance" (P = dU/di) 
where V is radiant energy and / is time. 

2. Radiant Intensity: Radiant intensity is measured hy the energy 
falling in unit time upon an area subtended by unit solid angle about an}' 
direction considered and at any distance from the source. This value is 

also called "steradiance" I ./ = , , ). The solid angle is represented 

by w. 

3. Steradiancy: Steradiancy is the radiant flux per unit solid angle per 

square centimeter of source w ^ = . , . . ). .4 is the area of the source. 

It is to be noted that the.se expressions contain no reference to wave 
length. For studies of the biological effects of radiation it is frequently 
necessary to know the value of one or more of the preceding intensity 
expressions w^th respect to wave length. The maimer in which such 
energy measurements are made will be considered on pages 130 to 142. 

CHOICE OF .\ SOURCE 

The choice of a source for a study of the biological elTects of radiation 
depends on a number of factors which are inherent in the exact nature of 
the experiment to be conducted. In a general way, decisions must be 
made as to (1) the size of the area to be irradiated, (2) the range of the 
ultraviolet spectrum to be covered, (3) the size of the band of energy wdth 
respect to wave length, (4) the time in which the largest amount of energy 
is to be delivered to the irradiated area. These factors are to some extent 



ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 123 

independent and yet they are often interdependent in a way which is not 
always completely understood. This situation can probably be best pre- 
sented by examples. Assume the practical problem of the sterilization of 
large volumes of a liquid by ultraviolet radiation. This is to be done by 
flowing the liquid in a thin film of large area exposed to the total radiation 
exclusive of the infrared. For practical reasons it is decided to use the 
mercury-vapor discharge lamp. The amount of ultraviolet energy 
needed has been established by previous experiments. 

The mercury discharge lamp has a relatively large fraction of the total 
ultraviolet output in the biologically potent region around 2600 A but 
the intrinsic brilliance (steradiancy) is quite low compared with other 
lamps. This particular problem allows a large area into which the 
required energy can be delivered. Thus what the mercury discharge tube 
lacks in steradiancy can be made up by extending the emitting area, which 
is quite easily accomplished, until the total amount of energy received 
by a unit volume of liquid during exposure meets the experimental 
requirement. 

The broad band of radiation to be used in this experiment is most 
easily isolated by a filter system which can usually also be extended in 
area at will (see p. 142). 

It is quite clear that, in this case, it would have been uneconomical and 
difficult to have attempted the use of a source which was very bright, 
i.e., of high steradiancy. Such sources usually attain brilliance by high 
current densities in small volumes. The total energy output may there- 
fore be less than a greatly extended source of low steradiancy. Indeed 
one of the highest rates of total ultraviolet output has been achieved with 
such a source of low steradiancy. Furthermore, as can be seen in the 
general references cited, a high current density is often obtained at the 
expense of simplicity and ease of operation. 

The steradiancy of a source becomes a matter of importance when for 
any reason it becomes necessary to use an image of the source for irradia- 
tion. The whole matter of power transmission through image-forming 
systems has been considered in detail by Loofbourow (1950) and Blout 
et al. (1950). 

The importance of a careful study of these principles may be indicated 
by the example which follows. Assume that only the cytoplasm of a cell 
is to be irradiated and a study made of the effects of such irradiation on 
the nucleus. Such an experiment will require the formation of a reduced 
image of the source or a portion thereof within the cytoplasm. This 
would probably be accomplished by use of a reflecting objective as a con- 
denser. In these experiments also the use of the total emission of a 
source will be assumed. The following relation, known as Lagrange's 
Law (see Hardy and Perrin, 1932, p. 43), has been shown to hold by a 
number of writers including those cited. 



r_> I RADIATION BIOLOGY 

i'lie ratio of area in object (source) space A , to that in image space Ao is 
equal to the ratio of the solid angle of rays forming the image and leaving 
the source, or expressed in terms of linear dimensions, 

L.NAi = L,NA,, 

which says that the product of a linear dimension of the source and the 
numerical aperture of the rays leaving the source collected by the imaging 
system is equal to the product of the same dimension of the image of the 
source and the numerical aperture of the image-forming rays. 

Let us say that we wish to form an image of the source which is 0.005 
mm in diameter with a condenser lens of NA = 0.5. Thus, 

0.005 X 0.5 = 0.0025. 

We assume a field lens for the source which has as high an aperture as the 
condenser. Then. 0.0025 = LiO.5. 

Li = 0.005 mm. 

From this it is clear that only an area of the source of diameter 0.005 
mm is contributing to the energy flowing into the cytoplasm. Should a 
collecting lens of smaller numerical aperture be used, a larger area of the 
source would contribute, but through a smaller solid angle and, assuming 
the source to be uniform, the total energy would be the same. In order to 
increase the amount of energy delivered into the cytoplasm in a given 
time the product L2NA2 must be increased, the steradiancy of the source 
must be increased, or both. 

This fundamental relation has been demonstrated in an example of sim- 
ple image formation. It has been shown to hold as the limit no matter 
how many image-forming steps are interposed between the source and the 
final image used for irradiation. 

Consider, for example, a possible arrangement of optical components 
(Fig. 4-1) for the determination of the action spectrum (see p. 384) of the 
effects of ultraviolet radiation found in the preceding experiment. In this 
system S is the source focused by a collecting lens on »S'Li, the entrance 
slit of the monochromator .1/. .S'L,. is the exit slit of the monochromator 
and / the image formed in the cytoplasm by the condenser. Then, 

LsNAs = Lsl.NAm = Lsl^NAm = L,NA,. 

In this case the slits of the monochromator serve as secondary sources. 
The width of the slits {Lsl,, Lsl,) is determined by the dispersion of the 
monochromator and the required width of the band of radiation to be iso- 
lated (p. 148). This may establish a limiting value for the products 
including these terms because of limitations inherent in available mono- 
chromators. It is again emphasized that the establishment of a value for 
any one of the products L^NA^ by experimental re(iuiremeuts or l)y 



ULTRAVIOLET SPECTROSCOPIC TECHNIQUE V2') 

instrumental limitations establishes the value for the remaining products 
and thereby the technical nMjuirements for all other optical components 
in the system. 

This treatment has been limited but is intended to indicate the frame- 
work within which an intelligent choice of a source of radiant energy is 
made. 





\ 




-M ^ / 

sz, / 

Fig. 4-1 

PRACTICAL ASPECTS 

The last important group of factors in the choice of a particular source 
are the practical considerations which include simplicity of construction 
and operation, ruggedness, useful life, availability, and cost. In the past 
two decades the commercial availability of many ultraviolet sources 
together with the power supplies for their operation from domestic mains 
has increased greatly. Because of the importance of the ready avail- 
ability of many sources a separate section has been given over to the 
description of these (see p. 126). 

To avoid repetition of the descriptive data contained in the general 
references noted earlier in this section, Table 4-1 has been prepared. 
This table constitutes a summary of the various types of experimental 
and laboratory commercial light sources which have been found useful in 
radiation studies in the ultraviolet range of the spectrum. No effort has 
been made to summarize all the data on the subject, but rather to give 
leads to the literature on some of the older sources which have certain 
useful characteristics and to give more detailed data on the more recent 
developments. The reader is again advised to consult the following 
references for a detailed description of the older sources of ultraviolet 
light: Forsythe, 1937; Ellis ct al., 1941; Harrison et al., 1948; and Roller, 
1952. 

DETECTORS OF ULTRAVIOLET RADIATIONS 

Ultraviolet radiations may be observed by the use of fluorescent 
screens, or such radiations may be detected and quantitatively measured 
by means of the thermal, chemical, or electrical effects they produce. 
Visual detection is of value in instances where qualitative observations are 
adequate, as in the alignment of optical systems; thermal and photochem- 
ical effects may be employed to determine absolute quantities of radia- 
tion; because of their susceptibility to amplification, some photochemical 



1 20 



HADIATION BIOLOGY 



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ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 129 

(photographic) effects and the electrical effects make possible the meas- 
urement of small (quantities of such radiations. 

FLUORESCENT SCREENS 

A great variety of substances fluoresce under ultraviolet irradiation 
(DeMent, 19-15). The properties of certain of these, of especial interest 
to the lamp industry, have been studied in considerable detail (Kniger, 
1948; Fonda and Seitz, 1948). 

Zinc silicate (willemite) powder can be used to make very satisfactory 
fluorescent screens over the wave-length region 1000-3000 A (Beese, 1939; 
Lui, 1945). Maximum excitation is obtained with radiation near 2500 
A, for which the quantum efficiency of fluorescence is nearly unity 
(Fonda, 1939; Schulman, 1946). The emission spectrum of manganese- 
activated zinc silicate peaks at 5250 A but the emission can be shifted 
throughout the visible spectrum by addition of beryllium and of various 
activators (Leverenz and Seitz, 1939). Throughout the region 2200- 
3000 A nonabsorbent silicone resins, such as General Electric #9980, may 
be used as a binder. Magnesium tungstate may also be used as a phos- 
phor throughout the wave-length region 2200-3000 A with a quantum 
eflficiency nearly unity (Fonda, 1944; Oszy, 1951). Data on other phos- 
phors useful on this spectral region are summarized by Thayer and 
Barnes (1939). 

For the 3000-4000 A region, sulfide phosphors are quite effective 
(Klasens et al., 1948; Studer and Larson, 1948; Pringsheim, 1949, pp. 
582^., pp. 594^".) with high quantum efficiency about 3650 A (Leverenz 
and Seitz, 1939). A large number of varicolored pigments are known 
which respond to radiation in this region (Barnett and Grady, 1949). 

Special phosphors have been developed to convert ultraviolet radiation 
at 2537 A to ultraviolet radiation at other wave lengths for particular pur- 
poses (Froelich, 1947). Thus ultraviolet-sensitive phosphors emitting 
radiation in the erythemal region (2900-3200 A) (Clapp and Ginther, 
1947; Nagy et al., 1950) and the "black-fight" region (ca. 3600 A) (Beegs, 
1943; Clapp and Ginther, 1947) have been described. 

Fluorescent coatings may be employed to extend the usefulness of 
phototubes to wave-length regions shorter than the transmission limits of 
their envelopes. Dejardin and Schwegler (1934) used sodium salicylate 
to extend the effective range of a potassium hydride surface phototube 
from 3400 to 2200 A. A constant quantum efficiency of fluorescence was 
obtained over this region. Coatings of salicylate and other materials 
have been used to extend the sensitivity of photomultiplier tubes to 900 A 
in the vacuum ultraviolet (Johnson et al., 1951). Again a constant quan- 
tum efficiency was found with salicylate, independent of the wave length 
of excitation. 

It would seem quite feasible to make use of the threshold wave lengths, 



130 RADIATION BIOLOGY 

niul spectral variation of sensitivity of various phosphors, in conjunction 
with the spectral response of phototubes, to make wave-length-selective 
detectors (e.g., Luckiesh and Taylor, 1940; Kerr, 1947j. 

THERMAL DKTECTORS 

The measurement of radiant energy in absolute units is most com- 
monly accomplished by the total absorption of such energy in an appro- 
priate substance, accompanied by a measurement of the increase in tem- 
perature of the absorber. If the heat capacity of the absorber is known, 
the energy content of the radiation may then be readily calculated. In 
practice, the increase in temperature of the absorber may be measured as a 
resultant change in electrical resistance (bolometer), as an electromotive 
force (thermocouple), or as a mechanical deformation induced by gas 
expansion (Golay cell). Radiation detectors based on a measurement of 
the change in temperature consequent to radiation absorption are called 
thermal detectors. 

To absorb totally the radiant energy, the detector must be "black" to 
all wave lengths represented in the radiation to be measured. It is pos- 
sible to prepare such "black" surfaces by vacuum deposition, under 
appropriate circumstances, of such substances as bismuth, zinc, platinum, 
or gold (Pfund, 1930, 1933, 1937a,b; Harris and McGinnies, 1948). Such 
surfaces are known to absorb all incident radiation from 0.2 m to beyond 

15 ^l. 

These detectors may be calibrated by the use of a radiation beam of 
known energy content, thereby avoiding the necessity of a direct measure- 
ment of their heat capacity. Such a defined beam may be obtained from 
standard lamps, available from the U.S. Bureau of Standards (Goblentz 
and Stair, 1933), operated under precisely defined conditions. 

Bolometer. Measurements of the change in electrical resistance of a 
detector, consequent to the absorption of radiant energy, are conveniently 
carried out by using the detecting bolometer as one arm of an initially 
balanced resistance bridge. The change in resistance leads to unbalance 
of the bridge with a resultant unbalance voltage which may be amplified 
to a readily meterable magnitude. A similar bolometer, shielded from 
the radiation, may be placed in an appropriate arm of the bridge to com- 
pensate for variations in ambient temperature. If the thermal capacity 
of the detector is low, it may be used with chopped radiation, with a 
resultant oscillatory unbalance voltage, which may be amplified by an 
alternating-current amplifier; in such cases the amplifier may well be 
.sharply tuned to the chopping frequency to improve the signal-noise 

ratio. 

To produce a bolometer detector of high sensitivity, it is desirable to 
deposit the absorbing coating upon a substance with a high temperature 
coefficient of resistance. Among the metallic substances, nickel and 



ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 131 

platinum (0.3 and 0.6 per cent per degree Centigrade, respectively) have 
been used. The semiconductor thermistors have appreciably larger, 
negative coefficients of resistance (to —5 per cent per degree Centigrade) 
(Becker et al., 1940) and are widely used (Dodd, 1951). Both thermistor 
and metallic bolometers hav-e been made with a limiting sensitivity of 
about 10~* watt, with a response time of a few milliseconds (Baker and 
Robb, 1943; Jones, 1946; Billings, Barr, and Hyde, 1947; Billings, Hyde, 
and Barr, 1947; Schlesman and Brockman, 1945), or increasing sensi- 
tivity to 10"^" watt with response time of a few seconds (Jones, 1949). 

Thermocouple. By placing the absorbing surface in good thermal con- 
tact with a bimetallic junction, a change in the temperature of the 
absorber may be measured as a change in the potential difference across 
the junction. To minimize the effects of ambient temperature changes 
on such a thermocouple detector, this potential is usually measured with 
reference to the potential across a similar junction, in thermal contact 
with a second absorbing surface, adjacent to the first, but not exposed to 
the radiation beam. If several such pairs of junctions are connected in 
series to produce a larger total change in potential difference, the device is 
called a thermopile. 

The potential difference thus developed may be amplified in a direct- 
current amplifier, or it may be mechanically interrupted in a "breaker" 
amplifier and thereby converted into an oscillatory signal to be amplified 
in an alternating-current amplifier (Liston et al, 1946). Alternatively, 
if the absorber has a small heat capacity, the radiation beam may be 
mechanically chopped to provide a cyclic voltage. This latter method 
has the additional advantage that it minimizes the effects of slow "drifts" 
between the potentials of the measuring and reference junctions. 

The rate of change of potential difference across such a junction with 
change of temperature is the thermoelectric power of the junction. The 
highest thermoelectric powers are obtained with junctions between bis- 
muth-antimony and bismuth-tin alloys (Pfund, 1937a; Hornig and 
O'Keefe, 1947); with such junctions, powers of 120 mv/°C may be 
obtained. However, because of the fragility of bismuth alloy junctions, 
other metallic couples such as constantan-chromel (77 mv/°C) are occa- 
sionally used (Launer, 1940). 

With such bimetallic couples and with careful design, it is possible to 
measure a rise in temperature of the absorber of the order of 10~*°C. 
Such radiation detectors can provide a sensitivity of 50 mv/mw or greater 
(Schwarz, 1949; Jones, 1949). 

For use in photochemical experiments a thermopile with a large receiv- 
ing area may be desired (Crane and Blacet, 1950). 

Golay Detector. The Golay radiation detector is characterized by both 
a high sensitivity and a relatively rapid response time (Zahl and Golay, 
1946; Golay, 1947a, b). The radiation is absorbed in a blackened surface 



132 UADIATION IJIOLOGY 

of low Ileal capacity ininuT.scd in an atmosphere of xenon in a .small cham- 
ber. The rear wall of the chamber is a thin collodion film, .silvered on the 
outside surface. When exposed to radiation, the absorbed heat is 
rapidly transferred to the gas, which expands, deforming the collodion 
wall. This slight deformation is readily detected by an optical system 
whereby an image of a grid is cau.sed to move across another grid, vary- 
ing the light received on the face of a photocell, as the membrane is 
deformed. The detector is intended to be used with chopped radiation. 
With .such a cell, energies of 5 X 10~" watt may be detected (Golay, 
1949). 

Ultimate Sensitivity of Thermal Detectors. The sensitivity of any ther- 
mal detector of radiation is ultimately limited by the random fluctuations 
to be expected on thermodynamic grounds, in the temperature of any 
body in equilibrium with its environment (Myers, 1946; Jones, 1947). It 
would be impractical to attempt to measure a temperature change due to 
incident radiation, which is small compared to these random fluctuations. 
Alternatively, from a different but equivalent point of view, one may 
regard the sensitivity as limited by the inevitable statistical fluctuations 
in the heat radiation emitted and received at all times by any body. 
Since a thermal detector is sensitive to radiation of all wave lengths, it is 
sensitive to the thermal radiation emitted by its surroundings. Any 
attempt to detect a radiation beam of energy less than the fluctuations to 
be expected in the thermal radiation energy received from (and emitted 
to) the surroundings (Fellgett, 1949) would be impractical. 

Such considerations set a lower limit to the sensitivity of thermal 
detectors expo.sed to a surround at ordinary temperatures, at about 
3 X 10~'"^ watt for a detector of area 1 mm- and response time of 1 sec 
(Jones, 1947). To achieve higher sen.sitivity in any radiation detector, 
it is necessary to limit the wave-length region to which it is sensitive, in 
order thereby to reduce the fluctuation in the detector output, due to the 
fluctuation in incident thermal radiation energy. Thus, for instance, for 
objects at ordinary laboratory temperatures, the intensity of emitted 
thermal radiation of wave length less than 6000 A is negligible. There- 
fore, a photoelectric detector which is .sensitive only to radiations of wave 
length less than 6000 A is entirely insensitive to the fluctuations in ther- 
mal radiation. 

PHOTOCHEMIC.\L DETECTORS 

If monochromatic radiation is employed or if the spectral energy dis- 
tribution of the radiation concerned is known, photochemical processes 
may be conveniently used as a mea.sure of radiation intensity. For 
absolute determinations, the quantum yield of the photochemical reaction 
must fir.st have been determined at all wave lengths of interest by calibra- 
tion again.st a standard thermal detector. The use of a photochemical 



ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 133 

detector, instead of direct use of a thermal detector, is frequently found 
advantageous for reproducing the geometry of an irradiation experiment, 
or for reasons of economy and convenience. 

In ultraviolet, the photochemical decomposition of uranyl oxalate is 
widely used as an actinometer (Leighton and Forbes, 1930; Forbes and 
Heidt, 1934; Bowen, 1946; Launer, 1949). The quantum yield for this 
decomposition has been carefully measured at a series of wave lengths 
from 208 to 434 m/x (Leighton and Forbes, 1930; Brackett and Forbes, 
1933). The initial oxalate content of the stock solution, and the residual 
oxalate after irradiation, are measured by permanganate titration and the 
decomposition determined by difference. From the quantity of oxalate 
decomposed and the quantum yield, the number of photons absorbed can 
be calculated, and from this figure, the incident intensity determined, 
knowing the absorption of the solution and making appropriate correc- 
tions for such factors as reflections at cell windows. It is convenient to 
use a solution of sufficient concentration to absorb all the incident radia- 
tion; adequate stirring must be provided to equalize the irradiation of all 
volumes of the solution. The photochemical reaction has been shown 
to have nearly unity temperature coefficient. 

A rather unusual photochemical transformation of certain triphenyl- 
methane dyes (Lifschitz, 1919; Lifschitz and Joffe, 1921; Harris et al., 
1935) from a colorless to colored form has been used as a means of com- 
paring ultraviolet intensities in the spectral region 2400-3200 A (Calvert 
and Rechen, 1952; Harris and Kaminsky, 1935; Weyde, 1930; Weyde et 
al., 1930; Miyake, 1949). The quantum yield for the transformation has 
been shown to be unity over this spectral region and is independent of 
temperature (Weyde and Frankenburger, 1931; Calvert and Rechen, 
1952). This actinometer can be used at considerably lower light inten- 
sities than can the uranyl oxalate. Under appropriate conditions, the 
transformation is quantitatively autoreversible, permitting the same 
solution to be used repeatedly (Weyde, 1930; Weyde et al., 1932). 

PHOTOGRAPHIC DETECTORS 

The photographic plate or film has a number of advantages as a radia- 
tion detector. It is a sensitive detector and can integrate the radiation 
received. Furthermore, it is unique ^ among ultraviolet radiation detec- 
tors in that it is a two-dimensional detector, thus permitting an entire 
spectrum or microscopic image to be recorded in one exposure. While 
it cannot be regarded as a precision instrument for the measurement of 
radiation, with appropriate auxiliary techniques, accuracies of the order 
of ± 3 per cent can be obtained. 

Since individual plates or films vary unavoidably in sensitivity and in 
contrast, for quantitative work it is necessary to standardize each plate 

^ With the potential exception of the image orthicon tube. 



134 RADIATION mOLOGY 

(for a general description of the properties of photographic materials, 
see James and Higgins, 1948). Furthermore, since the sensitivity and 
contrast vary with wave length, the plate must be calibrated at all wave 
lengths for which measurement is desired. (For data on the ultraviolet 
characteristics of spectroscopic plates, see Harrison, 1925a; Jones and 
Sandvik, 1920; Johnson and Hancock, 1933; Amstein, 1944. Fraser, 
1950, has obtained data on the ultraviolet characteristics of several types 
of motion picture film.) 

For homochromatic photometry, in which the only desire is to compare 
radiation intensities at a given wave length, the plate or film may be cali- 
brated by means of any device which produces a scale of plate blackening 
versus source intensity on which the desired unknown intensities may be 





(a) (b) 

Fig. 4-2. Rotating sectors: (a) stepped, (b) continuously variable. (Reproduction 
from Practical Spectroscopy, by G. Harrison, R. Lord, and J. R. Loofbourow, Prentice- 
Hall, Inc., 1948.) 

read (Harrison, 1934b). Calibrated step wedges or rotating sectors are 
commonly used for this purpose. Thin films of platinum (Merton, 1924; 
O'Brien and Russel, 1934; Uber, 1939) or Chromel A evaporated onto 
quartz (Banning, 1947a) are frequently used for the former, since they 
are nearly constant in optical density over a wide range of wave lengths, 
but for accurate work they must be calibrated. The rotating sector may 
either be stepped or it may vary continuously in exposure time (Fig. 
4-2). It is truly a "neutral-density" device. 

For heterochromatic photometry, in which it is desired to compare 
radiation intensities at different wave lengths, the relative sensitivity of 
the plate as a function of wave length must be determined. This is most 
easily done with a source of previously determined spectral energy distri- 
bution, preferably one with a spectral continuum, such as the hydrogen 
discharge tube. 

Ordinary photographic plates are sensitive to ultraviolet radiations to 
wave lengths as short as 2300 A. The faster of these plates reciuire a net 
exposure to 0.1 1.0 ergs/cm- to produce a plate density of 0.5-1.0. 
Below 2300 A, the absorption of the gelatin matrix for the silver halide 
grains prevents the radiation from penetrating beyond the upp«n- layer of 



ULTHAVIOLET SPECTROSCOPIC TECHNIQUE 135 

emulsion and thereby greatly reduces the plate sensitivity. For work at 
shorter wave lengths, very thin emulsions heavily laden with silver halide 
grains may be used, such as the Ilford "Q" plates or the Eastman Kodak 
SWR film (Schoen and Hodge, 1950). As an alternative, the surface of 
the film may be coated with material that will fluoresce under the short- 
wave-length radiation, so that the exposure is actually produced by the 
fluorescent radiation (Harrison, 1925b). Such thin-emulsion or fluores- 
cent-coated plates may be used far into the vacuum ultraviolet (Harrison 
and Leighton, 1930). By the use of a fluorescent coating with constant 
quantum yield of fluorescence, independent of exciting wave length, 
problems of heterochromatic photometry may be greatly simplified. 
Such coatings also eliminate the variation of contrast with wave length 
(Harrison and Leighton, 1931). For photomicrography, however, the 
use of fluorescent coatings generally leads to some loss of plate resolution. 

PHOTOELECTRIC DETECTORS 

Photoelectric detectors useful in the ultraviolet are of two general 
types: the photovoltaic or barrier-layer cell, and the photoemissive 
detector. The photovoltaic cells, which do not require an external power 
source, are convenient and useful in instances where relatively large 
amounts of radiant energy are available. The photoemissive detectors 
require more elaborate accessory equipment but are far more sensitive 
and are effective in a wide variety of applications. 

Photovoltaic Detectors. Upon illumination of a photovoltaic cell, a 
potential difference appears across a semiconductor (usually iron sele- 
nide), which potential can be used to drive a current through an external 
circuit (Lange, 1938; Zworykin and Ramberg, 1949, Chap. 11). Elec- 
trically, the photovoltaic cell acts as a source of current which is shunted 
by an internal resistance and capacitance. The shunting internal resist- 
ance is not constant, but decreases with increasing illumination and with 
increasing current flow. Although the photocurrent generated within the 
semiconductor is, at moderate light levels, linearly dependent on light 
intensity, because of the internal resistance and its variation with light 
intensity, the external current is a linear function of light intensity only 
if very low external resistance is employed (Wood, 1934). As a conse- 
quence, the output of a barrier-layer cell as a function of fight intensity, 
with various external resistances, is as shown in Fig. 4-3. 

Electronic circuits have been developed to permit the use of larger 
external resistance, if desired for purposes of amplification, without 
introducing appreciable nonlinearity. Such circuits (Rittner, 1947) 
employ negative feedback to effectively reduce the apparent resistance 
external to the photocell. 

The internal capacitance of the photovoltaic cell also acts to shunt the 
external resistance if an oscillatory photocurrent is produced by a modu- 



130 



RADIATION BIOLOGY 



700 



600 



500 



400 



300 



200 



100 



lated light beam. This capacitance siuiiit Hmits the useful range of 
modulation frcciuencies to below 10, ()()() cycles/sec. 

Although all commercially available barrier-layer cells have peak sensi- 
tivity in the \isible spectral region, they are available in quartz envelopes 
which permit apprecial)le response to wave lengths as short as 270 m/i. 
These cells are somewhat temperature sensitive, and may display an 
initial "fatigue" for 15-20 min on exposure to radiation (Lange, 1938; 

Barbrow, 1940). For use in appro- 
priate applications, matched pairs of 
cells are available. 

Photoemissive Detectors. Because 
of their high sensitivity, linearity and 
speed of response, and convenience 
of operation, photoemissive detectors 
have become the most widely used 
means for quantitative measurement 
of ultraviolet radiation. The elec- 
tric currents deri\^ed from these de- 
vices are easily amplified and may 
then be used to operate meters or 
au}^ of various kinds of automatic 
recording devices. With modern 
techniques (Engstrom, 1947a; Som- 
mer and Turk, 1950) it is possible to 
reduce the extraneous sources of elec- 
trical fluctuation, such as the thermal 
emission of electrons from the photo- 
cathode and the thermal motion of 
electrons in the amplifier input cir- 
cuit, to levels sufficiently low for the 
principal limitation on the precision 
of measurement of w'eak beams of radiation to arise from the (juantized 
nature of the radiation itself and from the concomitant statistical fluctua- 
tions in radiation intensity (Johnson and Llewellyn, 1934). 

The operation of photoemissive cells depends on the release of electrons 
from a photosensitive surface on incidence of quanta of adequate energy. 
Since the energy of a quantum is proportional to the frequency of the 
radiation, there is for any surface a minimum value of frequency — or a 
maximum value of wave length — below (or above) which the quanta will 
not have sufficient energy to release electrons. This maximum wave 
length is known as the threshold wave length for the photosurface in 
ciuestion. 

For many metallic svafaces, the threshold wave length lies in the ultra- 
violet. This circumstance has made possible the design of photocells 













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l( 


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50 100 150 200 250 300 350 
ILLUMINATION, ft-C 

Fig. 4-;J. Influence of external circuit 
resistance upon current output of 
photovoltaic cell. Photocurrent char- 
acteristics with several external resist- 
ances; rectangular cell Model lOA; 
active area 0.70 sq in. Figures on 
curves, ohms. {Bradley Laboratories, 
Inc.) 



ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 137 

which are sensitive only within well-defined spectral regions; the upper 
wave-length limit is defined by the quantum threshold and the lower by 
the absorption properties of the photocell envelope. 

Thus a cell with a cadmium-magnesium surface and a Corex-D window 
has a spectral response curve closely paralleling the action spectrum for 
erythema production, and is of considerable utility in the measurement of 
the erythemal effectiveness of various sources (KoUer and Taylor, 1935; 
Kerr, 1947; Taylor, 1944). Other cells with magnesium (Coblentz and 
Cashman, 1940), titanium (Coblentz and Stair, 1935; Kuper et al., 1941), 
or uranium (Rentschler, 1930) surfaces have found use for the measure- 
ment of the intensity of extreme ultraviolet radiation from the sun. 

Other metals such as zirconium, thorium, tantalum, platinum, and 
alloys such as beryllium-copper have threshold wave lengths at various 
places in the ultraviolet spectrum and might be used to provide photocells 
with specific spectral response characteristics (Rentschler et al., 1932; 
Glover, 1941; Andrews, 1945; Morrish et al., 1950; Piore et al., 1951). 
The precise threshold wave length and spectral response curves of these 
metallic surfaces depend considerably on the particular method of prepa- 
ration (Dejardin, 1933). 

Most metallic surfaces, however, have relatively low quantum effi- 
ciency, emitting one electron per lO'^-lO^ incident quanta (Sommer, 1947). 
Hence, most modern photocells are made with composite surfaces, such 
as the cesium-antimony surface which has a quantum yield of approxi- 
mately 0.1-0.3 at the wave length of maximum response (400 m/x) (Janes 
and Glover, 1941; Sommer, 1947; Morton, 1949; Zworykin and Ramberg, 
1949, Chaps. 5 and 6) and maintain a high yield well into the ultraviolet 
(Fig. 4-4). 

Glass-jacketed photocells begin to decline in sensitivity at wave lengths 
less than 3500 A. Commercially available ultraviolet-sensitive photo- 
cells have envelopes of Corex-D, Corning 9741, or Vycor glass. The 
Vycor glass provides good transmission to approximately 210 m/x, but 
begins to absorb appreciably at shorter wave lengths (Nordberg, 1947). 
Some response may be obtained to wave lengths as short as 160 m^ 
(Dunkelman and Lock, 1951), owing in part to fluorescence of the glass. 
Special quartz-jacketed photocells have high sensitivity to 175 mu and 
will respond to wave lengths as short as 155 m^t. 

In the vacuum-type photoemissive cell, the current developed is directly 
proportional to radiation intensity over several decades of intensity range. 
The current developed, obtained in a typical photoemissive cell as a func- 
tion of anode voltage, is shown for several radiation intensities in Fig. 4-5. 
Evidently at any anode voltage greater than 25 volts, the full photocur- 
rent is collected, and the current is thus substantially independent of 
anode voltage. The total current which may be drawn from a photo- 
surface without damage is limited to values of 5-10 ^a/cm-. 



138 



100 



2000 



RADIATION UIOLOGY 




4000 6000 

WAVE LENGTH, A 

(C) 



8000 



Fig. 4-4. Spectral response characteristics for three types of photosurface: (a) type 
1P22, S-8 response; (6) types 931-A and 1P21, S-4 response; (c) type 1P28, S-5 
response. (Engsirom, 1947a; Journal Optical Society of America.) 




250 



150 
AI\JO0E, volts 

Fig. 4-5. Current-voltage characteristic of RCA 935 (ultraviolet sensitive) 
(Radio Corporation of America.) 



photocell. 



ULTRAVIOLET SPECTKOSCOPIC TECHNIQUE 



139 



At low levels of radiation intensity, the photociirrent generated will be 
small. This current may be amplified external to the cell by conven- 
tional vacuum tube circuits (Zworykin and Ramberg, 1949, Chaps. 12-14) 
or it may be amplified within the cell, either by gas multiplication or by 
the use of secondary emission, as in the photomuitiplier tubes. The 




30 



50 70 



10,000 



100 200 300 500 700 1000 2000 4000 

ACCELERATING VOLTAGE OF PRIMARY ELECTRONS 

Fig. 4-6. Secondary-emission characteristics of typical photosurface materials. 
{Reproduction from Photoelectricity and Its Application, by V. K. Zioorykin and E. G. 
Romberg, John Wiley d- Sons, Inc., 1949.) 

advent of photomuitiplier tubes has largely supplanted the use of gas- 
filled tubes. The direct or amplified photocurrent may be measured with 
a galvanometer or ammeter, may be recorded, may be integrated in dis- 
crete quantities and counted (Douglas, 1947; Launer, 1949), or may be 
used to operate such devices as relays and motors. 

Photomuitiplier Tubes. In the 
photomuitiplier tubes, the primary 
current from the photosurface is 
multiplied by a factor which may 
be as large as 10^ by repeated use 
of multiplication at secondary emis- 
sion surfaces. Many surfaces, in- 
cluding those commonly used as 
photosurfaces, will, when struck by 
an electron of appropriate energy, 
emit several electrons. The num- 
ber given off per primary electron 
depends on the surface and the 
voltage applied to the primary elec- 
tron (Fig. 4-6) (Zworykin et at., 
1936; Morton, 1949). 

In the focused photomuitiplier tubes (Rajchman and Snyder, 1940), 
the primary photocurrent is focused by an electrostatic field onto such a 
surface, called a dynode. This process is repeated nine or ten times until 
the vastly amplified current from the last secondary emitting surface is 
collected on an anode (Fig. 4-7). 




FOCUSING 
GRILL 



MICA SHIELD' 

Fig. 4-7. Construction of nine-dynode 
focused photomuitiplier tube. 0, photo- 
cathode; 10, anode; 1-9, dynodes. 
{Engstrom, 1947a; Journal Optical Society 
of America.) 



10 



RADIATION HIOLOGY 



An alternative design, such as is used in the " Venetian bhnd" photo- 
multiplier tubes (Sommer and Turk, 1950), does not attempt to focus the 
electrons from each dynode upon the next, but merely uses an accelerating 
field to draw the majority of secondary electrons (70-8o per cent) to the 
succeeding dynode (Fig. -4-8). 

Thus a conventional photomultiplier tube consists of a photocathode, 
a series of 9-11 secondary emission dynodes, the first of which is main- 
tained at a potential 75-150 volts above that 
of the photocathode while each succeeding 
dynode is elevated another 75-150 volts in 
potential in sequence, and a final anode which 
is maintained 50-100 volts above the potential 
of the last dynode. With a multiplication of 
3-5 per dynode, the over-all amplification of a 



3- 



2- 



0-1 



I 



—v/yy/'/'T- 
— KSNWssr- 
—ryyyyy'yr- 

— kWVCsV^— 

—vyyyyyyr- 
— K\\\x\si— 
—vyyyyy^~ 

— KX\\\V1— 

—Y///y/r- 

— KVX\XV\I — 



> i?| to 27„ 



or 







^ 



umM 



9 dynode tube can range from 3* to . 
20,000 to 2,000,000. 

Because the current capacity of the last 
dynode or anode is limited, there is, at normal 
gain, a maximum current which may be drawn 
from the photosurface, and hence a maximum 
illumination to which it should be exposed. 
This limit, which will be less than 1 /xw for a 
1P28 photomultiplier tube operated at a cur- 
rent amplification of 10^, may be raised if the 
voltage applied per stage is reduced. 

The response of photomultiplier tubes is a 
linear function of light intensity over many 
decades. Fatigue is inappreciable at low light 
Jevels. Because of the variations in secondary 
emission with dynode voltage, the voltage 
supply for the photomultiplier tube potentials 
must be held stable to an order of magnitude better than the stability 
desired in the output current. Batteries may be used, or regulated elec- 
tronic supplies have been described (Ply male and Hansen, 1950; 
Higinbotham, 1951; Hill, 1945; Mautner, 1947). 

The over-all amplification of the photomultiplier is very closely a 
logarithmic function of the voltage applied per dynode, over several 
decades of gain (Fig. 4-9). As a consequence of this circumstance, it can 
be shown that, for varying levels of illumination, the voltage per dynode 
necessary to maintain a constant output current is proportional to the 
logarithm of the reciprocal of the intensity of the illumination. This 
property may be used in the design of circuits intended to measure absorp- 
tion directly in terms of optical density (Sweet, 1946). 

The time resolution of a photomultiplier tube is limited only by the 



Fifi. 4-8. Design of "Vene- 
tian blind" type photomul- 
tiplier tube: T, photosensi- 
tive surface; D, dynodes; E, 
collecting anode. (Sommer 
and Turk, 1950; Journal of 
Scientific Instruments.) 



ULTRAVIOLET SPECTUOSCOPIC TECHNIQUE 



141 



variations in time of transit of electrons from photosurface to anode, which 
are of the order of 6 X 10~^ sec (Morton, 1949). As a consequence, the 
photomultiplier tube will faithfully respond to very brief pulses of light, 
as short as 10~^ sec. 

The amplified photocurrent from the photomultiplier tube easily over- 
whelms the random fluctuations in electric current arising in the external 
circuit, as a result of thermal agitation, so that the only limitations on the 
sensitivity of a photomultiplier detector are those arising from the ran- 
dom fluctuation of the "dark current" which is actually the thermal 
emission of electrons from the photosurface, and from those inherent in 



1.000,000 




125 



150 



75 100 

VOLTS PER STAGE 

Fig. 4-9. Amplification characteristic of focused type photomultiplier. (Ent/strom, 
1947a; Journal Optical Society of America.) 

the statistical nature of the radiation intensity itself. The thermal- 
emission dark current and its corresponding fluctuations may be reduced 
by choice of a photocell with a small photosensitive surface, or it may 
be minimized by refrigeration of the photocell, without appreciably 
influencing the sensitivity to radiation (Engstrom, 1947a, b). 

It should be recognized that photoelectric detectors vary considerably 
from tube to tube (of the same design) with regard to sensitivity, to 
variation of sensitivity with wave length, and to the dark current. 
Because of these variations, if it is desired to use photoelectric detectors 
for the comparison of two beams of radiation, one of two courses is neces- 
sary: (1) some artifice whereby one detector may be used must be 
employed, or (2) if two detectors are used, either matched tubes must be 
found, or some means of compensating for their differences (which may be 
expected to be reasonably stable) must be provided. A single detector 
may be used, if it is alternately exposed to the two beam.s in time, or if 
the electrical signal arising from each beam can be distijiguished by virtue 
of a frequency or phase modulation (Wright and Herscher, 1947; Savit- 
zky and Halford, 1950; Wyckoff, 1952). 



142 RADIATION BIOLOGY 

The use of modulated radiation, giving rise to an oscillatory current, 
permits the use of alternating-current amplifiers and thus simplifies the 
associated electronic circuitry. Alternatively, a steady photocurrent 
may be converted to an oscillatory current, either by magnetic modula- 
tion (Kalmus and Striker, 1948) of the photocurrent, or by mechanical 
interruption (Liston et al., 1946; Lash, 1949). 

Image Orthicon. The application of television techniques (Zworykin 
and Ramberg, 1949, Chaps. 16, 17) to ultraviolet spectroscopy through 
the medium of ultraviolet-sensitive image orthicon tubes has significant 
potentialities. The orthicon is in effect a two-dimensional photoelectric 
detector which permits the application of photoelectric techniciues to 
problems that previously could be adecjuately approached only by photo- 
graphic means. The use of the image orthicon by Pai-part and Flory for 
the \isualization of ultraviolet microscope images (Purpart, 1950; Flory, 
19")1). and the study of source spectral characteristics (Benn el al., 
1949; Agiiew et al., 1949) may be cited as examples of the potential 
applications. 

METHODS OF SPECTRAL ISOLATION 

One of the important data in any problem in radiation biology is the 
variation of the subject under investigation (absorption, fluorescence, 
photobiological or photochemical effect) with the wave length of the 
radiation concerned. To obtain these data, spectrally defined beams of 
radiation must be available. A wide variety of devices have been devel- 
oped to provide such spectrally defined beams ; these devices differ in basic 
principles and in range of application and, in general, may have specific 
advantages or disadvantages for a particular application. For the iso- 
lation of well-separated spectrum lines from a discontinuous source, much 
simpler technicjues can be employed than are necessary to isolate narrow 
spectral band widths from a source of spectral continuum. For some pur- 
poses, high intensity or large total energy of radiation are more important 
than purity of wave length. For others, flexibility and the possibility of 
easy, rapid change of wave length are important. The optimum means of 
spectral isolation can be chosen only after the research objectives are 
clearly defined. 

The various means employed for spectral isolation may be somewhat 
arbitrarily grouped into two classes: filters, which by one means or 
another block or prevent transmission of all save the selected band of 
wave lengths, and dispersing systems, which transmit all wave lengths, 
but disperse them in space so that })arti('ulai' regions may be selected. 

FILTERS 

Absorption Filters. The simplest filters are absorption filters, liy 
virtue of the al)sorption spectra of their components these filters absorb, 



ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 

TaBI.K 4-2. Ul.TRAVlOLKT FILTERS 

M.I 11(1 pass, transmission Reference 

iiiyu: 



143 



Wave-length logion transiiiitti'i 

190-290 

230-265 

230-330 

230-420 

240-280: >350 

245-275 

245-290; >340 

255-290 

270-325 

290-340 

295-330 

300-340 

300-400 

320-360 

320-390 

340-390 
For specific spectral lines, mn: 

llg 254 

Hg 254 + 265 

Hg 280 

Hg 313 



Hg 334 
Hg 366 

Cd326 
Band pass, absorption 

Wave-length region absorbed, niju: 
280-390 (CI,) 

350-540 (Br,) 
290-360 (CSo) 
340-800 
Long-wave pass 

Approximate cnt-off wave length, niju: 
190-200 
210-230 
220-260 
230-250 
245-260 
260-280 
260-310 
2t)5-275 
270-280 



Heidt, 1939 

Backstrom, 1940 

Backstrom, 1940: Mazza, 11)40 

Corning, 1948 

Kasha, 1948 

Kasha, 1948 

Kasha, 1948 

Kasha, 1948 

Dorcas and Forbes, 1927 

Kasha, 1948 

Backstrom, 1940 

Kasha, 1948 

Corning, 1948: Schott-Jena, 1952 

Kasha, 1948 

Corning, 1948 

Kasha, 1948 

West, 1946 

Bowen, 1946 

Backstrom, 1940: Bi'icher and Kaspers, 

1946 
Backstrom, 1940; Bowen, 1946; Biicher 

and Kaspers, 1946; Hunt and Davis, 

1947; West, 1946 
Bowen, 1946; Biicher and Kaspers, 1946 
Bowen, 1946; Biicher and Kaspers, 1946; 

Corning, 1948; West, 1946 
Bowen, 1946 



Gibson and Bayliss, 1933; von Halban and 

Siedentopf, 1922 
Acton et al., 1936 
Bowen, 1946 
Backstrom, 1940 



Haas, 1935 
Maclean et al., 1945 
Corning, 1948 

Bass, 1948; McLaren and Pearson, 1949 
Bass, 1948 
Kasha, 1948 
Corning, 1948 

Bass, 1948; Maclean et al., 1945 
Bass, 1948; Bowen, 1940; Maclean et al., 
1945 



144 RADIATION moLOGY 

Tabke 4-2. Ui/rRAVioLET Filters. — (Continued) 
l-oiiH-wavc |>ass Reference 

280-300 Bass, 1948; Itowcn, lltKi; Maclean el al., 

1045 

280-:i20 Corning, 1U18; Scliult-.lena, 1952 

290-310 Polaroid, 1951 

300-310 Kasha, 1948; Ley ami Wingdien, 1934; 

Saiiiidcrs, 1928 

300-330 Corning, 1948; 8chott-Jena, 1952 

310-330 Kasha, 1948; Polaroid, 1951; Schott-Jena, 

1952 

315-365 Maclean et al, 1945; Schott-.Iena, 1952 

340-300 Kaslui, 1948; Schott-Jena, li)52 

340-380 Corning, 1948; Schott-Jena, 1952 

;^50-380 Polaroid, 1951 

360-400 Bowen, 1946; Corning, 1948; Schott-Jena, 

1952 

365-430 Corning, 1948; Schott-Jena, 1952 

380-410 Eisenlirand and von Hallian, 1930; Pola- 

roid, 1951 

420 Bowen, 1946 

more or less strongly, all wave lengths other than those of the selected 
region. Such filters are simple to use, may be made in large dimensions, 
and place no limitations on the angular spread of the radiation to be 
transmitted. On the other hand, it is difficult to obtain absorption filters 
which can provide both a narrow transmission band and high transmis- 
sion within the band; further the design of an absorption filter for any 
particular spectral region is a wholly empirical enterprise. 

Absorption filters may be made of glass, of liquid cells, or of gas-filled 
cells (chlorine and bromine) or combinations of these. In general, a 
filter need not (and will not) transmit only a narrow band of wave lengths 
out of the entire electromagnetic spectrum. Consideration must be given 
to the characteristics of the radiation source and the radiation detector, 
or biological subject, to be employed. Transmission bands in far-removed 
wave-length regions, such as the infrared, might well be of no consequence 
in particular investigations. 

References to band-pass and long-wave-length-pass absorption filters 
for various regions of the ultra\'iolet are summarized in Table 4-2. 
Appropriate combinations of these may be employed for isolation of par- 
ticular spectral lines from various sources. 

If the absorption of the ultraviolet radiation involves a photochemical 
decomposition of some component of the filter, the filter may have to be 
renewed frequently. This is particularly likely with liciuid filters includ- 
ing organic components. Such decompo.sition may sometimes be mini- 
mized by placing the sensitive component farthest from the source in the 
sequence of filter elements. 



ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 



145 



Interference Filter. The action of an interference filter is based on the 
cancellation of coherent light waves when they are added together in 
phase opposition. In its action, each ray of the light beam is divided 
into a large number of weaker rays, with regular shifts in phase between 
adjacent rays, by the use of multiple reflections between lightly silvered 
surfaces appropriately spaced by dielectric, these surfaces allowing a 
slight transmission of energy at each contact (Fig. 4-10). When these 
rays are recombined by a lens, the resultant intensity at the focus of the 
lens will depend on the phase difference between adjacent rays, being 
maximal when all rays are in phase, and minimal when adjacent rays are 
exactly out of phase. If a stop is then placed about the focus of the lens, 




Fig. 4-10. Figure illustrating the splitting and multiple reflection of light rays origi- 
nating from Pi by the silvered surfaces Ei and £"2, to provide a monochromatic image 
of Pi at P-i. {Reproduction from. Fundamentals of Optics, 2d ed., by F. A. Jenkins and 
H. E. White, McGraw-Hill Book Company, Inc., 1950.) 

only those wave lengths for which adjacent rays are exactly or nearly in 
phase will be transmitted in appreciable intensity. By controlling the 
thickness of dielectric between the reflecting surfaces, the variation of 
phase difference between adjacent rays with wave length can be controlled 
and thus the wave length or wave lengths of maximum transmission 
selected. The simple interference filter can be regarded essentially as a 
crude Fabry-Perot etalon (Jenkins and White^ 1950, Chap. 14). 

Thus a simple interference filter consists of two lightly silvered reflect- 
ing surfaces, spaced by an appropriate thickness of dielectric (frequently 
magnesium fluoride). The spectral selectivity of such a filter is depend- 
ent extrinsically on the angular aperture of the radiation with which it is 
employed (increasing with decreasing aperture), and intrinsically on the 
reflectivity of the reflecting surfaces and the number of wave length paths 
in the dielectric spacer (Mooney, 1946; Hadley and Dennison, 1947, 
1948). Under favorable optical conditions, such filters can provide a 
peak transmission of about 35 per cent with a band width of about 100 A 
(at half-maximum transmission) when peaked for various wave lengths 
in the visible region. The transmission of interference filters does not, 
however, drop to zero outside the transmission band (or bands) but to a 
minimum of about 1 per cent. The wave length of peak transmission 



146 RADIATION HIOLOGY 

is specified on the assumption that the filter will be used with radiation at 
normal incidence (Buc and Stearns, 1950). 

More complex "multilayer" interference filters (Banning, 1947b; 
Bolster, 1949, 1952), which rely on the cancellation of rays multiply 
reflected between sandwiches of dielectric layers of appropriate thickness 
aiul refractive index (replacinji; the silvered surfaces), can provide higher 
transmission (70 80 per cent) and narrower band widths (50-00 A) at 
half-maximum transmission) . 

The simple interference filters cannot be satisfactorily made for wave 
lengths less than 3600 A because of the decline in the reflectivity of silver. 
Aluminum reflectors have not proved satisfactory. It would seem possi- 
ble to extend the range of the multiple layer dielectric filters farther into 
the ultraviolet if dielectrics combining proper refractive indices and ultra- 
violet transparency can be found. 

Christiansen Filters. If rough chips of transparent dielectric are sus- 
pended in a cell containing a transparent liquid, the resultant mass will be 
highly scattering and hence of low transmission, except at or near the 
wave length at which the refractive index of the liquid matches that of 
the dielectric. When employed with an appropriate optical system, such 
a cell constitutes a Christiansen filter (Christiansen, 1884). 

Such filters can be made with large cross section. Their spectral selec- 
tivity depends inversely on the angular divergence of the radiation pass- 
ing through them, directly on the difference in the slopes of the refractive 
index versus wave-length curves of the liquid and solid at their point of 
intersection (Raman, 1949) (the curve for the liquid always has the 
greater slope), directly on the thickness of the cell, and also on the size of 
the dielectric chips, for which there appears to be an optimum (Denmark 
and Cady, 1935). As the refractive indices of liquid and dielectric gen- 
erally vary at different rates with temperature, the wave length of peak 
transmission of Christiansen filters is strongly temperature dependent. 

Appropriate dielectric and liquid mixtures have been described for the 
visible region by MacAlister (1935), for the 3100 4000 A region by Kohn 
and von Fragstein (1932), and for the 2300-3100 A region by Sinsheimer 
and Loofbourow (1947). A filter for the mercury 2537 A line has been 
described by Minkoff and Gaydon (194G); von Fragstein (1938) mentions 
filters centered at 2610 and 2450 A. It should be emphasized that the 
transmission and spectral selectivity of these filters depend strongly on 
the optical system in which they are employed (Weigert and Staude, 
1927; von Fragstein, 1938). The transmission of Christiansen filters 
does not decline to zero outside the transmission band but to a minimum 
d(;pendent on the opti(!al system employed. 

Focal Isolation Fillers. The focal length of a simple uncorrected lens 
depends on its refractive index and hence on the wave length of the radia- 
tion. At the focal plane of any gixcii wave length, radiation of othei' 



ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 147 

wave lengths is necessarily not in focus and is spread out more or less dif- 
fusely. If the image of a small source is sharply masked at the focal 
plane of the wave length desired, this wave lenj^th will be favored in the 
radiation transmitted. If this process is repeated several times (the 
same lens may be used in autocollimating schemes), quite narrow spectral 
band widths may be obtained. Such a device is known as a focal isolation 
filter. 

The selectivity of such a filter will depend on the dispersive power of the 
lens material, on the number of lenses employed, and on the size of source 
and angular aperture of transmitted radiation. Fluorite or quartz lenses 
have been employed in this fashion to isolate spectrum lines in the vacuum 
ultraviolet (Forbes et al., 1934; Duncan, 1940). 

Miscellaneous Filters. A simple ultraviolet filter which can be used in 
well-collimated light to reject all wave lengths greater than an arbitrarily 
chosen boundary, has been described by Regener (1936). A thin film of 
paraffin oil is sandwiched between the long sides of two 90° quartz prisms. 
Since the refractive index of the paraffin oil is less than that of the quartz, 
total internal reflection can occur at the first oil-quartz interface; 
owing to the greater refractive dispersion of the paraffin oil, there will be, 
for any angle between the entrant beam and the oil-quartz interface, 
some critical wave length above w^hich all wave lengths will undergo 
internal reflection. This critical wave length can be varied by rotation 
of the interface. Transmission is not complete at wave lengths imme- 
diately less than the critical w^ave length, but increases rapidly with 
decreasing wave lengths. 

Various types of light filters have found employment for special pur- 
poses in the visible portion of the spectrum, and could undoubtedly be 
adapted for use in the ultraviolet, but for one reason or another have not 
been so used. Among these might be mentioned the polarization inter- 
ference filter and the rotary dispersion filter. 

The former is based on the interference between two orthogonal com- 
ponents of a beam of polarized light after passage through a birefringent 
crystal; the retardation (in wave lengths) of the one component relative 
to the other will depend on the thickness and birefringence of the crystal 
and on the actual wave length, and hence varies wdth wave length, pro- 
ducing maxima and minima of transmission throughout the spectrum, as 
the interfering Avaves combine constructively or destructively (Billings, 
1947; Evans, 1949a, b). Filters of this type, using cascaded birefringent 
elements of appropriate sequence of thickness, have been made with a 
band width of 1 A at half-maximum transmission (Billings et at., 1951). 
Such filters are designed for use at a particular w^ave length. 

Rotary dispersion filters rely for their action on the variation in rotary 
power of an opticall}^ active material, such as quartz, with wave length. 
A piece of such material, placed between similarly oriented polarizing 



148 RADIATION niOLOGY 

elements, will transniil coinplotely only those wave lengths for which the 
total rotation <it tlx- plane of polaiizalion is an integral nniltiple of 180°, 
and will reject completely those wave lengths for which the total rotation 
is an odd mnlti|)le of 90°. By cascading a few such elements of appro- 
priately chosen secjuence of thickness, an over-all transmission band 
width of 100-150 A at half-maximum transmission may be obtained. 
The wa\e length of maximum transmission may be varied over a con- 
siderable spectral region by a programmed rotation of the various polariz- 
ing elements (Cambridge Thermionic Corp., 1952). 

This extension of the application of such filters to wave lengths as short 
as 3000 A would be straightforward, since Polaroid will transmit well to 
such wave lengths (Barer, 1949). Below 3000 A it would be necessary to 
use prism polarizing elements of limited aperture. 

DISPERSING SYSTEMS 

Prism Instruments. Because of the variation of its refractive index 
with wave length, a prism will deviate rays of different wave length 
through dilTerent angles. If the angular spread of the radiation incident 
on the prism is limited (by means of an entrance slit, or equivalent, and 
collimating lens), the radiation emergent in any given direction will con- 
tain a limited range of wave lengths. The emergent radiation may be 
focused by a telescope lens to form a spectrum consisting of a continuous 
series of images of the entrance slit in light of successively increasing 
wave length. 

The width of the spectral band contained in any one image of the 
entrance slit will depend on the angular divergence of the radiation inci- 
dent on the prism, the rate of change of angular deviation produced 
by the prism with wave length, and the physical breadth (in wave 
lengths) of the beam emergent from the prism. The first (luantity is 
determined by the entrance slit width and the collimator focal length. 
The second quantity depends on the dispersive power (dn/d\) of the prism 
material and the length of the base of the prism. The breadth of the 
emergent beam determines the size of the diffraction disc to which it is 
focused. 

By the use of an appropriately placed exit slit, any portion of the 
emergent spectrum may be selected. The width of slit used can control 
the spectral width of the radiation band transmitted, except that it is 
inefficient to reduce the band width to less than the spread of wave lengths 
contained in any single image of the entrance slit. 

In general, the band width, at half-maximum transmission, of radia- 
tion emergent from a monochromator used with symmetrical entrance 
and exit slits and similar collimator and telescope lenses, is given by the 
formula 

AX = ^^ 

/•' X dd/d\ 



ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 149 

where W = width of exit sHt 

F = focal length of telescope lens 
d6/d\ = angnlar dispersion of prism, 

or by 

W 
AX = 



/„ X t dn/d\ 

where /„ = /-number of telescope lens 
t = thickness of prism base 

dn/d\ = dispersive power of prism. 

The quantity of radiant energy transmitted through the dispersing 
system depends intrinsically on the size of the entrance slit, the angular 
aperture of the collimating lens, the height of the prism, and the size of 
the exit slit. Increased energy may be obtained at the cost of spectral 
purity by the use of wider entrance and exit slits. - 

In the ultraviolet region, 2000 4000 A, quartz (crystal or fused) is the 
most commonly used prism material. Its dispersion increases rapidly 
with decreasing wave length, which results in a corresponding increase in 
spectroscopic resolving power in quartz prism instruments. Crystal 
quartz of good quality may be used in prisms to wave lengths as short as 
1850 A and in windows to about 1600 A (Powell, 1934b; Terrien, 1936; 
Boyce, 1941; Gilles et al, 1949). 

Liquid-filled prisms may also be used in the ultraviolet (Forsythe, 1937, 
pp. 88-89; Cannon and Rice, 1942), although these are temperature 
sensitive and subject to such difficulties as convection currents due to 
excess heating of the liquid near the entrant surface. Harrison (1934a) 
has described some ingenious dispersing systems, employing water as the 
dispersing element, in which convection is minimized by the use of the 
upper surface of a water trough as the first surface of the dispersing 
element. Such a device can be cheaply made in almost any desired size. 
The dispersive power of water is within 50 per cent that of quartz. 

Below 2000 A, prisms of calcium or lithium fluoride may be used to 
wave lengths of approximately 1300 and 1200 A, respectively (Powell, 
1934a; Schneider, 1934, 1936, 1937; Kremers, 1940; Boyce, 1941; Stock- 
barger, 1949). 

If uncorrected quartz lenses are used as collimator or telescope, these 
must be refocused for different wave lengths. Achromatic quartz- 
lithium fluoride pairs have been developed which are adequatelj^ cor- 
rected throughout the ultraviolet and visible regions (Perry, 1932; Stock- 

^ An ingenious means for increasing the radiant flux transmitted through the dis- 
persing system, while retaining high spectral purity, has been proposed by Shurcliff 
(1949). In this proposal, which necessitates the use of two monochromators in 
tandem, adroitly spaced, multiple entrance slits are employed, thus markedly increas- 
ing the light input to the first monochromator. Appropriately placed secondary slits 
in the spectrum plane between the two monochromators, and exit slits in the final 
spectrum, then serve to exclude all save the desired wave-length region which is 
obtained in high energy with essentially double monochromator purity. 



l-)0 



KADIATIOX IU()L()(;Y 



barKor and Cartwright, 1939; Cart\vri<>;lit , 1939). Minor optics may also 
be employed to solve the problem of achromatization. 

Multiple prism cascades may be used etTectively io obtain greater 
prism l)nse and hence greater resohing power. Double monochromators, 
employing essentially two single monociiromators in tandem, may be used 
tor greater purity of radiation and freedom from scattered radiation at the 



COLLIMATING AND 
TELESCOPE LENS 




REFLECTING 
PRISM 



FOCAL 
PLANE 



REFLECTING SURFACE 

Fig. 4-11. Littrow moiiiiiiiig for prism iiistnunciit. {Reproduction from Practical 
Spectroscopy, by G. Harrison, li. Lord, and J. li. Loofbourow, Prentice-Hall, Inc., 1948.) 





(a) 



Fig. 4-12. Constant dovialion prism instrum('nt.s usiii^: (a) Pcllin-Broca prism; (h) 
\\ adsworth mounting for prism. {Reproduction from Practical^ Spectroscopy, by 
G. Harrison, R. Lord, and J . R. Loofbourow, Prentice- Hall, Inc., 1948.) 

expense of energy transmission (Sawyer, 1951; Harrison ef al., 1948). 
Cascaded Pellin-Broca prisms may also be employed in such a way as to 
minimize stray radiation (Benford, 1936). 

By reflecting the radiation back through the prism, as in the Littrow 
moimting (Fig. 4-11), twice the dispersion and resolving power may be 
obtained. The collimating lens then may serve also as telescope lens. 
With this arrangement the exit slit is spatially near to the entrance slit, 
and scattered radiation may be a problem. 

Constant deviation monochromators may be made with the Pellin- 
Broca prism (Fig. 4-12a), with the Wadsworth mounting for the ordinary 



ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 



151 



prism (Fig. 4-126), with the Young and ThoUon spht-prism arrangemont 
(Kurtz, 1926), or with an adroit mirror arrangement recently described by 
Makishima et al. (1951). In these instruments, the entrance and exit 
slits (and hence the source and monochromatic image) are maintained 
constant in position while the dispersing element(s) is rotated to vary 
the emergent wave length. 

Grating Instruments. If a wave front of radiation is broken into a 
number of narrow, parallel zones evenly spaced by appropriate distanc-es, 
the waves propagating from each zone will interfere with those from all 
other zones so as to produce a diffraction pattern. For any given wave 
length, there will be some direction 
or directions in which the waves from 
each zone will all be in phase to pro- 
duce a maximum of intensity. In 
another direction, waves of another 
wave length will be in phase to yield 
a maximum of intensity, whereas the 
waves of the first wave length will 
largely cancel each other. A device 
to thus disrupt a wave front is known 
as a diffraction grating. By thus 
deviating radiation of different wave 
lengths into different angles, a grat- 
ing can serve as a dispersing element. 

A grating may consist of a large 
number of thin, parallel slits, in 
which case it is a transmission grat- 
ing, or of a similar number of thin, parallel reflecting strips, in which case 
it is a reflection grating. 

If, in a direction of maximum intensity for a given wave length, the 
waves from one slit (or strip) are exactly one wave length retarded or 
advanced with respect to those from the two adjacent slits, this direction 
is referred to as that of the first-order maximum. If the phase difference 
between waves from two adjacent slits is just two wave lengths, the direc- 
tion is that of the second-order maximum. In general, directions of 
maximum intensity will occur whenever 

/)X = f/(sin a + sin i3) 

where n — order number (an integer) 

X = wave length 

d = spacing between slits. 
(See Fig. 4-13). 

For a given wave length, the distribution of intensity in angle al)Out a 
direction of maximum intensity, will d(^p(Mid ultimately (Hi iho ratio of th(^ 




Fic. 4-13. Diffraction by a plane trans- 
mission grating. 



152 HADIATION BIOLOGY 

ovtM-all width of tlio grating to the wave length, or more specifically on 
the number of wave lengths of phase dilTerence between the waves, pro- 
ceeding in a given direction from the extreme slits of the grating. As 
this phase dilTerence increases, as with increasing order number, the 
width of the angular intensity distribution decreases, and hence the 
spectroscopic resolving power increases. 

The angular dispersion of a grating — the variation of wave length of 
maximum intensity with angle — likewise increases with increasing order 
number, and also w-ith decreasing spacing between the slits. Analytically, 

where jS = angle of diifracted beam with grating normal 
n — order number 
d = spacing between slits. 

While both spectral resolution and angular dispersion are favored by 
the use of higher ditTraction orders, the problem of overlapping orders 
becomes acute for high-order numbers. The third diffraction order of 
Xi will overlie the second order of 1.5Xi and the first order of 3Xi, etc. 
Fre(iuently filters or elementary prism devices may be added to sur- 
mount this difficulty and permit the use of second- or third-order spectra. 

The grating, transmission or reflection, may be on a plane surface, in 
which case it is illuminated with parallel light from a collimating lens or 
mirror, and the emergent beams are focused to a spectrum with a tele- 
scope lens or mirror. Or the grating, if reflecting, may be ruled on a 
concave surface, in which case it will serve as its own focusing element, 
permitting the elimination of the collimating and telescope elements 
(Beutler, 1945). Such concave gratings are then effective throughout 
any wave-length region for which a reflecting surface may be made, since 
the need for any transparent dielectric is eliminated. The images formed 
by such gratings are, however, generally astigmatic, unless the grating is 
illuminated with a parallel beam, as in the Wadsworth mounting (Sawyer, 
1951, Chap. 6; Harrison et al., 1948, Chap. 4). 

Gratings may be produced with higher resolving power than any prism 
instrument. By the use of replica technicjues, many copies can be made 
from one master at moderate cost. However, grating instruments neces- 
sarily waste light in unused orders, although this drawback can be mini- 
mized by proper ruling of the reflecting strips which can serve to direct 
most of the energy into one order (Wood, 1944; Babcock, 1944; Stamm 
and Whalen, 1946). 

The use of grating dispersing elements in monochromators is a rela- 
tively recent development (French et al., 1947), although commercial 
designs employing plane reflection gratings are now available (Bausch 
and Lomb, 19.")1) (see Fig. 4-14). The u.se of such monochromators may 



ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 



153 



LIGHT SOURCE 




GRATING 



OBJECTIVE MIRROR, 




33-86-40 GRATING MONOCHROMATOR 

Fig. 4-14. Optical path of Bausrh and Lomb grating monochromator. {Bausch ami 
Lomh Optical Compani).) 

become more widespread as high-quality gratings become more generally 
available. 

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156 RADIATION HIOLOGY 

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ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 157 

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160 RADIATION HIOLOGY 

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102 UADIATION HlOLOtiV 

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ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 163 

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John Wiley & Sons, Inc., New York. 

Manuscript received by the editor Sept. 22, 1952 



CHAPTER 5 

Ultraviolet Absorption Spectra 

Robert L. Sinsheimer 

Department of Physics, Iowa State College 
Ames, Iowa 

Paravieters of absorption spectra: Position in the electromagnetic spectrum — Width of 
absorption band — Intensity. Absorption and chemical constitution: Empirical correla- 
tions — Theoretical developments. Specification of absorption: Intensity — Spectral posi- 
tion — Band undth. Environmental factors influencing absorption: Solvent — pH value 
— Concentration — -Temperature — Orientation — Scattering — Local concentrations. Ultra- 
violet absorption spectra of important biological substances: Proteins and amino acids — 
Nucleic acids and nucleotides — Steroids — Carotenoids — Porphyrins — Flavins — Pterins — 
Vitamins — Plant pigments. References. 

The absorption of ultraviolet radiation by a molecule results in a change 
in the electronic configuration of that molecule and therefore in a change, 
usually transient and reductive, in the stability of the molecule. The 
ability of any molecule to absorb ultraviolet radiation of a particular fre- 
quency is dependent on the electronic configuration of the molecule and 
the electronic configurations of the possible higher energy states of the 
molecule. This absorptive ability is thus intimately related to the 
detailed molecular structure (Lewis and Calvin, 1939; Ferguson, 1948; 
Bowen, 1946, 1950; Maccoll, 1947). An absorption spectrum of any 
substance is a quantitative description of the absorptive ability of 
the molecules of that substance over some range of electromagnetic 
frequencies. 

A knowledge of the absorption spectra of the major components of a 
living organism makes it possible to limit the number of possible primary 
receptors of radiation that are found to produce some biological effect. 
Only those substances that absorb the radiation found to be responsible 
for the effect need be considered. The correlation of the measured rela- 
tive efficacy of radiations of various wave lengths in producing the effect, 
with the variation of absorptive power with wave length of the substances 
under consideration as primary receptors, can, under favorable circum- 
stances, further limit the possibilities as to the nature of the receptor. 
A knowledge of the influence of absorption on the stability of the 

165 



166 



RADIATION BIOLOGY 



\arious substances considered may aid still further in identification of 
the primary receptor or receptors. 

PARAMETERS OF ABSORPTION SPECTRA 

Any absori)tion spectrum, however complex, may he regarded as a 
summation of a set of individual absorption bands, each corresponding to 
a transition between two particular electronic configurations (Fig. 5-1) 
(Sheppard et al., 1941; Wulf and Dcming, 1938). It is usually possible 
to group these individual bands, each group consisting of transitions 
invoking nearly the same energy difference. The dilTcrcnt bands within 
a group then represent transitions involving a common change in basic 
electronic configuration, together with varied associated secondary 
changes in the distribution of energy among the molecular vibrations. 




475 500 525 



550 



575 600 625 650 675 700 725 750 
FREQUENCY X IO-'2 

Fig. 5-1. Resolution of the absorption spectrum of merocyanine in hexane into six 
bands, each representing a particular vibrational transition associated with the funda- 
mental electronic transition. (Sheppard et al., 1941.) 



The parameters of an absorption spectrum are properly the sum of the 
parameters of the individual bands. Any individual band (representing 
a transition between two distinct electronic states plus vibrational states) 
may be described by three parameters: (1) the position in the electro- 
magnetic spectrum, (2) the breadth of the electromagnetic spectrum 
occupied by the band, and (3) the intensity of absorption. 

POSITION IN THE ELECTROMAGNETIC SPECTRUM 

The position of an absorption band in the electromagnetic spectrum Js 
dependent on the energy difference between the initial and the e.xcited 
electronic configurations since this energy dilTerence must be supplied by 
the absorbed photon, the energy of which is related to its frequency (v) 
by Planck's relation 

E = hv = ^^ (5-1) 



ULTRAVIOLET ABSORPTION SPECTRA l(i7 

where h = 6.61 X 10~" erg-sec 

h = 4.13 X 10-'-^ ev-sec 

c = 3 X 10'" cm/sec. 
The energy differences corresponding to absorption bands in the ultra- 
violet region (wave length <4000 A) are of the magnitude of 3.1 ev 
or greater. Since these energies are greater than those that corre- 
spond to the energy of formation of many chemical bonds (C — C bond 
energy = 2.54 ev; C— N bond energy = 2.11 ev) (Pauling, 1945), the 
rupture of such bonds in molecules raised to an excited level by absorp- 
tion of an ultraviolet photon is energetically possible. Such rupture may 
lead to the formation of free radicals or of oppositely charged groups or. 
in molecules containing atoms with unbonded electron pairs, to photo- 
oxidation and semiquinone formation (Waters, 1948; Lewis and Lipkin, 
1942; Lewis and Bigeleisen, 1943b). The farther into the ultraviolet the 
absorption band is located, the greater is the excess of excitation energy 
over the minimum necessary for bond rupture. With absorption bands 
in the far ultraviolet (wave length <2000 A) the absorbed energies gen- 
erally become adequate to produce molecular ionization (8-12 ev) 
(Price, 1947). 

WIDTH OF ABSORPTION BAND 

The width of an individual absorption band is dependent intramolecu- 
larly on the duration of the excited electronic state (Heitler, 1944, pp. 
110#) and extramolecularly on the statistical distribution of the fre- 
quencies of the particular absorption band among the assemblage of 
absorbing molecules, each exposed to a certain randomness of molecular 
environment. 

Considering any one molecule in a given molecular environment, the 
width of its absorption band is inversely dependent on the duration 
(mean lifetime) of the excited electronic state. This may be formulated 
by the "uncertainty principle" 

AE At ^ ^ 

ZTT 

where, in this instance, AE is the uncertainty, i.e., variation, in the energy 
difference accompanying the transition and A^ is the duration of the 
transition. For the usual absorptive process in an isolated mole- 
cule, At is of the order of magnitude of IQ-^ sec, A^ is about 10"' ev, 
AE/E = 10-^ per cent as is AX/X. 

However, any of several processes may shorten the duration of the 
excited state (A^, thus increasing the uncertainty in energy of the transi- 
tion {AE), and hence may broaden the absorption band. Disruption of 
the molecule may take place within the duration of a single moleculai' 
vibration and thus reduce the excitation lifetime to as short as 10"'^ sec. 



108 



RADIATION BIOLOGY 



II' disrupt ion is less likel}', tlir inolccule may remuiu iiituct for several 
luiiulrod or thousand vibrational periods, or lO^'^-lO"'^ sec. The spec- 
tral hroadeniii}; in this case will not he so great and will give rise to 
so-called "predissociation " l)ands (Rice and Teller, 1949). 

The duration of the excited state may be reduced because of a high 
probability of a transition to a third electronic state (Fig. 5-2). The dura- 
tion of this state will have no influ- 
ence on the width of the original 
absorption band. A number of such 
instances of transitions to a "triplet" 
state have been reported (Kasha, 
1947). Transitions from this triplet 
state to the original ground state are 
of very low probability so that the 
molecule may retain energy as an 
excited triplet state for appreciable 
lengths (seconds) of time (McClure, 
1949). 

Under conditions of appreciable 
intermolecular contact (solutions and 
solids) the energy of excitation may 
be rapidly dissipated by conversion 
to vibrational energy which, in turn, 
is simply transferred by collisions or 
electromagnetic damping to neigh- 
boring molecules and ultimately ap- 
pears as thermal energy (Massey, 
1949). Such dissipative effects, 
which reduce the duration of the ex- 
cited state, are in part responsible 
for the broadening of absorption 
bands of substances in solution as 
compared to their vapor absorption 
spectra. 

If the molecule retains its excita- 
tion energy for a time comparable 
with the probability^ of transition 
from the excited to the ground state, 
the energy will be reradiated as "fluorescence." This fluorescence radia- 
tion may then escape, or it may, under appropriate conditions, be reab- 
sorbed by other chromophores within the solution or biological system 
(Arnold and Oppenheimer, 1950; Forster, 1948; Franck and Livingston, 
1949). 

In addition to these primarily intramolecular factors, the electronic 




INTERATOMIC DISTANCE ALONG 
CRITICAL COORDINATE 

Fig. 5-2. Illustration of the possibility 
of a radiationless transition from the 
initial excited electronic state (T*) to a 
second excited state (T), in this 
instance a triplet state which would 
have a long duration and from whicih 
return to the ground state could occur 
by delayed emission of radiation or 
phosphorescence (P). The curves rep- 
resent the variation of potential energy 
of the molecule as a function of the inter- 
atomic separation for a diatomic mole- 
cule. (Kasha, 1947; copyright, 1947, 
by The Williams and Wilkins Company.) 



ultraviol?:t absorption spectra 



169 



configuration of the individual molecules and thus the energy associated 
with a particular electronic transition will be influenced in a condensed 
system by the electric and magnetic fields associated with nearby mole- 
cules. Since the spatial orientations involved will be random (except in 
crystals) and will be varying, owing to thermal motion, there will result 
a statistical distribution of electronic configurations and of transition 
energies of the absorbing molecules, thus broadening the observed absorp- 



60.000 



60,000 



40,000 






20,000 - 




12 3 4 5 

INTERATOMIC DISTANCE r, A 

Fig. 5-3. Illustration of the Franck-Condon principle. Horizontal line.s within the 
well of each potential-energy curve represent various vibrational-energy levels. A 
transition from the ground state (V") to the excited state (V) would most probably 
leave the molecule in the second excited vibrational level (point A) since the inter- 
atomic distance cannot change appreciably within the duration of the transition. 
(Reproduced by permission of the publishers front Practical Spectroscopy, by George R. 
Harrison, Richard C. Lord, and John R. Loofbourow, copyright, 1948, by Prentice-Hall. 
Inc.) 



tion band. These effects will be reduced if the fields involved are reduced 
(as in nonpolar solvents) or if the extent of the variations due to thermal 
motion is reduced, as in spectra of substances at low temperatures (Sin- 
sheimer et al., 1950a). 

For many substances in solution, the effects described widen the indi- 
vidual absorption bands associated with a given electronic transition so 
as to produce a fusion of these bands into an apparently single band of 
considerable breadth. The individual bands, representing transitions 
from vibrational energy states accompanying the normal (lowest energy) 
electronic state to various vibrational states accompanying the excited 



170 RADIATION HIOLOGY 

electronic conligurutioii, are thus concealed, reducing the amount of infor- 
mation available in the spectrum. The possible vibrational transitions 
are limited by the Franck-Condon principle (Rice and Teller, 1949), 
which simply recognizes that the duration of the electronic transition is 
brief compared to the duration of a molecular vibration, so that the posi- 
tion of the atoms camiot change appreciably during the act of absorption. 
Hence only transitions to excited-state vibrational levels, involving 
atomic configurations similar to those in the vibrational levels associated 
with the ground state, are probable (Fig. 5-3). 

Broadening of this type may also be reduced by a reduction of the 
temperature of the absorbing substance; the reduction in thermal molecu- 
lar energy decreases the molecular population in the higher vibra- 
tional energy levels and thus reduces the number of possible transitions. 
Indeed, at li(iuid-air temperature or below, all molecules must commence 
a transition from the lowest vibrational energy level. 

INTENSITY 

The total intensity of an absorption band, i.e., the integrated absorp- 
tion over the band, is dependent on the difference in scale and symmetry 
of the electronic configurations for the initial and the excited states 
(Heitler, 1944). A net time-average displacement of charge along some 
molecular axis must accompany the absorption of radiation. If the elec- 
tronic configurations of the two energy levels are of such a symmetry 
that a transition from one to the other does not provide such a time- 
average displacement, then a transition between these levels cannot be 
induced by radiation, i.e., absorption cannot occur. Such a transition is 
said to be "forbidden." 

In benzene, the electronic configurations of the ground and the first 
excited singlet energy levels are of such a symmetry that a transition 
between them is forbidden (Sklar, 1942). This transition, which is 
associated with the benzene absorption maximum at 2550 A, can occur 
only if accompanied by a particular molecular vibration which so dis- 
torts the molecule as to alter the symmetry of either the ground or the 
first excited energy levels and thus gives rise to a small time-average dis- 
placement of charge. The intensity of such forbidden absorption bands, 
which re(iuire the participation of a molecular vibration, is generally low. 
Thus for the benzene absorption maximum at 2550 A, € = 120; this may 
be contrasted with the intensity of the "allowed" benzene absorption 
band at 1835 A which is about 380 times as great (c = 4G,000) (Piatt and 
Klevens, 1947). In general, the greater the time-average displacement 
of charge, the greater the integrated absorption. 

For a given integrated absorption, the intensity of the al)sorption maxi- 
mum will ob\iously depend in\ersely on the width of the l)and and thus 
directlv on the duration of the excited state. 



ULTRAVIOLET ABSORPTION SPECTRA 



171 



WAVE LENGTH, A 
3250 3000 2750 2500 



2250 



3.0 - 



2.5 ■ 



2 - 



o 
o 



ABSORPTION AND CHEMICAL CONSTITUTION 
EMPIRICAL CORRELATIONS 

The empirical correlation of the spectral position of absorption bands 
in the ultraviolet with certain chemical structures was begun about 1885 
with the work of Hartley (1885) and has been steadily continued and 
expanded as improvements in technique have simplified the task of 
measuring absorption spectra (Braude, 1945; Lewis and Calvin, 1939; 
Ferguson, 1948; Erode, 1943; Jones, 1943). The broader long-recog- 
nized empirical correlations have now been given a theoretical basis by 
the development of approximate 
wave-mechanical methods of calcu- 
lation of electronic-energy levels in 
complex molecules. 

These correlations early indi- 
cated that the absorption bands 
of compounds, composed exclu- 
sively of saturated linkages, oc- 
curred generally below 2000 A, 
usually in the vacuum ultraviolet 
below 1850 A. The long-wave 
arms of such peaks extended above 
2000 A, increasing in amplitude 
with increase in size of the molecule. 
Because many spectrographs and 
spectrophotometers do not record 
below 2000-2200 A, these long- 
wave limbs of bands of saturated 
compounds, rising in absorption 
with decreasing wave length, are 
often referred to as "end" 
absorption. 

Compounds with single unsatu- 
rated bonds such as C'=C, C=0, 
or C=N were found to have ab- 
sorption bands, usually weak, in 
the region 1900-3000 A, the actual 
wave length being dependent on the adjacent parts of the molecule. 

Strong absorption bands in the region 2000-4000 A are always corre- 
lated with molecular structures containing chains or rings of conjugated 
double bonds; in general, the larger the conjugated structure, the stronger 
is the absorption and the longer the wave length of the maximum absorp- 
tion. Ring structures with conjugated double bonds, as in aromatic 
compounds, often possess particularly high absorption. 




0.5 - 



30,000 



35,000 



40,000 



45,000 



Fig. 5-4. Ultraviolet absorption spectra 
of some .simple derivatives of benzene. 
I, aniline; II, phenol; III, chlorobenzene ; 
IV, benzene; all in heptane. (Adapted 
from Wolf and Herold, 1931.) 



72 



RADIATION BIOLOGY 



The absorption o{ coiijusatcd-boiul groups separated within a {^ivcn 
molecule by two or more saturated bonds is usually independent and 
simply additive. The absorption of such groups can, of course, be 
affected by the addition of side chains or auxiliary groups, especially if 
the latter may be charged (Nils and OH). Such groups may distinctly 
affect the spectral position and intensity of absorption of a given conju- 
gated system (Fig. 5-4). 

In large polar macromolecules, such as proteins and nucleic acids, the 
near-ultraviolet absorption spectrum of the polymer is often not strictly 



WAVE LENGTH, A 
6000 4000 3000 2500 
I I I 



2000 



6000 



WAVE LENGTH, A 
4000 3000 2500 



2000 




20 

>- 
(/) I 5 



u I 



Q. 
O 



05 



1 1 


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_ 










// 


- 


- 








/'N 


// 


- 


- 








u\ 


/ 


- 


- 








f\ 


/ 


- 


- 








' 




- 


: 












- 


- 












- 


_ 












- 


1 J 1 1 


1 1 1 1 


1 1 1 1 


1 1 1 1 


1 1 1 1 


1 J 1 1 


1 1 1 1 



15 20 25 30 35 40 45 50 

-3 



Q I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 

15 20 25 30 35 40 45 50 

WAVE NUMBER, Cm"'xi0"' WAVE NUMBER, Cm''xiO" 

(a) (b) 

Fig. 5-5. (a) The ultraviolet dichroism of hexamethylbenzene crystals. I, Electric 
vector perpendicular to the plane of the ring; II, electric vector parallel to the plane of 
the ring, (b) The ultraviolet dichroism of tobacco mosaic virus particles, oriented by 
.streaming. I, Electric vector perpendicular to the direction of streaming; II, electric 
vector parallel to the direction of streaming. (Scheibe et al., 1943; see also Wilkins 
et al, 1950.) 



the linear sum of the absorption of its component conjugated groups, 
even though these component groups are separated by appreciable 
lengths of saturated bonds. This nonadditivity appears to be due to the 
formation of labile intergroup bonds to the conjugated groups, either of 
ionic or hydrogen-bond type. Thus the ultraviolet absorption spectra 
of proteins are often not simply the sum of the absorption of the compo- 
nent amino acids. The phenolic group of tyrosine, for instance, fre- 
quently appears to be involved in some type of loose bond (Crammer and 
Neuberger, 1943; Sizer and Peacock, 1947; Finkelstein and McLaren, 
1949; Schauenstein and Treiber, 1950). The ultraviolet absorption of 
highly polymerized deoxyril^onucleic acid is about 25-30 per cent less 
than that of the depolymerized form, which form is very nearly the linear 
sum of the absorption of the component nucleotides (Kunitz, 1950; 



ULTRAVIOLET ABSORPTION SPECTRA 



173 



Tsuboi, 1950; Loofbourow, 1940; Siiisheimer, 1954). The absorption 
spectrum of ribonucleic acid also increases in intensity and shifts slightly 
toward shorter wave lengths during depolymerization (Kunitz, 1946; 
Tsuboi, 1950). 

If the radiant energy is plane polarized, absorption will be greatest 
when the plane of the electric vector is parallel to the direction (s) of 
greatest electron mobility and maximal induced dipole moment, i.e., 
parallel to the direction of a chain of conjugated bonds, as in carotene, or 
parallel to the plane of the ring in a planar aromatic or heterocyclic mole- 
cule, as in benzene (Fig. 5-5). Spectrally distinct absorption bands may 
appear, corresponding to transitions involving mutually perpendicular 
changes in dipole moment (Lewis and Bigeleisen, 1943b; Scheibe and 
Kandler, 1938; Scheibe et al., 1943; Coulson, 1948; Nakamoto, 1952). 
In solution the random orientation of molecules will prevent detection 
of any such preferred directions; however, in cellular structures or in 
crystals, uniform molecular orientation may permit a preferential absorp- 
tion for light polarized in these directions (Butenandt et al., 1942; 



X^ ^\ 



\/ \/ 



/\ /\ /\ 



\ 



KEKULE DEWAR 

Fig. 5-6. The canonical structures of benzene. {Sklar, 1937.) 

Schauenstein et al., 1949). This phenomenon of differential absorption 
dependent on the plane of polarization is known as dichroism and can be 
useful as an indication of molecular orientation. 



THEORETICAL DEVELOPMENTS 

As indicated, these empirical correlations have received support from 
modern theories of the electronic structure of organic molecules and of 
the change in electronic configuration attendant on the absorption of 
radiation. These treatments have developed along two lines, the valence- 
bond concept (MaccoU, 1947; Heitler, 1945; Pauling, 1945; Van Vleck 
and Sherman, 1935; Sklar, 1937) and the molecular-orbital concept 
(Coulson, 1947; Herzfeld, 1947; Mulliken and Rieke, 1941). 

In the valence-bond concept, the electrons involved in chemical bonds 
are assumed to remain in atomic orbitals, which overlap with orbitals of 
the neighboring atoms, and the energy of each bond may be calculated 
from wave-mechanical principles. The energy level of the molecule is 
dependent on the summation of the energy levels of each bond. It is 
recognized that, with conjugated structures, the formulas usually written 
represent but one of several possible canonical forms (Fig. 5-6), all of 
which may be considered to contribute in varying degree to the actual 



174 RADIATION BIOLOGY 

structure. By combining these possible forms in various proportions, a 
combination may be found which produces the lowest energy state. The 
energy level of this state is generally less than that of any of the indi- 
vidual canonical forms, a result known as "resonance stabilization" 
(Whohind, 1911) and due to "exchange energy" (Hcitler, 1945). Other 
combinations, with varying proportions of the possible canonical forms, 
give rise to higher energy levels to whi('h the molecule may be excited on 
the absorption of radiation. 

Resonance among possible structures in the excited state can lower the 
energy of the excited state and thus reduce the energy difference between 
it and the ground state, thereby increasing the wave length of the absorp- 
tion associated with the electronic transition. The intensity of absorp- 
tion will be greatest for transitions between states involving resonating 
structures which have appreciable dipole moments. 

The molecular-orbital method, which has been the more succes.sful in 
regard to the correlation of calculated with ob.served spectra, has been 
based on the assumption of molecular orbitals for the valence electrons of 
the atoms involved in the chemical bonds. In this development the 
molecular orbitals are usually made up formally of linear combinations of 
appropriately chosen atomic orbitals — LCAO method' (Mulliken and 
Reike, 1941; Chirgwin and Coulson, 1950; Lennard-Jones, 1949; Matsen, 
1950; Dewar, 1950; Piatt, 1950; Longuet-Higgins d al, 1950). The con- 
struction of the molecular orbitals may involve only the atomic orbitals 
of two atoms, as is usually the case with single bonds and with isolated 
double bonds, in which case they are referred to as "localized orbitals." 
or, as in the case of conjugated chains of double bonds, the molecular 
orbitals may involve contributions from the tt orbitals of all the atoms 
involved in the chain. In this latter case, the orbital is said to be "unlo- 
calized," and electrons in such orbitals are considered to migrate freely 
along the chain (Fig. 5-7). 

Varying the combinations of atomic orbitals will produce molecular 
orbitals of various energy levels. The electrons available for bonding 
(all in the outer atomic shell) are then disposed in successively higher 
energy levels, two to a molecular orbital with spins opposed, until all 
electrons are accounted for. Absorption of radiation may then cause 
an excitation of an electron from the highest filled molecular orbital to the 
lowest unfilled orbital. The energy difference between orbital levels 

' Refinements of this procedure involve the ii.se of "antisyiiuiietrized inoiccuhir 
orbitals" to reduee the apparent contribution of configurations including multiply 
ionized atom.s ((loepixTt-Mayer and Skiar, 1938; Roothaan, 1951) and recognition of 
configurational interaction (Jacobs, 1919; Craig, 1950). .\notiier theoretical ajiproach 
which has had considerable success is the "free-electron model" in which all the ir 
electrons are considered to be able to migrate freely throughout the molecule, along the 
atomic bonds, in a jiotential field that is constant, or, in some instances, sinusoidally 
varying (Bayliss, 1918; Piatt, 1949; Kulm, 1949; Simpson, 1949). 



ULTRAVIOLET ABSOUI'TION ttl'lOCTUA 



t .) 



decreases with increasing); orbital energy. As a result, the transitions 
induced by absorption in large conjugated systems, which are transitions 
between high-level orbitals, re((uire less energy, and thus a longer wave 
photon, than the transitions induced by absorption in smaller conjugated 
systems. This deduction accounts generally for the increase in wave 
length of the absorption peaks with increase in size of the conjugated 
system. 

Absorption of radiation may also induce transitions from the highest 
filled molecular orbital to some of the higher unfilled orbitals; these tran- 




hiiiljiMiMiiiiiiiiiiiiiiriirpiii 
*''y iiiiiiiiiiiiiiiiiriiiii'^ 

(C) 

Fi<i. 5-7. Illustration of the a and tt atomic orbitals of benzene and the fusion of the n 
atomic orbitals to form the lowest energy molecular orbital, (a) a orbitals; ih) ir 
atomic orbitals; (c) tt molecular orbitals. (Coulson, 1947.) 

sitions correspond to absorption bands at shorter wave length than those 
of the bands just described. 

The excitation induced by absorption usually involves the transition 
of an electron from a "bonding" to an "antibonding" orbital without 
change of spin direction.- The antibonding orbital introduces an addi- 
tional nodal plane into the function specifying the probability distribution 
for the position of the electron. The absorption of double bonds or of 
conjugated chains involves excitation of a tt electron, an electron of which 
the probability distribution already contains a node in the plane of the 
bond or chain. Hence excitation introduces a new node, which lies 
between the atoms of the bond or two of the atoms of the chain. Thus 

^ Transitions involving a change in the net electron spin of the molecule, i.e., 
"singlet-triplet transitions," are usually of low probability (Kasha, 1947). 



170 



RADIATION BIOLOGY 



I'or a (limbic bond, in (he cxcitod .state, there is a greater proliahility of the 
electron hciiifj; on one or llic other atom of the bond than of ils Ix-inj!; in 
the interatomic region. The probability distributions and associated 



/ + 



+ 



+ 



+ 



c -< 



+ 



yc 



' 1 1 



/ \ 



c -< 



>- c 



- - I + 



+ 



! + 1 - 



>-c 



A 



>C' 



V + 



:a 



+ 



•c-^ 



+ )! - 



>-c 



li 
I \ 



I / 
w 

II 



X 



- )1 + 1! - 



c -< 






c -< 



- ; + 



Fig. 5-8. A schematic representation of the wave functions describing the four molecxi- 
hir orbitals that may be synthesized by Hnear combinations of the four tt atomic 
orbitals of butadiene CHo=CH— CH=CH.,. On and outside the dotted Hues the 
wave functions are practically zero. Within the dotted lines the wave functions have 
finite values, with sign as indicated, and thus these are the regions with an appreciable 
probability for the presence of an electron (the pluses and minuses refer only to the 
sign of the electronic wave function and not to the charge). (Reproduced from 
Chemical Aspects of Light, by E. J . Bowen, copyright, 1940, by Oxford University Press.) 



./^v 



^n/ V 






> \ 



/^s 



V 



) (, 



.-s 



1 ( 

-* I- 



V 



1 < 


\y 


V 


\ 
\ 


V 


,-, 




; 
/ 
/ 




Fig. 5-9. The molecidar orbitals of anthracene. (Bmren, 1950.) 



nodal planes for the molecular orbitals of a simple conjugated chain 
(butadiene) are shown in Fig. 5-8 (Bowen, 1940). Absorption induces a 
transition of an electron from orbital 2 to orbital 3. 

In aromatic molecules the new nodal plane may intersect the molecular 



ULTRAVIOLET ABSORPTION SPECTRA 177 

plane along either of two mutually perpendicular axes (.r, y), producing 
different molecular orbitals. If the molecule is asymmetric (as in naph- 
thalene), these orbitals will represent different energy levels, and transi- 
tions from the ground state to these orbitals will be associated with spec- 
troscopically distinct absorption bands, which will be strongly dichroic 
(Coulson, 1948). The intersections of the nodal planes with the molec- 
ular plane for the molecular orbitals of anthracene are indicated in Fig. 
5-9. The transitions that correspond to the two prominent ultraviolet 
absorption bands of anthracene are labeled with the band wave length. 
It can be shown that these two approaches, valence-bond and molec- 
ular-orbital, in their simple forms, probably bracket the correct solution; 
the valence-bond method does not allow enough weight to possible ionic 
structures, i.e., those in which two or more of the bonding electrons may 
be concentrated on one atom; the molecular-orbital method allows too 
much. More advanced developments of both theories have tended to 
narrow the gap between them. 

SPECIFICATION OF ABSORPTION 

INTENSITY 

The ultraviolet absorption spectra of substances of biochemical interest 
are usually obtained with solutions of these substances in transparent 
solvents. The measured absorption at any wave length will then be 
dependent on the concentration of the substance in the solution and the 
length of the light path in the solution. The specification of absorption 
spectra may be standardized by referring all measured spectra to the 
spectrum that would be obtained from a solution of a standard concentra- 
tion and a standard light path. This conversion of measured to standard 
spectrum is rendered easy by the simple nature of the formula relating 
absorption to concentration and light path. 

Since the absorption of a photon by a molecule is an all-or-none act and 
since all molecules may be assumed to have, statistically, the same proba- 
bility of absorption of an incident photon of a given wave length, any 
layer of a solution of thickness dl, transverse to the light beam, may be 
expected to absorb the same fraction of radiant energy of one wave length 
as any other such layer, and if dl is small, this fraction will be proportional 
to dl. Thus 

jT ^ dt. 

This statement is known variously as Lambert's or Bouguer's law. 

If the absorbing molecules may be assumed to act independently, the 
fraction of incident energy absorbed in a given layer will be expected to be 
proportional to the concentration of absorl)ing molecules in the solution. 



178 RADIATION BIOLOCY 

Combining this with the previous expectation, 

— -,- a cl 

is ohtained. This icljilion is known variously as the LainlxTt-Hccr or 
Bouf2;uer-]ieer law. 

Integratiiifz;, it is t'ouiid that 



(J I 



ell, 



or also 



In -J- *^ c/, 



logiu Y °^ ^^• 



The proportionality constant in this etiuation, which applies at eacii 
wave length, is, of course, characteristic of the absorbing substance. 
Numerically, it will depend on the logarithmic base employed and on the 



T.vble 5-1. Symbols for the Proportionality Co.nsta.nt 


Log base 


Optical path length (/), cm 


c, g /liter 


c, moles/liter 


r, moles /ml 


e 
10 


k; a, (specific extinction) 


E 

t\ a„, (molar extinction) 


13 



units for concentration (c) and optical path length [1). X'arious com- 
binations of base and units have been employed, and the most commonly 
used symbols for the proportionality constant in the various systems are 
indicated in Table 5-1. Thus 

^ logiu jh/I) 
' cl 

where c is in moles per liter and / is in centimeters. 

The (juantit}' logio (lo/I) = eel is often referred to as the optical den- 
sity^ (/)) of the solution. Thus e is the optical density that would be 
measured for a 1-cm path of a solution containing 1 mole/liter.''-^ 

' Also referred to as the extinction or the absorhance (Gibson, 1949; Brode, 1949). 

' Another symbol occasionally used to specify absorption is Ef which signifies the 
optical density of an /-cm path of a solution containing p per cent of tlie absorbing 
substance. 

' In some circumstances, l<tgiu * <>r logu, J) may be plotted either to compress a ui(h- 
pinge of vahies into rea.soiial)U' dimensions or to obtain a curve the shape of whicii is 
independent of the concentration or path length, wliich may be unknown. Since 
logio D = logii) t f- logio (• + logiu X, the latter will enter only as additive constants. 



ULTRA VKJLKT ABSOKPTION SPKCTRA 170 

The Lambert or Boiiguer law is valid under all foiiditions of normal use. 
The modification due to Beer is accurate as long as the condition for its 
validity is met — that the absorbing molecules act independently. In 
concentrated solutions there is often a tendency toward dimerization or 
other forms of molecular association. In these instances the nature of the 
absorbing entity really changes with changing concentration, so that 
Beer's law will not apply. 

Another method of indication of the absorbing power of a substance is 
the specification of the "absorption cross section" (a) of molecules of the 
substance as a function of wave length. The absorption cross section is a 
measure of the probability of absorption of a photon, known to be crossing 
a unit area transverse to the beam, by a single molecule known to be con- 
fined within that area. This quantity is useful in calculations concerning 
the possibility of radiative-energy transfer from the primary receptor to 
other receptors within a cell (Arnold and Oppenheimer, 1950). 

The absorption cross section is related to e by the following formula: 

a = 3.83 X 10-21^ 

where a is in square centimeters. The cross section a does not necessarily 
bear simple relation to the physical cross-sectional area of the molecule, 
although in certain instances a good correlation has been demonstrated 
between an "effective" geometrical cross section and absorptive power 
(Braude, 1950). 

SPECTRAL POSITION 

The position of an absorption band in the electromagnetic spectrum is 
usually defined by the position of the absorption maximum. This posi- 
tion may be expressed in terms of (1) wave length, in Angstroms (1 A = 
10-« cm) or millimicrons (1 m^ = lO"" cm); (2) wave number,^ in cm-^ 
or mm-i; or (3) frequency, in vibrations per second or in fresnels (1 f = 
10'2 vps). 

Thus the longer wave maximum in the absorption spectrum of methyl- 
cholanthrene at 77°K occurs at 2995 A. 299.5 m/x, 33,390 cm-', 10.02 X 
101^ vps, or 10,020 f. 

BAND WIDTH 

The width of an absorption band is usually considered to be the spectral 
separation between the points of half-maximal absorption. This separa- 
tion may be expressed in any of the units used to express the position of 
the absorption maximum. 

'• The wave number is defined as the number of wave lengths per centimeter (or 
millimeter) of path in vacuo. 



180 



RADIATION inOLOGY 



ENVIRONMENTAL FACTORS INFLUENCING ABSORPTION 

In general, a variation of any factor that influen(;e^s the electronic (^on- 
fi}z;in'ation of the absorbing molecules, either uniformly or with a statistical 
(listrii)ution, will affect the absorption spectrum (Sheppard, 1942). In 
addition, certain factors may alter the technical conditions of the absorp- 
tion measurement and thereby affect the spectrum. 

SOLVENT 

The choice of solvent can influence the position, width, and intensity of 
absorption bands. Changes in position are to some degree correlated 



4 8 




I 6 

V = 900 1000 HOC 1200 1300 1400 

I/X= 30,000 33,330 36,670 40,000 43,330 46,660 

A = 3333 3000 2727 2500 2308 2143 



1500 1600 f 

50,000 cm'' 
2000 A 



Fig. 5-10. The absorption spectrum of phthalic anhydride. I, in hexane; IT, in 
alcohol. {Mcnrzcl 1927.) 



with the dielectric constant of the solvent according to Kundt's rule which 
states that, with increasing dispersion of the solvent, the absorption 
maximum is shifted toward longer wave lengths. Although Kundt's rule 
is generally valid for nonpolar solvents, there are serious deviations with 
polar solvents (Sheppard, 1942). 

This shift may be interpreted as indicative of the increased role played 
by ionized structures in resonance stabilization of the excited state in 
media of high dielectric constant (Wheland, 1944) or in terms of the 
influence of the reaction field of the oscillating dipole on the electric field 



ULTRAVIOLET ABiSOllPTION SPECTRA 



181 



of the light wave in a dielectric medium (Bayliss, 1950; Hartmann and 
Schlafcr, 1950). 

As indicated, absorption bands are widened in polar solvents because of 
increased molecular interaction with conseciuent perturbation of the elec- 
tronic configurations (Fig. 5-10). 

The total absorption intensity, the je dv, can also vary with the solvent 
and would be expected, in general, to increase moderately with increasing 
solvent refractive index (Chako, 1934) owing to augmentation of the 




220 230 240 250 260 270 280 290 300 320 340 
WAVE LENGTH, m/( 

Fig. 5-11. The absorption spectrum of cytosine at three different vahies of pH 
(« = 8.0G X lO'D). I,pH 1.2;II, pH6.0;III, pHl2.7. (Scott, unpublished data, 1951.) 



exciting electric field by the field of the induced dipoles in the medium. 
However, this expectation is frequently not fulfilled for unknown reasons 
(Jacobs and Piatt, 1948). 

pH VALUE 

In aqueous solutions of substances containing dissociable groups, the 
pH of the solution will usually have a marked effect on the absorption 
spectrum. Ionization of any such group, resulting in gain or loss of 
charge, will certainly alter the basic electronic configuration of the mole- 
cule and thereby the spectral distribution of absorption. An example is 
the absorption of a solution of cytosine at various pH values (Fig. 5-11), 



lJ^-2 



UADIATION mOLOGY 



iiulicatiiifi; the olTccts of ionization of the amino sroup (i)K' = 4.()0) and 
of the enolic group (pK = 12.16) (Levenc and Bass. I'.Kil). 

CONCENTRATION 

As was mentioned in the discussion of Beer's law, in concentrated solu- 
tions the association of solute molecules may cause modification of their 
absorption spectrum. This elTect may give rise to a nonlinear relation 
between the optical density of such solutions at certain wave lengths and 




Fig. 5-12. Variation of the absorption spectrum of pinacyanol chlorides in water at 
20.0°C with concentration. I, 4.44 X 10"^ M; II, \.••^'^ X 10"^ M ; III, 4.44 X 10"^ M; 
IV, l.:^3 X 10-'' .1/; V, 4.44 X 10-« M . (Scheibe, 1938; reproduced from Kolloid- 
Zeitschrift. ) 

the solute concentration, as is observed with the Nessler test for ammonia 
(Hawk etal, 1947). 

In more extreme cases, extensive molecular association, possibly involv- 
ing electron transfer through intermolecular hydration, may cause the 
development of entirely new absorption bands. An example of this is the 
"mesophase" J band (Fig. 5-12) of the cyanine dyes (Sheppard, 1942). 

TEMPERATURE 

The temperature of an absorbing substance significantly affects its 
absorption spectrum by controlling the statistical distribution of mole- 
cules among various vibrational energy states associated with the lower 
energy electronic state and by influencing the velocity of Brownian 



ULTRAVIOLET ABSORPTION SPECTRA 



183 



motion, which in turn determines the frequency of molecular collision. 
The latter influences the duration of the excited state and the extent of 
the distortion of the molecular electronic configurations by the electro- 
magnetic fields of neighboring molecules. In addition, variation of tem- 
perature may vary the relative statistical contribution of various possible 
tautomers (Freed and Sancier, 1951) or resonating states. In general, 
reduction of the temperature, by reduction of the variety of initial energy 
levels in an electronic transition induced by absorption and by reduction 
of the perturbing effect of extramolecular fields, will reduce the width of 
the individual and fused absorption bands (Fig. 5-13). This effect may 
be particularly marked if the absorbing substance is in a crystalline 



WAVE LENGTH, A 
2600 2800 3000 3200 2400 2600 2800 3000 3200 

1— 



1 — I — I — I — r 

THYMINE 
(FILM) 




— I r 

H 

I I 

rHN^^CHj 



T 



— 1 — I — i — I — I — r 

THYMINE (EPA) 




r/oK 



(b) 



42 4.0 3.8 3.6 



32 3.0 



3.4 3.2 3.0 4.2 4.0 3.8 3.6 3.4 
WAVE NUMBER, cm"' X 10"^ 

Fig. 5-13. Effect of reduced temperature on the absorption spectrum of thymine. 
{Sinsheimer et al., 1950b.) 



form so that the molecules have a uniform environment (Scott et al., 
1952). 

ORIENTATION 

If the molecules of a dichroic substance are uniformly orientated, as 
might occur in a cellular structure, the absorption spectrum would depend 
on the plane of polarization of the incident radiation. If unpolarized 
light is used, as is ordinarily the case, the influence of the molecular 
orientation on the observed spectrum will depend on the degree of 
dichroism. In an extreme case, such as might occur if there were no 
absorption at all of light for which the electric vector lay in a particular 
plane, the maximum possible light absorption would be 50 per cent, cor- 
responding to an optical density of 0.3 at that wave length (Commoner 
and Lipkin, 1949). Obviously, intermediate cases would permit various 
maximal values of optical density. 



SCATTERING 

If the absorl)ing solution (or living cell) contains objects of dimensions 
comparable with those of the wave length of light employed, appreciable 
quantities of light may be lost from the l)eam by scattering out, as well as 



184 RADIATION HIOLOGY 

by absorption. For simple spherical particles of diameter <X/1() such 
scattering may be expected to vary as l/\*. For larger and for more 
irregularly shaped particles, the variation of scattering power with angle 
and with wave length depend intimately on the particle size and shape; 
this is, in fact, the basis of molecular size and shape determination by 
means of light scattering (Oster, 11)48; Doty and Steiner, 1950). 

In any event, scatt(M'ing will tend to obscure seriously the true absorp- 
tion spectrum (Schramm and Dannenburg, 1944). If it is necessary to 
determine the absorption of turbid media or coarse structures, the effects 
of scattering may be minimized by use of a detector designed to capture 
as much scattered light as possible (Caspersson, 1950) and by use of a fluid 
medium with a refractive index as closely matched to that of the scatter- 
ing substance as possible (Mitchell, 1950). In some instances it is pos- 
sible to introduce a reasonable correction factor by extrapolation of data 
from wave length outside the absorption band (Trciber and Schauen- 

stein, 1949). 

LOCAL CONCENTRATIONS 

In general, solutions will have a uniform distribution of the absorbing 
substance, but this situation is not necessarily true of cellular structures. 
If the substance tends to molecular association and deviation from Beer's 
law, the presence of local concentrations may appreciably alter the 
absorption spectrum. In addition to this potential effect, the aggregation 
of the absorbing molecules into discrete groups (possibly submicroscopic) 
will influence the absorption by virtue of the nonabsorbing "holes" left 
between the absorbing groups. The effect on the spectrum in this 
instance is similar to that described in the contingency of marked dichro- 
ism. If there is an appreciable chance of a photon passing through the 
specimen without encountering one of the postulated absorbing centers, 
then there will be a maximum possible absorption, independent of the 
amount of absorbing substance present. 

ULTRAVIOLET ABSORPTION SPECTRA OF IMPORTANT BIOLOGICAL 

SUBSTANCES 

Since the principal role of absorption spectra in radiation biology is to 
serve, within the limits suggested in the preceding section, as a key to the 
interpretation of the action spectra for various photobiological effects, it 
is useful to have a summary of the absorption characteristics of the 
principal known ultraviolet chromophorcs in living systems (Erode, 
1946; Morton, 1942; Loofbourow, 1940, 1943; Kllinger, 1937, 1938; 
Miller, 1939). 

PROTEINS AND AMINO ACIDS 

It is convenient to consider separately the ultraviolet absorption of 
proteins in the region below 2400 A and in the region abo\'e — generally 



ULTRAVIOLET ABSORPTION SPECTRA 



185 



6000 



2400-3000 A. All proteins show absorption below 2400 A, increasing 
rapidly toward shorter wave lengths. Although certain amino acids, 
histidine, and trj^ptophane (Coulter el al., 1936) and tyrosine (Smith, 
1928) show characteristic peaks in this region, these peaks are generally 
submerged by the fast rising "end" absorption; this absorption is due in 
large part to the peptide bond (Magill et al., 1937; Setlow and Guild, 
1951 ; Goldfarb et al., 1951) and to a lesser degree to absorption represent- 
ing the long wave limb of the vac- 
uum ultraviolet absorption peaks of 
single C — C, C — N, etc., bonds 
(Marenzi and Vilallonga, 1941a). 
The 223-m;u peak of tyrosine, which 
shifts to 242 m/x in alkaline medium 
(Kretchmer and Taylor, 1950), should 
then become apparent, but it does not 
always become apparent, probably 
because of binding of the phenolic 
group within the protein (Sizer 
and Peacock, 1947; Finkelstein and 
McLaren, 1949). 

Absorption in the region 2400 3000 
A is due to the presence of the aro- 
matic amino acids, phenylalanine, 
tyrosine, and tryptophane, especially 
the last two (Figs. 5-14, 15) (Goodwin 
and Morton, 1946). Proteins and 
polypeptides that lack these amino 
acids, such as gelatin (Loofbourow 
et al., 1949) and clupein (McLaren, 
1949), have negligible absorption in 
this region, although the peptide bond does have a very weak band at 
about 2800 A (e = 1-5) (Setlow and Guild, 1951). 

lodination of tyrosine, as in diiodotyrosine, induces a shift of the alka- 
line tyrosine band to 3115 A (Marenzi and Vilallonga, 1941b) ; the alkaline 
absorption maximum of thyroxine is still further displaced to 3310 A 
(Heidt, 1936; Marenzi and Vilallonga, 1941b, c). 




2400 



2600 2800 3000 
WAVE LENGTH. A 



Fig. 5-14. Ultraviolet absorption 
spectra of the aromatic amino acids. 
I, tryptophane in AVlO HCl; II, 
tyrosine in iV/10 NaOH; III, tyrosine 
in AVlO HCl; IV, DL-0-phenylalanine, 
(Loofhoiiroiv, 1940.) 



NUCLEIC ACIDS AND NUCLEOTIDES 

The ultraviolet absorption spectrum of nucleic acids and polynucleo- 
tides is characterized by a strong absorption maximum at about 2600 A, 
a minimum at about 2300 A, and continuously rising "end" absorption at 
below 2300 A. (Loofbourow, 1940; Hotchkiss, 1948; Ploeser and Loring, 
1949; Schlenk, 1949). As has been noted, the spectra of the highly 
polymerized nucleic acids are not the linear sums of the spectra of their 



18() 



RADIATIOX BIOLOGY 



conipoiiciit iiuclcotidos hut are appnM-iably les.s, siig}z;(\stinjj; wcjik iutcr- 
mitl(>i)litl(' liiikjit?(>s, alT(>ctiiiK the absorhing .structuros (Fi^j;. .'»-l(i). 

The spectra of the iiuli\ idiial miclcot ides are easily disliuKuished (Kig. 
5-17); they are generally similar to the spectra of the component purine 
and pyrimidiiic hases, which hases are primarily responsible for the 
absorption of these compounds in the spectral region above 2300 A. The 




200 



240 



320 



280 
WAVE LENGTH, rr\ p. 

Fig. 5-15. Absorption spectrum of trypsin in acid and in alkaline sohition, and of the 

trypsin-trypsin inhibitor complex (e = :3fi,700 K). • •, trypsin in .V/lOO 

H2SO4; O O, trypsin in A/10 HCl; ® ®, trypsin after 24 hours in A710 HCl; 

3 9, heat-inactivated trypsin; O O, trypsin in A/10 NaOH; O O, 

trypsin inhibitor complex. (Schonnuller, 1949.) 

spectra are sensitive to changes in pH, especnally in the regions of the pK 
vahies of the functional groups attached to the purines and pyrimidines 
(Stimson, 1949) . Absorption in these bases is considered to be dependent 
on the presence of — C^C— C=N— or — C=C— C=0 groupings 
(Cavalieri and Bendich, 1950). 

STEROIDS 

Although all steroids will exhibit end absorption in the region below 
2100 A, only those steroids that contain sequences of conjugated double 



ULTRAVIOLET ABSORPTION SPECTRA 



187 



bonds will show appreciable absorption in the region 2200 3000 A 
(Dannenberg, 1939; Fieser and Fieser, 1949). Thus steroids that contain 
only isolated ketone groups (androsterone) have only a very weak absorp- 



1.2 



1.0 



8- 



o 



0.6 






-^ 



02 



\ 




-1 




- \\ 


/"^•^. : 




:V<^ 


1 


V 



220 



230 



240 



290 



300 



310 



250 260 270 280 

WAVE LENGTH, mjl 

Fig. 5-16. Change in ultraviolet absorption spectrum of thymu.s deoxyribonucleic acid 
upon digestion with deoxyribonuclease. I, digested; II, undigested. {Kunitz, 1950, 
reproduced from the Journal of General Physiology.) 



16.000 



14.000 



12.000 



10.000 



o 
o 

2 



8000 - 



< 6000 

-J 

o 

s 

4000 - 



2000 




210 



220 



230 



240 



290 



300 



310 



320 



250 260 270 280 

WAVE LENGTH, mji 

Fig. 5-17. Absorption spectra of five deoxyribonucleotides at pH 4.30. I, deoxy- 
guanj'lic acid; II, deoxyadenylic acid; III, deoxycytidylic acid; IV, deoxy-5-methyl 
cytidylic acid; V, thymidylic acid. {Sinsheimer, 1954.) 

tion at about 2900 A (e = 43). Steroids with a and /3 unsaturated 
ketones will have a strong absorption in the region 2300-2000 A (for pro- 
gesterone, Xmax. = 2370 A, and e = 17,000) and a much weaker band near 
3200 A (e ~ 10-20) (Morton, 1942). 



188 



RADIATION BIOLOGY 



N 
I 

o 

















/I r\ 




100 


: 










s n 


V 
















Av/ 


\\ 20 


'" 


\ 






^ 








If 


\y 




! 


, \-^ 


















A 320 


360 












Jr^ 






m/i 




■ 








/ 








bO 


- 


1 


1 


1 1 


1 1 




1 1 


V 



220 



240 



280 



300 



260 
WAVE LENGTH, mjd 

Yu\. 5-18. Absorption speetrum of orgostorol. , ergosterol in CsHsOH; , 

crgostcrol in isooctane; X, T-dchydrtx-liolostcrol. {Ajler Hognrss et al., reproduced 
from The AppUeation of Absorption Speelra to the Stinlii of Vitamins, Honnones and 
Coenzymes, by R. A. Morton, 1942.) 





2200 3000 3800 

WAVE LENGTH. A 

Fig. 5-19. Absorption spectrum 
ofequilenin. (Jones, 1948.) 



300 



500 



400 
WAVE LENGTH, rr\ju 

Fig. 5-20. Absorption spectrum of lycopcuc with 
varying degree of cis-lrans isomerization. I, mix- 
tvu-e of stereoi.somers after iodine catalysis at room 
temperature in light; II, fresh solution of the all- 
trans compound ; III, mixture of stereoisomers after 
refluxiiig in darkness for 45 min. {Zeehmeister, 
1944; eopyriijht, 1944, by the Williams and Wilkins 
Company.) 



ULTRAVIOLET ABSORPTION SPECTRA 



189 



5.70 



5.50 



5,00 - 



Steroids with diene and triene chains, such as ergosterol (Fig. 5-18) and 
calciferol, will have strong absorption maxima in the region 2200 2900 A 
(for calciferol, \,„^^. = 2650 A, and e = 18,200). Steroids that contain 
aromatic rings, such as the estrogens, will, in general, have strong absorp- 
tion bands in the region 2400-2800 A and may have absorption intensity- 
well above 3000 A if the aromatic grouping includes more than one ring, 
as in equilenin (Fig. 5-19) (Morton, 1942; Jones, 1948). 

CAROTENOIDS 

The long conjugated double-bond chains of the carotenoid compounds 
give rise to from one to three ultraviolet absorption bands at varying posi- 
tions (Karrer and Jucker, 1948). 
One band is usually found in the 
region 260-320 m^u. Absorption 
in the region 320-380 m/x has 
been demonstrated to be depend- 
ent on the number and position 
of cts-configurations in the chain 
(Fig. 5-20) (Zechmeister, 1944). 
These ultraviolet bands 

(e = 10,000 50,000) 

are considerably weaker than the 
intense set of three bands com- 
monly found in the visible absorp- 
tion spectra of these compounds 
(e = 50,000-200,000). 

PORPHYRINS 

The intense Soret absorption 
band of the porphyrins is found 
in the near ultraviolet in the 
simpler members of the group, 
such as porphyrin itself (Fig. 
5-21). With increasing substitu- 
tion of the tetrapyrrole ring, this 
band moves into the visible-spec- 
trum region. A number of shoul- 
ders or weak maxima are usually 
to be found on the descending short wave limb of this peak. Porphyrins 
usually show a minimum of absorption in the region 2500-3000 A and 
then increasing absorption again at wave lengths <2500 A (Pruckner 
and Stern, 1937; Theorell, 1947; Holden, 1941). 

The Soret band of the dihydroporphyrines, such as the chlorophylls, is 



o 
o 



4 50 - 



4 00 - 



- 






r, 

■ 1 

! 1 
1 1 

1 1 
I 1 

: 1 
1 


- 




/i 
/ V 


1 

1 






t\\\ 






1 '\\ 


- 


// 


^l i ! 

'11 






' i 


'r— . ™ 


n/ 


; 


i "^ 


•■-^1 


i \ 


- \ 


• 1 


in 


\l. 


/ 1 


■\ 


\ ^^ 


\^ 


; \\ 




1 




; N 



3,50 



450 



250 300 350 400 
WAVE LENGTH, m/i 

Fig. 5-21. Ultraviolet absorption spectra 
of several porphyrins in dioxane. I, 
porphyrin; II, aetioporphyrin; III, copro- 
porphyrin-II-tetramethyl ester; IV, rhodo- 
porphyrin-XV-dimethyl ester. {Pruckner 
and Stern, 1937.) 



100 



RADIATION HIOLOGY 



moved woll out into the visible reRion ((Iranick aiul (Jilder. 1047). 'i'iie 
ehlorophylls show several minor absorption peaks in the speetral region 
•28tK) :i8(H) A (Fis- .V22). Chlort)phylls a and b differ notably in their 
absorption at about 3S(H) A (Harris and Zscheile. 1043). 



76 



68 - 



60 - 



52 - 



gAA 



X 

UJ 



^ 36H 
u 

Ul 

a. 

V) 



28 - 



20 



12 



C-~V?0\£\T i 





o c FS£FARAriO\ 8 


<i — < PREPARATION 10 


F 


©■ZSCHEILE AND COMAR 


i 


COMPONENT B 


i 


• • PREPARATION 8 


t 


_ V* PREPARATION 10 


I 


^ZSCHEILE AND COMAR 


I 


- 


i*o ^<l** 




V 


... 1 1 1 1 1 1 1 1 1 1 


1 1 1 1 1 



2600 



2800 



3000 



3200 3400 3600 3800 4000 4200 

WAVE LENGTH, A 

Fig. 5-22. I'ltraviolet absorption spectra of chlorophylls .\ aiul R in ethyl ether solu- 
tion. (Harris and Zscheile, 1943; reproduced from the Botanical Gazette, puhlished hy 
the University of Chicago Press.) 

FLAVINS 

As with the porphyrins, more attention has been devoted to the visible 
absorption band of the flavins than to their ultra\ iolet spectra, which do, 
however, possess strong bands at 223, 2(55, and 370 m/z, as well as the 
visible band at 445 m/x (Fig. 5-23) (Warburg and Christian, 1938; Morton, 
1942; Dagli-sh et al., 1948). 

PTERINS 

The renewed interest in the pterins has focused attention on their ultra- 
violet absorption spectra, which generally contain one strong band in 



ULTUAVIOLET ABSOKFTION SPECTRA 



191 



th(; rrfrioM 24(XJ 2800 A (e = 20,(K)0j and a second weaker band at 3400- 
3800 A (Fif^. 5-24) (Jacoh.son and Simpson, 1940; Totter, 1944; Mowat el 
al., 1948; Ilitchinj^s and Klion, 1949; Cain el al., 1948; Elion el a/., 1950;. 



PH 


04 


— 





— 




08 


— 





— 




2 5 


















90 
l?6 


















200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 

WAVE LENGTH, m/Z 

Fi<;. 5-23. Absorption spectra of riboflavin in solutions at different pH values. {Dag- 
lish el al., 1948.) 



800 



600 



^S 



400 



200 



A 








' * T 








A \ 
/ ^ \ 
/ * \ 

/ * I 
/ * I 










1 


/ 
/ 


'S, 
N 
\ 
\ 
\ 
\ 




•U 




L \ 



240 



440 



290 340 390 

AAVE LENGTH, m_// 

Fio. .5-24. .\bsorption spoctruni of xanthopterin. I, in 0.1 .V .sodium hydroxide, .50.1 
mg/liter; II, in glacial acetic acid, 49.4 nig/liter. {Totter, 1944.) 

VITAMINS 

Reduced nicotinamide iodomelliylate has a strong absorption band at 
345 mn which (lisapp(!ars upon o.xidation (Karrer and Warburg, 1936; 



192 



RADIATION HIOLOGY 



Karrer et al., 1936) and may then be restored by reduction. The dis- 
appearance and reappearance of this band in the spectra of coenzymes I 
and II (Fig. 5-25) (Euler et al., 1936; Warburg et al., 1935; Horecker and 
Kornberg, 1948) have been made the basis of elegant studies of the 
respiratory and fermentative enzymes by Warburg (1949). 

Pyridoxine in neutral solution has absorption bands with maxima at 
330 m/i (e = 5500) and 255 m/x (e = 2800). In alkaline solution these 

CH2OH 
I 

HOHpC — nS— OH 



15 



10 

















1 


1 






















) 


•■ 


/ 


/ 

CM 


y 

n 


^ 


\ 


Jj 









N 



-CH, 



6 r 



4 - 



2 - 



400 



360 320 

WAVE LENGTH, 



280 



240 




Fig. 5-25. Absorption spectra of re- 
duced and oxidized cozymase. I, 
reduced cozymase; II, oxidized co- 
zymase. (Euler et al., 1936; repro- 
duced from Hoppe-Seyler's Zeitschrift 
fur physiologische Chemie.) 



300 340 370 

WAVE LENGTH, mu 

Fig. 5-26. Influence of pH on the absorption 
spectrum of pyridoxine (vitamin Be). I, pH 
2; II, pH 4; III, pH 5; IV, pH 7. {From W. R. 
Erode, 1946, The Absorption Spectra of Vita- 
mins, Hormones, and Enzymes. In, Advances 
in Enzymolocjy, Vol. IV, F. F. Nord and C. H. 
Werkman, ed., copyright, 1944, by Interscience 
Publishers, Inc., New York.) 



maxima are displaced toward shorter wave lengths, but in acid solution 
l)oth maxima disappear and are replaced by a new peak at 292 mp, 
(e = ()()00) (Fig. 5-26) (Morton, 1942; Brode, 1946; Stiller el al., 1939). 

Vitamin B12 has two strong ultraviolet absorption bands with maxima 
at 2780 A (E^Z = 115) and 3610 A iE\2, = 204) in addition to the 
weaker band in the visible spectrum at 5500 A (fi'lJ,^ = 63) (Brink et al., 
1949). Vitamins B12,. and B121, have similar spectra (Kaczka et al., 1949; 
Brockman et al., 1950). 

Ascorbic acid in neutral solution has a strong absorption band 
(e = 9300) at 265 n\p, which shifts to 245 m/i in acid (Morton, 1942). 



ULTRAVIOLET ABSORPTION SPECTRA 



193 



The presence of this band has at times been mistaken as an indication of 
the presence of nucleic acid (Strait et al., 1947). Confirmation of the 
presence of ascorbic acid can be obtained by disappearance of the band 
upon oxidation, which can be induced by simply raising the pH above 10. 

Vitamin E (a-tocopherol) has a distinct absorption maximum at 2940 A 
(e = 3200). The 13- and 7-tocopherols have similar spectra (Smith, 1940) . 

Vitamin Ki has a strong, five-peaked absorption band in the region 
2400-2800 A and a weaker broad secondary maximum at 320 m^ (Fig. 
5-27). The peaks of the former band occur at 239, 243, 249, 260, and 
270 m/x, and for the 249-m/x peak, e = 19,600 (Ewing et al., 1943). Vita- 



.«i 



400 




4 














f 


r/ 


m 












300 




V 


\j 














/ 
















200 


/ 


































100 
























1 






-^ 


\ 









\ 


^ 






\ 



240 



280 320 

WAVE LENGTH, m// 

Fig. 5-27. Absorption spectrum of vitamin Ki in hexane. 



360 



{Ewing et ah, 1943.) 



min K2 has a similar, but slightly weaker, absorption spectrum (Ewing 
et al., 1939). 

PLANT PIGMENTS 

The common plant pigments such as the anthocyanins and flavones 
have strong ultraviolet absorption bands. In acid solution, all antho- 
cyanidins and anthocyanins have one or two strong absorption maxima 
in the region 2650-2800 A (e = 10,000-20,000) (Fig. 5-28). Some have 
additional bands at ~2450 and ~3300 A. In basic solution these ultra- 
violet absorption bands, as Avell as the visible bands responsible for the 
color of these pigments, are displaced a few hundred Angstroms toward 
the longer wave lengths (Schou, 1927; Hayashi, 1934, 1936). 

The flavones have two ultraviolet absorption bands at 2500 and 3000 A 
(e « 10,000). Hydrogenation of the chromene ring, as in the flavonones, 



194 



RADIATION BIOLOGY 



mt)\ I's (1k' position of (lie loiif^cr wjivc hand lo '.V2{)() A, without affect ing 
the '2')()() A hand. Addition of a .S-liy(h<)xy f2;rouj) to flax'onc, as in llu; 
flavonols causes thi- appcai'ance of an additional hand at XioO 3400 A 
(Fig. 5-29). Hydroxy or mothoxy side groups in various positions may 



45 






.^"■v 










1 1 






/ \ 
























1 / 












I 

A 


m 

i/h 










' / /^ 


•A 










/ \ 


1 / 










'// 


i 








/ 


^V// 1 


40 






■7 


\ 








I 


^^ 






/ 


/ 


i 


r 


V 




Y 


1 


/ 








/ 


1 


V 


s. 


\ 




T 












/ 


1 


\ 


s 


\ 


1 














ml 

/I 


I 


\ 


V% 


\ 


4 










3,5 










N, 


A 


1 \ 1 




















\\ 


n 






















\ 


[y 











6000 5500 5000 4500 4000 3500 3000 2500 2000 

WAVE LENGTH, A 

Fig. 5-28. Absorption spectra of I, pclargonidin ; II, cyanidin; and III, delphinidin. 
{Schou, 1927.) 




2.6 



2300 2500 2700 2900 3100 3300 3500 3700 3900 
WAVE LENGTH. A 



Fig. 5-29. Absorption spectra of I, flavone; II, 3-oxy-flavone (fiavonol); and III, 
flavonone. (Skarzynski, 1939.) 

shift these maxima by as much as 100-200 A (AronofT, 1940; Skarzynski, 
1939). 

The catechins, which occur widely in woods and leaves, may be 
regarded as partially reduced anthocyanidins. The reduction has 
destroyed the extensive conjugation, and the absorption of catechins may 



ULTRAVIOLET ABSORPTION SPECTRA 195 

be regarded as the sura of the absorption of the phloroglucinol nucleus and 
the polyphenoUc residue. Catechins generally have absorption maxima 
in the region 2700-2800 A (e = 1000-3000). Secondary maxima may 
appear about 2150 2200 A (Bradfield and Penney, 1948; Morton and 
Sawires, 1940; Klingstedt, 1922). 

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Chem., 5: 561-571. 
Bayliss, N. S. (1948) A "metallic" model for the spectra of conjugated polyenes. 

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(1950) Light absorption and photochemistry. Quart. Revs. London, 4: 

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Bradfield, \. E., and M. Penney (1948) The catechins of green tea. Part II. J. 
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Braude, E. A. (1945) Ultra-violet light absorption and the structure of organic com- 
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Brink, N. G., D. E. Wolf, E. Kaczka, E. L. Rickes, F. R. Koniuszy, T. R. Wood, and 
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Brockman, J. A., Jr., J. V. Pierce, E. L. R. Stokstad, H. P. Broquist, and T. H. Jukes 
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Erode, W. R. (1943) Chemical spectroscopy. 2d ed., John Wiley & Sons, Inc., New 
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— (1946) The absorption spectra of vitamins, hormones, and enzymes. Ad- 
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(1949) The presentation of absorption spectra data. J. Opt. Soc Amer., 



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Butenandt, A., H. Friedrich-Freksa, S. Hartwig, and G. Scheibe (1942) Beitrag zur 

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pyrimidiues and purines. J. Am. Chem. Soc, 72: 2587-2594. 
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2: 644-653. 



196 RADIATION BIOLOGY 

Chirgwiii, 1). II., and C A. Coulsou (1950) The electronic structure of conjugated 

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ultraviolet ah.sorption of naphthalene, anthracene, and homologs. I'roc. Thys. 

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absorption spectra of certain proteins and amino acids. J. Gen. Physiol., 19: 

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37: 302-310. 
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Doty, P., and R. F. Steiner (1950) Light scattering and spectrophotometry of 

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and 7-hydroxypteridines from 4,5-diaminopyrimidines and a-ketoacids and esters. 

J. Am. Chem. Soc., 72: 78-81. 
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241: 239-272. 
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vitamin Ki and the effect of light on the vitamin. J. Biol. Ch(>m., 147: 233-241. 
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vitamins Ki, K>, and some related compounds. J. Biol. Chem., 131: 345-356. 
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constitution of organic molecules. Chem. Revs., 43: 385-446. 
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ed., Rcinhold Publishing Corporation, New York. Pp. 184-198. 
Finkelstein, P., and A. D. McLaren (1949) Photochemistry of proteins. VL pH 

dependence of quantum yield and ultraviolet ab.sorption spectrum of chymo- 

trypsin. J. Polymer Sci., 4: 573-582. 



ULTRAVIOLET ABSORPTION SPECTRA 197 

Forster, T. (1948) Zwischenmolekulare Energiewanderung und Jluoreszenz. Ann. 

Physik, 2: 55-75. 
Franck, J., and R. Livingston (1949) Remarks on intra- and intermolecular migra- 
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Freed, S., and K. M. Sancier (1951) Absorption spectra of chlorophyll in .solutions 

at low temperatures — equilibria between isomers. Science, 114: 275-276. 
Gibson, K. S. (1949) Spectrophotometry. Natl. Bur. Standards (U.S.) Circ. No. 

484. 
Goeppert-Mayer, M., and A. L. Sklar (1938) Calculations of the lower excited levels 

of benzene. J. Chem. Phys., 6: 645-652. 
Goldfarb, A. R., L. J. Saidel, and E. Mosovitch (1951) The ultraviolet absorption 

spectra of proteins. J. Biol. Chem., 193: 397-404. 
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198 RADIATION BIOLOGY 

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ULTRAVIOLKT ABSORPTION SI'l-X'TRA 190 

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200 RADIATION BIOLOGY 

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ULTRAVIOLET ABSORPTION SPECTRA 201 

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Manuscript received by the editor Feb. 20, 1952 



CHAPTER 

A Critique of Cytochemical Methods 

Arthur W. Pollister 
Department of Zoology, Columbia University, New York, New York 

Introduction. Lairs of absorption. Localization of substances in cells: Preservation 
of intracellular substances in situ — Nucleic acid staining and tests — Protein staining 
and tests — Ultraviolet absorption of nucleic acids and proteins. Quantitative micro- 
scopical methods: Visual comparison — Photometric technique — Some errors of quantita- 
tive microspectrophotometry — Quantitative applications, absolute and relative. References. 

1. INTRODUCTION 

The bulk of the extensive researches in cytology has been aimed pri- 
marily at demonstrating the morphological features of the cell. By 1875 
modern microscopes had become available which reached close to theo- 
retical limits imposed by the properties of visible light, and within less 
than two decades the application of this tool, in conjunction with an 
increasing number of special microtechniciues for preparing cells for 
examination, had demonstrated a wide diversity in intracellular mor- 
phology and many striking correlations of cell structure with physiology. 
From these studies the concept of the histological unit, the cell, emerged 
with increasing clarity (Figs. 6-1, 2A). 

A similar morphological picture of most cell types could be drawn from 
the researches of three-quarters of a century of cytology. Such a descrip- 
tion, of course, is compfetely unsatisfactory from the standpoint of 
intracellular biochemistry; indeed, it is merely an invitation to further 
research which might lead to more nearly complete understanding of the 
physiological processes by which such a cell can synthesize a secretory 
granule or can elaborate the material to duplicate itself. One obvious 
way in which to complete the picture is to attempt to determine the 
chemical composition of the cell. To a cytologist this problem emerges 
as one of demonstrating how the various substances, which biochemists 
obtain by such procedures as extraction from minced organs, are dis- 
tributed among the various visible cellular constitutents and in the 
apparently structureless material which fills spaces between the iniclei, 
mitochondria, granules, and other formed elements of the cell. Broadlj' 
regarded, this appears to be the field of what has come to be called 

203 



204 RADIATION BIOLOGY 

"cytochemistry," and in this chapter an attempt will be made to describe 
and to evalnate critically some of the approaches to this problem of chem- 
ical organization of the cell. 



2. LAWS OF ABSORPTION 



Since cytological stndies are necessarily carried out at high magnifica- 
tion with a compound microscope the preparation, a section or smear of 
an organ, is always examined by transmitted light. For this reason the 



Fig. 6-1. Diagram of the structural features of a serous glandular cell at an early stage 
in restitution of the secretory granules, c, centriole; mg, mature zymogenic granule; 
ig, immature zymogenic granule; n, nvideus with masses of chromatin and a large 
spherical nucleolus; m, mitochondrion. 

technique of cytological microscopy is readilj^ adapted to photometric 
chemical analysis, in which the nature and amount of material may be 
determined from the spectral characteristics and intensity of the light 
which emerges from a semitransparent mass. This fraction of the light 
is said to be "transmitted"; that portion which entered the object 
but did not emerge is said to have been "absorbed." Application oi 
laws of absorption (see Chap. 5, this volume) not only make it possi- 
ble to use on slides qualitative and (juantitative methods of chemi- 
cal analysis, but these laws also apply directly to the visual examination 
of cells since the visibility of a natural or artificial color in a cell depends 
on whether the colored object absorbs enough light to make it distin- 
guishable from the surrounding nonal^sorhing regions. 



A CRITIQUE OF CYTOCHEMICAL METHODS 



205 




Fig. 6-2. A series of photomicrographs of pancreatic glandular cells, showing structure 
and the results of various techniques of localization of nucleic acids and proteins. 
(A) phase contrast, no stain or reaction; (B) Millon reaction for total protein; (C) 
nucleic acids stained specifically by basic dye, azure A; (D) ultraviolet photograph 
(254 m/u) showing absorption in regions of nucleic acid concentration; (E) digested 
with ribonuclease before staining with azure A [compare with (C)]; (F) Feulgen's 
nucieal reaction for deoxypentose. {Pollister et al., 1951.) 

Inferences from the more general laws of relation of mass to absorption 
of radiant energy are conveniently summarized by some simple equations, 
in which the following symbols are used: 

/o = light intensity (galvanometer reading) when no absorbing 
object is in the optical path (in cytological measurements, the 
reading through an empty part of the slide) ; 
Ix = intensity when the absorbing object or sample is in the optical 
pathway (in cytological measurements, the reading through 
the cell component) ; 
T = transmission, h/h (7c T = \00T); 



•JOG UADIATIU.N lllULOGV 

E = extinction, also called optical density, logio 1/7' or logio I d h; 
X = wave length; 
E\ = extinction at a given wave length, e.g.. A'b-.o niyu; 
{E\)2 = extinction for unit amount per unit area; 
c = concentration; 
/ = length of absorbing pathway; 
.1 — area of absorbing mass in plane perpendicular to the absorbing 

pathway; 
V = volume of absorbing mass; 
A- = specific e.xtinction, e.g., (^^x)^^ cm- 
Beer's law deals with the relation of light lo.ss to concentration (c) ; 
Lambert's law deals with the relation of light loss to absorption path (/). 
A useful simple e(iuation which expresses the fact that extinction is 
directly proportional to the number of specifically absorbing chromo- 
phores (k) in the absorbing path, as determined by concentration and 
thickness is 

E = kcl. 

In most routine (juantitative photometric chemical analj'sis the sample 
is in a carefully measured cuvette or absorption cell (generally 1 cm 
thick). The unknown concentration (Cx) may be computed in terms of 
the extinction of a standard of unit thickness and concentration, e.g., 
(■^x)i.ncra» where E^ is the extinction of the unknown from the following 
formula. 



^x % 



('E'x)l.'oc 



In cytological preparations the thickness is always but a few microns. 
When this thickness has been measured, computation from a standard 
extinction must take into account the relative thickness. An equation 
by which, assuming the validity of Beer's law over the concentration 
range of which the two measurements are the extremes, the percentage 
concentration in a cytological preparation may be computed from the 
standard (£/x)L^°e„ is 

10,000 (£:x), 

(^x /c ~ 7/Tn\l% 



h(E,)\, 



cm 



where l^ is the thickness of the cytological structure, in microns. 

Instead of using the .standards ol)tained in the cuvettes of a colorimeter 
or spectrophotometer, it is more convenient to compute special cyto- 
logical standards. Most useful is Ea computed as the extinction for 10"'" 
rfig/M", {E\)li ""~', in which Caspersson has expressed the results of his 
(juantitative cell analyses. In a thickness of 1 ^ this is equivalent to 10 
per cent, to 100 mg^cc, or to 10~'" mg/;u^. This standard has been com- 



A CRITIQUE OF CYTOCHEMICAL METHODS 207 

putcd from in vitro (l;it;i tor a number of naturally colorc^d .substances 
commonly found within cells [\\\i\ foi- some speeitic stains and tests for 
proteins and nucleic acids (columns 1, 2, and 3, Tables G-l, 2). The 
sources of the \alues from which computations were carried out are indi- 
cated in column 5.' 

These values in Tables 6-1 and 2 are not claimed to be necessarily close 
approximations of any physical constants of the intracellular substance. 
They are almost certainly subject to considerable revision as more is 
learned of the effects on absorption of high concentrations and of special 
intermolecular associations within the cell (see p. 215 and Chaps. 1 and 5, 
this volume). In the meantime, they are useful relative values from 
which the possibilities of seeing, or measuring photometrically, an intra- 
cellular substance under the microscope can readily be estimated; they 
are likewise the only method of translating results of intracellular absorp- 
tion measurements into the familiar values of chemical analysis, and in 
radiation experiments they can serve as the basis for an approximate esti- 
mate of the amount of energy absorbed per cell or cell part. Since the 
errors introduced by high concentrations and other special conditions 
within the cell tend, as a rule, to reduce the specific extinctions, it seems 
fairly safe to assume that the values in column 3 are maximal and those 
in column 4 are minimal. The great usefulness of the cytological stand- 
ard, {E\)a, arises from the fact that of itself extinction is a direct measure 
of the number of molecules in the absorbing path and can be used as such 
when neither concentration nor thickness of the absorbing area is known. 
From the standard {E\)lt '""'* the amount per total area of the part of 
the cell measured (^4) can be computed simply as {E\)a-A {A being meas- 
ured in square microns). Likewise, because extinction times area is the 
equivalent of amount, it can be used in simple arithmetical compu- 
tations to compare compositions of cytological objects in purely arbitrary 
amounts (see Swift, 1950). 

So far W'C have been considering photometric methods with measure- 
ment in a more or less restricted spectral region, a procedure which, some- 
what paradoxically, is often called colorimetry. .When measurements are 
made at many wave lengths, a picture is obtained of the etTect of the 
absorbing substance on the light, which expresses in objective data the 
phenomena which cause the visual sensation of color. Such data are 
often plotted as absorption curves, with some measure of relative light 

^ The cytological standard, {E\)ll ^"'"^ is computed as follows, from a cuvette 
standard, e.g., for deoxyribonucleic acid (DXA) where {E2i4)l',o^ "" is 20. It is 
given that 20 is the E^hi of 1 yu^ of a standard solution in a thickness of 1.0 cm, lO"* m 
(for extinction depends on thickness and concentration and is independent of area). 
Each cubic centimeter of cuvette standard contains 1.0 mg of DX.\; 1 cc is 10'^ fx^, and 
the volume of a mass which is 1 m^ in area and 1 cm thick is 10^ n^. This volume 
of the standard solution then contains lOVlO'^, or lO"**, mg of DNA. Therefore, 
(Ei,,)lV'"'"^ is 20/102 or 0.200 (Table 6-1). 



20S RADIATION HIOLOGY 

Tabi.k ()-i. N.\n K.M, Absokption of Substances Occurring in Cells 





Wave 




Concentration 




8iil>.-;t;iii('i! 


length, 
m^ 


(E^y.r""' 


(%) in 1 M to 
give E\ of 0.030 


Kcfcicncc 


Deoxyribonucleic acid . . 


254 


0.200 


1.5 


Ris (1947) 


DeoxvrilxJiiucleic acid. . 


260 


0.220 


1.4 


Caspersson (1940a) 


Deoxyril)()imcl('ic acitl , . 


275 


0.180 


1.7 


Caspersson (1940a) 


Ribonucleic acid 


260 


. 230 


1.3 


Thorell (1947) 


Tryptophane 


275 


0.270 


1.1 


Fig. ()-2 1 ; Caspersson 










(1940a) 


Tyrosine (acid) 


275 


0.065 


4.6 


Fig. 6-21; Caspersson 
(1940a) 


Tyrosine (alkali) 


290 


090 


3.3 


Fig. 6-21 ; Caspersson 
(1940a) 


Serum albumen 


275 


0.006 


50.0 


Fig. 6-5 


Serum albumen 


260 


0.0016 


Not detectable 


Fig. 6-5 


Ascorbic acid 


265 


0.355 


0.85 


Stearns (1950) 


Thiamine 


232 
265 


0.312 
1.78 


. 9() 
0.17 


Stearns (1950) 


Ril)()tlavin 


Stearns (1950) 


Vitamin A 


324 


1.82 


0. 16 


Stearns (1950) 


Oxyhcinofrlobin 


413 


. 069 


4.3 


Thorell (1947) 


Chlorophyll a 


665 


2.90 


0.10 


Zscheile (1934) 


Chlorophyll b 


640 


1.22 


0.25 


Zscheile (1934) 


Cytochrome c 


415 


0.665 


. 46 


Dixon et al. (1931) 



Table 6-2. 


Absorption of Cytochemical 


St.\ins and Tests 








Concen- 






Wave 




tration 




Substance 


length, 
m/x 


/ 7,1 NlO^lOinK 


(%)inla 

to give E\ 

of 0.030 


Reference 


Deoxyribonucleic acid, 










Feulgen reaction. . . . 


546 


200 


1.5 


Alfert, 1950 


Tryosine, Millon reac- 










tion 


365 


0.220 


1.4 


Pollister (1950) 


Protein, Millon reac- 




tion (6.25% tyrosine) 


365 


0.014 


21 


Pollister (1950) 


Protein, Millon reac- 










tion (6.25% tyrosine) 


490 


0.007 


43.8 


Pollister (1950) 


Fast green, pH 2.0 ... . 


625 


1.6 


0. 19 


Bryan (1951) 


Egg albumen, fast 


625 


0.568 


0.53 


Computed from Fraenkel- 


green 








Conrat and Cooper (1944); 
Bryan, 1951 



A CRITIQUE OF CYTOCHEMICAL METHODS 209 

loss on the vertical axis, and wave length or frefjuency on the horizontal 
axis. The shape of the absorption curve is of qualitative value in identi- 
fying the specific absorbing atomic configuration, the chromophore. 
When a sample contains two or more nonreacting absorbing substances, 
the compound absorption curve results from addition of the individual 
components. If the curves of the chromophores are considerably differ- 
ent in absorption coefficient at some wave lengths, the compound curve 
can be analyzed into the individual curves of its components by solving 
simultaneous equations (Stearns, 1950). 

As Tables 6-1 and 2 indicate, in order to be detectable in the cell by 
absorption (i.e., by contrast), substances must reach a concentration 
many thousand times that which is sufficient for analysis in an absorption 
cuvette. This imposes a fundamental limitation on the whole method of 
interpreting the chemical composition of cellular components from visual 
microscopical appearance or, indeed, from the most careful microscopical 
absorption measurements with an objective photometer. Very small 
quantities can be detected, as little as 10~''* g in a single small granule, 
which may be perhaps no more than a millionth of the entire cell volume. 
Therefore, in one sense, these are very "sensitive" techniques in the 
vocabulary of the microchemist (see Benedetti-Pichler and Rachele, 
1940). But, as a means of demonstrating a complete picture of distribu- 
tion of a substance within the cell, these microscopic methods are deplor- 
ably inadequate. Obviously, if the concentration within a small granule 
just reaches the threshold for detection (which experience shows to be 
roughly equivalent to an extinction of 0.03 or about 7 per cent absorption; 
see Tables 6-1 and 2, column 4), then outside this spot of high local con- 
centration there can be a relatively enormous amount of substance which 
is below the detectable absorption or contrast. It is a simple matter to 
compute in any cell, from data like those of Tables 6-1 and 2 and the 
volume relations within the cell, the maximum possible amount of sub- 
stance that could escape visualization or measurement. Conclusions 
about localization and distribution of intracellular substances from micro- 
scopical data must always take into account this interrelation between 
absorption and intracellular geometry. It must be emphasized that 
microscopic methods alone can prove neither the exclusive localization of 
a substance within a small intracellular structure nor the complete absence 
of a substance from any part of the cell.- Such conclusions can come 
only from a combination of methods of chemical analysis of cell isolates 
and cytology, as pointed out by Pollister, Himes, and Ornstein (1951). 

^ One escape from tliis limitation on microscopic detection and estimation lies in 
developing stains which are fluorescent and tests based on fluorescence. Since 
fluorescence is seen or measure4 as total intensity against a dark field, i.e., zero inten- 
sity (instead of by subtraction from a field of high intensity), the dye or color reaction 
can be detected readily in concentrations as low as one one-thousandth of the minimum 



210 UADlATlU.N JJlOLUCiV 

3. LOCALIZATION OF SUBSTANCES IN CELLS 

It imisl not l)c' supposed that an awareness of the difficulties just suin- 
mari/ed lias operated to inhibit the development of a chemical cytology. 
(Juite the contrary has been the case; from the earliest days of cytology 
there have been attempts to supplement the morphological descriptions 
of cells by some idea of the chemical composition. 

A few substances such as hemoglobin or chlorophyll are visible in the 
living cell because of the natural color, but in most cases a substance can 
become visible only because of a color reaction carried out on a micro- 
scopic slide. To be useful cytochemically such a test or stain must ful- 
fill certain criteria: 

1. It must be possible to carry out the test under conditions which will 
not seriously distort the cell morphology, a requirement which excludes 
a great many of the color reactions of analytical chemistry. 

2. The specificity of the reaction must be known from data obtained 
in vitro. 

3. The reaction must be one which will proceed without interference 
in the presence of large amounts of proteins, and often in the presence of 
nucleic acids or lipids. 

3-1. PRESERVATION OF INTRACELLULAR SUBSTANCES IN SITU 

Lison (193G) has considered critically many of these so-called "histo- 
chemical" techniques; additional ones are briefly mentioned by Glick 
(1949). The microchemical tests for inorganic ions, such as ferric, 
chloride, and phosphate, and for smaller organic molecules, such as uric 
acid and oxalic acid, are for the most part closely analogous to those of 
microchemistry and cause the appearance on the slide of a colored pre- 
cipitate or crystals only when the reacting group is in solution. For 
cytological studies this means that the group will not react Avhen it is a 
part of a large molecule, such as that of protein, but only after it has been 
split off. These tests can undoubtedly be interpreted as an indication 
that the reacting substance is present in the section of tissue, but this is 
but a poor imitation of the precise conclusions which are possible from the 
methods of analysis of tissue masses (Hogeboom, 1951) or from refine- 
ment of microchemical methods to reach down to the level of a single 
cell (Xorberg, 1942). For cytological studies the intracellular localiza- 
tion is most important, and there are good reasons to (juestion the validity 
of methods of microscopical demonstration of small diffusible chemical 

detoetable by absorption. Tlic |)ossiI)iliti('s have not been widely explored, but a 
fluoiosceiit Scliiff reatfciit lias hccii dcNcloiM'd and found to dcnioustrate marked 
aldfliyde (jjlasnial?) reaction in cells where the test api)eared couipletely negative by 
ab.sorption contrast technique (L. Ornstein, unpublished). Fluorescent cytological 
techniques also offer one possibility of avoiding the distributional error (p. 235). 



A CRITIQUE OF CYTOCHEMICAL METHODS 211 

entities by color reactions. Within the dimensions of the cell, diffusion — 
11 slow process at the macroscopic lev^el — is practically instantaneous, and 
there is every reason to suppose that extensive redistribution takes place, 
either as tissue is being fixed or as the test is carried out on the tissue. 
This is particularly misleading because the precipitates tend to be 
adsorbed on the extensive internal surfaces of the denser parts of the cell 
such as nuclei, myofibrillae, or the thick distal borders of some epithelial 
cells. Thus many early observers erroneously reported that the nucleus 
contained considerable iron, an element in which it is actually notably 
deficient. The nucleus was repeatedly described as the site of the enzyme 
alkaline phosphatase, since phosphate split off from a substrate, glycero- 
phosphate, appeared as an intranuclear precipitate, but Jacoby and 
Martin (1949) have demonstrated that this is largely a secondary accumu- 
lation of the phosphate (see also Novikoff, 1951, 1952). 

Redistribution during the fixation process is effectively prevented by 
the freeze-dry method of preparing sections of tissue, which was suggested 
by Altmann (1890) and elaborated by Gersh (1932), Hoerr (1936), Simp- 
son (1941), and others. In this technique fresh tissue is quickly frozen 
at such low temperatures ( — 190°C) that ice crystals do not form (the 
water appears rather to be practically ^^itrified). Tissue is then dehy- 
drated at low pressure and temperature and is finally embedded in paraf- 
fin and sectioned. Up to this stage, it is generally agreed that little 
redistribution of intracellular chemical constituents can have taken place, 
and two excellent methods of elementary chemical analysis apparently 
can be carried out without producing any essential change in this dis- 
tribution. The paraffin sections may be burned in an electric furnace 
(microincinerated; Policard, 1923; Scott, 1943), and the appearance and 
amount of the ash indicate the distribution of the mineral elements in the 
cells (e.g., iron is a yellow to red ash, silicates are crystalline, and calcium 
and magnesium are amorphous and dense white ashes.) In another 
method the paraffin is removed from the section, and the tissue is dried 
and subjected in vacuo to X-ray absorption analysis (Engstrom, 1946, 
1950). Although it involves immense technical difficulties, the latter 
appears to be an extremely promising approach since it offers the possi- 
bility both of determination of cell mass from polychromatic X-ray 
absorption data and of analysis for a large number of individual elements 
from absorption of monochromatic X rays in the wave length range 
2-50 A. 

The distribution in frozen-dried sections would also be expected to 
remain essentially unchanged, except for lipoidal constituents, if the 
paraffin is removed — for instance, by chloroform — and the section kept 
in nonaqueous solvents such as alcohol and glycerin. This procedure has 
been recommended in preparing material for ultraviolet absorption 
studies of intracellular proteins (Caspersson, 1947) principally because 



212 UADIATIO.N HlOLOGV 

the optical coiKlitioiis within the tissue arc tlicii more i'ax'orablc loi' tlu-.so. 
measurcnuMits. This aclvaiitufj;c would appear to ho somewhat olTset by 
the confusion which is introduced into the absorption picture hy the 
probable presence, in unextracted material, of many absorbinji, com- 
pounds of low molecular wci<>;lit, a situation like that which makes the 
results of ultraviolet absorption stud}'' of li\iiiji; cells so inconclusive. 

When precipitation or a color reaction on the microscopic section 
necessarily invoh'es use of a(|ueous reagents, then the frozen-dried sec- 
tions must be rehydrated. This in effect appears to eliminate about every 
advantage of the freeze-drying technicjue because extensive redistribution 
can take place at once and can continue dining any subsecjuent steps of 
the technifjue (Iloerr, 1943). Unless these technical difficulties can be 
overcome, the sound view seems to be to admit that, except for micro- 
incineration and X-ray absorption, precise intracellular localization is 
practically limited to substances of high molecular weight, which diffuse 
slowly and are easily converted into relatively insoluble masses, and to 
the use of tests which demonstrate the smaller chemical groupings when 
they are parts of these large molecules. Two substances of this charac- 
ter, proteins and nucleic acids, bulk large in the composition of all cells. 
Indeed, it is easy to see that, if these two constituents are removed, as 
can be done with enzymes, the remainder is but an unrecognizable ghost 
of a cell. As a matter of fact, the whole concept of the fixed cell is mainly 
that of a nucleoprotein mass. Except for some of the special mito- 
chondrial methods which preserve lipids, the vast majority of cellular 
studies have been made on cells fixed in strongly acid fluids which, while 
precipitating admirably proteins and nucleic acids, at the same time must 
wash out smaller unattached cellular constituents, organic or inorganic, 
to such an extent that the residue can hardly be great enough to be cyto- 
logically detectable (see Pollister, 1952a). This predominance of nucleo- 
protein in cellular composition has always been so obvious that it is possi- 
ble to overlook its significance to cytochemistry. For example, in 
Lison's Histochemie animale (1936) this point is not stressed, and there is a 
distinct impression that the primary concern of histochemists should l)e 
the localization of simple chemicals such as iron and amino acids. 

Whatever the special objectives of a study of intracellular localization, 
a very obvious fact about cellular composition is that, in all cells, proteins 
and nucleic acids occur in high enough concentration so that tests for 
them fall within the visible, or measurable, range, and consequently tech- 
nifiues for nucleoprotein demonstration are applicable to a great variety 
of problems. 

3-2. NUCLEIC ACID STAINING AND TESTS 

Although the major features of morphology are distinct in living cells 
and in fixed uncolored cells, the bulk of cytological researches have been 



A CRITIQUE OF CYTOCHEMICAL METHODS 213 

made with the advantage of artificial contrast which is introduced by 
.staining or developing a metallic precipitate in c(>ll c()mpf)ncnts.'' One 
of the oldest of these techniques is l)asic staining, which involves the use 
of dye salts which, upon dissociation, carry the ctjlor, the chromophore, 
in the cation and which therefore form colored salts with the anions of 
strongly acid substances within the cell (Fig. 6-2C). It was early recog- 
nized (e.g., Mathews, 1898) that, if basic dyes were applied in acid solu- 
tion (after appropriate fixation), this property of cell substances, which 
is included in the general term "basophilia" of tissues, constituted in 
effect a test for strongly acidic substances. Specifically, in animal cells 
these include the relatively uncommon sulfuric acid esters of polysac- 
charides and the phosphoproteins plus the universal cell constituents 
nucleic acids, w^hich are orthophosphoric esters of nucleosides. There 
were many early cytochemical researches based on the supposition that 
basophilia indicated the intracellular distribution of nucleic acid. One 
outstanding example is the so-called "chromidial hypothesis," which as 
applied to metazoan cells held that the basophilia of cytoplasmic struc- 
tures was evidence of their origin from the nuclear chromatin (see Wilson, 
1925, pp. 700^.). However, most cytologists used basic staining so as to 
achieve maximum contrast for morphological studies (e.g., iron hema- 
toxylin, applied to material which had been fixed in reagents containing 
chromic acid). Such technique departed widely from the strict criteria 
laid down by Alathews for specific staining by salt formation between 
basic dyes and nucleic acids, and undoubtedly the increased contrast was 
to a large extent due to adsorption of dye rather than chemical staining 
(Pollister, 1952a). This distinction was rarely appreciated, however, 
either by cytologists themselves or by others interested in the chemistry 
of the cell, and as a result there developed a widespread distrust of 
attaching any chemical significance whatsoever to the basophilic reac- 
tion. This was not dissipated even when van Herwerden (1913, 1914) 
developed the nuclease technique for identification of intracellular 
nucleic acid basophilia. The modern use of basophilia for localization 
of nucleic acids (Mazia and Jaeger, 1939; Brachet, 1942; PoUister, 1950; 
Kaufmann et al., 1951) stems directly from van Herwerden's work 
but rests on a much firmer biochemical basis since it is now known 
that: 

1. There are actually two nucleic acids, the pentose type — ribose 
(RNA), plasmonucleic acid — found in the cytoplasm, nucleolus, and to 
some extent in chromatin, and the deoxypentose type — deoxyribose 
(DNA), chromonucleic acid — which is normally restricted to chromatin 
of the nucleus (Davidson, 1950). 

■^ The development of phase contrast microscopy fsee Fip;. 6-2.4 and Bennett et al., 
1951) has nearly freed cytologists from the necessity of introducing artificial contrast 
by these methods. 



211 K \1)1 AI'lON HI()I,<)(;V 

■J. Thcic arc specific enzymes, rihoiiuclease and deoxyrihonuclease, 
which act to detiiadc each type of nncieic acid. 

3. The total nucleic- acid content may he renio\c(l specifically hy chemi- 
cal ajj;ents such as trichloroacetic acid. 

Thus, sites of nucleic acid are readily identifiable a.s parts of the 
cell with basophilia which is removable by acid extraction, and the type 
of nuclease susceptibility shows which of the two nucleic acids is present 
(cf. Figs. 6-2C, F and 5a, /;). 

Such specific nucleic acid basophilia adds to cell morphology an impor- 
tant chemical datum, showiiif^ that, in a cell such as that of Fig. G-1, for 
example, there is undoubtedly considerable nucleic acid in the basal zone 
and nucleolus as well as in the chromatin. More precisely, from such a 
cytological preparation as Fig. (3-20 it can be concluded that, in these 
parts of the cell, the nucleic acid concentration is so high that the dye 
bound as dye nucleate is in high enough concentration to appear as strong 
visible color in structures no more than o ij. thick (p. 209). To what extent 
does this approach a complete picture of the distribution of major polynu- 
cleotide concentrations within the cell? It must be emphasized that basic 
staining does not lead to localization of nucleic acid by any of its natural 
physical properties in the same manner as the natural green color indicates 
the sites of chlorophyll. Instead, visualization by basophilia depends 
on the capacity of nucleic acid to bind the cations of basic dyes, which 
may be mainly through displacement of protein from its natural combina- 
tion with the residual phosphoric acid valencies of the polynucleotide. 
This staining reaction may therefore be very complex, and the relation 
between color and amount of substrate may by no means necessarily be 
a simple one. From basophilia alone it is impossible to answer such 
questions as: How strict is the proportionality between basophilia and 
nucleic acid concentration? Is this proportionality constant or highly 
variable? What interpretations may be assigned to negative basophilia? 
Can there be considerable accumulations of polynucleotide which are 
unaccompanied by any basophilia? Is an increase or decrease of baso- 
philia due to change in amount of nucleic acid or to change in the number 
of phosphoric acid valences which are available for dye binding? The 
transition from a cytological to a cytochemical viewpoint poses all such 
questions and immediately reveals the danger inherent in uncontrolled 
cytochemical use of staining reactions. Only by an independent method 
which measures nucleic acid directly can these questions be answered, and 
in natural ultraviolet absorption of nucleic acid such a method is available 
(see Sect. 3-4). In general, regions of strong nucleic acid ultraviolet 
absorption have been found to coincide with those of pronounced baso- 
philia (see Figs. 6-2C, D), but there are indications that the amount of 
dye bound in cells for a given amount of pentose polynucleotide is variable 
(M. H. Flax, unpublished data), and one extreme case has been reported 



A CRITIQUE OF CYTOCHEMICAL METHODS 215 

ill which there was nearly negative cytoplasmic basophilia in cells which, 
by ultraviolet absorption, were shown to contain considerable nucleic acid 
(Pollister . . . Breakstone, 1951). In view of these difficulties it is per- 
haps best to regard basophilia as a useful indicator of sites of major nucleic 
acid concentration, which then becomes a guide to application of more 
satisfactory (lualitative and c^uantitative methods. In fact, this has been 
the role of l)asophilia in development of modern concepts of the intracel- 
lular distribution of nucleic acid. Although the methods of identification 
by the ultraviolet absorption spectrum measure nucleic acid directly and 
are therefore potentially applicable to situations where polynucleotide 
basophilia might be misleading, for the most part ultraviolet studies have 
proceeded along lines which were clearly foreshadowed by old findings of 
the basophilic reactions of cells. This is strikingly emphasized also by 
the fact that practically all the fundamental conclusions about intracellu- 
lar distribution of nucleic acids which the Caspersson school reached by 
use of ultraviolet absorption techniques and used in elaborating compre- 
hensive theories of cell function (see Caspersson, 1950) were arrived at 
independently by Brachet and his collaborators (Braehet, 1944) with 
only nuclease-digestible basophilia as a guide. Indeed, since Brachet and 
coworkers used nucleases in combination with basophilia, they were able 
to detect ribonucleic acid in chromatin, while it was necessarily overlooked 
in the less specific ultraviolet absorption studies. 

There are certain applications of basophilia to (lualitative cytochem- 
istry which are of special interest because not only do they demonstrate 
the presence of strongly acidic substances, but also by specific color 
changes they appear to indicate something of the intramolecular structure 
of the acidic substrate with which they combine. The best known of 
these is the so-called "metachromatic" basophilia (metachromasia) by 
which certain cellular structures stain red with dyes which appear blue in 
solution (e.g., toluidine blue and azure). This method was empirically 
recognized long ago (Ehrlich, 1877; Hoyer, 1890). It has been the sub- 
ject of a number of chemical and spectrophotometric studies (e.g., Kelley 
and Miller, 1935a, b; Lison, 1935; Bank and Bungenberg de Jong, 1939; 
Wislocki et al., 1947; Michaelis and Granick, 1945). Spectrophotometric 
analysis shows that, whenever these dyes are in water solution, three 
states are in ecjuilibrium. An a absorption peak in the red part of the 
spectrum represents unaggregated dye in the "monomeric" state; a 
absorption peak (green) is believed to represent dye in the two-molecule 
aggregate, or "dimer," state. A /x absorption peak (in the blue-green) 
supposedly represents highei- states of aggregation than the dimer which 
may for conv^enience be called a "polymer" state. The aggregation, as 
would be expected, is dependent on concentration, with the result that 
these basic dyes notably fail to follow Beer's law. In stained cells, basic 
dyes are, of course, removed from solution and combined with the solid 



210 RADIATION BIOLOGY 

coagulu or i)iecipitates of the substrates. This suggests that the sub- 
strates \\ liich are colored red have bound the dye in such steric relation 
that the dimor and polymer association occur, while those colored blue 
have the dyr molecules more widely separated so that substantially all the 
color is due to the blue monomer. According to this interpretation, the 
color of the dye is in effect a reflection of the intramolecular structure of 
the substrate. The polysaccharide sulfuric esters (e.g., chondroitin sul- 
fate of connective tissue) are decidedly metachromatic. A careful spec- 
trophotometric analysis has shown that a metachromasia distinguishes 
RXA from DNA, a difference which presumably is related to the highly 
branched structure of the former (Flax and Himes, 1950, 1952). 

Methyl green basophilia is another staining reaction which appears 
to reflect the intramolecular configuration of the substrate. This dye 
stains normal DNA; with i-are exceptions it does not stain UNA. In 
vitro the formation of the salt methyl green-deoxyribonucleate is depend- 
ent on the nucleic acid being in a state which forms highly \'iscous solu- 
tions (Kurnick, 1947, 1949; Kurnick and Mirsky, 1949), and therefore 
reduction or loss of methyl green basophilia of nuclei has been interpreted 
as evidence of a physical change in the DNA molecule which is similar to 
that which is accompanied by loss of viscosity of solutions of the acid, 
a change which is usually called " depolymerization " (Pollister and 
Leuchtenberger, 1949; Leuchtenberger, 1950; Leuchtenberger e/ a/., 1949; 
Harrington and Koza, 1951). Such changes in methyl green basophilia of 
nuclei have been noted to result from experimental treatment (heat, 
deoxyribonuclease digestion, ionizing radiation) and also to accompany 
pathological nuclear degeneration. 

3-2a. Nucleal Reaction. Goldschmidt (1904) and the other adherents 
of the views embodied in what was called the "chromidial hypothesis" 
believed that they had in basophilia a sort of qualitative test for chro- 
matin by which they could detect this substance even after its extrusion 
from the nuclei into the cytoplasm. This was an over-optimistic point of 
view and led to widespread distrust of cytochemical conclusions from 
staining I'esults. Feulgen and Uossenbeck (1924) developed a specific 
cytochemical test foi' chromatin (Fig. i\-'2F) which not onl,y finally dis- 
posed of the chromidial hypothesis, sensu strictu, but also led to great 
strides in clarification of the whole problem of intracellular distribution of 
luicleic acids. For nearly thirty years two different nucleic acids had 
been recognized by chemists. One, obtainable in (juantity from yeast 
and often called yeast nucleic acid, had been shown to contain a pentose; 
the other, identical with the acidic component of Miescher's iniclein but 
usually later obtained from the thymus gland, contained a sugar that was 
clearly not a pentose and was generally considered to be sort of hexose 
(see Le\'ene and Bass, 1931; Dax'idson, 1950). Feulgen and Rossenbcck 
discovered that mild acid hydrolysis, which was known to split off the 



A CUlTUjUE OF CYTOCHEMICAL METHODS 217 

purine bases from nucleic acid, changed the thymus nucleic acid so that it 
gave a positive Schiff reaction for aldehydes — a restoration of color to a 
reduced leukofuchsin, and the}^ demonstrated that this reaction could he 
carried out not onl}^ in vitro but also on tissue sections in which it colored 
brilliantly the chromatin of the cell nuclei. For this reason they called it 
the " nucleal reaction. " C'ytologists were quick to try this new technique, 
and in a few years the reaction had been demonstrated to be positive on 
the tissues of a wide variety of animals and plants (Milovidov, 1936). 
This is an ideal (lualitative chemical reaction, highly specific (Fig. 6-2F) 
for the unique substance of chromatin which Levene et al. (1930) even- 
tually showed to be deoxypentonucleic acid (DNA), not a hexose polynu- 
cleotide. The intense color (Table 6-2) is one of its important character- 
istics. The Feulgen reaction at once demonstrated that the major 
basophilic component of chromatin and chromosomes of both plants and 
animals is always DNA and that the cytoplasm never contains this in 
detectable amount. This was eventually fully confirmed by the analysis 
of isolated nuclei and cytoplasm (Feulgen et al., 1937; Hogeboom et al., 
1948). It thus became quite clear that, whatever the nature of the 
basophilic substance in the cytoplasm, it was certainly not the same as the 
nucleinic acid of Miescher and Altmann. When, therefore, as a result of 
the earlier discoveries of pentoses and purine bases in the cytoplasm of 
developing eggs, Brachet (1942) was led to reintroduce on a wide scale 
van Herwerden's (1914) long-forgotten nuclease technique and conclude 
therefrom that the Feulgen-negative basophilic substance of the cyto- 
plasm was a pentose nucleic acid, it was not seriously questioned; like- 
wise, no objections were raised when Caspersson and Schultz (1939) 
stressed the fact that the ultraviolet absorption spectrum of these same 
basophilic areas must be due to pentose nucleic acid because these parts 
of the cell were Feulgen negative. Like basophilia, the Feulgen reaction 
is not directly dependent on a physical property of nucleic acid but on a 
chemical reaction which the acid can give after removal of the purines 
from a part of the nucleotides. (From the results of Stacey et al, 1946, 
it appears that the deoxypentose undergoes considerable intramolecular 
change to become a substance which is capable of recolorizing the Schiff 
reagent.) It must be understood that the very useful specificity, which 
rarely has been seriously cjuestioned, resides in the release of reactive 
groups as a result of the hydrolytic process, and it is customary to stain 
simultaneously a control slide which has not been hydrolyzed. 

3-3. PROTEIN STAINING AND TESTS 

Although the bulk of the solid matter of the cell is protein, the prospect 
for fruitful cytochemical protein analysis is by no means as bright as for 
nucleic acids. It is certain that this protein mass must be a very hetero- 
geneous mixture, including countless intracellular enzymes and other pro- 



218 RADIATION HIOLOGY 

tein.s of special fuiiction. Most of these may never be hifi,hly coiiceiitrated 
ill aii>' one part of (he cell. Even if such an accinnuhitioii did occui, the 
correlation of specific function with icadily accessihh' asjiects of the 
chemistry of the protein molecuk! is rarely so definite! as to offer hope of 
localizing many specific proteins by techni(|ues similar to those which have 
just l)een tlescribed for nucleic acids. Natural color, as in hemoglobin, of 
course, ofTers one opportunity for a microscopic approach (Thorell, 1947). 
For the most part, however, methods of microscopic analysis of proteins 
cannot be expected to give more than information concerning the approxi- 
mate total amount of the protein mass, the fractionation of which on a 
microscopic slide is possible to but a very limited extent. For example, a 
considerable proportion of the histone of chromatin is split off readily 
(PoUister and Ris, 1947). Nevertheless, the approximate analysis of 
total protein is information of considerable importance to the broad fiucs- 
tion of protein synthesis as the prime chemical achievement in growth, 
cell division, and secretory activity (Caspersson, 1950; Pollister, 1954). 

Most methods for protein are not nearly so sensitive as are basophilia 
and the Feulgen reaction for nucleic acids since the special reactions are 
almost entirely those of groups at the omega ends of the amino acid resi- 
dues, and in most proteins (protamine being one exception) no specific 
reacting group makes up more than a small fraction of the total rmmber of 
amino acid residvies. The reactive groups which have been used cyto- 
logically are (a) the dibasic amino acids arginine (Serra, 1944; Thomas, 
1946) and histidine (acidophilia, p. 219); (6) the dicarboxylic amino acid 
glutamic acid (by alkaline basophilia, Dempsey and Singer, 1946) ; (c) 
the sulfur-containing amino acids cystine and cysteine (see Lison, 1936; 
Bennett, 1948) ; and (d) the aromatic amino acids tyrosine, tryptophane, 
and phenylalanine (see p. 222). 

Specific reactions for proteins in cytological material are all adapta- 
tions of well-known spot tests. One of the oldest of these is the Millon 
reaction for tyrosine and tryptophane (Fig. 6-2B), which was usedbyLeit- 
geb to identify the nature of crystals in plant cells as early as 1888. No 
Millon test is impressive under the microscope, partly because the protein 
cannot possibly be concentrated enough to give a strong visible reaction 
on a slide and partly because, since everything is colored, the observer 
does not have the benefit of the contrast to which he is accustomed in a 
stained preparation. The test material, if present, is, of course, readily 
detectable microscopically by objective photometric measurements. 
The sensitivity of the Millon reaction in visible light is low (Table 6-2). 
At the visible absorption peak (490 m/x), for a protein assumed to give a 
Millon reaction ecjuivalent to 6.25 per cent tyrosine, the £"490 is 0.007, and 
the protein would have to reach a concentration of over 40 per cent to give 
a detectable extinction, 0.030, in a thickness of 1 m- (At the natural 
ultraviolet absorption peak, 275 m^i, the absorption is no more intense.) 



A CRITIQUE OF CYTOCHEMICAL METHODS 219 

The sensitivity of the Milloii reaction is nearly doubled if it is measured at 
365 mn, near the peak in the near ultraviolet (Table 6-2 and Fig. 6-13). 

By contrast with the specific spot tests, the widely used method of con- 
trast by acid dyes (in which the color is carried in the anion) gives readily 
detectable color in parts of the cell where protein is concentrated (Table 
6-2). For this reason, a dye of color roughly complementary to the basic 
dye for polynucleotide is often used in preparing microscopic slides for 
histological or pathological examination (e.g., basic methyl green and 
acid fuchsin or basic hematoxylin and acid eosin). It has long been 
known that this acid dye staining probably has a sound chemical basis 
(Mathews, 1898), that it is essentially the binding of the dye anions by 
the cationic groups (NH^) of the diamino acids of the protein to form a 
salt (for example, what we may call, for convenience, protein fuchsinate 
with acid fuchsin). There is evidence for the chemical basis of this stain- 
ing reaction in demonstrations that in vitro protein binds acid dyes 
stoichiometrically (Chapman et al., 1927; Fraenkel-Conrat and Cooper, 
1944). The specificity of acidophilia for the amino groups of protein has 
been demonstrated by Monne and Slautterback (1951), who showed that 
deamination removes the acidophilia. As with basophilia the staining 
must be carried out in acid solution (pH 1.5-2.0) so as to preclude binding 
of the dye in other than salt formation (Mathews, 1898; Leuchtenberger 
and Schrader, 1950). The basic amino acids constitute so large a propor- 
tion of the composition of nearly every protein that acidophilia should be 
a more sensitive test for proteins than the Feulgen reaction is for DNA 
(e.g., fast green in Table 6-2). Since protein acidophilia is a measure of 
diamino acids, it can be employed, in conjunction with either the Millon 
reaction or ultraviolet absorption, to localize proteins of basic character 
(Leuchtenberger and Schrader, 1950). 

All the protein methods just discussed determine the presence of pro- 
tein only indirectly, through using one of its side groups to develop color 
in a reagent, or to bind a dye. Hence, any semiquantitative conclusions 
drawn therefrom are subject to all the possible errors which have been 
discussed earlier (p. 214). Protein can be measured directly by the 
natural ultraviolet absorption spectrum of its aromatic amino acids 
(Chap. 5, this volume, and Fig. 6-3). The microscopic techniciue for this 
has been developed by Caspersson (1940a, 1950), and, as usually carried 
out, the data obtained are absorption curves of masses of nucleoprotein. 
The qualitative and semiquantitative ultraviolet methods for these two 
major cell components, nucleic acid and protein, are therefore discussed 
together in the next section. 

3-4. ULTRAVIOLET ABSORPTION OF NUCLEIC ACIDS AND PROTEINS 

While chromatin is quite colorless, in the ultraviolet spectrum — at a 
wave length a little shorter than the region transmitted b}'- glass — it can 



220 



RADIATION BIOLOGY 



be seen that it absorbs heavily, as first shown by Kohler (1904). Thus, if 
the cell is studied \isually on the fluorescent screen or phot(jf^raphed by 
ultraviolet lifiht, the nucleus is dark and is often fully as sharply con- 
trasted with the lighter background as it would be if the chromatin had 
been stained with a basic dye (Fig. (i-ID). Of the many observers who 
wvvv im})iessed with the strikinj^ contrast shown by chromatin under 
ultra\iolet examination, none seems to have realized that this was due to 
a physical property of luicleic acid until Caspersson published his thesis 
on the chemical composition of structures of the cell nucleus (1930). In 




2200 



2400 



2600 2800 
WAVE LENGTH, A 



3000 



220 



300 



Fig. 6-3. Ultraviolet absorption curve of a 
0.02 per cent deoxyribonucleic acid solu- 
tion (I) compared with that of a 0.2 per 
cent solution of serum albumen (II). 
Curves were measured with a Beckman 
spectrophotometer in a cuvette 1 mm thick. 
(After Thorell, 1947.) 



240 260 280 

WAVE LENGTH, m/^ 

Fig. 6-4. Ultraviolet absorption curves 
of the salivary gland of Dro.sophila. 
Curve I is through a chromosome band ; 
II is through an adjacent part of the 
cell outside the chromosome; III is 
computed for a 10 per cent solution of 
nucleic acid. (After Caspersson, 1936.) 



this landmark in cytochemistry, Caspersson pointed out that nucleic 
acid has so strong a natural specific ultraviolet absorption that it can 
account for the great contrast of ultraviolet pictures (Chap. 5, this vol- 
ume; Table 6-3 and Fig. 6-3); he showed that the absorption curve 
through a single chromosome (Fig. 6-4) closelj^ resembles that of nucleic 
acid, not protein; and he confirmed the opinion that protein could account 
for but little of the absorption, by digesting the protein with little effect 
upon the ultraviolet contrast. Xumerous subseriuent publications of 
Caspersson and his coworkers have made it amply clear that regions of 
strong ultraviolet contrast in cells are, as a rule, sites of high nucleic acid 
con('entration (as seen in Figs. 6-2C, D). However, it is a mistake to sup- 
pose that such ultraviolet contrast is necessarily an accurate reflection of 
the intracellular nucleic acid distribution, for in each region the density 



A CRITIQUE OF CYTOCHEMICAL METHODS 



221 



.shows only the total light loss. In ultraviolet light of wave length near 
that of nucleic acid absorption a large part of the light lo.ss, particularly 
in fixed preparations, may he of nonspecific character mainly as a result 

Table (i-3. Beer's Law for Nucleic Acid 
(After Thorell, 1947.) 



Deoxyribonucleic acid 


Ribonucleic acid 


Concen- 
tration, 
% 


Layer 

thickness, 

mm 


E 


k 


Concen- 
tration, 

% 


Layer 

thickness, 

mm 


E 


k 


2 
0.01 


0.01 
3.17 


. 37.3 
0.600 


1.86 
1.89 


2.5 
0.005 


0.01 

3.17 


0.550 
0.340 


2.20 
2.15 



The extinction {E) and absorption constant (A-) of RNA and DNA at concentra- 
tions of 0.005-2.5 per cent. Preparation according to Hammarsten (1924). 

Table 6-4. Ultraviolet Absorption of Maize Nucleoli 
(After Polli.ster and Leuchtenberger, 1949b.) 



Experiment 
No. 


Number 
measured 


Nucleotides 
removed by 


Em 


Percentage 

reduction of 

extinction 


1428E-3 


30 
30 

23 
10 

31 
31 
31 

17 




0.690 ± 0.011 
0.347 + 0.011 

0.847 ± 0.010 
0.407 ± 0.009 

1.000 ± 0.014 
0.860 + 0,011 
0.472 ± 0.008 

0.843 ± 0.016 
0.395 + 0.010 




1428E-3 

1428E-7 


Hot TCA 


50.0 


1428E-7 
1428E-5 


Hot TCA 


53.4 


1428E-5 
1428E-5 

1428E-6 


Cold TCA 
Hot TCA 


52.8 


1428E-6 


Ribonuclease 


52.8 



The reproducibility of the effect of the enzymatic and chemical (trichloroacetic 
acid, TCA) removal of nucleic acid upon the extinction {E-za) of whole nucleoli of 
pollen mother cells of Zea mays. (In experiment 1428E-3 the extinction is lower 
because a very large central cylinder was measured; in 1428E-5 the higher extinction 
results from using a smaller cylinder.) In each experiment the percentage reduction 
of extinction indicates the proportion of the light loss (specific and nonspecific) due 
to ribose polynucleotide. The residual light loss (46-50 per cent) is largely non- 
specific, owing to high protein concentration. 

of scattering by the dense protein mass (see Table 6-4 and Fig. 6-56). 
Specific nucleic acid absorption may be recognized from the shape of the 
absorption curve (Caspersson, 1950, and Fig. 6-3). Also, the specific 
polynucleotide absorption can readily be dissociated from the absorption 



OO') 



UADIA rioX UK )!.()(; Y 



aiul scattering of protein by making two photographs or absorption 
measurements of the same cell, one before and the other after enzymatic 
or chemical rem()\al of the nucleic acid (see p. 231, Table 0-4, and Fig. 
6-5a, 6). Since the difference between the two is (hrectly dependent upon 
the natural absorption of the purine and pyrimidine components of the 
nucleic acid, the latter technicjue provides an easy and sure method of 
obtaining evidence of intracellular nucleic acid distribution. The sensi- 
tivity is comparable with that of the Feulgen reaction (Tables 6-1, 2). 
In the specificity of the nucleases lies the possibility of overcoming the 




(a) (6) 

Fig. 6-5. Test, left, and blank for nucleic acid determination by ultraviolet absorption. 
Photographs, at 254 mn, of a maize pollen mother cell (No. 14281'>3-54) taken before 
(a) and after (b) the section had been subjected to hot 5 per cent trichloroacetic acid 
to extract all polj'nucleotide. The change in density of the spherical nucleolus is 
marked. By direct measurement of this nucleolus (Pollister and Leuchtenberger, 
1949b) it was found that the extinction of a central cylinder through a was 0.750, that 
through b was 0.405, the difference being 0.345. These three values are assumed to 
represent, respectively: (1) total specific and nonspecific light loss in the part of the 
nucleolus measured; (2) light loss due to protein, mainly nonspecific; and (3) the light 
lo.ss due to polynucleotide, mainly specific absorption. 

major disadvantage that ultraviolet absorption alone does not discrimi- 
nate between RNA and DXA (Davidson, 1947; Pollister, 1950). 

None of the ultraviolet absorption curves of cell structures is exactly 
like the curve of pure nucleic acid; there is always distortion, certainly 
due in part to the associated protein, which characteristically exhibits 
specific absorption in the region of 2750 A owing to its content of aromatic 
amino acids (Chap. 5, this volume). Since these constitute but a small 
percentage of the total amino acid content, the specific absorption of 
proteins is very low in comparison with that of nucleic acid (Table 6-1 
and Fig. 6-3), and, within the region of nucleoprotein absorption which 
has been most studied (2500-2800 A), protein must be present in 20-50 



A CRITIQUE OF CYTOCHEMICAL METHODS 



223 



times the coiicentratioii of the nucleic acid to cause an equivalent ultra- 
violet light loss. The distortion of the nucleic acid curve means therefore 
that in the cell the nucleic acid is always accompanied by at least several 
times as much protein. 

The intracellular nucleoprotein curves published by the Caspersson 
group are of two distinctly different types 
(I and II, Fig. 6-6). In type I the nucleic 
acid peak is broadened, and the whole 
right shoulder is shifted toward the longer 
wave lengths. This shape is not unex- 
pected for nucleoprotein; it seems to be 
simply the summation of a nucleic acid 
curve and that of a common protein type, 
hke serum albumen (Fig. 6-3). On this 
basis, cell regions showing this type ab- 
sorption have been interpreted as sites 
where nucleic acid and a typical acid pro- 
tein ("globulin type") occur together. 
The type II curves are very different and 
puzzling; there is less broadening of the 
nucleic acid peak, and within the long- 
wave-length slope a second peak is indi- 
cated by a distinct shoulder. For a variety 
of reasons (see Caspersson and Thorell, 
1941), curve II has been held to localize 
nucleic acid accompanied by markedly 
basic protein, called "histone type" or 
"diamino-acid-rich" protein (Caspersson, 
1940a, 1950). The strongest evidence for 
this interpretation of the type II curves 
was that certain nucleohistone prepara- 
tions showed a protein peak apparently 
shifted toward 2900 A. However, when 
histones cjuite free of nucleic acid were 
finally obtained, it was found that they did 
not show such a shift of absorption, the 
peak being near 2750 A as in typical pro- 
teins (Mirsky and Pollister, 1943, 1946). 
There remains therefore no certain expla- 
nation of the peculiar shape of the type II curves. 

The nucleoprotein curves have been the basis for detailed speculations 
concerning the roles of basic and acid proteins in cellular physiology 
(Caspersson, 1950). For the general cytologist, perhaps the greatest 
significance of these curves is that they emphasize unmistakably what 




2400 2600 2800 3000 
WAVE LENGTH, A 

Fig. 6-6. Absorption curves of 
the cytoplasm of cells of dif- 
ferentiated (I) and undifferen- 
tiated (II) renal tubules of the 
chick embryo. Curves of the 
I type are considered to repre- 
sent ribonucleic acid with pro- 
tein of the "globulin type"; the 
type II curves are believed to 
indicate the presence of ribonu- 
cleic acid accompanied by con- 
siderable basic protein, "histone 
type." {After Caspersson and 
Thorell, 1941.) 



'22 \ RADIATION HIOLOGY 

inifj;lit otherwise be overlooked, namely, thiit, however striking the baso- 
philia and ultraviolet absorption of nuclcotide-rich parts of the cell may 
be. this by no means signifies that they are strongly acidic pools of nearly 
pun^ nucleic acid. Instead, the al)sorption curves clearly show that pro- 
tein is usually present in much greater (|uantity than the nucleic acid. 
This situation must always be recalled in any attempt to interpret chem- 
ically the results of staining reactions, and it must be the starting point 
for all speculations concerning the role of nucleoproteins in intracellular 
physiology (see Pollister, 1952b). 

4. QUANTITATIVE MICROSCOPICAL METHODS 

l-l. VISUAL COMPARISOX 

The (jualitativc c,ytological methods which have just been discussed 
lead to localization of a substance within a cell by its absorption, "con- 
trast" in the language of a microscopist, which is detectable visually. 
Cytologists often speak of the intensity of a stain or color reaction as 
weak, strong, very dark, etc. Of course these terms imply semicjuantita- 
tive evaluation of the concentration of the component which is responsible 
for the color. When two similar objects are side by side in an evenly 
illuminated microscopic field, or in the two half fields of a comparison eye- 
piece, visual matching appears to be as accurate as objective photometric 
measurements. For example, with the comparison eyepiece, Bauer 
(1932) arranged a series of slides in order of intensity and was thus able to 
work out the relation of intensity of the Feulgen reaction to time of 
hydrolysis, which was essentially like that later worked out by Di Stefano 
(1949) from photoelectric measurements. For objects of the same size 
proper visual comparison is, then, a rough indication of relative amounts 
— if two objects are equally dark the}^ may be assumed to have approx- 
imately the same amount, and if they are different, the darker one may 
be said to contain more reacting substance. The same conclusions 
regarding relative concentrations may be drawn of two bodies of eqwdl 
vertical thickness (equal absorbing path). More often, the (juantitative 
question which faces a eytologist cannot be answered, even roughly, by 
visual comparison. For example, one often wishes to know relative 
amounts in two objects of very different size. It is uncertain to what 
extent by visual study a microscopist can determine whether two such 
une(iual objects have the same intensity (a rare condition probably); for 
the relati\'e sizes of the contrasting surroundings introduce considerable 
difficulty. If this match could be accurately made, a fairly good estimate 
of how much more the larger object contained could be computed from 
the dimensions of the objects. 

The examples cited illustrate the range of visual microscopic com- 
parison. If, to mention a very common experience, one cytological 



A CRITigi'E OF CYTOCIIEMICAL METHODS 225 

object is both larger and more lightly colored than another, the cytolosist 
is almost completely helpless to answer the obvious (juestion of whether 
the decrease of color is entirely due to dilution in the larger mass. The 
relation of volume to light absorption is easily computed from an actual 
figure, a measurement of extinction, but such a (juantitative datum is 
absolutely necessary. No amount of experience can train a cyto- 
logist's eye to operate as a microscopic photometric device. These 
measurements must be made with objective photometers, with which a 
transmission is measured from which concentration may be estimated and 
amount computed (where the form and homogeneity of the cytological 
object are favorable). 

4-2. PHOTOMETRIC TECHNIQUE 

The simplest photometer is a photographic plate, which can be used to 
determine relative intensities, from comparison with a density-intensity 
calibration curve. This curve may be independently measured and used 
for a whole series of plates (Caspersson, 1936; Pierce and Nachtrieb, 1941) 
or may be measured from a series of intensities through a rotating sector 
(Cole and Brackett, 1940) or a calibrated wedge (Uber, 1939) which is 
photographed on the same plate with the cells. The photographic 
method seems at first glance easy and obvious, a simple modification of 
the technique of photomicrography. For accurate results, however, it is 
far more complicated. Plate exposure and development must be rigidly 
standardized, and the negative density must be measured with a fairly 
elaborate photoelectric apparatus. The latter must, in fact, be nearly 
as sensitive as a photometer for direct measurement of microscopic slides 
— hence, in most cytological w^ork, the latter is an easier technique. 
Photographic photometry is indispensable for some problems, for 
example, where ultraviolet absorption measurements are to be made upon 
living cells (Thorell, 1947; Malmgren and Heden, 1947; Mellors et al, 
1950). 

For direct absorption measurements of fixed preparations at a single 
wave length, the relatively simple device indicated in Fig. 6-7 is adequate 
(Pollister and Moses, 1949). Photomultiplier tubes are sensitive enough 
to allow measurement of areas less than 1 m', tor all methods for nucleic 
acid and protein. Other devices employing photomultiplier tubes have 
been described by Lison and Pasteels (1951) and by Pollister (1952c). 
The data obtained are pairs of measurements, a first (h) through part of 
the cell, a second (h) through an empty part of the slide, outside the sec- 
tion. From these, transmission (7') can be computed (as Ix/h) and 
extinction either computed (as logio /o /x) or obtained from a conversion 
table (Erode, 1943). 

If absorption curves are to be measured, the apparatus described must 
be supplemented by means of dispersing the spectrum either before or 



22G 



RADIATION HIOLOGY 



after the microscopic preparation. There are many possible types of 
apparatus for this sort of procedure (see Caspersson, 1950; Loofl)()urow, 
1950; Mellors et al., 1950; Blout et al., 1950). Since in the visible spec- 
trum there are objectives and condensers, corrected both for chromatic 
and spherical aberrations, the measurement of visible absorption spectra 
is relatively simple, involving merely movement of the wave-length drum 
of the monochromator. For ultraviolet absorption measurements, 
Caspersson (193G) originally used the Zeiss-Kohler apparatus and meas- 
ured absorption at each wave length by photography. This whole 
instrument was designed for photography at one wave length, quite 



MAGNIFIER 

PHOTOTUBE 



POWER 
SUPPLY 




Fig. 6-7. Diagram of a simplified apparatus for microphotometric study of cytological 
preparations. {After Swift, 1950.) 

uncorrected for chromatic aberration, and its use for absorption curve 
measurement by photography is extremely tedious. For each wave 
length both condenser and objective must be refocused, and it is necessary 
to carry out two measurements or make two photographs at each wave 
length. This uncorrected optical system has been used for all the exten- 
sive work of the Caspersson school, with many additions (Fig. 6-8), such 
as an achromatic grating monochromator, extremely sensitive photore- 
ceivers (measuring currents of the order of lO"'^ amp) and a polarizing 
prism and special mechanical stages (Caspersson, 1950). The develop- 
ment of achromatic reflecting objectives and condensers (Fig. 6-9) 
(Brumberg, 1943; Burch, 1947; Grey, 1950; Norris et al, 1951; Barer, 
1951) makes the problem of optical apparatus for ultraviolet absorption 
measurements essentially as straightforward as in the visible spectrum. 
With the instrument which has been developed by Sinsheimer a com- 



A CRITIQUE OF CYTOCHEMICAL METHODS 



227 



plete absorption curve can be run mechanically, without refocusing; 
a density-wave length curve is recorded on a drum; and by a beam- 
splitting mechanism and chopping the beams at two frec^uencies it is 
possible to compensate for the transmission of the empty part of the slide 
so that no second curve is necessary (Loofbourow, 1950). Many details 
of techni(iue will be found in such references as Caspersson (1936, 1950); 




Fig. 6-8. Diagram of main instrument used in measuring ultraviolet absorption with 
high accuracy and stability. A, mercury lamp; B, tungsten band lamp; C, mono- 
chromator; D, exit slit of monochromator; E, lens; F, movable 90° quartz prism; G, 
quartz plate, used with photocell to compensate for changes in the lamp; H, condenser; 
/, object on slide; K, objective; L, ocular with adjustable diaphragm; il/, accurately 
movable prism of fused quartz; N, rotating sector; 0, telescope for centering; P, 
Kohler's rotating spark gap arrangement; R, photocell; *S, electrometer; T, leakage 
resistance; U, four-step potentiometer; X, camera; F, Kohler focuser for the ultra- 
violet, interchangeable with prism M. (After Caspersson, 1950.) 

Gersh and Baker (1943); Thorell (1947); Pollister and Ris (1947); Pol- 
lister and Moses (1949); Swift (1950); and Pollister (1952c). 

An extreme simplification of the problem of instrumentation for 
microspectrophotometry is to regard the whole apparatus as merely a 
somewhat more complicated optical pathway than that in the conven- 
tional devices which use absorption cuvettes, and to consider the micro- 
scope as no more than an aid to locating an extremely small analytical 
sample symmetrically in the optical pathwa}^ and delimiting the area to be 
measured. If computations of concentrations and amounts are to be 



228 



RADIATION UIOLOGY 



made, it is obviously necessary to take into account the problems raised 
by such factors as the angle of the illuminating cone from the condenser, 
the probable variations of path Icnj^th, and scatter into or outside the 
area of measurements as this cone is changed. These questions are con- 



IMAGE OF SOURCE 
4-nim DIA. HERE 
31-10-02 OBJECTIVE \ CONDENSER 

SHOULDER 



31-15-02 EYEPIECE 

J. 




DIAPHRAGM 



Fig. 6-9. (a) Diagram of the optical components of a microscope with reflecting- 
refracting condenser and objective, for use in the visible and ultraviolet spectrum. 
(The Bausch and Lomb Optical Company.) (b) Diagram of a totally reflecting objec- 
tive, a design with chromatic correction over a wide extent of the visible, ultraviolet, 
and infrared regions of the optical spectrum. (Courtesy of A. J. Kavanagh and The 
American Optical Company.) 



sidered at length, from both the theoretical and experimental viewpoints, 
in such references as C^aspersson (193G, 1950); Uber (1939); Thoroll 
(1947); Swift (1950); and Davies and Walker (1953). 

Caspersson, especially, has discussed at some length the problems raised 
by image formation, a treatment which seems to make the whole ques- 



A CRITIQUE OF CYTOCHEMICAL METHODS 



229 



tion of microspectrophotometry considerably more difficult than in the 
simplification suggested. He believes that if the intensity distribution 
in the microscopic image is to correspond in every detail with that in the 
cell, a requirement for absorption measurements, then the demands on 
the microscope are essentiall}^ identi('al with those set forth by Abbe for 
highest resolution. Others have suggested that an absorption micro- 
scope can perhaps be a compromise between the simplified optical system 
of such instruments as colorimeters and that for the sharpest images at 
high magnification (Norris et al., 1951; Grey, 1952; Kavanagh, 1952). 



4-3. SOME ERRORS OF QUANTITATIVE 
MICROSPECTROPHOTOMETRY 

In practice, an apparatus for microscopic absorption is indeed much like 
a colorimeter or a spectrophotometer, except for the introduction of a 
microscope into the optical pathway, and the actual absorption measure- 
ment is essentially the same — Ix is 
intensity measured through the cell 
while /o is a second reading through 
an empty part of the slide, outside the 
section. Such quantitative absorp- 
tion data are easily obtained, but the 
successful evaluation of the results 
must take into consideration many 
possible sources of error which arise 
from the nature of cytological mate- 
rial and the fact that the microscope 
is used. Most substances within the 
cell are in a physical state very differ- 
ent from the dilute solutions meas- 
ured in a colorimeter or spectropho- 
tometer. The proteins and nucleic 
acids are very concentrated and if 
fixed, possibly even when unfixed, are 
more like solid precipitates or gels 
than solutions. Very little is actu- 
ally known about the extent to which 
this physical state can affect the 
operation of Beer's law because no 
extracellular model for such a study 
is available. Solutions of nucleic acid which approach that which occurs 
in cells (1-5 per cent) appear to give the same k value as dilute solutions 
(Table 6-3), and the absorption curve of such concentrated solutions meas- 
ured in the microspectrophotometer matches very closely that obtained on 
dilute solutions (Fig. 6-10). Nevertheless, it is difficult to escape a sus- 




2400 



3000 



2600 2800 

WAVE LENGTH, A 

Fig. 6-10. Absorption spectrum of a 
2.5 per cent ribonucleic acid solution, 
obtained (1) in a cellophane bag with 
a microspectrographieal arrangement 
(soHd circles) and (2) in a 10-/x Scheibe 
cuvette with a photoelectrical absorp- 
tion spectrograph according to Warburg- 
Negelein (open circles). (Redrawn 
after Thorell, 1947.) 



230 RADIATION BIOLOGY 

picion that, in the cuinplex iiucleoproteiii association within the cell, the 
resonance conditions may be significantly different from those of the iso- 
lated nucleic acid and protein in solutions (see Chap. 5, this volume), and 
hence any computations of absolute concentration or amounts from stand- 
ards obtained on solutions must be considered provisional. For relative 
concentrations this is less important, for it is a much less unlikely assump- 
tion that A- is constant throughout the limited range under study. 

Deviations from Lambert's law that extinction is proportional to thick- 
ness are uncommon sources of error if photometric analysis is properly 
carried out, since there are no conditions within a thick sample which 
differ from those within a thin one. It has been suggested that, within 
a cytological preparation, error may arise because the color reaction 
occurs only at the surface of the section, or because a colored product 
piles up to form a sort of opacjue screen on the surface. This has not 
yet been found. Conformity to Lambert's law is easily tested in cyto- 
logical preparations, and it has been repeatedly demonstrated that light 
loss is proportional to thickness of the absorbing layer (Pollister and Ris, 
19-1:7; Pollister and Swift, 1950). A possible source of failure is dichroism 
as a result of orientation of chromophores (Commoner, 1949; Commoner 
and Lipkin, 1949). No case of such error has yet been detected in visible 
and ultraviolet studies (Pollister and Swift, 1950), and there is evidence 
that moderate nucleic acid orientation would have little effect on the 
ultraviolet absorption measurements of cells carried out with unpolarized 
light (Thorell and Ruch, 1951). It is perhaps a safe rule that dichroism 
is unlikely to be a complicating factor except in objects which are con- 
spicuously birefringent (e.g., skeletal muscle). Marked dichroism is a 
potential tool for study of molecular orientation within the cell. For 
example, Caspersson (1940b) has demonstrated that the ultraviolet 
dichroism of grasshopper sperm heads is due to orientation of the pyrimi- 
dine chromophores, and infrared dichroism has been employed to detect 
orientation of protein polypeptide chains (Goldstein, 1950). 

When carrying out in vitro photometric analysis, the usual method of 
isolating specific absorption from nonspecific is by subtracting the light 
loss of a blank, which is either the solvent alo?ie, or a solution of the sample 
substance in which a color test, which is the basis of the photometric 
analysis, has not been developed. The wide usefulness of the photo- 
metric approach in ([uantitative chemical analysis depends largely upon 
these simple methods of extracting the essential datum from what is 
actually in most cases an extremely complicated optical phenomenon 
(see Chaps. 1 and 5, this volume). The cytological use of photometric 
analysis likewise depends on, in one way or another, the relation of light 
loss within the cell to specific absorption of a given chemical substance. 
The elimination of the nonspecific component has been l)est achiexed in 
methods of photometry of color reactions for nucleic acids and proteins, 



A CRITIQUE OF CYTOCHEMICAL METHODS 231 

which may be measured by visible light (Pollister, 1950, 1952a). It 
happens that when tissues are so fixed that the cell consists of little more 
than nucleoprotein, all cellular structures have very nearly the same 
optical dispersion, and it thus becomes possible to mount the specimen in 
a medium (an oil) which matches the refractive index at any wave length. 
Under these conditions unstained structures are invisible even by dark- 
field or phase contrast, showing that nonspecific light loss is negligible. 
If such material, colored by a reaction, is measured while mounted in oil at 
or near the appropriate refractive index, practically all the light loss may 
be assumed to be due to specific absorption by the chromophore of the test. 
[One possible source of error is that of anomalous dispersion near the 
absorption peak of the chromophore as pointed out by Scott (1952) and 
Ornstein (1952).] Another approach is to measure a cell twice, first before 
(a blank) then after (a test) development of color, a procedure which has 
been followed with the Millon reaction for proteins (Pollister and Mirsky, 
1946; Pollister, 1950). Another method is that used in photometry of the 
natural absorption of nucleic acids, in which the blank is the second meas- 
urement made after removal of the nucleic acid by nuclease digestion or 
chemical extraction (Fig. 6-5 and Table 6-4). This is somewhat less 
satisfactory than the protein blank because the component of the non- 
specific light loss due to nucleic acid is also removed and thus becomes 
added to the apparent specific chromophore absorption. 

When the refractive index of the mounting medium is markedly differ- 
ent from that of the section (e.g., when an unstained section is in water), 
the nonspecific light losses become appreciable. The methods of ultra- 
violet microspectrophotometry have been applied either to living cells or 
to sections which are mounted in glycerin, after either freeze-drying or 
fixation (e.g., in acetic alcohol). In the two former materials nonspecific 
light loss is believed by Caspersson (1950) to be minimized in some cases 
by the absence of sharp phase boundaries. In nearly all fixed material 
the nonspecific light loss is always considerable (Fig. 6-2D and 6-5). 
Apparently no mounting medium for ultraviolet studies closely matches 
the refractive index of such fixed sections. Hence ultraviolet absorption 
studies must always grapple with the problem of estimating the scatter 
and internal reflections. As Caspersson (1950) has said, "the most 
important of all conditioning factors for quantitative microspectrograph}^ 
is the elimination of the sources of errors caused by these factors." Cas- 
persson elected to estimate the nonspecific light losses, where appreciable, 
in the preparations, not by a measured blank as described, but by the 
unusual method of computing them from analogy with the light losses 
which he had previously studied in solutions of colorless salts — in which, 
of course, all light loss was nonspecific. In spite of the urgency and 
priority of this problem for any quantitative interpretation of the nucleo- 
protein ultraviolet absorption curves, there has never been a complete 



232 RADIATION UIOLOGY 

explanation of how llic nonspccilic li^ilit losses can be estimated and .sul>- 
lract(Kl from the compound measured curve of intracellular nucleopro- 
lein. The nonspccilic li<ilit loss in a cell becomes evident as apparent 
absorption outside the spectral region of specific absorption, in the case of 
luicleoproteius abo\e 300 m/x. When extinction approaches zero near 
this point, it is clear that one is perhaps justified in the assumption that 
scatteriufi; and leHections cause no light losses in the shorter wave length 
i-egion of strong specific absorption, although the possil)ility of anomalous 
ilispersion near the absorption peak can by no means be ignored (Scott, 
1952). Often, however, the light loss at the nonspecific zone is from one- 
third to one-half that at the maximum of the specific absorption. It is 
not clear why in one such case "the loss of light was assumed to depend 
ecjually upon reflection and Kayleigh's light scattering" (Hyden and 
Ilartelius, 1948), while in another instance it was assumed that "at 3100 
A light-dispersion conditions the whole absorption and is inversely pro- 
portional to the fourth power of the wave length" (Caspersson and 
Thorell, 1942). Where there is obviously considerable nonspecific light 
loss, it seems logical to expect to see, side by side, the uncorrected and 
corrected curves, but there has never appeared an intracellular nucleo- 
protein curve from which nonspecific light loss has been subtracted. In 
some cases a curve of "light-dispersion" has been published with the 
measured light. 

In photometric analysis it is a very uncommon procedure to attempt 
to account for a substantial amount of the nonspecific light loss by com- 
putation, probably because it is often difficult to determine the optical 
constants to be used even with simple solutions or suspensions. Caspers- 
son has chosen to do this with an immensely complex unknown sample, a 
nucleoprotein mass in a cell, and a full evaluation of the success of this 
attempt must await more complete details of the computations in specific 
cases. 

The possibility of a change in light loss or in cell structure as an effect 
of the radiant energy is not very gi-eat in measurement of absorption in 
the visible spectrum. On the other hand, such an effect is rather to be 
expected when working in the middle ultraviolet range, since it is a com- 
mon experience that many substances rapidly lose their specific absorp- 
tion upon exposure to this higher energy radiation. Caspersson was 
aware at the very start of his researches (1936) that this might occur, and 
presented experimental evidence that, in vitro, the absorption of nucleic 
acid was less sensitive to ultraviolet radiation than were free guanine 
and adenine. Caspersson (193G, p. 22) remarked that, "Die Messung 
dieses Effects ist im mikroscop technisch ausserordentlich schwer, da im 
mikroskopischen Priiparat Deformationen auftreten." Sections of fixed 
material mounted in glycerin are extraordinarily stable, and an exposure 
to intense 254 m/u radiation for many hours causes no measurable reduc- 



A CRITIQUE OF CYTOCHEMICAL METHODS 233 

tioii of extinction or noticeable change of appearance. When living cells 
are studied, however, the possibility of both types of alteration must 
always be kept in mind. The physical changes induced in nuclei by 
radiation, presumably of the sort Caspersson noted in 1936, were later 
pointed out by Brumberg and Larionow (1946), and considered in much 
more detail by Ris and Mirsky (1949), who believed that nearly all pub- 
lished ultraviolet photographs of living cells show signs of radiation 
injury, in that the nuclear details are too conspicuous. 

Although there is some room for dispute about the exact nature of the 
effects of ultraviolet radiation on the structure and absorption of living 
cells, there is little question but that "the living cell is, as a rule, an 
unsuitable object for microspectrophotometric studies," because of its 
great motility and because great structural changes occur during irradia- 
tion (Caspersson, 1950, p. 57). This point of view is different from that of 
earlier years, when great emphasis was placed on the advantages of 
applying these methods to living cells (e.g., Caspersson, 1947, p. 127: "It 
must be possible to apply the results directly or indirectly to the living 
cell itself"). Mellors et al. (1950) have shown that these particular 
obstacles to ultraviolet studies of living cells are by no means insurmount- 
able. Using a microscope with achromatic reflecting optics, they dis- 
persed the light after it had passed through the microscope, and photo- 
graphed the whole ultraviolet spectrum of a mercury vapor lamp on one 
negative. With very sensitive emulsions, it was found that up to 85 
such photographs could be taken before the cell became injured to the 
extent that mitotic division could not proceed. There may be many 
physiological problems which can be profitably attacked directly by this 
method. For studying the question of the nucleoprotein composition of 
cells, however, the wide range of specific absorption in living cells seems 
an unnecessary complication, and "for most problems suitably extracted 
objects give the cleanest data" (Caspersson, 1950, p. 57). 

A major task in making significant absorption studies of cells is that of 
selecting the region of the cell to be measured. All of the cytologist's 
experience and special craft are sometimes called upon in overcoming this 
difficulty. How can the absorption of particular cellular structures be 
isolated from that of adjacent structures and from a surrounding visibly 
structureless sort of background? This is accomplished easily enough if 
a highly specific absorption is localized in a particular structure, as is 
chlorophyll in chloroplasts or the Feulgen reaction in the nucleus. The 
successful quantitative application of the latter (Pollister, Swift, and 
Alfert, 1951) is in large part due to its high specificity and sharp localiza- 
tion (Fig. 6-2F) . When, on 1 he other hand, a reaction is widespread, often 
to nearly all parts of the cell, it is difficult to determine the absorption 
of one particular component. Examples are protein tests (Fig. 6-25), 
basophilia, and ultraviolet absorption, or cases where the cytoplasm 



234 RADIATION mOLOGY 

give a markt'cl plasmal reaction with SchifT's rcaf^ont. The structures 
may be isolated by crushing or smearing the cell, the method used l)y 
Caspersson (H)3()) with components of the salivary gland nucleus and by 
Ris and Mirsky (1949) with nuclei. This is, however, a procedure by no 
means always applicable. The problem is probably best solved by sec- 
tioning tissue approj)riately. Nuclear and luicleolar absorption are thus 
easily isolated if sections are cut approximately ecjual to or le.ss than the 
respective diameters of these cell components. The techni(|ue of sec- 
tioning material in a much lower range of thickness has been developed 
for electron microscopy (Hillier and Gettner, 1950); sections can be cut 
1 ^t or less, and thus the absorption of single mitochondria or secretory 
granules can possibly bo studied without interference from cytoplasmic 
overlay or underlay. Until now mitochondria have been studied only 
where a considerable number are fused to form a large body, the neben- 
kern of the insect spermatid (Leuchtenberger and Schrader, 1950). A 
similar advantageous concentration of small bodies (e.g., microsomes) 
can be accomplished by ultracentrifuging (Lagerstadt, 1949). 

In studies of living cells, this problem of isolating the absorption of a 
particular component seems nearly insolvable. Rarely, if ever, can it 
be arranged that the nucleus is relatively free of overlying or underlying 
cytoplasm; either this must be ignored (Caspersson, 1939; Thorell, 1947; 
Mellors et al., 1950) or a correction must be attempted by measuring the 
c3''toplasm, estimating the thickness above and below the nucleus, and 
subtracting the proportional cytoplasmic absorption, assuming that it is 
homogeneous (Caspersson, 193(5). This assumption is likely to be incor- 
rect since in many cells the ultraviolet absorbing material is especially 
concentrated just outside the nuclear membrane. Much more difficult is 
any sort of measurement of absorption of a nucleolus, and it seems inad- 
visable to attach much significance to the apparently uncorrected absorp- 
tions of small nucleoli in living cells (Thorell, 1947). 

The problem of fixation for chemical cytology is of major importance, 
even though it has not received much attention, cytochemistry having 
merely taken over some orthodox cytological technicjues. The.se are by 
no means all suitable methods of observing tissue for cytochemical study. 
For example, the best fixation of the complete structural details of cells 
is accomplished by use of mixtures which include osmium tetroxide 
(osmic acid, Os2()4) and chromium trioxide (chromic acid, CrOs), (Fig. 
6-2^4), but these fluids are of extremely limited application in cytochem- 
istry. The chrom-osmium mixtures always tend to overemphasize phase 
boundaries, they introduce color into the section, and, after their use, 
practically no fractions can be removed chemically or enzymatically. 
C'ytochemical fixation, as a rule, has been a compromise between the 
requirements of accurate preservation of morphology and tho.se of photo- 
metric analysis and fractionation (Pollister, 1952a). Two colorless 



A CUITlgUE OF CYTOCHEMICAL METHODS 235 

reagents have been most widely used, acetic acid-alcohol and neutral 
formalin. The former has the advantages of conserving little but 
nucleoprotein, and this in a state where fractions are readily lemovable; 
but acetic acid-alcohol certainly introduces gross morphological artifacts 
especially in lipid-containing structures such as mitochondria. Formalin 
introduces less artifacts, but it does preserve many other substances in 
addition to nucleoprotein and has a tendency to react vigorously with 
proteins (French and Edsall, 1945) which may quite possibly lead to con- 
siderable confusion in interpretation of staining and tests, or even of ultra- 
violet absorption. 

The selection of the exact spot to be measured in the cell is also a diffi- 
cult task. If the material is homogeneous over a considerable area, the 
size of the sample is relatively unimportant. The Caspersson group, as a 
rule, measures a very narrow cylinder (less than 1 /x- in area). This has 
the merit of being so small that in most cases great variations of absorp- 
tion across the field are unlikely. It has the disadvantage that the spot 
may not be representative of any considerable part of the cell. All other 
workers (e.g., Gersh and Bodian, 1943; Pollister and Ris, 1947; Leuchten- 
berger, 1950; Swift, 1950; Lison and Pasteels, 1951; Fanijel, 1951) have 
measured the transmission of larger areas, often entire nuclei. Some- 
times these larger areas are fairly homogeneous, but more often the 
absorbing material is to some extent concentrated in scattered masses 
among which there is a sort of continuum of relatively lower absorption. 
This heterogeneity introduces into the absorption measurements an 
obvious unavoidable error (conveniently called the "distributional error") 
which was first pointed out by Caspersson (1940a). This leads to lower 
extinction values, never to higher. The error is best understood from 
considering the actual conditions of measurement of two extreme con- 
ditions, one where absorbing material is evenly distributed throughout an 
area and another where the same amount of absorbing material is con- 
centrated in one-half the area, the remainder being free of absorbing mate- 
rial. Suppose the concentration in the first case is sufficient to cut the 
light down 90 per cent, i.e., the transmission is 10 per cent and the extinc- 
tion is 1.0. In the second case the light loss will be as follows: Since the 
concentration of absorbing material in the dark half is doubled, its extinc- 
tion becomes 2. This means that this half will transmit 1 per cent of the 
light which falls upon it; and, since this darker portion is one-half the 
total area measured, the amount of light passing through this absorbing 
region to the photoreceiver is 0.5 per cent of the total light. The lighter, 
nonabsorbing half, however, transmits all hght, which is one-half the 
total amount; hence the reading on the instrument will indicate a trans- 
mission of 50.5 per cent. The ec}uivalent extinction is 0.306, less than 
one-third the true value (1.0) obtained when the material is evenly dis- 
tributed. This case is an example of extreme heterogeneity which 



230 



RADIATION HIOI.OGY 



ol)viously should ncxcr he incjisuiccl as a whole ohjeci. The ei'ior in 
moasuriii^ spurscly clistrihutcd chiomosomcs on a mctaphasc plate 
would j^orhaps he comparahle with this. As the heterogeneity decreases, 
and the darker and lighter parts of the object come to contain more nearly 
Hke amounts of absorbing maforial, the distributional error rapidly 
decreases. For an extensive theoretical treatment of the distributional 
error see Ornstein (1952) who considers several methods of minimizing or 
correcting for the error. 

There is an error involved in the summation of transmissions where 
intensity distribution is uneven, and also an error in converting the total 
or average transmission of a heterogeneous object into an average extinc- 
tion. It appears that, in direction and relative magnitude, these should 

run parallel w'ith the distributional 
eri'or which w^as just discussed (see 
Fano, 1947; Swift, 1950). 

The unexceptionable method for de- 
termining amount of absorbing mate- 
rial in large areas with varying density 
is that of making densitometer cross- 
tracings of negatives (Caspersson, 
1940a) or a series of scannings of a cell, 
in many azimuths, with the photo- 
metric apparatus (Thorell, 1947). 
Caspersson followed this procedure 
with living early meiotic prophase 
nuclei of the grasshopper; the cross- 
trace in one azimuth is shown in Fig. 
6-1 1 . He did not detect any distortion 
of what was to be expected from absorp- 
tion of a sphere aside from some margi- 
nal diffraction, i.e., there was no meas- 
urable heterogeneity although, visibly, 
there seemed somedensei' structures. Accordingly, he used the extinction 
through the center as a measure of concentration, and computed total 
amount in the nucleus from this datum. 

Other possible sources of error in microspectrophotometry of cells are 
discussed in such references as Caspersson (193(), 1940a, b, 1950) ; Thorell 
(1947); Swift (1950, 1953); Naora (1951); PoUister (1952a); and Davies 
and Walker (1953). 



0^0 




y^-^^ 




s? 






^ 






/J 


\\ 


■£ 




Ij 


\\ 


o 




y 


V\ 


P 60 


_ 


i 


Q. 




i 


tt 




I 


v 


o 




j 


>fl 




1 


\\ 


ID 




/ 


< 40 


- 


1 


« 


1- 




j 


1 


I 




1 


I 


o 


1 


i 


\ 


^20 


\ 


j ^ 


i\ 

1 i Ll_ 



20 30 

MICRONS 



40 



50 



Fig. 6-11. intraviolot absorption 
measureiucnts at a series of points 
across the diameter of a grass- 
hopper spermatocyte (soHd line), 
compared with the computed ab- 
sorption (uirve of an absorbing 
sphere (broken line). (Redranm 
from Caspersson, 1939.) 



4-4. QUANTITATIVE APPLIC.\TIONS, ABSOLUTE AND REL.\TIVE 

Because of the potential errors in photometric analysis of cells it is 
evident that a straightforward computation of absolute amount of sub- 
stance by referring light loss in a cell to any standard value obtained on 



A CRITIQUE OF CYTOCHEMICAL METHODS 



237 




solutions in an absorption cuvette (as described in Sect. 2) is by no means 
a reliable procedure, although this has frequently been done. 

The first such attempt at absolute quantitation was made by Caspers- 
son (1939) who computed the amount of DNA in 24 nuclei of living grass- 
hopper spermatocytes, assuming that distribution was homogeneous (p. 
235 and Fig. 6-10) and that all light loss was due to specific DNA absorp- 
tion. No correction was made for possible protein or RNA absorption, 
or for nonspecific light loss, but it now 
seems evident that these first two must 
have contributed to the total weaken- 
ing of the light (Ris, 1947; Caspersson, 
1950, Fig. 36). 

It will be recalled that two kinds of 
ultraviolet absorption curves (curves 
I and II, Fig. 6-6) have been obtained 
for intracellular nucleoprotein. No 
absolute computations have been made 
from type I curves except the semi- 
quantitative nucleic acid-protein ratio, 
from extinctions near the nucleic acid 
and protein peaks (best described by 
Thorell, 1947). Caspersson, however, 
has made an elaborate effort to com- 
pute quantities of nucleic acid, tyrosine, 
and tryptophane from the type II 
curves, which were formerly supposed 
to characterize regions of nucleic acid 
accompanied by histone type or diam- 
ino acid-rich protein (Fig. 6-12). The 
method of computation has not been 
described completely enough so that 
readers may discover just how the val- 
ues were reached from the measured 
curve. It is clear, however, that the 
curve analysis depends on the validity 
of the assumption that the protein 
moiety is of basic type in which the 
presence of a large proportion of dibasic 
amino acids, as in histones, brings about a shift of the absorption peak 
of tyrosine (as suggested very tentatively by Stenstrom and Reinhard, 
1925). Therefore, this particular method of curve analysis is in effect 
invalidated by the demonstration that in histones the peak is not shifted 
toward longer wave lengths (p. 223). 

Pollister and Ris (1947) reported computation of the amount of DNA 



2500 



3100 



2700 2900 

WAVE LENGTH. A 

Fig. 6-12. An example of analysis 
of compound ultraviolet absorp- 
tion curve of a cell structure con- 
taining nucleoprotein. Curve I, 
measured absorption; curve III, 
nucleic acid component; curve 
IV, tyrosine coinponent; curve V, 
tryptophane component; curve 
II shows the sum of the compo- 
nents. Results: tyrosine, about 
0.1 X 10^"' mg/ix^; tryptophane, 
0.04 X 10 '" mg/M^; nucleic acid, 
0.6 X 10->" mg/ix\ Light refrac- 
tion and dilTraction are, in this 
special case, negligible, as special 
experiments have shown. (Redrawn 
after Caspersson, 1940, 1950.) 



238 RADIATION BIOLOGY 

ill isolated thymus nuclei, very much as Caspersson had done earlier for 
spermatocytes. They reported MX 10~^ mg per nucleus, and, for the 
first time in cytochemistry, compared a figure obtained by cytological 
photometry with one obtained by chemical analysis of a mass of 
known luimbor of isolated nuclei. The two values agreed within 10 per 
cent, which appeared to be excellent validation of the microspectrcjphoto- 
metric procedure. It later appeared that this was merely a fortuitous 
coml)ination of errors, since all other chemical studies indicated the 
amount per nucleus to be nearer 6.0 X 10~^ mg (see table in Davidson and 
Leslie, 1950b) ; hence the value computed by Pollister and Ris from ultra- 
violet absorption is probably considerably less than one-fifth the real 
amount. 

This type of study was later carried out under much more favorable 
conditions by LeiK'htenberger and coworkers (1951). The citrate-iso- 
lated nuclei were swollen in glycerin to minimize nonspecific light loss and 
to increase the homogeneity. The ultraviolet absorption was demon- 
strated by cross-scannings of the spherical nuclei, according to Caspers- 
son's earlier procedure for spermatocytes (p. 237). The biochemists then 
determined the DNA, RNA, and protein content, and showed that the 
last two could have only slight effect upon the absorption. The amount 
of DNA per nucleus computed from these cytological data (5.4 X 10~^ 
mg) is much closer to that found independently by the chemical analysis 
of the remainder of the same sample of isolated nuclei (6.1 X 10~^ mg). 

It must be pointed out that in such nuclei the DNA is determined under 
very special conditions of minimal RNA and protein, brought about by 
the citrate isolation. Such a condition is rarely, if ever, encountered in 
intact fixed tissue (see Pollister and Leuchtenberger, 1949a, Pollister, 
1952b). 

Di Stefano (1949) also computed DNA in frog cartilage nuclei from 
ultraviolet absorption data. By comparison with chemical analysis 
(Davidson and Leslie, 1950b), it appears that these values were deficient 
by about as much as those given for thymus nuclei by Pollister and 
Ris. 

Di Stefano likewise compared the value from ultraviolet absorption 
with that computed from the Feulgen reaction, on the assumptions that 
at maximum reaction all purines had been removed and that the fuchsin 
regeneration from Schiff's reagent proceeded according to the scheme pro- 
posed by Wieland and Scheuing (1921). The DNA amounts per nucleus 
computed by these two cytological methods were in satisfactory agree- 
ment, but, as just indicated, these are far below the value obtained by 
chemical analysis. Protein-DNA ratios for tissue nuclei were computed 
from Feulgen and Millon reactions, before the invalidation of the Pollister 
and Ris and the Di Stefano attemj)ts at absolute (juantitation became 
known (Pollister and Leuchtenberger, 1949). The Feulgen data of the 



A CRITIQUE OF CYTOCHEMICAL METHODS 



239 



last-named authors lead, by the Di Stefaiio type of computation, to DNA 
vahies about half those obtained by chemical analysis of isolated luiclei 
(see Pollister, Swift, and Alfert, 1951). 

As this brief summary shows, in practice the computation of absolute 
amounts from microscopic absorption measurements has been far from 
uniformly successful, a result not unexpected in view of the many poten- 
tial errors. It is apparent, however, that for many problems in cell chem- 
istry this dubious extrapolation is not necessary; an adeciuate answer is 





/ 


. 


■A 




^r\ 


1.000 


- 


\ \ 


A 


i ny 1 


\ 


2 


1 


I 








o 




ll 




\ It 




1- 




1 




\ / / 




u 




I 




\ /I 


1 


z 




1 




\ 1 




K 


/ 


\ 




\ / 




X 


/ 


1 




v / 


\ > 


UJ 500 


7 


1 




J, 


\ 



300 



600 



700 



400 500 

WAVE LENGTH, mu 

Fig. 6-13. Absorption curves, measured in a Beckman spectrophotometer, of various 
substances which are analyzed photometrically in cytological preparations. Unless 
otherwise indicated, the length of the absorption path was 1 cm. The light vertical 
line cutting each curve indicates the spectral region which is measured in the micro- 
scopical preparations. Curve I, natural absorption of deoxyribonucleic acid, concen- 
tration about 0.05 mg/cc (redrawn from Pollister and Mirsky, 1946); curve II, tyrosine- 
mercurial formed by the Millon reaction, tyrosine concentration 0.045 mg/cc; curve 

III, fuchsin regenerated from standard Schiff's reagent by addition of formalin; curve 

IV, azure A in distilled water, concentration 0.5 mg/cc, thin cuvette {data of M. 
Flax); curve V, methyl green (Natl. Anil., cert. No. NG35) in carbol-glycerin-alcohol 
mixture, concentration 0.01 mg/mm' {data of C. Leuchtenberger). 

obtainable if the question of relative amount or of relative change can 
be answered. Numerous quantitative studies with this aim have been 
published, some of which are reviewed in Pollister (1952a). The specific 
methods are those which have already been discussed in Sect. 3. The 
in vitro absorption curves and the wave lengths measured in microscopic 
material are shown in Fig. 6-13. Since these methods are colorimetric 
(that is, measurement at a single wave length) and the apparatus is simple 
(Fig. 6-7), large numbers of absorption measurements on individual 
nuclei, nucleoli, or other cell structures are readily obtained. On anj^ one 
type of structure the values are found to vary considerably, the 
highest extinctions being often as much as twice the lowest. When 



?to 



UADI A'l'ION Hl(H.(KiY 




0.200 



4 00 



600 



800 




0.600 



800 



100 
EXTINCTION 

Fig. 6-14. Typical distrilnition curves of microspectrophotometric data. Curve I, 
sections of nucleoli, corn pollen mother cells, Millon reaction ; curve II, nuclei of mouse 
sarcoma, methyl green; curve III, nucleoli, corn pollen mother cells, ultraviolet 
absorption; curve IV, mouse spermatocyte nuclcM, Feulgen reaction; curve V, pre- 
leptotene nuclei from mouse testis, Feulgen reaction. {After Pollister and Swift, 1950.) 



Table 6-5. Liver Nuclei, Ultraviolet Absorption 



Slide 


N 


umber 


Mean Eon 


no. 


measured 


A-6 




20 


0.489 + 0.015 


A-6 




10 


0.474 + 024 


A-4 




20 


0.466 ± 0.023 


A-9 




22 


0.507 + 0.016 


A-8 




25 


0.475 ± 0.012 


A-5 




23 


0.499 ± 027 
Mean = 0.485 



The reproducibility of ultraviolet absorption of 5.0-m sections of nuclei of the liver 
of the salamander, Amhh/stoma, is shown by data from different slides of the same 
organ, measured at different times. Fixation in acetic acid-alcohol, eml^edded in 
paraffin, mounted in glycerin, measured as absorption of 254 ni/u radiation isolated 
from a Hanovia SC2537 mercury-vapor lamp by a Zeiss two-prism quartz mono- 
chromatic illuminator, with glycerin immersion monochromatic objective, N.A. 0.85, 
condenser aperture about N.A. 0.40. 



A CRITIQUE OF CYTOCHEMICAL METHODS 241 

enough data are accumulated for statistical analysis it is found that these 
values group themselves into normal unimodal distribution curves (Fig. 
6-14). The cause of this variability is not fully known in any instance. 
The instrumental and cytological variability can account for but a small 
part; in many structures (e.g., interphase nuclei) a major portion appears 
to be due to the distributional error. Until these errors of tec^hnique and 
unavoidable errors due to cytological structure can be precisely estimated 
(Sect. 4-3) the magnitude of the biological variation in any one popu- 
lation of similar cells cannot be estimated; and therefore man}^ of the 
possible more subtle correlations of nucleoprotein composition with 
physiological, developmental, pathological, or experimental phenomena 
cannot be investigated by microspectrophotometry. In the meantime, 
it has been found possible to make many fruitful studies by utilizing the 
fact that with proper cytological and photometric techniques the mean 
values of any population are reproducible to about 10 per cent. This 
holds when comparing different slides of one material (Table 6-5), when 
examining the same animal at different times (Table 6-6) , or when study- 
ing different animals of the same species (Table 6-6). This variability 
of the means presumably is a measure of the over-all error of the technique 
of preparing tissues for microscopic examination and of the photometric 
procedure. In this type of relative quantitative analysis, a major 
change of composition which may accompany cyclical, experimental, 
or pathological events is detected as a change of mean value, outside 
the normal range of variability. This reproducibility of the means 
has been shown repeatedly for each of the techniques indicated in Fig. 
6-13; indeed, it is customary to reexamine it for each new material 
studied.^ 

An important aspect of these relative quantitative studies is that in 
manj'^ cases it has been possible to compare them with results by inde- 
pendent methods, such as biochemical analysis of samples of known num- 
bers of isolated cell components. For example, the correlation of 
amount of DNA with the chromosome complement of nuclei, first indi- 
cated by biochemical analysis, has been confirmed and greatly extended 
by cytological studies made with the Feulgen reaction, whereas the pro- 
tein composition of the nucleolus, suggested by results with the Millon 
reaction, has been confirmed by both X-ray absorption data and analysis 
of masses of isolated nucleoli. Many of these essential validations of the 
cytochemical analyses are summarized in PoUister (1952a). 

* The approach of the Caspersson school to microspectrophotometry has ])een 
almost entirely qualitative and semiquantitative. This is emphasized particularly 
by the fact that in the total of nearly a hundred publications (see list in Caspersson, 
1950) there is not one demonstration of the statistical reprodui'il)iIity of the ultra- 
violet absorption terhiii(|U(', nor is the urgency and priority of such a demonstration 
ever recognized. 



242 



RADIATION mOLOGY 



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A CRITiyUK OF CYTUCHKMICAL AIKTJlOD.S 243 

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i 



A CRITIQUE OF CYTOCHEMICAL METHODS 245 

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344-349. 



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Manuscript received by the editor Mar. 12, 1952 



CHAPTER 7 

The Effect of Ultraviolet Radiation on the Genes 
and Chromosomes of Higher Organisms 

C. P. SWANSON 

The Johns Hopkins University 
Baltimore, Maryland 

L. J. Stadler 

The University of Missouri and U.S. Department of Agriculture 

Columbia, Missouri^ 

Introduction. Experimental procedures. Genetic effects of ultraviolet radiation. 
Direct effects of ultraviolet radiation on chromosomes. Spectral relations. References. 

INTRODUCTION 

This review will be concerned with the effects of ultraviolet radiation 
on the genes and chromosomes of organisms above the microbiological 
level. The group of organisms thus included is relatively small in num- 
bers, and the literature is scanty in comparison to that devoted to radia- 
tion studies on the fungi, bacteria, and viruses. But cytogenetic studies 
can be made only on organisms with suitable chromosomes. The ultra- 
violet results obtained with Drosophila 7nelanogaster and Zea mays may be 
evaluated against an extensive background of X-ray data bearing on 
problems of cytogenetic interest. Supplementing the data from these 
organisms are those from Antirrhinum and Sphaerocarpus, which relate 
to the genetic effectiveness of various wave lengths within the ultra- 
violet spectrum, and those from Tradescantia and Gasteria, which deal 
only with induced chromosomal aberrations. 

Each of these species possesses certain disadvantages, none being 
wholly satisfactory as a test organism because of difficulties of radiation 
penetration, accurate dosage measurements at the site of genetic altera- 
tion, or critical analysis of induced effects. Despite these shortcomings, 
however, the accumulated evidence from ultraviolet studies has been 

1 Cooperative investigations of the Division of Cereal Crops and Diseases, Bureau of 
Plant Industry, U.S. Department of Agriculture, and Department of Field Crops, 
University of Missouri. Missouri Agricultural Experiment Station Journal Series 
No. 1470. 

249 



250 UADIATIO.N lilol.oCY 

sufficient to advance malctinllx' tlic uiKicrstandiiiji; of I lie nature of 
induced hereditary chanjies. 

'I'li<> genetic action ot ult ia\iolel radiation hears on threi- major proh- 
li-nis. riie lirst ot" these is the analysis of the coniph'x of }:;<*"<'tie varia- 
tions induced l)y the ioni/ins radiations. It is invariably found, where 
adeciuate cytogenetic tests can he made, that hoth mutations and chro- 
mosomal aberrations are induced by X radiation. A clear-cut separation 
of the two phenomena has not yet been accomplished, and the question of 
their similar or dissimilar nature and origin remains unanswered. If the 
induction of mutations and chiomosomal rearrangements by the ionizing 
radiations results from some common, fundamental effect, the two types 
of genetic alteration should have a common spectral limit. The analysis 
of induced hereditary variations would then involve determining the 
alternate pathways of reaction which culminate in a variety of genetical 
and cytological expressions. If, on the contrary, the diverse effects of X 
radiation are of independent origin, it is possible that their spectral rela- 
tions may be sufficiently different to permit the separation and perhaps 
the selective induction of one type of variation to the exclusion of others. 
The individual phenomena should then pro\e to lie more amenable to 
analysis. 

The second problem is concerned with the determination of genetic 
effectiveness of specific wave lengths of the ultraviolet spectrum, as a 
ckie to the chemical nature of the substance within which the energy 
absorption leading to genetic change takes place. With ionizing radia- 
tions such a study is not possible since their absorption is independent 
of molecular organization. 

The third problem is concerned with the nature of mutations in general, 
whether spontaneous or induced. Mutations as experimentally identified 
are a residual class, identified by negative criteria. The analysis of spe- 
cific X-ray-induced mutations has shown that character changes inherited 
as if due to gene mutations may be in some instances the result of chromo- 
somal rather than genie alteration. The category "mutations," as 
experimentally defined, must therefore be a complex one, including vari- 
ous extragenic as well as intragenic alterations. Since the mode of action 
of ultraviolet radiations is so different from that of ionizing radiations, the 
comparativ^e study of mutations induced by these agents is promising. 
The relative infrequency of chromosomal derangements induced by 
ultraviolet suggests that certain extragenic alterations simulating gene 
mutation may be infre(|uent or absent among the mutations from ultra- 
violet treatment. The possibility of qualitative differences in the chro- 
mosomal alterations must also be considered. 

The spectral analysis of the complex of genetic effects induced by X 
rays, as outlined above, implies the as.sumption that the mutations and 
chromosomal alterations induced by X rays are qualitatively analogous 



GENETIC AND CYTOLOGICAL EFFECTS 251 

to those induced by ultraviolet radiation. If they are not, it is possible 
that the spectral limit of both classes of X-ray alterations is far below 
the shortest ultraviolet wave length that can be used in biological experi- 
ments, and that the mutations and chromosomal alterations characteristic 
of the ultraviolet are of a type not occurring in appreciable frequency in 
the X-ray progenies. 



EXPERIMENTAL PROCEDURES 

Genetic and cytological studies of the effects of ultraviolet radiation on 
higher organisms are limited by the techni(iues which permit the investi- 
gator to irradiate the germ cells effectively without killing the organism 
or the cells being treated. Ultraviolet produces considerable physio- 
logical damage, setting limits to the dosage; it is also low in penetrating 
power, a disadvantage when deep-seated sex organs are being treated. 
Both of these phenomena result from the high absorption of ultraviolet by 
extranuclear constituents of the cells as well as by the nucleoproteins of 
the chromosomes. A number of techniques are available, however, and it 
is fortunate that they permit the irradiation of the germ cells of Droso- 
pfiila and maize, thus making possible genetic and cytological comparison 
with other mutagenic agents. Ultraviolet radiation and X rays have also 
been used extensively with Neurospora, in which significant cytogenetic 
comparisons could be made, but no comparative studies of the induced 
alterations have been reported. 

Perfection of the technique of artificial insemination of Drosophila 
females with treated spermatozoa would circumvent the difficulties of 
reaching the sex cells of adult males with ultraviolet radiation, but 
apparently all attempts to duplicate Gottschewski's (1937) artificial 
insemination results have so far been unsuccessful. Two other methods, 
however, have been developed: (1) irradiation of the pole cells of the 
early embryo in the egg, and (2) irradiation of mature spermatozoa 
through the ventral side of compressed abdomens of adult males. 

The polar cap technique was developed by Geigy (1931) after the early 
work of Guyenot (1914) and Altenburg (1928) had demonstrated that 
ultraviolet treatment of adult males gave only inconclusive results. The 
pole cells are destined to enter the germ tract. At 75 min after fertiliza- 
tion of the egg (at 24°C) the pole cells appear close to the surface at the 
amicropolar end, w^here they remain for approximately 1 hr (Altenburg, 
1934). In this position their nuclei can be readily reached by ultraviolet 
radiation. The early experiments of Altenburg (1933, 1934) were made 
with the angle of incidence of the ultraviolet at right angles to the vertical 
axis of the egg, the lower portion cjf which could be shielded to reduce 
injury to the developing embryo. More recently, it has been found that 
a shift in the angle of incidence from a plane of 90° to one more nearly 



252 RADIATION mOLOGY 

parallel to the axis of the egK fj;reatly increases the j;;enetic elTectivencss of 
a fiiveii ultraviolet dose while lessening the physiological damage to the 
embryo (Altenhurg et al., I'JoO; Meyer et al., 1950). Dechorionation of 
the eggs by immersion in a 5 per cent solution of sodium hypochlorite also 
facilitates penetration of the radiation (Clark, 1948). 

At the time of irradiation, the pole cells number about 20. Their later 
incorporation into the germ tract is accompanied by an increase in the 
number of cells as the sex organs and the reproductive cells are formed. 
Therefore, if a mutation is induced in one gene at the polar cap stage, it 
theoretically should appear replicated in about 5 per cent or more of the 
sex cells from that particular individual. Should the mutation occur 
after the process of multiplication has begun, a smaller proportion of the 
germ cells would receive it. 

Two assumptions are involved if the 5 per cent level of mutation repli- 
cation among the Fi offspring provides the distinction between induced 
and spontaneous changes. In the first place, the pole cells at the time of 
exposure to radiation must be 20 or less in number, and second, an ecjual 
rate of multiplication of mutated and normal pole cells must be assumed 
to take place up to the time of formation of the reproductive cells. 
Unequal exposure of the pole cells through unavoidable shielding as well 
as through differences in the degree of penetration of radiation would sug- 
gest that variations in cellular injury are to be expected. These varia- 
tions would be expressed in unequal rates of cell multiplication, with the 
most heavily injured cells having the slowest rate of division. The latter 
cells would also be most likely to possess mutated genes. As a means for 
demonstrating that ultraviolet is effective in inducing mutations, the 
technique is entirely satisfactory, but it is unsuitable for accurate deter- 
minations of the frequency of mutation as a function of dosage. 

Recessive lethals induced in the X chromosome by the pole cap method 
of exposure may be determined by testing, through the CIB technique, 
the Fi daughters of males arising from irradiated eggs. Replicated 
lethals from any single male must be further tested for identity because 
of the possibility of two or more coincident lethals. Visible mutations 
in the X chromosome may be detected in Fi males by breeding the 

treated Pi males to XX females. 

Reuss (1935) developed an effective method for the exposure of mature 
spermatozoa to ultraviolet radiation. The abdomens of adult male 
Drosophila are compressed gently between (luartz plates, the radiation 
being applied ventrally. Since the testes are superficially located, the 
amount of overlying tissue is at a minimum and consists of the chitinous 
exoskeleton, the wall of the testis, and the intervening connective tissues. 
Clear areas of chitin, which has chemical and absorptive properties char- 
acteristic of polysaccharides, are in general highly penetrable by wave 
lengths from 250-400 m^, with the degree of absorption increasing rapidly 



GENETIC AND CYTOLOGICAL EFFECTS 253 

at shorter wave lengths (Durand el al., 1941). The method, while useful 
in ascertaining the kinds of gene and chromosome changes which may be 
induced by ultraviolet in the mature spermatozoa, is not suitable for the 
accurate (juantitative determination of dosage-mutation frequency rela- 
tions and wave-length dependence yields. The chitin is of uneven trans- 
parency and the intervening tissues vary in thickness and position, with 
the result that considerable differences in the amount of radiation reach- 
ing the spermatozoa must be expected from one individual to another. 
The variations in penetration of radiation can be lessened somewhat by 
the use of light-colored mutant stocks, but there remains the difficulty 
of accurately determining the amount of energy absorbed by the se.x cells 
at the site of effective action. Also, the practical limits of dosage are 
determined largely by the tolerance of the adults to physiological damage 
leading to sterility. Demerec et al. (1942) have reported induced sex- 
linked recessive lethals in frequencies as high as 50 per cent, but such high 
proportions of mutations are a rare exception. The usual frequencies of 
recessive lethals obtained from optimal abdominal exposures range from 
0-5 per cent, with a wide variation among similarly treated flies. 

The higher plants offer somewhat better technical possibilities for 
studies of the genetic effects of ultraviolet radiation. The male cells are 
of minute size, readily accessible, and free from extraneous tissues. 
Three types of cells have been used: (1) swimming spermatozoids, (2) 
pollen grains, and (3) generative cells in developing pollen tubes. 

Swimming spermatozoids of the liverworts, for example those of 
Sphaerocarpus donnelUi, are particularly favorable objects for ultraviolet 
studies, since they consist essentially of a naked nucleus approximately 
0.5 n in diameter (Knapp, 1938). Absorption of the radiant energy by 
extranuclear materials is at a minimum, and treatment of the cells can 
be carried out in water. Their use in the determination of dosage rela- 
tions has not been exploited to the extent warranted by the excellence of 
the material. 

Exposure of the male cells of angiosperms can be readily made by treat- 
ing a monolayer of loose, dry pollen. Pollens of maize, Antirrhinum, and 
Gasteria have been successfully employed in ultraviolet studies, the 
irradiated grains being used to fertilize untreated plants. The maize 
pollen grain has both sperm cells fully formed at the time of anthesis. 
One sperm fertilizes the egg to produce the zygote while the other unites 
with the fusion nucleus to give rise to the endosperm. Maize also pos- 
sesses the added advantage of a goodly number of clear-cut endosperm 
marker genes whose presence or absence (or mutated state) can be 
directly determined by examination of the kernels produced on ears pol- 
linated by the treated pollen. These markers facilitate the collection of 
massed data and provide a convenient measure of genetic effectiveness 
when comparative studies are being made of wave-length and dosage 



254 RADIATION BIOLOGY 

Illations. In both maize and Antirrhinum, mutation data may bo 
()btaiiu>d throuj^h tho detection of sefi;re}j;atin}i; characters in F-j popula- 
tions, (instrria has been used only tor the study of induced chromosomal 
changes appearing in the cells of I*\ embryos. 

Compared with the spermatozoids of Hverworts, the pollen grains of 
angiosperms are relatively large, those of maize, for example, being almost 
100 n in diameter. AVhen comparisons are being made of the relative 
effects of dilTerent wave lengths or of different doses of the same wave 
length, it becomes necessary to take into account the factor of internal 
filtration, since the energy incident at the surface of the pollen grain is 
very greatly reduced by absorption in the extranuclear material. Inter- 
nal filtration varies greatly with the wave length of radiation employed 
(ll)er, 1939; Stadler and Uber, 1942), and failure to correct for these 
ditYerences of penetration may lead to gross error in wave-length com- 
parisons. The filtration factor can be roughly calculated, as Stadler and 
Uber have shown, but the difficulties involved stress the need for better 
genetic materials in this area of investigation. 

Cytological studies of ultraviolet-irradiated chromosomes have been 
carried out in the Fi progeny of maize, Gasteria, and Drosophila. The 
chromosomes of surviving Fi individuals represent a selected group from 
which all inviable aberrations have been screened. Through genetic 
technicjues, the types of chromosomal rearrangements induced by ultra- 
violet may be inferred without cytological examination, but there remains 
the possibility that certain aberrations may be eliminated after the pas- 
sage of several cell generations. The pollen-tube technique overcomes 
this difficulty in that it permits a direct examination of irradiated chromo- 
somes before the elimination of inviable changes can take place. The 
technique involves the culturing of pollen tubes on an agar-coated slide, 
with sucrose or lactose added to the agar as a carbon source. The 
generative cell, after passing from the pollen grain into the tube, is 
covered only by a thin cytoplasmic layer and a thin tube wall. Since 
the pollen tube is narrow (approximately 5 ^ in Tradescantia) , the amount 
of radiation absorbed before it reaches the nucleus is not great. The 
chromosomes, undergoing mitotic division in the tube, may therefore be 
readily exposed to ultraviolet, and an analysis of structural changes may 
be made at metaphase by blocking the division with colchicine (Swanson, 
1940, 1942). The method, as first employed with Tra(l(t<can(ia, has been 
materially improved by Bishop (1949). Certain limitations in the tech- 
ni<iue must be recognized, however, if the derived data are to be logically 
compared with those obtained from other organisms. In the first place, 
cytological analysis is made on the heavily condensed metaphase chromo- 
somes; small aberrations such as interstitial deficiencies, if present, are 
quite likely to pass unnoticed. Second, the chromosomes cannot be 
maintained and studied beyond the metaphase stage. Any aberrations, 



GENETIC AND CYTOLOGICAL EFFECTS 255 

therefore, which are reuUzed only at a later stage in cell division would 
not be detected. The absence of ultraviolet-induced translocations in 
the pollen tube chromosomes of Tradcscantia, as contrasted to their 
occurrence in the Fi populations of maize and Gasteria, may well be due 
to the formation of these aberrations at later stages of division or during 
the process of fertilization. 



GENETIC EFFECTS OF ULTRAVIOLET RADIATION 

Guyenot's (1914) early attempt to induce mutations with ultraviolet 
radiation was unsuccessful. Adequate techniques for the quantitative 
screening of mutations were not a\'ailable at the time, and genetic 
knowledge was too scanty to provide a background against which such 
studies could be properly evaluated. After Muller's discovery (1927) 
of the significance of X rays as a mutagenic agent, the question of the 
spectral limits of genetic effectiveness arose. Genetic studies with 
ultraviolet radiation by Altenburg (1928) on Drosophila and Stubbe 
(1930) on Antirrhinum gave no positive indication that this radiation 
could induce mutation. As the techniques of irradiation were improved, 
however, it became apparent that under favorable conditions of exposure 
mutations could be induced in both plants and animals. Early indi- 
cations of effectiveness were reported by Altenburg (1930, 1931), Geigy 
(1931), and Promptov (1932) in Drosophila. Largely as a result of the 
development of the polar cap technique of exposure by Geigy, unequivocal 
confirmation of the mutagenic action of ultraviolet in Drosophila was 
provided by Altenburg (1933, 1934). Noethling and Stubbe (1934) also 
demonstrated in Antirrhinum that exposure of the pollen grains to ultra- 
violet could significantly increase the mutation freciuency. 

Experiments with Drosophila. A summary of Altenburg's Drosophila 
data is given in Table 7-1. From these figures it is clear that a significant 
increase in the frequency of recessive lethals can be obtained by exposure 
to ultraviolet. As might be expected on the basis of penetration, eggs in 
the polar cap stage are readily affected. The replicated lethals appearing 
in 5 per cent or more of the Fi females from a single male represent mutations 
occurring in pole cells at the time of treatment, and their frequency is 
clearly increased by irradiation. The distinct increase in "isolated" 
lethals (i.e., lethals that appear singly) is assumed to be due, at least in 
part, to the inclusion of some eggs beyond the polar cap stage at the time 
of treatment. 

The results of Geigy (1931) and Promptov (1932), on the induction of 
recessive lethals in eggs of Drosophila, are in essential agreement with 
those reported by Altenburg (1934). Both Altenburg and Promptov 
noted an increased incidence of visil)le mutations, in frequencies con- 
siderably lower than for se.x-linked lethals. 



256 



RADIATION HIOLOGY 



Each of the 79 lothals rccordctl in 'I'ahle 7-1 was t(^stod for position on 
the X chromosome and found to he unaccompanied by disturbances of 
crossover frequency with neifj;hboring loci. The following conclusion 
was drawn: "Ultraviolet light therefore produces no inversions or other 
chromosomal changes that are detectable from changed crossover values. 
In this respect, the effect of ultraviolet light differs markedly from that of 
X rays" (Altenburg, 1934). 

Table 7-1. Summary of Ai.tenburg'.s (1934) Drosophila Data in Which 
Ultravioi.kt Radiation Was Shown' to Be Mutagenic in Nature 





Number 
treated 


Tyi)e.s of recessive lethals in Fi otTsi)ring 


Total 
lethals 


Stage 
treated 


Induced in 
5% or more 


Spontaneous in 
5% or less 


Isolated 


Adults: 

Control 

Treated 

Larvae : 

Control 


8694 
92.39 

3094 
3098 

222" 
239" 


1 
13 


2 
3 


13 
32 


2 
24 

1 


Treated 

Eggs: 

Control 


7 
16 


Treated 


48 







" From th(> 222 control eggs, 13,063 Fi females were tested; from the 239 treated 
eggs, 14,059 Fi females were tested. 

The development of the abdominal exposure techiii(iue by Reu.ss (1935) 
demonstrated the feasibility of inducing mutations in the matiu-e sperma- 
tozoa of Drosophila. Mackenzie and :\Iuller, using this method, have 
confirmed and extended the earlier findings of Altenburg, particularly as 
concerns the comparison of the effects of ultraviolet and X rays (MuUer 
and Mackenzie, 1939; Mackenzie and Muller, 1940; Mackenzie, 1941). 
The filtered radiation employed consisted of wave lengths above 280 m^u, 
a (luality of radiation less damaging physiologically than the shorter 
wave lengths. A dose of 2 X 10'^ ergs/mm^ w^as found to be optimum 
for the study, since an appreciable frequency of sex-linked lethals (about 
3 per cent) was induced without an accompanying high degree of sterility 
or mortality. Higher doses raised the frequency of lethals to 9 per cent, 
but the sterility was disproportionately increased, making extensive 
observations difficult and (juantitative comparisons unreliable. The 
principal results of these studies were the following: 

1. No translocations affecting the Y, II, and III chromosomes were 
found in a population which had a frequency of sex-linked lethals of 4.3 
per cent. Such a fre(iuency would be induced by an X-ray treatment of 
about 1300 r. The authors estimate that, in the number of culttn-es 



GENETIC AND CYTOLOGICAL EFFECTS 257 

tested, 40 or more translocations of type II-III alone would have resulted 
from this X-ray dose. I'hese data were in agreement with an earlier 
trial in which no translocations were found from treatments which induced 
sex-linked lethals in such numbers as to indicate an expectancy of at least 
25 detectable translocations, on the basis of the relation found with 
X rays. The discriminatory action of the ultraviolet was thus shown by 
the absence of detectable translocations in cultures in which at least 65 
were to be expected if the relation of mutation and gross chromosomal 
rearrangement were the same with ultraviolet as with X rays. 

2. Similar evidence relating to the occurrence of minute rearrangements 
was obtained by the use of a special technique, with w^hich minute rear- 
rangements are induced by X rays in relatively large numbers and are 
recognizable by mutants at specific loci. In ultraviolet-treated cultures 
yielding sex-linked lethals at a rate corresponding to an X-ray dose of 
1000 r, no mutants at these loci were found. In an X-ray experiment by 
MuUer and Makki (Mackenzie and MuUer, 1940), these had occurred at 
significant freciuencies following dosage of 1000 r. MuUer and Mackenzie 
concluded provisionally that the ultraviolet does not produce minute 
rearrangements, or at least that it is far less efficacious in this respect than 
X rays. 

3. Wave lengths above 320 m^ were found to be ineffective in inducing 

mutations. 

4. The frequency of mutations was higher when mating of the irradi- 
ated males followed immediately after treatment. No mutations were 
transmitted 5 days after irradiation although the supply of mature sperm 
would not have been exhausted for some days thereafter. Those irradi- 
ated flies which bred but* died early had higher frequencies of mutations 
than those which continued to breed over longer periods of time. A 
correlation was established therefore between the frequency of induced 
mutations and the amount of physiological damage as determined by the 
duration of the fecund period. Both phenomena are undoubtedly 
affected by the degree of penetration of ultraviolet, a factor which varies 
widely among similarly exposed individuals. 

5. Dose fractionation was without effect on the frequency of mutations 
or the degree of sterility. 

The occurrence of minute deletions among the mutants induced by 
ultraviolet was cytologically demonstrated by Slizynski (1942). Among 
21 of the sex-linked lethals produced in the experiments of Mackenzie and 
Muller, 5 Avere found to be cytologically detectable deficiencies, 1 involv- 
ing the loss of 1 band, 3 the loss of 2 bands, and 1 the loss of 14 bands. All 
were interstitial deficiencies. 

Results of studies by McQuate (1950) support the hypothesis of 
Mackenzie and Muller that terminal deficiencies, if produced, are not 
recovered in Drosophila populations derived from mature spermatozoa 



258 RADIATION BIOLOGY 

which have boon exposed to ultraviolet, r.sing a stock containing a 
spcci.-i! ^■ chromosome (y''Y') marked with the normal allele of achaeto, 
he matccl irradiated males to achacte females, 'rerminal l«»ss of th(> nor- 
mal allele of acliaet(>, with retention of the remainder of the special Y 
chromosome, would yield fertile achaete males in the Fi population, 
l-'rom a total of H),;i01) h\ males, 23 sterile achaete exceptions were found. 
Such males, however, result from a loss of the paternal X chromosome, or 
of all or part of both arms of the y-'-Y^ chromosome, losses which could 
arise through lagging of the chromosomes in division, or by breakage 
followed by fusion of broken ends to give acentric and dicentric portions 
having a low survival probability. Breakage of the y^-Y^ chromosome, 
with loss of the y^ region (which also includes the normal allele of achaete) 
and foUow-ed by healing, did not occur. The fertile achaete exceptions, 
two in number, were no more frequent than in the control population. 

Experiments with Plants. In many species of plants the pollen grain 
may be effectively treated with ultraviolet radiation. It is therefore 
feasible to make somewhat simpler tests of the genetic effects of the treat- 
ment than can be made in the experiments with Drosophila. The indirect 
analysis required by the polar cap technique is avoided, and the difficul- 
ties from internal filtration, while serious, are not nearly so great as in 
the irradiation of the sperm within the body of the adult fly. In the cul- 
tures grown from seeds produced by the use of the irradiated pollen (which 
we may, for convenience, refer to as the Fi cultures), each plant provides 
the material for testing the effects of the treatment on one irradiated 
gamete. Dominant effects of chromosome or gene changes induced by 
the treatment may be observed in the Fi plants, and each F2 culture pro- 
duced by self-fertilization of one of these plants shows segregation for any 
haplo-viable recessive alteration induced in the gamete tested. 

The results reported by Noethling and Stubbe (1934) clearly demon- 
strated the effectiveness of ultraviolet in inducing mutations. These 
were detected in segregating F2 populations of Aniirrhinum. 

A similar increase in the frecjuency of point mutations was found in 
maize by Stadler and Sprague (193(m), together with evidence of certain 
chromosomal effects of the treatment and further indications of differ- 
ences in the genetic action of ultraviolet radiation and X rays. The 
mutations identified were only those affecting seed and seedling charac- 
ters. Progenies representing various doses of unfiltered ultraviolet radia- 
tion yielded 31 mutations from 830 gametes tested, and control progenies 
yielded 6 mutations from 557 gametes. 

This increase in mutation rate, while clearly significant, is not large. 
But among the 31 mutants detected (of which 9 would have been expected 
without irradiation), there were two cases in which 2 unlinked mutants 
occurred in a single F2 progeny, and one case w^ith 3 unlinked mutants in a 
single progeny. These represent cases of two or three presumably unre- 



GENETIC AND CYTOLOGICAL EFFECTS 259 

lated mutants in a single treated gamete. In addition, three of the other 
mutants occurred in the progeny of plants segregating for defective pol- 
len. The independent inheritance of the mutants, in these and several 
similar cases, has been shown by Sprague (1942). The degree of coinci- 
dence is far beyond that expected by chance, if all the tested gametes 
received an equal dose. But the treated gametes must receive 
quite unequal doses, for the sperm nuclei are eccentrically located 
in the spheroidal pollen grains, and the loss by internal filtration must 
vary widelj^ with the casual orientation of the individual grains. If this 
is the explanation of the coincidences observed, the frequency of mutation 
in the most effectively exposed pollen grains must be very high. In later 
experiments with more effective ultraviolet treatments, mutation rates of 
about 20 per cent have been reported (Stadler, 1941b). These are sub- 
ject to the same limitation by casual orientation of the pollen grains 
treated, and the results suggest that the effect of ultraviolet radiation on 
mutation frequency in maize, in individuals that are effectively treated, 
may be well beyond that produced by X rays. 

A technical advantage of the maize material is the availability of many 
endosperm characters of known inheritance. In matings with appro- 
priate marker genes, the loss of the effect of dominant alleles present in 
the male parent may be detected at once by phenotypic changes in the 
endosperms of the seeds produced. Linked endosperm characters, deter- 
mined by genes located on one arm of one of the chromosomes, permit the 
detection of deficiencies in this region. 

Ultraviolet treatment applied to the pollen greatly increased the fre- 
quency of endosperm deficiencies, as detected by loss of the linked factors 
C and Wx. The frequency of loss of other dominant genes for endosperm 
characters {A, Pr, Su) was similarly increased. There are no linked 
genes for endosperm characters suitable for determining whether A, Pr, 
and Su losses represent deficiencies. The fact that ultraviolet-induced 
loss of C and Wx is usually coincidental indicates that the endosperm 
effect is due usually, if not always, to deficiency rather than to gene muta- 
tion. Since no genetic analysis can be made, the identification of an 
individual case as a recessive mutation rather than a deficiency could not 
be positively established. The cases are referred to as "endosperm 
deficiencies," with the reservation that there may be included among 
them an unknown proportion of losses of dominant characters as a result 
of gene mutation. 

The endosperm deficiencies resulting from ultraviolet treatment of pol- 
len included a large proportion of fractionals, in contrast to those resulting 
from X-ray treatment of pollen, which are largely deficiencies affecting 
the entire endosperm. 

The occurrence of deficiency in the Fi plants was not determined by 
means of marker genes or cytological examination in this experiment. 



260 RADIATION BIOLOGY 

The ahsonco of gross deficiency or translocation could Ix' assumed it' all 
the Fi plants were free from segregation for defective pollen. It was 
found, however, that, among about lOOO Fi jjlants examined, almost 4 
per cent, showed segregation for defective pollen, ascrihahle to deficiency 
or to mutations affecting pdjlcn dcxclopment. Later studies showed 
that mutations affecting pollen development are a frecjuent result of 
ultraviolet treatment, and lh;it there are also cytologically demonstrable 
deficiencies induced by the treatment. 

No translocations were found among the Fi plants characterized by 
defective pollen segregation. In later studies, however, translocations 
were found, though in very low freciuency. The rarity of translocations 
was given special study because of their very high frequency in compara- 
ble X-ray progenies. Since the frequency of deficiency was lower under 
the ultraviolet doses used than under the X-ray doses commonly applied, 
it is possible that the difference in effect on translocation is incidental to 
dosage. If translocations result from chromosome breakage followed by 
reattachment of broken ends in new combinations, the rarity of transloca- 
tion following ultraviolet treatment might be due to the smaller number of 
iireaks produced by the ultraviolet dose applied. A further trial was 
made (Stadler and Sprague, 1937) in which a maximal dose of ultraviolet 
was compared with a rather low dose of X rays, these doses being approxi- 
mately equal in total frequency of induced deficiencies for the endosperm 
genes A and Pr. The frequency of translocation was determined for each 
treatment by direct cytological examination of about 100 unselected 
plants of the Fi progeny. Only one translocation was found in the ultra- 
violet progeny, while 44 per cent of the plants of the X-ray progeny showed 
translocations, several of them two or more independent translocations. 

Since it w^as subsequently found that the frecjuency of deficiencies in 
endosperm and embryo is very different under ultraviolet treatment, this 
comparison represents doses very unequal in frequency of induced defi- 
ciencies in the embryos. It therefore does not test the possibility that 
the rarity of translocations under ultraviolet treatment may be due to the 
smaller number of chromosome breaks produced. It is clear, however, 
that, for doses equal in frequency of induced mutation, the frequency of 
induced translocation is much greater with X rays than with ultraviolet 
radiation. 

Preliminary comparisons of w^ave-length effectiveness were made in the 
early maize experiments (Stadler and Sprague, 193Gb, c), using the radia- 
tion from a commercial mercury-vapor arc with three filters of mercuric 
chloride solution of varying concentration, and using also the radiation 
from a commercial mercury discharge tube. The relative effectiveness of 
the various wave lengths was inferred from the frequency of induced 
endosperm deficiencies, in terms of the spectral distribution of energy in 
the filtered radiation applied. The indications regarding wave-length 



GENETIC AND CYTOLOGIC AL EFFECTS 261 

dependence were quite different from those found in the experiments of 
Noethling and Stubbe (1936) with monochromatic irradiation of Antir- 
rhinum pollen. The results indicated (1) that wave lengths 313 m^ and 
longer were relatively ineffective, (2) that wave length 302 m^ was geneti- 
cally effective but less effective than wave lengths 297 m/x and shorter, 
and (3) that wave length 254 m^u was much more effective than 297 mju. 
Subsequent studies with monochromatic radiation, reviewed in a later 
section, confirmed these indications. The maize data are for the fre- 
quency of endosperm deficiency, while the Antirrhinum data are for the 
frequency of point mutations. However, tests of mutation rate in the F2 
progenies indicated the same spectral relations for mutations as for 
endosperm deficiency, as far as could be determined from filtered radia- 
tions (Stadler, 1941a). 

The frequency of induced embryo abortion was much lower with the 
longer ultraviolet wave lengths than with the shorter. Comparing doses 
approximately equal in frequency of induced endosperm deficiencies, the 
frequency of induced embryo abortion was about nine times as high for 
the discharge-tube radiation (chiefly wave length 254 m^u) as for the 
filtered radiation (chiefly wave lengths 297 mn and longer). 

The frequency of endosperm deficiencies induced by ultraviolet treat- 
ment of the pollen is very much higher than the frequency of deficiencies 
affecting the embryos of the same seeds (Stadler, 1941a). Both values 
can only be estimated, but the discrepancy is too great to be accounted 
vor by any possible error in the estimates. 

With maximal doses of the longer wave lengths, the frec^uency of endo- 
sperm deficiencies marked by A, Pr, and Su sometimes exceeds 40 per 
cent. Since these marker genes can detect only a part of the deficiencies 
occurring in 3 of the 20 chromosome arms, there are presumably several 
hundred endosperm deficiencies per hundred seeds. These represent the 
deficiencies (and perhaps gene mutations) realized under the conditions 
of endosperm development, from alterations induced in one of the sperm 
nuclei of the treated pollen grains. The embryos of the same seeds may 
be checked to provide a maximal estimate of the frec^uency of deficiencies 
realized under the conditions of embryo development, from alterations 
induced in the other sperm nuclei. By this check, every sperm nucleus 
in the tested population of treated pollen grains may be accounted for. 
Assuming that every aborted embryo, every plant which failed to yield a 
pollen specimen, and every plant which showed segregation of defective 
pollen represents an induced deficiency, the maximum estimate of defi- 
ciencies in the gametes tested by embryo constitution is only about 30 
per cent. 

A similar comparison may be made for endosperm deficiencies and 
embryo deficiencies marked by specific genes affecting both endosperm 
and plant characters. Endosperm deficiencies of .4 are very common, 



202 KAUIATION H1()L(KJY 

occurring at the doses here considered with fre(iueiicies as high as 20 per 
cent. Deficieney of A in the Fi seedlings is rare; a series of progenies 
grown from seeds which inchuled H)S endosperm deficiencies for A yielded 
only five -1 deficiencies in the Fi plants. 

Approximately three-fourths of the endosperm deficiencies are frac- 
lionals, commonly affecting about half of the endosperm. Deficiencies 
similarly affecting half of the pro-embryo would presumably be present 
in the resulting Fi plants in only about half of the alTected cases, and this 
proportion might be reduced by competitive development of the defective 
and nondefective sectors. But the proportion of fractionals, as shown 
by the endosperm deficiencies, cannot account for the discrepancy; 
among the seeds which yielded the five A deficiencies mentioned in the 
preceding paragraph, there were more than 100 with nonfractional A 
deficiencies in the endosperm. 

This pronounced disproportion between the frequency of deficiencies 
in the endosperm and the embryo does not occur wuth X-ray treatment. 
Ill cultures marked by specific genes affecting both endosperm and plant 
characters, deficiencies in the Fi plants are somewhat less frequent than 
in the endosperms, but not more so than might possibly be accounted for 
by reduced survival. 

Aside from possible differences in the exposure to ultraviolet of the two 
sperms in the treated pollen grain, a possible cause of the wide disparity is 
contrasting behavior of chromosome l)reaks in endosperm and embryo 
development. McClintock's study (1941) of the effects of mechanically 
broken chromosomes suggests that some alterations induced in the two 
sperms, though similar in character and in frequency, might have quite 
disproportionate effects upon the frequency of detectable changes in the 
embryos and endosperms of the resulting seeds. 

This possibility has l)een investigated by Schultz (1951), using a ring 
chromosome {Dp 3a) carrying the gene A''. The pollen treated was that 
of a Dp 3a stock homozygous for a deficiency at the A locus, a-X3, a type 
in which all functioning pollen carries the duplication (Stadler and Roman, 
1948). Pollinations on ears of aa constitution show the loss of A'' in 
endosperm and embryo tissue by the absence of anthocyanin pigment. 
With untreated pollen a small percentage of deficiencies occurs in both 
endosperm and embryo, owing to losses of the ring in late microspore or 
early endosperm or embryo divisions. The sporadic loss of the ring in 
subse(iuent mitoses results in variegation in the remaining endosperms 
and plants. 

The result of a break in the ring chromosome differs from that of a 
break in a rod chromosome chiefly in the effects of restitution. If the 
break may be followed alternatively by restoration t)f the linear order 
between the original chromatids or crosswise between the sister chroma- 
tids, the effect in a rod chromosome would be undetectable in both cases. 



GENETIC AND CYTOLOGICAL EFFECTS 2C3 

But, in the ring chromosome, reproduction must take place in a single 
plane; otherwise the daughters either will be interlocked or will form a 
double ring with two centromeres. "Crosswise" restitution in the ring 
chromosome is therefore likely to result in loss of the ring. 

Schultz (1951) found that ultraviolet treatment very greatly increased 
the frequency of loss of the ring, both in the endosperm and in the embryo. 
The frequency of deficiencies for A'' among the Fi plants was about as 
high as the frequency of entire (i.e., nonfractional) endosperm deficiencies. 
This indicates that the low frequency of broken chromosomes found in 
plants from irradiated pollen, as compared with the endosperm, is largely 
due to a higher rate of restitution in the plant. 

Another interesting result of the study was the absence of any evidence 
of the production of a rod chromosome from the ring. The type of break 
which would ordinarily result in a stable terminal deficiency should, if it 
occurred in a ring chromosome, convert the ring to a rod. This would be 
detectable as a nondeficient nonvariegated plant. Among more than a 
thousand seedlings in progenies from ultraviolet-treated pollen, no such 
plant was found, although the number of A* deficiencies presumably 
induced by the treatments was almost 200. This result is not in conflict 
with the observed occurrence of terminal deficiencies in other studies. 
The frequency of deficiencies for a given locus as observed in the Fi 
plants is extremely low, and the primary breaks or potential breaks which 
occur in a rod chromosome proximal to the locus concerned may be much 
more numerous than those which would occur within the small ring. 
Presumably the proportion of potential breaks resulting in deficiencies 
realized in the embryo may be well below one in 200. 

The results with the ring chromosome show that the frequency of poten- 
tial breaks induced by ultraviolet radiation is extraordinarily high, and 
that in the embryo all but an extremely small proportion of these are fol- 
lowed by restitution. The types of chromosomal alterations found in the 
deficient plants that make up this small proportion may be quite mislead- 
ing as to the primary chromosomal effects of the radiation. 

The frequency of deficiencies detectable in the endosperm following 
ultraviolet treatment is comparable with that found following X-ray 
treatment. The two agents differ widely in the relative frequency of 
deficiencies affecting the endosperm as a whole and deficiencies affecting 
only a part of the endosperm ("fractionals"). With ultraviolet, about 
75 per cent of the endosperm deficiencies observed are fractionals, and the 
fraction showing the deficiency is most frequently about one-half of the 
endosperm, as estimated by the surface area. The deficient sectors vary 
widely in form and in relative size, sometimes covering only a small frac- 
tion of the surface and sometimes covering the entire surface except for a 
small fraction. The frequency distribution of these fractions of varying 
size is approximately normal about the modal class of )^. Presumably 



2()4 RADIATION BIOLOGY 

they represent the distribution of tissue resulting from chjinoe variations 
in cleveh)pment, when one cell in the two-celled pro-endosperm is deficient. 

With X rays, the endosperm deficiencies affect the entire endosperm in 
most cases. There is a substantial minority of fractionals, but these in 
general are strikingly different in pattern from those observed with ultra- 
violet. In most of them, the deficient portion co\'ers the entire surface of 
the endosperm except for one or more islands of tissue, usually amounting 
in total to only a small fraction of the entire area. These cases in the 
X-ray material cannot be regarded as merely the extremes of a range of 
patterns resulting from random variations in development of the half- 
deficient endosperm; there is no corresponding frecjuency of half-and-half 
mosaics and their variants. The distribution of tissue in the fractionals, 
as observed in the mature endosperms, suggests that in the ultraviolet 
cases the nondeficient tissue is the sector derived from one cell of the two- 
celled pro-endosperm, but in the X-ray cases it is the sector derived from 
only one or a few cells at a more advanced stage of the pro-endosperm. 
The distribution of tissue is as would be expected if a chromosome frag- 
ment could occasionally escape elimination through one or more cell 
divisions, and then be restored to normal mitotic distribution. 

The elucidation of the breakage-fusion-bridge cycle in mechanically 
broken chromosomes by McClintock (1941) suggests plausible hypotheti- 
cal mechanisms by which such endosperm patterns could be produced, 
though the sequelae of mechanical l)reakage do not parallel those of 
either X-ray or ultraviolet alteration of the chromosome. 

A comprehensive study of the chromosomal effects of ultraviolet and of 
X rays, as shown by the mosaic patterns in the maize endosperm, is being 
made by Faberge (1951). Using the endosperm marker genes /, Sh, Bz, 
and Wx, all located in a single chromosome arm, a variety of chromosomal 
effects may be recognized, including rings, dicentric translocations, and 
inversions, if accompanied by a breakage-fusion-bridge cycle. Ultra- 
violet treatment produces all these aberrations in large numbers, as does 
X-ray treatment. 

Cytological studies of the chromosomal effects of ultraviolet in maize 
have been made by Singleton (1939), Singleton and Clark (1940), and 
De Boer (1945). The accounts of these studies have been published only 
in abstract form. Singleton (1939) examined Fi plants identified by the 
loss of dominant characters present in the treated male parent and found 
cytologically demonstrable deficiencies of the corresponding chromosome 
regions in four plants. In another series, cytological examination of Fi 
plants identified by segregation for defective pollen showed deficiencies 
in several cases, including one plant with deficiencies for parts of two 
chromosomes. All the deficiencies observed appeared to be terminal. 
No translocations were found. Singleton and Clark (1940) found, among 
16 Fi plants with segregation for defective pollen, 8 with observable 



GENETIC AND CYTOLOGICAL EFFECTS 265 

deficiencies and 8 without. In addition there was one "dehciency trans- 
location, . . . three-armed, the plant being deficient for parts of chromo- 
somes 1 and 10." De Boer (unpublished) found several similar cases of 
deficiency and deficiency translocation. 

Although all the deficiencies appeared to be terminal, the distinction 
between terminal and nonterminal deficiencies is not convincing in maize 
without critical cytological material. Nonhomologous pairing may give 
a known interstitial deficiency the appearance of a terminal deficiency. 
De Boer (19-15) has presented evidence of ultraviolet-induced terminal 
deficiency free from this difficulty. The gene bz is a plant color gene 
located on the short arm of chromosome 9, and stocks of maize are avail- 
able bearing a terminal knob on this arm. Ultraviolet- and X-ray- 
induced deficiencies of Bz, in a stock with the terminal knob, were exam- 
ined cytologically, the criterion of terminal deficiency being loss of the 
terminal segment including the knob. Nonhomologous pairing of an 
interstitial deficiency could result in a terminal unpaired region of the 
untreated chromosome, but would be recognizable by the presence of the 
knob, or a portion of the knob, on the deficient chromosome. Among six 
Bz deficiencies in the ultraviolet series, four showed terminal deficiency of 
the short arm of chromosome 9. The other two were deficiency translo- 
cations. Among nine Bz deficiencies in the X-ray series, none was a 
terminal deficiency. These alterations included one interstitial defi- 
ciency, three ring-9 configurations, and five deficiency translocations. 

Straub (1941), in a study of somatic metaphase chromosomes, has 
shown that ultraviolet-induced translocations occur with appreciable fre- 
quency in Gasteria. From some 1800 embryos obtained from the fertili- 
zation of untreated eggs by exposed sperm, 210 were selected for cytologi- 
cal study because their weak development suggested that they might 
possess chromosomal abnormalities. Of these, 75 showed chromosomal 
changes as contrasted to 1 from 300 control embryos. Four of the 
embryos were chimeras showing some cells with normal and some with 
altered chromosomes. Straub considered these to be similar to the frac- 
tional endosperm deficiencies induced by ultraviolet in maize. In the 
remaining 71 embryos, 72 apparently terminal deficiencies were detected 
in the long arms of the four G chromosomes, which could be recognized 
by their conspicuous satellites. The breaks giving rise to these deficien- 
cies were largely concentrated in the neighborhood of the centromere. Of 
the remaining cytological abnormalities, five were translocations, includ- 
ing one of an undefined but probably reciprocal type, two which were 
defined as "isochromosomes," one dicentric chromosome, and one ring 
chromosome. 

Barton (1954) has compared the chromosomal effects of X-ray and 
ultraviolet treatments in the tomato {Ly coper sicum esculentum Mill.). 
In this plant each of the 12 chromosomes at pachytene shows a densely 



200 RADIATION UIOLUGY 

staining chromatic n'nioii on cither .side of the chromomere and a Hghtly 
staiiiiiifj; achromatic rc}>ioii (list ally. Each chromosome arm terminates 
in a sinfile well-delined chromomere. This circum.stance is very favorable 
for the detection of terminal deiiciencies, especially those in which the 
break is in the achromatic region. 

Barton found a nnicli higher ratio of deficiencies to translocations with 
ultraviolet than with X rays. The two translocations found with ultra- 
violet were deficiency translocations. Mutations also showed a much 
higher ratio to translocations with ultraviolet. The deficiencies found 
with both ultraviolet and X-ray treatment included both terminal and 
interstitial deficiencies. Chromosome breakage was highly localized in 
the chromatic regions; however, the terminal deficiencies (observed in the 
ultraviolet series included one in which the break was in the achromatic 
region. 

The various contrasts in genetic effects which ha\e been mentioned 
indicate that the chromosome breaks induced by ultraviolet radiation are 
of a (lualitatively different kind from those induced by X rays. Accumu- 
lating evideiice from many sources has indicated that the mutations 
induced by X rays are in many cases, if not in all, extragenic alterations 
incidental to chromosome breakage. The indication that the chromo- 
somal effects of the ultraviolet are of a different kind encourages the hope 
that the induced mutations also may be (lualitatively different. 

This possibility may be investigated effectively only by the critical 
study of the mutation of specific genes, for there are no general criteria by 
which mutations induced at miscellaneous loci may be distinguished from 
the possible effects of known extragenic phenomena. AVith certain 
selected loci it may be possible to develop special criteria for the identifi- 
cation of gene mutations and for the recognition of alterations which, in 
experiments on the general mutation rate, would .simulate gene mutation. 

A comparison of X-ray and ultraviolet mutations of the gene .4 in 
maize (Stadler and Roman, 1943, 1948) indicates that the mutations 
induced by the two agents may be qualitatively different. Among about 
400 alterations affecting A, induced by X rays, and a much smaller num- 
ber induced by ultraviolet, those most nearly approaching the typical 
genetic behavior of gene mutations were selected for detailed study and 
comparison. Among the X-ray alterations, only two were normal plants 
free from segregating pollen defects. A third haplo-viable X-ray altera- 
tion, showing segregating for subnormal but not aborted pollen, was 
included for comparison. Among the ultraviolet alterations, normal 
plants with normal pollen were more frequent. Three cases with a pheno- 
type and one with intermediate phenotype (.4") were included in the 
comparisons. 

The three X-ray mutants were characterized in varying degree by 
reduced viability in haplopha.se and by reduction in the frequency of 



GENETIC AND CYTOLOGICAL EFFECTS 207 

crossing over, and were found to be inviable as homozygotes or com- 
pounds. These are attributes suggestix-e of deficiency, and all three 
mutants wei'e identified as deficiencies by f2;enetic evidence showing that 
the induced alteration in each case involved loss of the effects not only of 
A but of additional genes affecting chlorophjdl de\'elopment and viability. 
The four ultraviolet mutants gave no indication of deficiency by any of the 
criteria mentioned. 

The evidence for Drosophila comparing the genetic effects of X-ray and 
ultraviolet radiation contrasts somewhat in its general implications with 
that from plants. It would not be surprising to find actual contrasts, 
but the evidence from both sources is still too scanty to force the assump- 
tion of any basic difference in the nature of the chromosomal or genie 
alterations induced. The contrasting indications are briefly noted as 
follows : 

1. Gross chromosomal rearrangements. Asshownby Altenburg (1934) 
and by Muller and Mackenzie (1939), there is no appreciable frequency 
of gross chromosomal rearrangements in ultraviolet progenies which yield 
an abundance of sex-linked lethals. Demerec et al. (1942) found a single 
translocation in the progeny of ultraviolet-treated flies, but this single 
case cannot be considered evidence of an effect of the treatment. The 
absence of induced translocations in the Drosophila cultures tested does 
not represent a conflict in the evidence, for there is no necessary implica- 
tion that the radiation is unable to induce translocation in low frequency. 
The Drosophila evidence shows that the ratio of induced translocations to 
induced sex-linked lethals is far lower with ultraviolet than with X rays. 
The maize evidence also shows that the ratio of translocations to muta- 
tions is far lower than with X rays, and shows further that translocations 
are induced in very low frequency. 

A search for terminal deficiencies of the X chromosome, identifiable by 
genetic markers, was made by Mackenzie and Muller (1940) and by 
McQuate (1950), and none was found. The occurrence of ultraviolet- 
induced terminal deficiencies in maize was shown cytologically by De Boer 
(1945). 

2. Nature of the induced mutations. Although genetic experiments to 
detect minute rearrangements, by Mackenzie and Muller (1940), indi- 
cated that they were absent or very rare in ultraviolet progenies, the direct 
cytological study of induced lethals by Slizynski (1942) clearly showed 
that short deletions are included in this class. The frequency of cytologi- 
cally detectable deletions among the sex-linked lethals was not much lower 
among the ultraviolet than among the X-ray cases. They occur also, in 
considerable frequency, among sex-linked lethals arising in untreated 
material. 

The implication from the evidence in maize is that the ultraviolet 
mutants are distinctlv different from the X-rav mutants in that the 



268 RADIATION mOT-OOY 

former show no evidence of deliciency, while the hitter Iik ludc dearly 
detectable deficiencies. The evidence of deficiency here is genetic rather 
than cytolot!;ical. The only critical evidence is from the induced muta- 
tions of A studied by Stadler and Roman (1948), and here the number 
of cases is too small to imply the absence or extreme rarity of short 
deficiencies among ultraviolet mutations. The fact that no interstitial 
deficiencies have been found in cytological studies of ultraviolet progenies 
in maize cannot be considered evidence of their extreme rarity, for 
here again the only critical evidence, that of De Boer (1945) on Bz 
deficiencies, relates to a relatively small number of cases. It is dear 
that ultraviolet radiation induces terminal deficiencies in maize, but it 
is possible that it may induce interstitial deficiencies also, and that some 
of these may be included among the alterations genetically identified as 
mutations. 

It should be noted also that the alterations identified as mutations in 
Drosophila and in maize may not be analogous classes. The sex-linked 
lethals spontaneously occurring in Drosophila appear to be qualitatively 
identical with sex-linked lethals induced by X rays, and the ultraviolet 
lethals are not dearly distinguishable from either class. In maize, the 
evidence of difference in the type of mutation induced by the two agents 
comes from studies at a specific locus, at which the X-ray mutants are 
found to be distinctly different from the spontaneous mutants. Here the 
ultraviolet mutants are found to be dearly distinct from the X-ray 
mutants and similar to the spontaneous mutants. 

DIRECT EFFECTS OF ULTRAVIOLET RADIATION ON CHROMOSOMES 

The pollen-tube technique, as employed with Tradescantia pollen, per- 
mits a study of the direct effects of ultraviolet on chromosomes before 
inviable aberrations can be eliminated. As discussed earlier, the tech- 
nique has certain inherent limitations in that only those aberrations 
realized by the onset of metaphase can be recognized; any which would 
form at later stages of cell division, or during the process of fertilization, 
would escape detection. The chromosomes in the generative nucleus are 
effectively double to both X rays and ultraviolet, and the aberrations 
induced are consequently of the chromatid types. 

In the course of these studies, over 50,000 chromosomes have been 
examined, and approximately 700 terminal deletions have been identified 
(Swanson, 1940, 1942, 1943). With the exception of occasional iso- 
chromatid deletions or chromatid translocations, which were no more fre- 
quent than in untreated nuclei, the aberrations were all terminal. No 
interstitial deficiencies have been identified with certainty, but it is 
realized that the method of analysis is such as to preclude their positive 
identification. The great majority of the deficiencies involved the break- 



GENETIC AND CYTOLOGICAL EFFECTS 



269 



age of only one of the two sister chromatids, although infrequently half- 
chromatid deficiencies were observed (Swanson, 1947). 

The frequency of terminal deficiencies increases linearly with increasing 
dosage if the time of exposure after the pollen grains have germinated is 




15 30 



60 



240 



120 

EXPOSURE, sec 
Fig. 7-1. The relation of chromatid deficiencies (terminal deletions) to the dosage of 
ultraviolet (wave length 254 m/u) in the pollen tube chromosomes of Tradescantia. 
Radiation given at a distance of 20 cm, at an intensity of approximately 10^ ergs/ 
mm^/60 sec, and at 2 hr after germination. (Sivanson, 1942.) 




5 10 

TIME AFTER GERMINATION, hr 

Fig. 7-2. The relation of chromatid deficiencies (terminal deletions) induced by ultra- 
violet (wave length 254 lUfx) in the pollen tube chromosomes of Tradescantia to succes- 
sive prophase stages following germination. Dosage approximates 2 X 10' ergs/ 
mm^'/eO sec. (Swanson, 1943.) 

kept constant (Fig. 7-1). With increasing condensation of the chromo- 
somes as they pass through prophase development, the frequency of 
deficiencies induced by any given dose declines, however, suggesting that 
internal changes taking place within the chromosome play a role in deter- 
mining its susceptibility to breakage by ultraviolet (Fig. 7-2). A ques- 



270 RADIATION HIOLOGY 

lion may Ix' raised concerning the relali\c resistance of the chromosomes 
to breakage at the 0- and 1 -In- periods. At these time periods the genera- 
tive nuclei have not, as a rule, passed from the pollen grains into the pollen 
tubes, and some uncertainty exists as to the degree of absorption of ultra- 
\iolet by the heavily pigmented pollen wall. At the 2-hr period, however, 
the nuclei are in the pollen tube, where absorption by overlying materials 
is at a minimum. After the 10-hr period the fretiuency of induced 
aberrations does not exceed that found in untreated nuclei. This period 
corresponds roughly to late prophase. 

In addition to the terminal deficiencies induced by ultraviolet in the 
pollen tube chromosomes of Tradescantia, there is also found a type of 
aberration which, for want of a more definite term, has been called an 
" achromatic lesion " (Swanson, 19-40). This type of aberration is induced 
by X rays also. The lesions extend completely or partially across the 
chromatid in the form of a nonstainable gap. Their fre(iuency increases 
with increasing dosage. Whether they represent incompletely separated 
deficiencies, interstitial losses of chromatin, or merely separated coils 
within the chromosome is not known. Since many of them extend only a 
part of the way across the diameter of the chromatid, a large subjective 
error would be involved in any determination of frequency, and for this 
reason they have been omitted in the tabulated data. 

The nature of the ultraviolet-induced deficiencies in the pollen tube 
chromosomes of Tradescantia suggests that, structurally at least, they are 
comparable to the fractional endosperm deficiencies in maize even though 
the changes are induced in dissimilar types of nuclei. Each involves the 
loss of a portion of a chromatid. No aberrations were found in Trades- 
cantia, however, which involved both chromatids, and which would corre- 
spond to the entire endosperm deficiencies. Whether this difference can 
be ascribed to differences in the nuclei studied, to their different states of 
chromosome condensation, or to some unknown factor cannot be answered 
at present. ]Muller (1941) suggests that the preponderance of fractional 
endosperm deficiencies in maize treated with ultraviolet may result from 
a mutational process initiated in a single-stranded chromosome but 
delayed in completion until the chromosome has doubled, the effect being 
restricted ordinarily to only one of the two chromatids. This hypothesis 
appears unnecessary in light of the Tradescantia data. Since half- 
chromatid aberrations are found occasionally in treated pollen tube 
chromosomes, the chromosomes must have at least four strands, and 
chromatid deficiencies must therefore involve the fracture of t\vo half- 
chromatids at the same locus. If the chromosomes of the sperm cells of 
maize pollen have only two strands, the loss of both chromatids by simul- 
taneous breakage to give rise to entire endosperm deficiencies becomes 
understandable. 

P'igure 7-3 illustrates the effectiveness of X rays (370 r) in inducing 



GENETIC AND CVTOLOGICAL EFFECTS 



271 



aberrations in the pollen tube chromosomes of Tradescantia at successive 
prophase periods. A comparison can, therefore, be made with the ultra- 
\iolet data obtained under similar circumstances (Fig. 7-2). Isochroma- 
tid deficiencies and chromatid translocations are readily induced by 
X rays, and the close parallelism of the terminal deficiency and translo- 
cation curves supports the generally accepted hypothesis that a transloca- 
tion owes its origin to the illegitimate fusion of the broken ends of two 
independently induced deficiencies. It would appear that the lack of 
translocations in the pollen tube following exposure to ultraviolet cannot 




2 4 6 8 10 

TIME AFTER GERMINATION, hr 

Fig. 7-3. Relation of chromatid aberrations induced by X rays (370 r) in the pollen 
tube chromosomes of Tradescantia to successive prophase stages following germination. 
Curve I, terminal deficiencies; II, translocations; III, isochromatid deficiencies. 
(Swanson, 1943.) 

be ascribed to a lack of deficiencies. This is more clearly indicated in 
Table 7-2, where the frequencies of terminal deficiencies induced by the 
two types of radiation are similar, but those of translocations are not. 

The great majority of broken ends of chromosomes induced by ultra- 
violet clearly do not possess the capacity for subsequent reunion. This 
may result from a more rapid healing of the broken ends, or it may stem 
from a lack of maneuverability of broken ends imposed by the surrounding 
chromosomal matrix. The matrix seems not to be disrupted b}^ ultra- 
violet, and it may, following heavy doses, actually become more promi- 
nent in appearance (Swanson, 1942, 1943). 

The terminal deficiencies produced by both radiations in the pollen tube 
chromosomes of Tradescantia are indistinguishable in microscopic appear- 
ance, but there is good evidence here, as in maize endosperm, for believing 



272 



UADIATION HIOLOGY 



that they are quiihtativrly (UlTcicnt l)oth as to their nature and as to their 
mode of orij^in. \\'h(Mi ultra \iolet is used in combination with X rays as a 
pretreatment, the frecjuency of terminal deficiencies is no greater than that 

Tabi.k 7-2. Kkequencies of Chromatid Aberrations Induced in the 
Pollen Tube Chromosomes of Tradescantia by X Rays and 

Ulthavioi.ht 

(RwMiisoii, MM2.) 





Percentage aberrations per chromosome 


Treatment 


Chromatid 
deletions 


Isochroinatid 
deletions 


Translocations 


Total 
chromosomes 


Ultraviolet (2537 A) . . 
X ray (123 r) 


10 15 
10.00 




2.7 




4.4 


030 

180(i 



expected from the ultraviolet treatment alone (Table 7-3). It would 
appear that the circumstances which favor the induction of one kind of 
terminal deficiency actually suppress the appearance of the other kind. 

Table 7-3. Frequencies of X-ray-induced Chromatid Aberrations as 
Influenced by Pretreatment with Ultraviolet 

(Swanson, 1944.) 





Percentage aberrations per chromosome 


Treatment 


Chromatid 
deletions 


Isochroinatid 
deletions 


Translocations 


Total 
chromo- 
somes 




Ob- 
served 


Ex- 
pected 


Ob- 
served 


Ex- 
pected 


Ob- 
served 


Ex- 
pected 


Ultraviolet (2537 A) . . . . 

X ray (246 r) 

Ultraviolet -|- X ray . . . 


3.05 
3.51 
3.01 


6.56 


1.05 
0.35 


1.05 


1.89 
0.35 


1.89 


3540 
5304 
2256 



The X-ray-induced aberrations involved in illegitimate fusion, i.e., iso- 
chromatid deficiencies and translocations, are similarly suppressed. 
Since a like reduction in frequency of X-ray-induced al)errations is encoun- 
tered when ultraviolet is employed as a posttreatment, it seems reasonable 
to assume that the action of ultraviolet is not to prevent the initiation of 
X-ray-induced breaks, but rather to lessen their probability of realization. 
Kaufmann and Hollaender (1946) have demonstrated that a combination 
of the two radiations has a similar depressing effect on gross chromosomal 
aberrations in Drosophila, whereas Schultz (1951) has shown that the 
effects of X rays in maize, as judged by the freciuency of gcrmloss seeds, 
are completely inhibited by a posttreatment with wave length 297 m^t at 
the same time that the ultraviolet effects remain uninfluenced by X rays. 



GENETIC AND CYTOLOGICAL EFFECTS 273 

Since the ring-chromosome studies in maize also revealed that breakage 
by ultraviolet is frequent, a phenomenon obscured in rod chromosomes by 
restitution, it has been assumed that ultraviolet must have, in addition, a 
marked influence on the matrices of the chromosomes. Coagulation of 
the matrices by ultraviolet would thus not only prevent the realization of 
rearrangements from X-ray-induced breaks but also from those breaks 
which it itself induces, providing in this manner a mechanism which would 
lead to an apparent qualitative difference in behavior of the X-ray- and 
ultraviolet-induced breaks. 



SPECTRAL RELATIONS 

Significant comparison of the relative effectiveness of different wave 
lengths can be made only on the basis of rather precise estimates of the 
amount of energy reaching the site of the mutagenic reaction. Among 
higher organisms suited to the genetic analysis of the induced alterations, 
this is a serious difficulty, and all wave-length comparisons must be 
interpreted with due regard for the approximations involved in estimating 
the dose actually applied to the chromosomes whose reactions are 
determined. 

With Dr-osophila, using the technique of irradiation of the adult fly, the 
comparison of effectiveness of different wave lengths is not feasible. 
Mackenzie and Muller (1940) estimate that about 99.9 per cent of the 
ultraviolet energy is absorbed before the radiation reaches the germ plasm. 
Even slight differences in the relative loss for different wave lengths could 
make tremendous differences in their relative intensity at the site of 
genetic action. With the polar cap technique the absorption loss is very 
much less, but the technical requirements for the identification of the 
individual mutation make the method unsuitable for experiments on the 
scale required for wave-length comparisons. 

With seed plants adapted to genetic analysis, irradiation of the pollen 
presents some opportunity for the comparison of wave-length effective- 
ness. Extensive data on the effects of monochromatic radiations have 
been reported for Antirrhinum and for maize. But even within the single 
pollen grain, internal filtration results in large and variable differences in 
the penetration of different wave lengths to the site of the nucleus. 

The maize pollen grain is approximately spherical, with a diameter of 
about 93 n. Uber (1939) measured ultraviolet transmission, at different 
wave lengths, for the pollen grain wall and pollen grain contents of maize. 
The results indicated that, with equal incident energy at wave lengths 
297 and 265 m^u, for example, the dose penetrating to a point 16 fx beneath 
the wall is three times as large for the longer wave length as for the shorter, 
and at a depth of 32 n it is about fifteen times as large. In most of the 
pollen grains the sperm nuclei are located within this depth range. But, 



274 RADIATION HIOLOGY 

since the pollen, when treated, is oriented at random with reference to the 
radiation source, the sperm nuclei in most of the pollen grains treated are 
at a greater depth, and the filtration losses and ineciualities of filtration 
loss are mucli greater. Obviously, under such conditions any corrections 
for dosage must be at best very rough approximations. 

In the liverwort Sphacrocarpus donncllii, conditions for the comparison 
of wave-length effectiveness are incomparal)ly better, for the radiation 
may be applied to the spermatozoid, which is an almost naked nucleus 
about 0.5 n in thickness. Knapp and Schreiber (1939; see also Knapp 
et ai, 1939) have compared the effects of monochromatic radiations in 
this organism. 

For direct cytological comparison of chromosomal effects, the pollen- 
tube technique used by Swanson (1940) with Trade scantia is also well 
suited to the study of w^ave-length effectiveness, with minimal interference 
from internal filtration of the radiation. Studies of this kind have been 
exploratory only (Swanson, 1942), but they indicate that considerable 
wave-length differences are to be expected. 

Dosage Effect. The comparison of wave-length effects requires a care- 
ful consideration of the dosage effect for two reasons: (1) for evaluating 
the error in approximating equal dosage with the wave lengths compared, 
because of the varying internal filtration already discussed, and (2) for 
determining the actual form of the dosage curve, as a basis for interpreting 
the differences found \vith the wave lengths compared. 

The effect of variation in internal filtration among the individuals 
treated is to flatten the dosage curve for specific effects. The population 
treated consists of individuals varying in the proportion of the incident 
dose that will be received by the gamete nucleus. For example, in a 
population of maize pollen grains as described earlier, some may be so 
oriented that the gamete nucleus is reached by radiation that has pene- 
trated through 1() /i of overlying material, while in others the gamete 
nucleus can be reached only by radiation that has penetrated through 80 /i 
of overlying material. The first unit of dosage may produce the effect 
in any of the pollen grains, and its hits will tend to occur most frequently 
in the most favorably oriented ones. Added units of dosage may produce 
additional effects only in the unaffected individuals remaining, which 
offer a lower probability of hits because of the lowered dose reaching the 
gamete nucleus in these pollen grains. The flattening of the dosage curve 
resulting from this factor should occur at all ultraviolet wave lengths, Init 
should be more pronounced at the shorter wave lengths since these show 
higher absorption in the pollen grain contents. Using observed values 
for the position of the miclei within the pollen grain and for the trans- 
mission losses in jiollen wall and contents, the expected form of the dosage 
curve at wave lengths 254, 297, and 302 m/x was in fairly good agreement 
with that observed (Stadler and Uber, 1942). 



GENETIC AND CYTOLOGICAL EFFECTS 275 

In experiments with the discharge-tube radiation (largely wave length 
254 m^), it was possible to show the relation of internal filtration to the 
dosage curve directly (Table 7-4). A given dose may be applied with 

Table 7-4. Frequency of Endosperm Deficiencies from a GrvEN Dose 

When Applied to Both Sides of Pollen as Well as to One Side 

Only (Wave Length 254 M/j.) 

(Stadler and Uber, 1942.) 

Frequency, 
Dose per cent 

One unit (one side) 18.9 

Two units (one side) 24 . 9 

Two units (one from each side) 35.4 

equal effect from either above or below the layer of pollen. When the 
dose is doubled by applying a second unit of dose from the same direction, 
the added frecjuency of induced effects is considerably less than that from 
the first, and thus the yield from 2 units is considerably less than double 
the yield from 1. But if, instead, the second unit of dose is applied 
from the opposite side, the added yield of induced deficiencies is as great 
as that from the first unit, and thus the yield from 2 units of dose is 
double the yield from 1. 

When the measure of radiation effect is not a specific result (e.g., a 
given deficiency, death of the irradiated individual) but rather an indefi- 
nite group of results, any number of which may be observed in the single 
treated individual (e.g., mutations at miscellaneous loci), the result 
expected from internal filtration is not a flattening of the dosage curve. 
Instead it is a tendency toward coincidence of independent effects in the 
single treated gamete, such as was noted in the maize experiments men- 
tioned in an earlier section of this review (see also Meyer et al., 1950). A 
flattening of the dosage curve for mutation frequency would be expected 
as a result of variations in internal filtration only if the accumulation of 
mutants and other radiation effects is a factor in eliminating individuals 
from the population tested. 

In treated populations in which there are large variations in exposure 
to radiation injury, gross distortion of the dosage curve ma\^ occur. For 
example, if the treated population were a mass of pollen grains more than 
one layer deep, the lower layers would be almost wholly shielded from the 
radiation. With a sufficiently heavy dose, the pollen grains of the top 
layer might be largely eliminated from the population tested, and the fre- 
quency of genetic effects in the surviving population would be materially 
lower than that found with lighter doses. The correlation between 
genetic effects and killing would be a spurious correlation, but any correla- 
tion of genetic effects with elimination among the individuals treated 
would tend to flatten or reverse the dosage curve. 

The dosage data for maize pollen treatments are therefore of interest 



27G 



RADIATION BIOLOGY 



chiofly ill relation to the error involved in wave-length comparisons, rather 
Ihaii to the nature of the reaction of the chromosomes to increasing dose. 
The results of dosage trials at wave lengths 254, 297, and 302 mn are 
shown in Table 7-5. 

Tablk 7-5. l{ia,.\TioN of Do.sk to FHWiiENrY OK Endosperm Deficiency 

(Stndlcr ;in(l t'l.cr, I'.tt2.) 



Dose, 


Wave length, 


Kiulospenn 


deficiencies, 


X lO^ergs/nnn" 


m^t 


per cent ± S.E. 


1.0 


254 


3.0 


±0.6 


2.0 


254 


5.7 


±1.1 


4.0 


254 


8.2 


±1.6 


8.1 


254 


10.7 


±1.8 


17.1 


254 


16.8 


± 2.6 


4.0 


297 


2.6 


± 0.7 


7.9 


297 


10.7 


+ 1.1 


15.7 


297 


18.0 


± 2.2 


88.3 


297 


32.6 


± 2.3 


15.6 


302 


2.9 


± 0.7 


32.0 


802 


9.4 


± 1.0 


63.8 


302 


24.8 


± 1.8 


132.0 


302 


36 . 1 


± 2.7 



The relatively low yield of endosperm deficiencies at the lowest doses 
tested for wave lengths 297 and 302 suggests the possibility of a multiple- 
hit curve for the dosage relation. This possibility was suggested also by 
the results of treatments at nine wave lengths in the range 238-293 m/z, 
comparing the frequency of endosperm deficiencies from doses of approxi- 
mately 0.5 X 10^ ergs/mm- with that from doses of approximately 
2X10^ ergs/mm-. In every case the yield from the lower dose was less 
than one-fourth of that from the higher dose. Since these treatments 
were not seriated for the control of daily variation, a separate trial was 
made with wave length 2G5 m/x, using doses of 0.25, 0.50, 1.0, and 2.0 X 10^ 
ergs/mm'-. The frequency of induced endosperm deficiencies was again 
disproportionately low at the lower doses, but the deviation from linearity 
was not statistically significant. 

Indications of nonlinearity of ultraviolet effect on the frequency of 
mutations at low doses have also been found in lower organisms. The 
data of Emmons and Ilollaender (1939, Table 2; see also Hollaender and 
Emmons, 1941) on induced mutation frequencies in Trichophyton suggest 
that low doses are disproportionately less effective than higher doses, 
although again the departure from linearity is not always obvious or 
consistent. Similar results have been obtained in AspfrgiUns (Swanson 



GENETIC AND CYTOLOGICAL EFFECTS 



277 



et ai, 1948, Table 2) and Neurospora (Hollaender et ai, 1945). The 
statements of Lea (1946) and Catcheside (1948) to the effect that a linear 
proportionality holds up to the peak of the ultraviolet mutation curve 
therefore cannot be accepted without question. The need for more 
precise measurements of mutation effects at low doses is evident, since 
the interpretation of data on the comparative effects of different wave 
lengths must depend in part upon the shape of the dosage curve. 

The striking drop in mutation frequency observed at very high doses of 
ultraviolet in fungi (Emmons and Hollaender, 1939) has not been found 
in maize. This apparently is not related to factors of internal filtration, 
})ut rather to the selective elimination of individuals from the population. 
A somewhat similar drop in frequency has been reported in Drosophila. 
The study by Reuss (1935), in which ventral abdominal exposures of 
adult males w^as first employed, provided the first quantitative data on 
the effects of increasing increments of ultraviolet on the frequency of 
recessive lethal, semilethal, and visible mutations in Drosophila. Expo- 
sures of 15, 22.5, and 30 min were used, and a leveling in the dosage rela- 
tions was evident, but the data are inconclusive because of the small num- 
l>ers of flies observed. Those obtained by Sell-Beleites and Catsch 
(1942), following similar methods of exposure, are more striking. In two 
different experiments (Table 7-6), the mutation frequency rose to a peak 

Table 7-6. Types and Frequencies of Mutations in the X Chromosome 

OF Drosophila melanogaster Induced in Spermatozoa by Ventral 

Exposure of the Abdomen to Ultraviolet Radiation 

(Sell-Beleites and Catsch, 1942.) 





Duration 

of exposure, 

min 


Total no. 
of flies 


Lethals 


Visibles 


Mutations, 
per cent ± S.E. 


Expt. I 


6 


2431 


26 





0.92 ± 0.19 




12 


1195 


22 


1 


1.77 ± 0.38 




24 


963 


3 





0.16 ± 0.13 


Expt. II 


5 


2478 


8 





0.17 + 0.08 




10 


2002 


14 


1 


0.60 ±0.17 




15 


1443 


12 


4 


0.96 +0.26 




20 


1464 


6 





0.26 ± 0.13 



with increasing increments of ultraviolet and then dropped abruptly as 
added increments were given. 

Sell-Beleites and Catsch concluded that they were dealing with a 
greatly disturbed "one-hit" curve, and that the decrease in mutation 
frequency at high doses results from increasing sterility induced by the 
penetrating radiation. Many of the irradiated flies were completely 
sterile or yielded no mutations of any kind. The percentage of sterility 



278 KADI \'I'I<>N HIOLOCY 

increased witli increasing dosage and resnlted presumably from physio- 
l()gic;il damage unassociated with the mutation process. Once a maxi- 
mum mutagenic elTect was obtained in those indivi(hiais in which liic 
ra(hation had successfully penetrated, added increments of ultraviolet 
would be likely to contribute more rapidly to sterility than to the induc- 
tion of additional mutations. A drop in mutation frequency would there- 
fore be expected since the fertile flies with a high degree of filtration and a 
low frequency of mutations would contribute a disproportionately greater 
number of offspring to the next generation. 

Attempts to determine dosage relation in Drosophila by the polar cap 
techniciue have been reported. Altenburg ct al. (1950) indicated that a 
linearity of efTect obtains at low doses, but that a leveling of the curve is 
rapidly achieved, after which large increments of dose are relatively inef- 
fective in raising the frequency of mutation. Meyer ct al. (1950) also 
found that a clustering of mutations occurs in cells or chromosomes 
favorably oriented with respect to the radiation. A study of 1 1 chromo- 
some II lethals showed that 10 of these chromosomes carried additional 
mutations. Also, increases in dosage caused death to an increasing num- 
ber of pole cells, as evidenced by the greater proportion of Fi offspring 
carrying a particular mutation. 

Perhaps the best evidence oii the relation of ultraviolet effects to dosage, 
from the standpoint of the avoidance of internal filtration difficulties, is 
that from Tradcscaniia pollen-tube treatments. Chromatid deficiencies 
here provide the criterion of effect (Swanson, 1942). The data indicate 
that the relation is linear. Figure 7-1 illustrates the curve obtained with 
wave length 254 m/n. 

Wave-length Dependence Studies. The earliest studies of the differen- 
tial genetic effectiveness of various wave lengths of monochromatic light 
on the germinal material of higher plants were made by Noethling and 
Rtubbe (1934, 1936; see also Stubbe and Noethling, 1936). The dry 
pollen of Antirrhinum majus was treated, and the detection of induced 
mutations was made by observing the segregations in F2 populations. 
The control populations had a comparatively high frequency of spon- 
taneous mutations (1.6 per cent). From a study of Fo offspring from 
some 3000 pollen grains treated with wave lengths 265, 297, 302, and 
313 mn, the authors were able to show that each wave length was capable 
of increasing significantly the frequency of mutations, the highest rate 
obtained being approximately four times the control frequency. 

Several doses were compared at wave lengths 265 and 297 m^u. At 
wave length 265 m/x, doses of 2 X 10'^ ergs/mm' failed to raise the muta- 
tion frequency significantly, and the rise with increasing doses was slow. 
At doses of 2.7 X 10' ergs/mm^ the frequency w^as twice that of the 
controls, while doses of 14.6 X 10' and 50.3 X 10' ergs/mm''^ increased 
it onlv to three times that of the control. The use of wave length 297 m^ 



GENETIC AND CYTOLOGICAL EFFECTS 279 

in a wide range of doses (1.8 X lOMJo.O X lO'' ergs/mm^), gave signifi- 
cant increases over the control, but no significant differences between 
doses. A dose of 65 X 10^ ergs/mm- at wave length 302 m/x and one of 
120 X 10^ ergs/mm- at wave length 313 m/x quadrupled the spontaneous 
frequency. From these results the conclusion was drawn that the peak 
of genetic effectiveness was in the neighborhood of 300 mfx. Supporting 
this hypothesis was the demonstration that a suspension of Antirrhinum 
pollen in 30 per cent alcohol had a maximum of absorption around 300 vay.. 

Comparable data by Stadler and Uber (1938) for the frequency of 
endosperm deficiencies induced by monochromatic radiations also showed 
effects in large numbers for all wave lengths tested in the range 235-302 
mM- Longer wave lengths had no appreciable effect. The frequency of 
germless seeds was increased markedly by radiation of wave lengths 280 
van and shorter, but not by longer wave lengths, even at much higher 
doses. The tabular data on which these conclusions were based were 
later published (Stadler and Uber, 1942, Table I), together with the 
results of additional experiments on relative wave-length effectiveness. 

The frequency of induced effects was higher for wave length 297 m/x 
than for 2G5 m^i, as in the experiments of Noethling and Stubbe. But, 
though wave length 297 m/x significantly excelled 265 van at the higher 
doses, the relation was clearly reversed at the lower doses. Both wave 
lengths were tested at doses of approximately 1, 2, 3, 6, and 12 X 10' 
ergs/mm^ While the longer wave length was about twice as effective as 
the shorter in comparisons made at the heaviest dose, it showed no effect 
at the lowest dose, and only about one-fourth of the wave length 265 m^u 
effect at the second dose. The results as a whole showed that there were 
wide differences in the dosage relations at the different wave lengths 
tested, and that a study of the dosage relation and of internal filtration 
was needed before the relative effectiveness of the wave lengths could be 
estimated. The results of these studies were summarized in the preceding 
section. 

The most reliable indications of relative effectiveness come from com- 
parisons made at low doses. However, at the lowest doses filtration 
losses are still effective and require correction. The frequency of endo- 
sperm deficiencies produced in an experimental comparison of seven wave 
lengths, all at a dose of 2 X 10-^ ergs/mm^, seriated for the control of 
daily fluctuations, is shown in Table 7-7. 

At this dose, the endosperm deficiencies induced by wave lengths 265 
and 254 m^ix are about 3 times as frec^uent as those induced by wave 
length 297 m^i and about 15 times as frequent as those induced by wave 
length 302 m^u. When the dose is increased to about 8 X 10' ergs/mm', 
the differences are much less pronounced and in some cases hardly signifi- 
cant. The evidence on internal filtration, subject to the approximations 
which have been mentioned, indicates that wave lengths 265 and 254 in/u 



280 



UADIATION BIOLOGY 



are prohalily more than 10 times as effective as wave length 207 m^ and 
probably more than 100 times as efTective as wave length 802 in/i. in terms 
of ('(|ual int(Misity at the surface of the sperm inicleus. 

Tablk 7-7. Tnio Fbequency of Endosperm Deficiencies in Maize as a 

Function ok Wave Length at Two Different Doses 

(Stadlor and Tber, 1942.) 





Endosperm deficiencie.s, 


per cent ± S.E. 


Wave length, 














niM 


At 2 X 10* ergs/mm* 


At 8 X 10* ergs/mm* 


248 


9.9 ± 1.0 




22.9 ± 2 :i 


254 


15.5 ± 1.:^ 




23.2 + 2 2 


2(i5 


11.6 + 1.0 




15.2 ± 12 


270 


7.8 ± 1.6 




20.9 ± 19 


280 


9.3 ± 0.9 




23 . 7 ± 1 5 


289 


6.2 ± 0.8 




18.5 ± 1.5 


297 


4.7 ±0.5 




11.4 ± 1.2 



In the comparison of wave-length effects in Sphaerocarpns, complication 
from internal filtration is at a minimum, for the spermatozoid irradiated 
consists almost entirely of nuclear material. Knapp ei al. (1939) com- 
pared the effectiveness of six wave lengths in inducing genetic alterations 
in Sphacrocarpus donncllii. The spermatozoids were irradiated in water 
suspension, and the sporogonia produced by fertilization of untreated 

Table 7-8. The Frequency of Induced Mutations in Sphaerocarptis as a 

Function of Wave Length, Dosage 2 X 10' ergs/mm* 

(From Knapp ei al., 1939.) 



Wave length 


No. of sporogonia 


No. of 


Percentage 


m^u 


analyzed 


mutations 


mutations 


254 


61 


17 


27.8 


265 


53 


22 


41.8 


280 


64 


14 


21.9 


297 


52 


3 


5.8 


302 


71 


4 


5.6 


313 


65 





0.0 


Control 


64 





0.0 



female gametophytes were tested by analysis of the spore tetrads. 
Mendelizing mutations were identified by the production of two normal 
and two mutant plants; lethal "mutations" (i.e., all genetic alterations 
w'ith haplo-lethal efTect), by the production of only two instead of four 
plants by the spore tetrad. By the tetrad analysis of 50 to 75 sporogonia 
representing each treatment, it was possible to show sharp differences in 



GENETIC AND CYTOLOGIC AL EFFECTS 281 

the effectiveness of some of the wave lengths applied in inducing such 
alterations. Radiation injury was in general parallel to genetic effective- 
ness. The data are shown in Table 7-8. 

These data represent the most significant evidence now at hand, bear- 
ing upon the ac^tion spectrum of the ultraviolet in producing genetic 
effects. It is interesting to note that wave length 2()5 m/x is about seven 
times as effective as wave lengths 297 and 302 m^, and that the two latter 
wave lengths are not appreciably different in effectiveness. The effective- 
ness of wave length 254 m^t is about two-thirds that of 265 m/x though the 
significance of this difference is perhaps questionable. The effectiveness 
of wave length 280 mn is about half that of 265 m^. From the resem- 
blance between these values and the absorption spectrum of nucleic acid, 
the authors conclude that absorption by nucleic acid is of essential sig- 
nificance in determining the genetic effects of ultraviolet radiation. 

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284 UADIA'PION HIOI.OCY 

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Manuscript received by the editor July 5, 1951 



CHAPTER 8 



The Effects of Radiation on Protozoa and the Eggs of 
Invertebrates Other than Insects* 



R. F. Kimball 

Biology Division, Oak Ridge National Laboratory 
Oak Ridge, Tennessee 

Introduction. Lethal effects: Kind of death — Sensitivity — Recovery — Substances 
present in the medium during irradiation — Effect on the medium. Retardation of cell 
division: Recovery — Sensitivity — Localization. Inherited effects. Miscellaneous effects: 
Activation of eggs — Excystment of protozoa — Motility and behavior of protozoa — Sensiti- 
zation to heat — Miscellaneous microscopically visible changes — -Various physiological, 
biophysical, and biochemical effects. References. 



INTRODUCTION 

From the point of view of a biologist interested in the effects of radia- 
tion there is a certain unity between investigations with protozoa and with 
the eggs of marine invertebrates — in both instances, one is deahng with 
single cells in liquid medium; in both, the methods employed have tended 
to emphasize the cell as an individual rather than as a member of a popu- 
lation. So, while the connection between investigations with the two 
kinds of material has sometimes been slight, it seems proper to consider 
them together. 

The review could have been organized in many ways. The actual 
choice, a consideration of each kind of effect separately, declares the point 
of view to be that of an experimental biologist rather than a biochemist or 
biophysicist. It seems important to stress that such frequently employed 
radiobiological criteria as death and division delay may not be the same 
in all materials. Actual occurrence of death of cells may be observed, not 
just the end result, such as failure to produce daughter cells in large num- 
bers. Division delay may be examined in detail rather than as an over- 
all effect on the number of cells or mitoses in a population. From such 
studies, it is clear that a variety of phenomena are concerned. When 
investigations on many organisms have been made, it may be possible to 
recognize certain phenomena which are common to all. Until this is 

* Manuscript prepared and work at Oak Ridge performed under Contract No. 
W-7405-eng-26 for the Atomic Energy Commission. 

285 



'2R{\ KADI \ TlON HlOl.OfiY 

(loiic, any attempt to treat death, division delay, etc., as having the same 
cause in all cells is likely to he mislcadinji;. 

'This rc\ie\\ has l)e(Mi limited, for the most pari, to papers appearing 
from the year H).'^') through the year 1950. A few earlier pieces of work 
have been included when they seemed especially important for the topics 
discussed, or when (hey were early papers in a series which continued 
beyond 1935. A number of new lines of investigation have developed 
since this manuscript was submitted. It has not been feasible to revise 
the manuscript to include them. References to most of this work may 
be found in fliese (1953). Wichterman (1953). and in Xudear Science 
Abstracts. 

LETHAL EFFECTS 

Kind of Death. Many investigations have been concerned with the 
lethal effects of radiation. However, the nature of the death is certainly 
not the same in different cases. A variety of possible causes of death will 
be discussed in the following paragraphs. 

First, there is death which occurs during or shortly after irradiation. 
Numerous investigators have observed such death and described in more 
or less detail the accompanying phenomena, such as loss of motility, 
vacuolization and coagulation of the cytoplasm, and other changes. At 
least a part of the investigations recorded in Tables 8-2 and 3 involve 
phenomena preliminary to death. 

This type of death may occur very rapidly, as shown by the observa- 
tions of Rentschler and Giese (1941) and Harvey (1942), with intense 
flashes of ultraviolet. Some organisms, notably the ciliates, disintegrate 
within a few seconds. In other cases, some minutes or even an hour or 
so may elapse between irradiation and disintegration. 

The doses of ionizing radiation required to bring about this tj^pe of 
death are very high. Dognon and Piffault (1931a) estimate the imme- 
diate lethal dose for Paramecium to be about 500,000 r. Halberstaedter 
(1938) showed that a dose of more than 100,000 r of X rays was needed 
to immobilize Trypanosoma gambiense. Halberstaedter and Back (1942) 
report loss of motility and cytolysis of the colonial flagellate Pandorina 
morum after doses of 300,000-000,000 r. The very high doses required 
for immediate killing have sometimes been considered characteristic of 
the protozoa. However, Scott (1937) in his review points out that imme- 
diate, as contrasted to delayed, death requires doses of similar magnitude 
in other organisms. 

Giese and Leighton (1935a) report on the immobilization and vesicula- 
tion of Paramecium, by monochromatic ultraviolet. In both cases 2804 A 
appears to be more effective than other wave lengths. However, calcu- 
lations of quantum efficiency, based on measurement of absorption of 
paramecia. suggest that 2654, 2804, and 3025 A were about equally effec- 



PROTOZOA AND INVERTEBRATE EGGS 287 

tive, whereas 2537 A was less effective. McAulay and Taylor (1939) 
investigated the death (by bursting) of Paramecium as a result of exposure 
to monochromatic ultraviolet. They found that all wave lengths up to 
3000 A were quite effective but that effectiveness was much less at longer 
wave lengths. It may be doubted that their techniques were sufficient 
to detect differences in effectiveness among the shorter wave lengths. 
Shettles (1938), using the flagellate Peranema trichophorum, observed 
death after 2 hr exposure to 2537 A ultraviolet but no death after 8.5 hr 
exposure to 2650 A. Likewise, no death was found after comparable 
exposures to longer wave lengths. The intensities of the different wave 
lengths are said to have been eciualized by use of a photometer. Thus 
no general conclusions are possible about wave-length dependence of 
immediate death after ultraviolet. 

It is probable that immediate death is due to extensive damage to the 
cellular material. This is suggested by both the rapidity with which it 
can occur and the very large doses of ionizing radiation necessary to bring 
it about. It might be surmised from the microscopic observations that 
coagulation of the protoplasm or damage to the cell membrane, or per- 
haps both, are involved, but no really conclusive evidence is available. 
Possibly, different kinds of damage lead to death in different cells under 
different circumstances of irradiation. 

It is rather generally accepted (Lea, 1947) that irradiated cells may be 
able to survive until they divide, at which time death is caused by loss of 
parts of chromosomes as a result of chromosome aberrations induced by 
the radiation. Such a mechanism is suggested by Hoi week and Lacas- 
sagne's observation (1931a, b) of death at division in the flagellate 
Polytoma. At certain doses of a. particles, many of the cells remained 
normal until division and then disintegrated. Using the hit theory, they 
calculate that a two-particle event is involved with a target diameter of 
2.3 ju, corresponding roughly to the area presented to the particles by the 
pericaryosomal space in the nucleus, the region in which, presumably, the 
chromosomes are located. Similarly, Halberstaedter and Back (1942) 
report that Pandorina colonies exposed to between 3000 and 300,000 r 
remain normal until cell division, when cytolysis takes place. This was 
so regardless of whether old colonies that underwent cell division shortly 
after irradiation or young colonies that did not undergo cell division for 
some time were used. 

When the test of survival is the ability of the cell to multiply and pro- 
duce a culture, death due to mutations or chromosome aberrations must 
be considered as a possible explanation, although other causes cannot be 
excluded. Two cases showing the ability of free-living flagellates to 
produce cultures after X irradiation may be mentioned. Ralston (1939) 
distinguishes immediate from delayed death and reports the median lethal 
dose foi- delaved death in Dunaliella salina as between 10,000 and 13,000 r. 



288 RADIATION HIOLOGY 

S('h()enl)oni (1949) reports the median lethal dose for Astasia longa to be 
between 20,000 and 40,000 r. 

There have been a iiunil)er of investigations of the killing of parasitic 
protozoa by radiation; and in most of these the criterion was the abiUty to 
infect the host after irradiation in vitro. Thns chromosomal changes may 
have been invohed. Ilaberstaedter (1938) fonnd that a dose of 12,000 r 
of X rays to Trypanosoma gatnbiense, in vitro, was sufficient to prevent 
the infection of mice, whereas doses of more than 100, 000 )• were necessary 
to produce any obvious changes in the motility of the trypanosomes. 
Other investigations with parasitic protozoa are summarized in Table 8-1. 

There are a few cases of delayed death in which a genetic explanation is 
more difficult. Holweck and Lacassagne's case (1931a, b) with Polytoma 
in which death occurred after division has alicady been mentioned, and 
the interpretation has been suggested that chromosomal aberrations were 
involved. Using a particles, they distinguished other kinds of effects 
as follows: (1) the cells remain motile and grow in size but finally lyse 
without division; (2) the cells are immobilized but grow, yet they finally 
lyse without division; (3) the cells become immobile, fail to grow, and 
lyse without division. These workers (see also Lacassagne, 1934a, b; 
Holweck, 1934) made sensitive volume calculations for the first and the 
last two effects and found for the first a volume approximately the equiva- 
lent of the centrosome, whereas for the last two, the volume was approxi- 
mately that of the kinetosomes. These rather remarkable identifications 
presumably would offer an explanation for the failure to divide and for the 
immobilization, but would not in themselves account for the eventual 
death. Holweck and Lacassagne (1931b) suggest that death is due to the 
suppression of reproduction and of motihty. However, Polytorna cells are 
probably haploid and so lethal mutational changes could be invoked as an 
explanation on the assumption that a mutation can express itself in the 
cell in which it arises. Nevertheless, the possibility must be borne in 
mind that this is a case of delayed death due to causes other than muta- 
tional change. 

In the ciliate protozoan, Paramecium, a nongenetic explanation for 
delayed death appears by far the most likely one. Sonneborn (1947) has 
shown that the macronucleus in this ciliate contains many sets of genes. 
It is therefore improbable that loss of chromosomal material or gene 
mutation in either the macronucleus or micronucleus would have much 
effect on ciliates multiplying vegetatively. In order to allow for such an 
effect the unlikely assumption would have to be made that a given muta- 
tion or deficient chromosome is dominant over many normal genes or 
chromosomes. Alternatively, the radiation would have to be assumed 
to cause sufficiently extensive damage to the chromosomes to lead to 
death even with many sets. 

For this reason, Paramecium and other ciliates might be expected to be 



PROTOZOA AND INVERTEBRATE EGGS 289 

relatively well buffered against radiation damage and, in a sense, this is 
true. The continued reproduction of the flagellates is prevented by a 
few tens of thousands of roentgens of X rays, but hundreds of thousands 
are needed to prevent the vegetative multiplication of ciliates. Back 
(1939) found for Paramecium caudatum that a dose about two-thirds the 
immediate lethal dose, i.e., about 300,000 r, led to eventual death. The 
animals survived for several weeks but decreased in size, and finally died. 
Lacassagne (1934b), working with Glaucoma scintillans, had previously 
reported death after several days without division. However, he found 
that some increase in size occurred. Thus delayed death does occur in 
these organisms but it occurs without division. 

Kimball and Gaither (1951) have been able to distinguish at least two 
kinds of delayed death in Paramecium aurelia following exposure to ultra- 
violet. With higher doses, some of the animals survive a day or more 
without division, but eventually die. Long periods of the kind reported 
by Back (1939) for X rays were not observed for death without any divi- 
sion. However, at slightly lower doses, the animals pass through two or 
three divisions rather slowly (in 2 or 3 days) but then cease dividing 
entirely for several days. Some animals finally recover the normal divi- 
sion rate after remaining undivided for periods as long as three weeks, but 
others die during this "cessation" period. All animals, whether they 
eventually recover or die, become very small and thin during this cessa- 
tion of division, and apparently all come very near death. From the 
decrease in size of the animals, it seems probable that the delayed death of 
Paramecium involves an effect on the synthetic processes within the cell. 
Death could well result when the cell, no longer capable of making new 
material, comes to the end of its resources. In the case of ultraviolet, 
several divisions ordinarily elapse before the synthetic processes come to 
a halt, while with X rays. Back's study (1939) suggests that this is not 
so. Also, recovery has been found for the ultraviolet effect but no 
recovery has been reported for X rays. Whether these differences are 
real or only apparent remains for future investigation. In any case, it 
seems hardly possible that death during the cessation period after ultra- 
violet irradiation is due to mutational changes, since recovery can occur 
even in the sister animals of those which die. It also seems improbable 
that enough genetic damage could be done either by X rays or ultraviolet 
to cause the delayed death without division, although perhaps such an 
explanation is just possible with the large doses of X rays employed. 
Nonetheless, death due to mutational changes is known for Paramecium 
aurelia. Animals given doses of 10,000 r or less divide normally as long 
as they multiply vegetatively, but when they undergo the self-fertilization 
process of autogamy many of the exautogamous clones are inviable. This 
phenomenon will be discussed in more detail in the section on inherited 
effects. 



21)0 UADIKIION HIOl-OCY 

The killing of nematode cj^gs hy ladiaiion may involve, in addition to 
(l(>ath of (-('lis, rather diverse processes, such as abnormal development of 
embryos and failure of the younp; worms to hatch. Several invest ij^at ions 
with ultraviolet will be reviewed here. The eggs of Asrari.s have been, 
in the past, favorite subjects for radiobiological research with X rays but 
most of this work can be found summarized in Duggar (193G) and in the 
tables of Dognon and Biancani (1948). 

Wright and McAlister (1934) examined eggs of Toxocara canis and 
Toxascaris leonina for cmbryonation after exposure to 3()o0, 3130, 3022, 
29()7, 2804, and 2050 A monochromatic ultraviolet. At the lowest dose 
used (()840 ergs/mm-) effects were found only with the two shortest wave 
lengths. Effects were found with 3022 A at a dose of 274,000 ergs/mm', 
but even 1,370,000 ergs/mm^ of the two longest wave lengths had no 
effect. 

These results are in agreement with those of Jones and HoUaender 
(1944) with .4 scans lumhricoides. They found that energies of about 
6,000,000 8,000,000 ergs/mmr of ultraviolet of the wave-length band 
3500-4900 A were needed to prevent cmbryonation of a large part of the 
eggs. Slightly higher energies were needed to prevent the hatching of the 
eggs of the pinworm Enterobius vermicularis. These very long wave 
lengths do have a lethal effect but only at very high doses. In an earlier 
communication HoUaender ci al. (1940) show that the action spectrum for 
the prevention of hatching of Enterobius eggs has a small peak at 2804 A 
and then rises rapidly to the shortest wave length tested, 2280 A. This 
action spectrum is similar to the absorption spectra of some proteins and 
lipids. HoUaender and coworkers suggest, as possible modes of action, 
hardening of an outside protein layer of the shell, change in composition of 
the lipoid membrane, damage to the embryo, or production of toxic sub- 
stances within the egg. The last is considered unlikely at the energies 
used. 

In summarizing this section on kinds of death, it would appear that at 
least the following categories of cell death can be recognized: (1) death 
within a maximum of a few hours after irradiation, (2) death after con- 
siderable periods of time but without division, (3) death at or shortly 
after the first division, (4) death after several divisions, and (5) death fol- 
lowing sexual processes. It seems quite possible that both (3) and (5) are 
the result of gene mutations and chromosomal aberrations and so it is not 
surprising to find that they occur in detectable amounts even at quite low 
doses. The very high doses characteristic for (1), together with the 
immediate changes involved, suggest extensive damage to cellular mate- 
rials. The death in both (2) and (4) may involve disturbances in the 
synthetic processes of the cell which finally lead to death when the 
resources of the cell are exhausted. In some instances, mutational 
changes can be excluded as a probable explanation of (2) and (4). When 



PROTOZOA AND INVERTEHRATE EGGS 291 

the ability of the cells to produce cultures, or infections in the case of 
parasitic forms, is investigated, death will be due, obviously, to the most 
sensitive of these processes occurring within the part of the life cycle 
investigated. 

Sensitivity. The sensitivity of individual cells of the same species to 
the lethal effects of radiation varies considerably. Halberstaedter and 
Back (1942) found that all cells in a colony of Pandorina died at the same 
dose although different colonies required different doses. Secondly, they 
found, by giving repeated increments of 100,000 r and examining for 
immediate death after each increment, that the colonies of a small clone 
(16 colonies) all died at the same, or approximately the same dose; but 
colonies of different clones died at quite different doses. In this way some 
clones were found which died at 300,000 r and others which died at 
600,000 r. 

Back and Halberstaedter (1945) demonstrated a somewhat similar 
phenomenon with Paramecium caudatum. When groups of about eight 
paramecia from cultures derived from a single animal by at least six 
divisions were tested, it was found that the eight might die at quite 
different doses. However, the four to eight animals derived from one, by 
two or three divisions, were found to die almost invariably at the same 
dose, although different groups died at quite different doses. Therefore, 
the sensitivity is the same for closely related individuals but not for the 
more distantly related ones. 

This investigation of Back and Halberstaedter is of much interest, for 
it suggests that minor variations between cells may lead to rather marked 
changes in sensitivity. The evidence also suggests that these minor 
variations may be maintained over a few cell divisions. It is improbable 
that the similarity between products of a single Paramecium could be due 
to the products being in the same part of the fission cycle, for the numbers 
given in the tables suggest that Back and Halberstaedter (1945) often 
used groups in which some of the animals had divided once more than 
the others. 

Different stages in the life cycle of a given species may be of different 
sensitivity. Tang and Gaw (1937) find that older cultures of Paramecium 
bursaria are more susceptible to the immediate lethal effects of ultraviolet 
than younger ones. Brown et al. (1933) report that cysts of the ciliate 
Ewplotes taylori are killed by approximately 400,000 r of X rays, whereas 
the motile form requires approximately 460,000 r. The criterion in this 
case was the ability to survive 48 hr. However, they suggest that this 
difference may have been due to the different media in which the cysts 
and motile forms were kept. Bennison and Coatney (1945) found that 
8000 r of X rays prevented infection of chicks by a suspension of sporo- 
zoites oi Plasmodium gallinaccum, while 20,000 r was required to prevent 
infections by suspensions of trophozoites. Here, as in the work with 



•292 RADIATION HIOLor.Y 

Kiiplotcs, the possibility of an effect of the medium during irradiation must 
l)e considered. Packard (1924) reported what appears to have been a 
major difference in sensitivity to radium (principal effect said to have 
been due to slow /3 particles) between two species of ciliates. The lethal 
dose for Paramecium was 3 hr exposure to the source while that for the 
hypotrichous ciliate Styloni/chia was 15 hr exposure. Since Paramecium. 
is very resistant to ionizing radiation, this result is (}uite surprising. 
Packard attributes the difference to the lesser permeability of Stijlonijchia. 
Unfortunately, there has been no other work with Stylonychia; however, 
the related hypotrich, Euplotes, is killed at approximately the same dose 
as Paramecium (Brown et ai, 1933). More recently, Wichterman 
(1948a, b) has reported results which suggest a small difference in sus- 
ceptibility to X rays between Paramecium hursaria and P. calhinsi. He 
has slated that some P. hursaria survived doses between 400, 000 and 
(iOO.OOO r, whereas all P. calkinsi were killed by 400,000 r. 

More striking differences have been reported in the lethal doses of ultra- 
violet. Rather extensive comparisons between different species and 
strains of protozoa, primarily ciliates, are to be found in the works of 
Giese and Leighton (1953b) for long wave lengths, of Giese (1938b) for 
shorter wave lengths, and of Harvey (1942) for intense flashes of ultra- 
violet. Giese (1946b) has made similar comparisons for the eggs and 
sperm of a number of marine invertebrates. Shalimov (1935) reported 
that eggs of Ascaris equorum and Enterobius vermicularis were killed by 
ultraviolet in 5 min, whereas those of Sirongylus equinus were killed in 
only 3 min. Wright and McAlister (1934) found Toxascaris eggs to be 
more readily affected by monochromatic light than were the eggs of 
Toxocara. They suggest that this may be due to differences in the 
absorption of the shell. This emphasizes the difficulties which are inher- 
ent in interpreting differences in sensitivity to ultraviolet. Differences in 
absorption in the outer layers of cytoplasm or egg shells and differences 
in the action spectrum for superficially similar effects may be involved, as 
well as more su})tle differences in the biological organization of the organ- 
isms being compared. 

Recovery. The possibility that recovery may occur from changes which 
ordinarily lead to death has been investigated by use of fractionated doses. 
The method assumes that the effect is not due to a single "hit." If this 
is true and recovery does occur, then fractionated doses with sufficiently 
long rest periods in between the fractions should have less effect than the 
same total dose given as a single exposure in a brief period of time. Such 
a decreased effectiveness of fractionated doses of X rays has been reported 
by Crowther (1926) for the ciliate Colpidium colpoda and by Back (1939) 
for Paramecium caudatum. Growther (1926) found that one dose given 
in about 20 min produced death, whereas a dose one and a half times 
greater was required when given as three exposures at 2-hr intervals. It 



PROTOZOA AND INVKRTKBRATE EGGS 293 

can be estimated from Crowther's data that the doses used were of the 
order of 10* r. Back (1939) reported that about half the immediate lethal 
dose given every day for 3 to 4 successive days produced the same effect 
as two-thirds the immediate lethal dose given in a single exposure; i.e., 
the paramecia survived for some time without division, but eventually 
died. Since the final dose was several times the single exposure dose for 
immediate death, it can be concluded that fractionated exposures were 
less effective than single exposures. Quite different results were reported 
by Berner (1942) for immediate killing of Paramecium caudatum. He 
found that doses of X rays given in fractions, one fraction every 24 to 48 
hr, were considerably more effective than doses given at more frequent 
intervals. He presented evidence that X irradiation decreased the min- 
eral content as shown by ashed preparations and that recovery from this 
decrease was just complete in 48 hr. He believed that at this time the 
animals were more susceptible to X rays because their reserves had been 
depleted by the recovery process. The results of his investigations with 
ashed preparations and with death were quite variable, indicating a need 
for further investigation before these conclusions can be fully established. 
Nonetheless, the idea that a recovery process may lead to a temporary 
increase in sensitivity to radiation is an important one. 

Halberstaedter and Back (1942) found that fractionation into several 
parts with 1 or more days between had no noticeable effect on the action 
of X rays in Payidorina morum, either on immediate death or death after 
division. Halberstaedter and Luntz (1929) had previously found a simi- 
lar lack of effect of fractionation of the dose of radium rays on the related 
species Eudorina elegans. As just pointed out, death after division in 
Pandorina might be due to chromosomal aberrations. If a large propor- 
tion of the total were attributable to one-hit aberrations, the failure to 
find an effect of fractionation would be expected. However, the lack of 
an effect of fractionation on immediate death is surprising, especially since 
doses of 300,000 r and greater are necessary to bring it about. On the 
other hand, Forssberg (1933) found a marked effect of the intensity of 
X rays on killing and division delay in the single-cell algae, Chlorclla 
vulgaris, Scenedesmus hasiliensis, and Mesotaenium caldariorum. The 
effectiveness increased with intensity, and reached a maximum at about 
1600 r/min. Halberstaedter and Back (1942) used intensities of 9000 
r/min and greater. Thus it is possible that this intensity was too high to 
allow discovery of an effect of fractionation. 

Substances Present in the Medium during Irradiation. There have been 
a number of reports of the combined action of radiation and substances of 
one sort or another added to the medium. Dognon and Pift'ault (1931c) 
reported that the lethal dose of X rays for Paramecium was distinctly 
decreased in the presence of several dyes and toxic salts, e.g., potassium 
cyanide or iodide. Preliminary irradiation of the compounds had no 



294 RADIATION BIOLOGY 

clTccl l>ut irradiated paramenia added to the compounds died more rapidly 
tliaii the controls. Resorcinol and sodium hypo.sulfite protected against 
the combined action of radiation ;iii(l these compounds. These authors 
believed that the death was due to the easier penetration of toxic sub- 
stances broufiiht about by a radiation-induced increase in the permeability 
of the membrane. lilack (llK^ti) lias studied the effects of a numl)er of 
salts on the cytolysis of .1 mocha proteus by ultraviolet irradiation. 
Koehring (1940) has shown that the ameba, Chaos chaos, is more readily 
killed by a combination of neutral red and the radiation from radon than 
by either alone. Bohn (1941) found that paramecia in various salt solu- 
tions, dyes, etc., survived quite normally in the dark but were killed in a 
few hours on exposure to visible light. It is possible to consider all these 
effects as due to increased permeability to injurious substances, but it 
should be emphasized that there is no complete agreement that permea- 
bility is changed by irradiation (see Table 8-4). 

Levin and Piifault (1934a, b, c) have found that Paramecium aurelia 
placed in suspensions of lecithin or of cholesterol become resistant to the 
immediate killing action of X rays. Thus, after exposure for 3 days to a 
mixture of 1 part of lecithin emulsion to (^00 parts of culture fluid, a dose 
of X rays three and a half times the normal was needed to kill the animals. 
It is, of course, of interest that the substances concerned are considered to 
be important constituents of the cell membrane. However, it must be 
kept in mind that animals kept in emulsions of this sort for some days ma}^ 
change their nutritive condition. Giese and Heath (1948) have shown 
the importance of the nutritive condition for sensitivity to X rays. Like- 
wise, cholesterol and lecithin may have a protective action of the sort 
found by Evans et at. (1942) for sea-urchin sperm. 

Halberstaedter and Back (1943) found that pretreatment of Para- 
mecium caudatum with sublethal concentrations of colchicine for 2 days 
lowered the resistance to X rays. In controls, the dose required to 
produce immediate death of 50 per cent of the animals lay between 
250,000 and 300,000 r, while in the colchicine-treated animals it was 
between 100,000 and 150,000 r. They found no effect of colchicine on 
resistance to arsenic or ultraviolet. No explanation is offered for these 
findings. 

Effect on the Medium. There is considerable evidence that radiation 
may act indirectly on cells by way of an effect upon the medium surround- 
ing them. To what extent, then, can these effects on the medium account 
for the total effect of radiation upon the cell? It seems obvious that this 
is not a matter of mutually exclusive alternatives. Rather, it is a ques- 
tion of the relati\'e impoi'taiice, under the conditions employed, of dif- 
ferent mechanisms by which the radiation effect could be brought about. 
All discussion of effects on the medium will be inchided in this section 
even though other than lethal effects are involved. 



I'llOTOZOA AND INVERTEBRATE EGGS 295 

Taylor et al. (1933) found that an irradiated tap- water extract of com- 
mercial yeast killed Colpidiinn rampi/lutn when added after irradiation. 
They were able to demonstrate the presence of hydrogen peroxide in th^ 
irradiated water in concentrations sufficient to kill, and concluded that 
this substance probably played a major role in the death of irradiated 
protozoa, although they added that production of other toxic agents by 
irradiation of the yeast medium was not improbable. Since that time, 
several workers have reported that their media were not rendered toxic 
to unirradiated paramecia by doses of X rays sufficient to kill directly 
irradiated organisms (Piffault, 1939; Back and Halberstaedter, 1945; 
Giese and Heath, 1948; Wichterman, 1948a). However, Piffault (1939) 
reported that medium exposed to a dose about four times as great as that 
necessary to produce death of irradiated animals was toxic and gave posi- 
tive tests for peroxide. Giese and Heath (1948) reported that medium 
irradiated with 1,000,000 r was not toxic, while only 560,000 r, directly to 
the animals, led to complete death within 75 min. It would seem then 
that the lethal effect of the radiation cannot be ascribed to stable poison- 
ous substances produced in the medium. Obviously, an unstable poison 
of short half* life is not excluded, since an appreciable time had to 
elapse between the end of irradiation and the addition of cells to the 
medium. 

Mention may also be made here of a report by Heilbrunn and Young 
(1935) which states that eggs of sea urchins irradiated in the presence of 
minced ovarian tissue are more affected by X rays (cleavage delay) than 
eggs irradiated free of such materials. They believe that the irradiated 
ovarian material produced poisonous substances. This is in line with 
Loofbourow's finding (1948) of similar injurious substances from yeast and 
other organisms. Heilbrunn and Young were unable to demonstrate the 
production of such substances by organs other than ovaries. 

The problem of lethal substances in the medium has been carefully 
investigated with sea-urchin sperm by two groups of workers (Evans et al., 
1942; Evans, 1947; Barron et al., 1949a, b). Evans et al. (1942) showed 
that the effect of X rays on Arbacia sperm as measured by the percentage 
of fertilized eggs was markedly influenced by dilution of the sperm and by 
the addition of various protective substances to the medium in which 
irradiation was carried out. The more dilute the sperm suspension during 
irradiation, the more effective was a given dose of X rays. A wide variety 
of substances, such as egg albumin, gelatin, and egg water protected 
against X rays if present during the irradiation. No effect of protective 
substances was found on cleavage delay by irradiated sperm. Tests for 
hydrogen peroxide suggested that too little was formed to account for the 
effects. The investigators accepted Fricke's activated water as an expla- 
nation of the effects. 

On the other hand, Evans (1947) comes to the conclusion that hydrogen 



20() K ADI XriON lilOLoGY 

peroxide can ai'couiil for at least a part of the effects at very high doses. 
He shows that both the percentage of eggs fertilized and the time of first 
ciea\'age were affected by treating sperm with hydrogen peroxide; in about 
the concentration in which it is found in heavily irradiated sea water. 
Contrary to the conclusion of Evans ct al. (1942), cleavage delay is there- 
fore not necessarily a direct effect. Evans (1947) believed that the effect 
of hydrogen peroxide was slow, so that by irradiating a dilute suspension 
of sperm and removing the sperm ([uickly to fresh medium all or almost 
all this effect could be eliminated. The peroxide effect appears to be dif- 
ferent from the "activated water" effect in a number of respects such as 
the effect on cleavage as well as on fertilizing power of the sperm. Conse- 
quently, both these mechanisms of indirect action through the medium 
have to be taken into account. 

Barron et al., (1949a, b) have also studied this problem with Arbacia 
sperm by using respiration of the sperm to measure the radiation effect. 
Diluted sperm (1 -.200) showed that X rays, even at doses as low as 100 r, 
caused a measurable inhibition of respiration. These investigators 
pointed out that hydrogen peroxide in low concentration increased 
respiration and so the effect at low doses could not be caused by this sub- 
stance. Furthermore, they found that sea water exposed to 100,000 or 
200,000 r had a marked inhibitory effect on sperm respiration, and that 
the addition of catalase to the water before addition of sperm had no 
effect. Finally, they were not able to demonstrate any hydrogen 
peroxide in sea water exposed to 200,000 r although such water inhiljited 
respiration by about 60 per cent. They believe that stable organic 
peroxides which may be formed in sea water can account for this and other 
cases in which irradiated fluids have an effect. 

Attention should be drawn here to the finding of Stone and his coworkers 
(Wyss et al., 1950) that mutagenic substances are formed by ultraviolet 
irradiation of culture medium. 

In summary, there is a good deal of evidence that stable substances 
which produce biological effects can be formed in the medium as a result 
of irradiation. However, these substances do not appear to be formed in 
sufficient concentration to account to any large extent for such effects as 
death of paramecia, since medium treated with a dose which would have 
been lethal to the animals is not in itseff lethal. Evans et al. (1942) have 
presented evidence for the formation of very unstable substances in the 
medium; and such substances may be responsible for at least part of the 
effects produced. This group was unable to find evidence that cleavage 
delay was affected by such unstable substances. It can be concluded that 
stable and unstable substances produced in the medium all play a role 
but that direct effects in the cells are probably also involved. The rela- 
tive importance of these diverse pathways of action of the radiation m^ay 
not be the same for different effects. 



PROTOZOA AND INVERTEBRATE EGGS 297 

RETARDATION OF CELL DIVISION 

Giese (1947a) has reviewed in detail much of the work on the effects of 
radiation on cell division, and Hevesy (1945) has presented a review from 
a rather different point of view. Nevertheless, it seems desirable to sum- 
marize the major work on fission delay in the protozoa and cleavage delay 
in invertebrate eggs and to expand, somewhat, particular topics upon 
which the reviewer wishes to express opinions. It has been fully estab- 
lished by many investigators that ultraviolet and ionizing radiations, in 
sufficient dosage, retard cell division. In some cases at least, this retarda- 
tion may last for several divisions; but, unless death intervenes, recovery 
of the normal rate occurs sooner or later. Perhaps this recovery is one 
of the most interesting features of the effect. 

There are a few cases in which visible light has been reported to retard 
division. Most of these are the result of photodynamic action (Blum, 
1941, may be consulted for a review of this phenomenon). Tennent 
(1942) mentions delay in cleavage in the sea urchin by visible light in the 
presence of several photodynamic dyes. Giese (1946a) reports delay in 
cell division in Paramecium caudatum in the presence of eosin and in the 
ciliate Blepharisma, w^hich contains a naturally occurring photodynamic 
pigment. However, Zhalkovsky (1938) claims a reduction of cell division 
in Paramecium caudatum by visible light in the absence of a photodynamic 
dye. The delay was said to be more marked in direct than in reflected 
light. Phelps (1946) reports that the division rate of cultures of the 
colorless Tetrahymena geleii was lowered by exposure to sunlight. This 
has since been shown to result from destruction of necessary substances in 
the medium (Phelps, 1949). Perhaps a somewhat similar interpretation 
would be possible for Zhalkovsky's results. 

There have been a number of purported cases of acceleration of division 
by small doses of radiation. Giese (1947a) reviews these cases and comes 
to the conclusion that most of the evidence is of questionable significance. 
However, he apparently accepts several reports, mainly from the older 
literature, of acceleration by ionizing radiation. In all cases, the effects 
are small, and careful statistical analysis has not been made. Moreover, 
there would seem to be considerable inherent difficulty in being sure that 
there are no systematic differences between the controls and the experi- 
mentals other than in the exposure to radiation. Further investigation 
seems necessary before accepting stimulation of division by low doses of 
radiation as a real phenomenon. 

Although division delay is an extremely common result of irradiation, 
it is not universal. Halberstaedter and Luntz (1929) and Halberstaedter 
and Back (1942) were unable to find division delay in Eudorina or 
Pandorina at any sublethal dose of radium rays or X rays. 

Recovery. As far as the reviewer is aware, there is no adequate evidence 



298 RADIATION HIOLOGY 

that division ck'hiy by nidiatiou ever lasts for more than a few divisions, 
provided the cells survive at all. The eases in microorganisms in which 
lasting reduction in rate of multiplication have been found are probably 
the results of genetic changes quite independeni of the original retarda- 
tion of division. 

The time course of reccnery may vary greatly in ditferent cases. In 
some cases, retardation may last for several divisions before complete 
recovery occurs; in some, recovery may be complete, or nearly so, by the 
first division, while in others, there seem to be stages during which no 
recovery occurs. The studies have been concerned mainly with (lualita- 
tive and ciuantitative descriptions of the time course of recovery with only 
a small amount of attention being devoted to attempts to influence 
recovery experimentally. 

The most complete ([uantitative study of division delay has been car- 
ried out with echinoderm eggs and sperm, chiefly those of Arbacia. 
Most of the investigations with ionizing radiations have been concerned 
with the first cleavage only. However, Miwa et al. (1939a) irradiated 
unfertilized eggs of Pseudocentrotus depressus with /3 particles from radon 
and recorded the time to both the first and the second cleavage. The 
data suggest to the reviewer that the interval between the first and second 
cleavages may be slightly longer than normal at higher doses although the 
the authors say that ''there is little or no delay in the . . . second divi- 
sion" (see Fig. 8-la). Yamashita et al. (1939) exposed fertilized eggs of 
this same sea urchin to X, y, and rays and stated that they could find 
no evidence that irradiation during most of the period before the first 
division had any marked effect on later cleavages. However, Blum 
. . . Loos (1949) mention in an abstract that they have obtained the 
same results with X rays and with ultraviolet radiation in fertilized 
Arbacia eggs. Presumably, this includes delay in cleavages later than 
the first. The method used by Blum and his coworkers (see Blum and 
Price, 1950) is probably better designed to detect small difi'erences in 
clea^'age times than were those of previous investigators. It therefore 
seems probable that the effects of ionizing radiations last for more than 
one cleavage but are, in most experiments, of small importance in intervals 
beyond the first interval following the treatment. 

The recovery of sea urchin eggs from cleavage delay by ionizing radia- 
tions has been investigated by two groups of workers, a Japanese group 
(Miwa, Mori, and Yamashita) and Ilenshaw and his collaborators. 
Henshaw's results have been interpreted theoretically by Lea (1938a, b, 
1947). 

Ilenshaw (1932, 194()c) and Miwa d al. (1939a) found that the longer 
the period between irradiation of eggs and insemination with unirradiated 
sperm, the less the effect. In other words, recovery occuncd between 
irradiation and insemination. Irradiation of sperm also brings about 



PROTOZOA AND INVERTEBRATE EGGS 299 

cleavage delay, l)ut there is no recovery if sperm are kept for a time l)efore 
they are used for insemination (Henshaw, 1940a; Miwa et al., 1939a; 
Mori ct al., 1989). The cleavage delay produced by irradiated sperm can 
be shown to increase as a linear function of the dose. According to Lea 
(1947), the data of Henshaw (1940a) show an increase of 25 min in 
cleavage delay for each doubling of the dose. 

Lea (1938a, b) shows that the recovery in eggs can be adequately repre- 
sented by an exponential decay of the original effect with time according 
to the expression e~'^^, where / is the time after irradiation and T is a con- 
stant. The value of T was calculated to be 35 min for the fertilized egg 
and 104 min for the unfertilized egg. 

For Arbacia, Henshaw (1940b) has shown that there is little if any 
delay in stages before the prophase of the first cleavage. Most of the 
delay is in the prophase, with minor delays in the later stages of mitosis. 
Yamashita ct al. (1940) find major delays in the late nuclear fusion and 
prophase stages for Pseudocentrotus and Str'ongylocentrotus. Henshaw 
and Cohen (1940) irradiated eggs at different times after fertilization and 
found that the effect produced by a given dose increased for the first 
10-15 min and then declined, so that by the end of prophase there was 
little if any effect on the time of the first cleavage. The decline was not 
quite regular, there being a small secondary increase in sensitivity at 
about 25 min (early prophase). Henshaw and Cohen (1940) show that 
there is good agreement between the first peak in sensitivity and changes 
in viscosity and permeability, but point out that recovery in the egg pro- 
nucleus up to the time of fusion might also be involved. Lea (1947) also 
■ suggests that the early increase in sensitivity is due to a recovery process 
which he believes can continue up to prophase. Thus the time for 
recovery decreases as the time after insemination increases. The later 
drop in sensitivity may be due to irreversible changes leading to division 
which cannot be affected by radiation; but, as pointed out by Henshaw and 
Cohen (1940), this explanation fails to account for the secondary peak. 

Henshaw (1940d) has shown that low temperature (0°C) decreases the 
rate of recovery. Mori d al. (1939) found no effect of dilution of sperm 
immediately after irradiation, and conclude that failure of the sperm to 
recover is not due to something produced in the medium by the radiation. 

Recovery of the sort reported for Arbacia apparently does not occur 
for all invertebrate eggs. Henshaw ct al. (1933) treated Cumingia and 
Arbacia eggs simultaneously with X rays. The Arbacia eggs showed 
recovery but the Cumingia eggs did not. It should be noted that recov- 
ery, in the sense that the egg developed successfully, did occur. The 
Cumingia eggs simply showed the same delay in first cleavage whether the 
dose of X rays was given at low intensity over a long time or at high 
intensity for a brief period. Cook (1939) has also reported no recovery 
for Ascaris cquorum eggs exposed to X rays and then kept at 5°C for 



300 



RADIATION BIOLOGY 



periods of time ranfj;iiiK up to six 
(a) 



o 

H 
Z 

o 
o 

u. 

o 

UJ 

a 



3 
2 




0-1 



2-3 3-4 
INTERVAL 
(O) 
n DENDRASTER-CHASE- UV 

■ URECHIS -CHASE - UV 
STRONGYLOCENTROTUS-GIESE 

2804 A, 1244/ergs/mm^ 

Dl PSEUDOCENTROTUS- MIWA et al., 
BETA RAYS 

(b) 

■ P. AURELIA-KIMBALL a GAITHER 

2650 A, lOOO/ergs/mm^ 

□ R CAUDATUM -GIESE 

2650 A, 2000/ergs/mm 
Fig. 8-1. Bar diagrams to show the rela- 
tive importance of delay in various divi- 
sion intervals after irradiation. The 
data from the various authors v/as recal- 
culated as time for the division interval 
in question and this time was expressed 
as a nudtiple of the control time for the 
same interval, (a) Data (Chase, 1938; 
Miwa et al., 1939a; Giese, 1938c) for the 
first three cleavages of various marine 
eggs. The eggs were irnuliated sliortly 
before insemination, and 0-1 is the inter- 
val between insemination and the first 
cleavage. (6) Data for two different 
species of Paramecium. The interval 
0-1 is between irradiation and the first 
division thereafter. (Giese, 1945b; Kim- 
ball and Gaither, unpublished.) 



months. The e{i;frs kept in the cold 
.sho\v(!d the .siinu; dciUiy in the first 
fe\v di\isions as those allowed to 
devek)p immediately at 25*'C. 
Ho\vever, another effect, produc- 
tion of ahnormal embryos, showed 
recovery during the period in the 
cold. Evans (lOoO) has confirmed 
these results hut has found a some- 
what different situation with 
Arbacia eggs. Arbacia eggs irradi- 
ated \vith low-intensity X rays 
divide without further delay when 
the irradiation ceases, which sug- 
gests that recovery and inhibition 
occur at nearly equal rates. How- 
ever, the effects on later embryonic 
development are more pronounced 
after longer exposures, indicating 
that the rates of recovery for the 
two effects are quite different and 
that recovery of division delay is 
more rapid. 

There have been a number of re- 
ports that marine eggs exposed to 
ultraviolet show delay in cleavages 
later than the first one after treat- 
ment. Chase (1937, 1938), using a 
quartz mercury arc and the eggs of 
the marine worm Urechis caupo and 
the sand dollar Dendraster excen- 
tricus, found that several successive 
divisions were affected when radia- 
tion was given before fertilization. 
Not all of Chase's data demonstrate 
recovery but observations were ex- 
tended over only the first few 
cleavages. Giese (1938c), using 
monochromatic ultraviolet, found 
the same thing for Strongijloccnt- 
rotus purpuratus. Sample cases 
from Chase and Giese are shown 
in Fig. 8-la. However, Giese 
(1939b) irradiated the sperm of 



PROTOZOA AND INVERTEBRATE EGGS 



301 



S. purpuratus and obtained retardation of first cleavage with, at most, a 
very slight effect on later divisions. Marshak (1949b) reports that sperm 
exposed to 2537 A ultraviolet delay the first cleavage but have no appre- 
ciable effect upon the second. He also reports that there is less delay in 
division if the sperm are irradiated shortly before insemination than if 
they are irradiated ^-1^ hr before. Perhaps this increase of the effect 
with time between irradiation and insemination was due to some sub- 
stance produced in the medium. 

Blum and Price (1950) report a detailed study of recovery in Arbacia 
eggs irradiated with ultraviolet from a mercury arc. In most cases, the 




80- 



60- 



\. 



40- 



20- 



NORMAL CLEAVAGE INTERVAL 
i 



— r— 
50 



— I — 
100 



— 1 

150 







(6) 



NORMAL CLEAVAGE INTERVAL 

L 



— 1 — 

50 



100 150 

TIME (MIN) FROM IRRADIATION TO CLEAVAGE 

0,« I st TO 2nd CLEAVAGE 

A,A2ndT0 3rd cleavage 
□ .■3rdT0 4th cleavage 

Fig. 8-2. Graphs, modified from Blum and Price (1950), to show the recovery of 
Arbacia eggs from the effects of ultraviolet irradiation. Eggs were irradiated at 
various times and the time in minutes between a given pair of cleavages was plotted 
against the time from irradiation to the cleavage beginning the interval in question. 
The open and solid symbols represent different experiments in which radiation was 
given at different times. The points are approximately the center of distribution 
of a whole series of points given by Blum and Price (1950). (a) Irradiation after the 
first cleavage. (6) Irradiation h^efore the first cleavage. 

eggs were irradiated after fertilization, often after the first cleavage. 
Eggs irradiated early in a cleavage interval showed a maximum effect on 
the duration of that interval, while those irradiated late in an interval 
showed no effect until the succeeding intervals. By plotting the length 
of the interval against time from irradiation to the cleavage beginning the 
interval, a smooth curve showing recovery from the effect was obtained 
(see Fig. 8-2). The smooth form of this curve and its extension over more 
than one division interval suggests that the recovery process was inde- 
pendent of the occurrence of cleavage. Recovery was also demonstrated 
to occur in eggs irradiated before cleavage. Blum . . . Loos (1949) 
state in an abstract that X-irradiated eggs behave in the same way as 
those exposed to ultraviolet, but they give no details. 

The results of Blum and Price (1950) are in agreement with those of 



302 UADIATION lUULOGY 

Ilonshaw and Cohen (1940) in that sensitivity decreases as the time after 
fertilization increases. However, Ilenshaw and Cohen found a small 
secondary increase in sensitivity in the early prophase stage. Gross 
(1950) found that Chartoptcrus vg^s exposed to ultraviolet showed some- 
what the same sensitivity relations as did X-irradiated Arhacia eggs. 
'Hie eggs were (juite sensitive for the first 30 min, after which the sensi- 
tivity decreased hut increased again in the period from 40 to 50 min. The 
simplest explanation of the results of Blum and Price (1950) would be 
that there is a period after which division is irrevocably determined. 
However, the results of the other two investigators are not so easily 
explained, and it seems probable that rather complex factors are involved 
in the.se changes in sensiti\'ity. 

These reports make it clear that sponluncous recovery from the effects 
of ratliation occurs in the sea urchin. For ultraviolet, it appears estab- 
lished that this recovery is a gradual process extending over more than 
one division. Both Henshaw and Lea treat the X-ray data as thougli 
recovery were complete by the time division occurs, but Henshaw and 
his coworkers do not present evidence on later cleavages. In view of this 
and the statement of Blum . . . Loos (1949), that X- and ultraviolet- 
irradiated material showed the same behavior, it seems possible that in 
both cases the recovery process is independent of the occurrence of 
cleavage. If this is so, as suggested also in a brief statement in Blum 
et al. (1951), then radiation must affect something which controls the rate 
at which division occurs instead of simply destroying some material which 
must be restored to its original amount before any cleavage can take place. 

The separation between cell division and recovery is much more marked 
in the cihate protozoa. Giese (1939a, 1945b) has shown clearly for 
Paramecium caudatum that ultraviolet retards several divisions following 
the irradiation (see Fig. 8-lb). Giese and Reed (1940) have shown the 
same thing in somewhat less detail for several species of Paramecium. 
(Jiese (194(ia) has made similar findings for tfie retardation of division by 
visible light in Paramecium exposed to eosin, and in Blepharisma. Kim- 
ball et al. (1952) find the following pattern for P. awre/m exposed to mono- 
chromatic ultraviolet (see Fig. 8-16). The same pattern has been found 
for wave lengths 2378, 2537, 2()50, and 2804 A. The first division follow- 
ing irradiation is usually markedly delayed. The next division is also 
delayed, but less so. Either the third or fourth interval is often extremely 
long, lasting in some cases for two weeks or more. Finally, recovery of 
the normal rate is usually complete by the sixth division. It seems possi- 
ble that at least thn^e prcx'cs.ses should be recognized: (1) retardation of 
the first division after irradiation, (2) a long but not permanent cessation 
of division, usually setting in after two or three divisions have occurred, 
and (3) a relatively small increase in all other division intervals through 
about the sixth. The relative magnitude of these various effects appears 



I'UOTUZOA AND INVEKTEHRATE EGGS 30.3 

to (*hanji,v with dose, the second process becoming relatively more impor- 
tant as the (lose increases. However, even at quite low doses, elTects 
lasting through about six divisions can be recognized. Ivccovery from 
lower and higher doses appears to require about the same number of 
divisions. 

The effect of X rays on cell division in the ciliates has been investigated 
to only a small extent, mainly because the doses necessary to produce an 
appreciable delay are of the order of a hundred thousand roentgen units. 
Perhaps it is this feature more than any other which emphasizes the great 
radioresistance of protozoa, for most other cells are retarded by much 
smaller doses. Back (1939) reports that the X-ray dose for death within 
2 hr in P. caudatum lies between 400,000 and 600,000 r. A dose between 
two-thirds and five-sixths the lethal dose leads to a permanent cessation 
of division. About half the lethal dose leads to a retardation of the first 
division of some 36 to 48 hr after which the normal rate is restored, appar- 
ently with no effect on divisions later than the first. Giese and Heath 
(1948) report an effect only on the first division at lower doses but effects 
on later divisions at higher doses. Powers and Shefner (1950) report that 
650,000 r reduces by half the rate at which irradiated P. aurclia reaches 
the first division, but effects on later divisions are not mentioned. Kim- 
ball et al. (1952) found an effect of X rays on divisions later than the first 
in P. aurelia, but the effect was much smaller, relative to delay in the first 
division, than that for ultraviolet. 

Thus the results ior Paramecium and the sea urchin seem quite different. 
A guess might be made that cleavage delay in the sea urchin corresponds 
more nearly to the delay in the first division in Paramecium, while the 
other delays in this organism have no counterpart in the sea urchin. 
However, it is not quite certain that the latter is true. In Paramecium, 
the four products of the first two divisions of one treated animal may have 
cessation periods of rather different duration (Kimball et al, 1952). In 
the sea urchin, a similar occurrence would lead to abnormal cleavage, and 
perhaps development would finally stop. A number of workers have 
reported abnormal cleavages following irradiation of invertebrate eggs 
(see Giese, 1949, for review). 

Mention may be made here of the investigations of Robertson (1935a, b) 
with the flagellate Bodo caudatus. She found that continuous exposure to 
7 rays from radium led, at first, to a decrease in rate of cell division, but 
later a partial recovery toward the normal rate occurred even though the 
irradiation continued. Meanwhile, the flagellates became larger than 
normal in size. Following cessation of irradiation, they multiplied more 
rapidly for a time before the normal rate was restored. The partial 
resistance to radiation did not persist. 

Sensitivity. There has been a rather miscellaneous group of investiga- 
tions on the effects of various factors on retardation of cell division by 



;j()4 |{ ADl A'PIOX IIIOLOGY 

radiation. Alpatov and Xastiukova (iy34(') found that the effect of 
ultraviolet on Paramrriiim was intensified by exposure to slifi;htly unfa- 
\-oral>le temperatures after irradiation, the least effect hein^ evident when 
the animals were irradiated at temperatures near the middle of tlu; \ital 
range. They suggest tliat these changes in sensitivity may he related to 
protoplasmic viscosity. In another paper (1934h) they report that 
sodium sulfate and electrical stimulation, both of which increased the 
viscosity, decreased the effect of ultraviolet while potassium thiocyanate 
and mild narcosis, which decreased the viscosity, increased the sensitivity 
to ultraviolet. On the other hand, Wilbur and Recknegel (1943) have 
shown that treatment of Arbacia eggs with potassium citrate (0.35 M) 
only slightly decreased the retardation of cleavage by X rays, whereas 
addition of calcium or magnesium to the sea water had no effect at all. 
All these treatments affected the viscosity of the egg. These investiga- 
tors also report that doses of X rays (30,000 r) which markedly affected 
the rate of cleavage had no detectable effect on viscosity. They con- 
cluded that changes in ionic composition and viscosity cannot be impor- 
tant factors in division delay by X rays. Zirkle (1936) has shown that a 
high carbon dioxide content in the atmosphere at the time of irradiation 
increases the sensitivity of Paramechim to the division-retarding effects of 
X rays. Hutchings (1948) reported that Arbacia eggs suffer cleavage 
delay when briefly exposed to 36°C^ 10 min after insemination. She 
found that the cleavage delays produced by this temperature and by 
2537 A ultraviolet were additive, or nearly so, when the eggs w^ere exposed 
to the high temperature either before or after the irradiation. 

The phenomenon of photoreactivation has been studied for clea\'age 
delay in sea urchin eggs exposed to ultraviolet by Blum . . . Robinson 
(1949); Blum, Loos, and Robinson (1950); Blum, Robinson, and Loos 
(1950); Marshak (1949a, b); and Wells and Giese (1950). It has also 
been found for di\'ision delay in ultraviolet-irradiated P. aurclia by Kim- 
ball and Gaither (1951). The subject will not be discussed further here, 
since it will be reviewed in detail by Dulbecco in Chap. 12 of this volume. 

Differences in sensitivity between various strains and species have 
been reported by several investigators. Alpatov and Nastiukova 
(1934a) found distinct differences in sensitivity to ultraviolet between 
P. caudatum and P. bursaria. Giese and Reed (1940) made an extensive 
study of different species and stocks of Paramecium and found considera- 
ble difference in their sensitivity to the division delay produced by ultra- 
violet. They also found that starved pai'amecia were more susceptible 
than well-fed ones. Giese (1946b) reported a wide range in sensitivity to 
ultraviolet in the eggs and sperm of different marine invertebrates. The 
echinoderms, whose eggs have indeterminate cleavage, are mature when 
shed, and cleave radially, showed a consideral)le difference in sensitivity 
between egg and sperm, and cleaved abnormally only when given high 



PROTOZOA AND INVERTEBRATE EGGS 80") 

doses. 'I'he other organisms were from various phyla whose eggs have 
determinate cleavage, are immature when shed, and cleave spirally. 
They showed little difference in sensitivity between the egg and sperm 
and showed irregular cleavage at low doses. Henshaw et al. (1933) 
report marked differences in sensitivity to cleavage delay by X rays 
between Chaetoptenis, Nereis, Cumingia, and Arhacia eggs. 

Localization. A considerable amount of evidence in regard to the part 
of the cell which is responsible for radiation-induced cleavage delay has 
been accumulated. It rather strongly suggests a nuclear effect for the 
sea urchin but is not so clear for the protozoa. Of course, the delay need 
not be due to the same causes in such different types of cells, although 
such a unifying hypothesis would be attractive. Thus the details of the 
mitotic process in the ciliate protozoa are quite different from those in the 
sea urchin. Moreover, in the eggs, there is no growth in size between 
cell divisions, whereas in ciliates growth occurs, and is, perhaps, essential 
for later divisions. 

In the sea urchin, it has been repeatedly shown that irradiation of either 
the sperm or the unfertilized egg can bring about cleavage delay. Among 
reports on this subject may be mentioned those of Henshaw and Francis 
(193()) and Henshaw (1940a, b) on X-irradiated Arhacia; Marshak 
(19-l:9b) and Blum, Robinson, and Loos (1950) on Arhacia exposed to 
ultraviolet; Giese (1939b, c; 1946b) on a variety of marine invertebrates 
exposed to ultraviolet. The two gametes obviously contribute quite dif- 
ferently to the zygote. Thus the sperm contributes the male pronucleus 
and the centrosome which functions in the first cleavage (Henshaw and 
Francis, 1936). The egg contributes the female pronucleus and the bulk 
of the c^ytoplasm. As Henshaw and Francis (1936) point out, the delay 
produced by irradiation of the unfertilized egg indicates that injury to the 
centrosome is not involved since this gamete does not contribute a func- 
tional centrosome. The one portion of the zygote to which both gametes 
are known to contribute is the nucleus. Therefore, the simplest conclu- 
sion would be that the effect is on this structure. This conclusion is not 
absolutely demonstrated by such evidence since it is possible that the 
sperm contributes cytoplasmic elements which, though small in bulk, are 
important in division. Nonetheless, the very fact that irradiation of 
either gamete produces delay certainly suggests a nuclear effect. This 
conclusion is not affected by the difference in sensiti^'ity between egg and 
sperm, for the nuclei in the two gametes are in quite different physical 
states and are subject to different amounts of shielding in the case of 
ultraviolet. 

Supporting evidence for a nuclear site of the injury is furnished by 
experiments with eggs fragmented by centrifugation. Henshaw (1938) 
has shown that X irradiation of either the whole Arhacia egg or the 
nucleated half results in cleavage delay but X irradiation has no effect on 



30(i UAUlvriON lUOLC^GY 

eiuu'leute halves subscMiiuMitly fertilized with unirrudiated .sperm. Blum, 
R()l)iiis()ii, and Loos (1950, 19ol) carried out similar experiments with 
ultraviolet-irradiated Arharia ejiigs and found the same results. They 
also demonstratetl that other combinations, such as irradiated sperm with 
unirradiated enucleate halves of egs^, result in dela3^ Their conclusion 
was that the locus of the primary injury must he in the nucleus. Harding; 
and Thomas (1949, 1950) found that centrifuged Arbacia eggs irradiated 
unilaterally with ultraviolet through the fat cap were more affected than 
were those irradiated through the pigmented end. They draw no final 
conclusions from these results, but a nuclear effect seems to be fa\-ored 
since the nucleus would be displaced toward the fat cap. Marshak 
(1949b) suggests that the relative inefficiency of ultraviolet for the egg as 
compared to the sperm favors a nuclear effect. Otherwise, the high pro- 
portion of ultraviolet absorbed in the cytoplasm should make the egg 
more, not less, sensitive. 

Thus most of the evidence clearly favors a nuclear effect. However, 
there is certain evidence which is not in full agreement. Giese (1939b, 
1947a) has shown that the action spectrum for delay by ultraviolet-irradi- 
ated sperm resembles the absorption spectrum for nucleoprotein, whereas 
that for ultraviolet-irradiated eggs resembles the absorption spectrum for 
certain other proteins (Fig. 8-3). Giese (1939b, 1947a) discusses various 
explanations among which is the possibility that the effect is partially 
cytoplasmic in the case of the egg but entirely nuclear for the sperm. 
However, in the egg, the primary absorption might be in the cytoplasm 
with secondary effects on the nucleus or it might be by proteins, other 
than luicleoprotein's, in the nucleus. Since the nuclei in the tw'o gametes 
are not in the same state, such differences in importance between nucleic 
acid and protein absorption would be possible. This emphasizes that 
action spectra cannot be used to reach a clear decision between a nuclear 
and a cytoplasmic site of radiation injury since nucleic acids and several 
kinds of proteins are present in both. 

Blum and Price (1950) believe that the fact that recovery from ultra- 
violet-induced delay is independent of the occurrence of cleavage suggests 
a cytoplasmic locus, since the nucleus undergoes major changes at the 
time of cleavage. However, Blum, Robinson, and Loos (1950, 1951) pre- 
sent e\'idence that the primary absorption of the ultraviolet is in the 
luicleus. On the basis of their belief that the sperm cannot be photore- 
activated before fertilization, they conclude that recovery is a cytoplasmic 
process. The argument that cytoplasmic rather than niu'lear processes 
are suggested by a recovery independent of cleavage appears weak since 
the cytoplasm at cleavage may well undergo changes (luite as profoiuid as 
those in the nucleus. The evidence that sperm are not subject to photo- 
reactivation has been called in cjuestion by the finding of Wells and Giese 
(1950) of some photoreactivation of Stromiijloccntrotus sperm. Blum, 



( 



PROTOZOA AND INVERTEBRATE EGGS 



30: 



RoV)in.son, and Loos (1951 ) do not l)elieve that this applies to Arbacia. 
Thns there are compelling reasons for thinking that cleavage delay is dne 
to nuclear damage. The evidence that recovery depends on cytoplasmic 
events is suggestive but not very strong. 

The situation for the protozoa is not so clear. Hohveck and Lacassagne 
(1931a, b) found that one of the efTects which occurred in the flagellate 
Polytoma uvella when it was exposed to a particles was a cessation of divi- 
sion accompanied by an increase in cell size. The cells failed to recover 
from this effect and finally died, so it is not clear that the effect should be 
classified with division delay. Hohveck and Lacassagne do not give 
detailed data but state that the effect was due to a single-particle event 



100 



80- 



60 



>- 
o 



40 



20- 




2400 



2600 



2800 
WAVE LENGTH. A 



3000 



1 

3200 



Fig. 8-3. Action spectra for retardation of cleavage in the sea urchin for irradiated 
sperm and irradiated eggs, replotted from Giese (1947a). Solid circle = sperm 
irradiated. Open circle = egg irradiated. 

and that sensitive volume calculations suggested a body the size of the 
centrosome. Certainly, it would be expected that injury to the centro- 
some might lead to difficulties with division; but in the absence of 
detailed data it is hard to judge how compelling is the evidence for this 
identification. 

Using Amoeba proteus, Mazia and Hirshfield (1951) have found evidence 
for both nuclear and cytoplasmic effects on division delay by ultraviolet 
The nucleated halves of bisected amebae are more sensitive to division 



308 



T{ADI\TT<)N HIOLOCY 



delay by the nidiation than are whole amehae. This cannot be inter- 
preted as the result of shieldinp; of the nucleus by the larger amount of 
cytoplasm in whole amebae, for both whole and half amel)ae si)read over 
the substrate so that they are of approximately the same thickness. 
Mazia and Ilirshfield (H)51) sug{i;est that the increased sensitivity 
reflects an effect of the cytoplasm on recovery processes. They also 
find evidence for a cytoplasmic effect of the radiation in the fact that 
irradiated enucleate halves die more rapidly than the unirradiated halves. 



lOOn 



90- 



80- 



70 



o 60- 



50 



40- 



30- 



20- 



10 




2400 



— I 

3000 



2600 2800 

WAVE LENGTH, A 

Fig. 8-4. Action spectra for retardation of cell division in Paramecium, modified from 
Giese (1945bj. Solid circle = time to third division — starved. Open circle = time 
to recovery — well-fed. 

For Paramecium, the evidence as to localization of the effect is not ade- 
quate. Giese (1945b) has found an ultraviolet action spectrum for 
retardation of the third division in starved P. caudatum similar to the 
absorption spectrum for nucleoproteins (Fig. 8-4). A similar action 
spectrum was found for recovery of the normal rate in \vell-fed animals. 
However, the action spectrum for time to the third division in well- 
fed animals is rather nondescript, having a very slight maximum at 2804 A 
(Fig. 8-5). Kimball et al. (1952) have been able to confirm, although 
with differences in detail, the nondescript action spectrum for well-fed 
P. aurelia (Fig. 8-5) but have been unable to demonstrate a nucleoprotein- 
like spectrum for recovery of the normal rate. Giese (1947a) concludes 
that "the immediate effect is upon the cytoplasm but the more lasting 
effect is upon the nucleus." It does not seem to the reviewer that this 



PROTOZOA AND INVIOUTEBRATE EGGS 



309 



conclusion is justified. As has been pointed out in a preceding paragraph, 
a nucleoprotein-type action spectrum does not, by itself, indicate that the 
nucleus is involved. Effects upon cytoplasmic nucleic acids are just as 
possible. Moreover, the duration of an effect through a number of divi- 
sions before recovery does not necessarily mean that the effect is nuclear. 
Long-lasting cytoplasmic effects are also possible. It can only be con- 
cluded that there is no critical evidence on the localization of the changes 
leading to division delay in Paramecium. 



100-1 



80- 



60- 



40- 



20- 




2200 



2400 



2600 
WAVE LENGTH. A. 



2800 



3000 



Fig. 8-5. Action spectra for retardation of cell division in well-fed paramecia, modified 
from Giese (1945b), and from Kimball, Geckler, and Gaither (1952). Open circle = 
time to third division (Giese) — Paramecium, well-fed. Solid circle = time to sLxth 
division (Kimball, Geckler, and Gaither) — Paramecium, well-fed. 



INHERITED EFFECTS 

There have been scattered reports of mutation induction in inverte- 
brates, other than insects, but such studies add nothing basically new to 
the studies of mutation which are to be considered elsewhere in this series. 
This section will be concerned almost entirely with the protozoa. For 
this group a rather different point of view from that in classical mutation 
investigations must be taken where inherited effects are concerned. 
Work reviewed by Sonneborn (1947, 1949) has made it clear that watch 
must be kept for kinds of inherited differences which are not dependent on 



310 HAOIATION niOLOGY 

clirt'ciciices ill the genos for tlioir maiiitciiaiico. In tlio protozoa, iiih(!ril- 
ancc ill iiii<'a}2;(>s of cells niultiplyiiif? vegctatixciy ;is \v(»ll as iiihcrit.ancc 
over the sexual processes can l)e investigated. Thus, in addition to the 
classical mutation approach, (jther lines of investifi;ation may prove fruit- 
ful. Actually, the inxestigations availal)le for this review ha\'(> added 
only scattered bits of information along these lines. 

To the reviewer's knowledge, MacDougall (1929, 1931) was the first to 
report the induction by radiation of inherited changes in protozoa. She 
exposed mass cultures of the ciliate Chilodonella uncinatus to ultraviolet 
from a (luartz merciiiv aic for brief periods on several successive days. 
In some cultures abnormal animals appeared. A few of the abnormalities 
proved to be inherited for many generations of asexual reproduction, and 
in some cases through conjugation. These included apj)arent tetraploid 
and triploid forms as well as others exhil)iting only changes in form and 
size. Since the mutant forms appeared in only a few of the irradiated 
cultures, the conclusion that they were induced by the radiation can 
hardly l)e considered established, especially since the number of control 
cultures is not given, but the aim of this woik was to obtain mutant forms, 
not to investigate their origin. 

Alottram (1941, 1942) exposed cultures of an amicronucleate Col- 
pidinm (said in a footnote to belong to this genus though called Para- 
mecium in most of the text) to daily doses of ultraviolet or y radiation 
(also to low and high temperatvn-es and to carcinogenic hydrocarbons). 
After 4 to 62 days, a few abnormal animals were found. The doses of 
ultraviolet are not given. The doses of y rays ranged from 800 to 
12,1()0 r. The abnormals continued to produce abnormal descendants 
though not necessarily of the same type. Somewhat similar changes 
were produced by ultraviolet irradiation of Glaucoma sctosa. Investiga- 
tions with Paramecium caudatum and Aspidisca sp. are also reported, but 
it is not stated that radiation was employed. 

Mottram (1942) suggests that changes in viscosity are invoUed, that 
the changes are cytoplasmic, not nuclear, and that they are similar to 
those involved in carcinogenesis in higher forms. The arguments Mot- 
tram gives for cytoplasmic and not nuclear change are inconclusive. 
Nonetheless, it is hard to see how chromosomal changes could be involved 
in an amicronucleate Colpidium. However, further investigation is 
recjuired of both the origin and inheritance of these changes. It is espe- 
cially necessary that the experiments permit a quantitative study of the 
origin of the abnormalities followed by a careful genetic analysis of the 
inheritance. 

Spencer and Calnan (1945), working with P. multimicronucleatum, 
report a long-term deleterious effect of continuous sublethal exposures to 
radium, to a number of dyes, and to methylcholanthrene. The animals 
were grown in mass culture with continuous exposure to the agent. 



PROTOZOA AND INVERTEBRATE EGGS 311 

Transfers were made every 10-12 days. The division rate was about one 
per day. Although the exposed cultures continued to multiply through 
many transfers, they eventually died out while control cultures survived. 
Thus the cultures exposed to radium succumbed at the 190th transfer 
while 16 control series were still aHve after more than 20(i transfers. It 
would appear that the effects of sublethal exposure to radium and the 
other agents were accumulati^•e o\'er the course of many generations of 
cell division. However, no genetic analysis of the material was made, 
and the method allows autogamy, and so gene recombination, to occur. 
Various complex processes of selection of both the paramecia and their 
accompanying bacteria are also possible. An interpretation in terms of 
mechanism seems impossible without further analysis. 

Schaeffer (1946) reported an inherited change in size induced in the 
giant multinuclear ameba Chaos chaos. Some of the amebae broke into 
fragments following X irradiation. The largest fragments developed into 
clones of normal size and the smallest died. However, some of the 
medium-sized fragments grew into clones whose average volume was 
about 60 per cent of the parent clone. One such clone was maintained 
for four years. When this small clone was exposed to X rays, clones were 
obtained which were still smaller. These latter clones had been main- 
tained for three months at the time of the report. Schaeffer does not 
propose a mechanism to explain these results. In the light of the multi- 
nuclear condition of this species, it seems difficult to suppose that gene 
mutations or chromosomal aberrations were involved. If fragmentation 
into medium-sized pieces is really a necessary first step, interesting specu- 
lations concerning the determination of size in such multinucleate proto- 
plasmic masses might be made. However, no data on the frequency with 
which the change has occurred are given, so that it is difficult to evaluate 
the apparent correlation l)etween the size of the fragment and the occur- 
rence of the variant. 

The self-reproducing cytoplasmic particle, kappa, of P. aurelia has been 
shown to be inactivated by X rays (Freer, 1948, 1950), by nitrogen 
mustard (Geckler, 1949), and by 2537 A ultraviolet (Kimball, 1950). 
Freer (1950) finds that the curve of the logarithm of the number of par- 
ticles against dose is not quite linear and suggests several sources of diffi- 
culty which might explain the departure, since he believes that the inac- 
tivation of kappa is basically a single-event phenomenon. Depending on 
the interpretation, the true inactivation dose (37 per cent dose) is con- 
sidered to lie between 3400 and 4000 r or at approximately 10,000 r. 
Sensitive volumes calculated on this basis are in reasonable agreement 
with the size of the particles which can be observed under the microscope. 
Freer reports that microscopic examination showed that it takes some 2 
to 3 days in the absence of cell division for the visible kappa particles to 
disappear following X irradiation. 



312 RADIATION 1U()I-<JGY 

DippoU (1948) lias reported finding spontaneous mutations of kappa. 
So far lit) reports of radiation-induced mutations of this entity have been 
made. 

Lee Oi)4l)) lound that X inaxhatioii of I', hmsnria led to changes in 
mating type wliich became more fre(iuent as the dose was increased. He 
suggests that X rays may induce autogamy which may in tiun cause an 
increased frequency of mating type change. However, as he points out, 
tlie comicctioii between chang(> of mating type and autogamy has not been 
fully established for this species. Thus some other pathway of action of 
the X rays is possible. 

A series of reports on genetic changes in /^ aitrclia induced by 0, ultra- 
violet, and X radiation and by nitrogen mustard ha\-e been made by 
Geckler, Kimball, and Powers and their coworkers. The method used 
by all these workers was basically the same and depended on the fact that 
autogamy makes the animals completely homozygous. The paramecia 
were exposed to the radiation, and a number of the exposed animals were 
isolated. After a period of vegetative multiplication autogamy was 
induced, and a number of autogamous animals were isolated from the 
progeny of each treated animal. Each autogamous animal was allowed 
to multiply for a period of several days and was then checked for survival 
(Powers) or for survival and amount of growth (Kimball, Geckler). The 
percentage surx^iving with normal growth can be taken as a measure of the 
effect. 

When ionizing radiations were used, it was found that at doses much 
too low to have immediately detectable effects either on survival or rate 
of division of vegetative animals, many of the autogamous clones were not 
viable or, if viable, divided more slowly than usual. The dose range used 
has been from about 300 to about 20,000 r ; above the latter dose almost 
all the exautogamous clones were affected. Such observations have been 
reported by Kimball (19-l:9a, b) for /3 particles from P'^' outside the culture 
medium and for X rays, by Powers (1948) for P'*- and for a mixture of Sr^^ 
Sr^", and Y^" in the medium, and by Powers and Shefner (1948, 1950) for 
X rays. Geckler (1950) reported similar findings for nitrogen mustard; 
Powers and Shefner (1950) and Powers and Raper (1950) reported on 
doses of X rays and nitrogen mustard which were sufficiently high to have 
a distinct immediate effect on the animals in addition to the effect after 
autogamy. Kimball and Gaither (1951) report that doses of 2650 A 
ultra\iolet, which an^ sufficient to produce a detectable effect after autog- 
amy, also cause a temporary retardation of the first few cell divisions 
following irradiation. 

The simplest interpretation for effects which do not appear until after 
autogamy is that they are due to gene mutations or chromosomal aberra- 
tions in the micronuclei. Sonneborn has shown that autogamy results in 
the formation of a completely homozygous synkaryon from which the new 



PROTOZOA AND INVERTEBRATE EGGS 313 

macroniiclei and the micromicloi of tlie exautogamous clone are derived. 
Sonnehorn also has given evidence that the macronucleus is a compound 
structure in which each gene and chromosome are represented many 
times. The evidence is reviewed by Soinieborn (1947). Under the cir- 
cumstances, mutations or chromosomal aberrations would not be expected 
to express themselves immediately following irradiation but only after 
homozygosity of both the macro- and micronuclei had been brought about 

by autogamy. 

On this basis and from the results of several breeding experiments, 
Kimball (1949b) came to the conclusion that most of the death and low 
rate of multiplication in the exautogamous progeny of irradiated animals 
were the results of gene mutations or chromosomal aberrations. Kimball 
(1949a) showed that a given total dose of particles divided into several 
small daily fractions, with the animals undergoing several cell divisions 
between each fraction, was as effective as the same dose given in a single 
exposure of a half-hour's duration or less. This was taken to mean that 
the mutational changes must have been gene mutations or one-break 
chromosomal aberrations rather than two-break aberrations. However, 
the dosage curve was more nearly typical of a "multiple hit" than a "one- 
hit" curve. For this reason, Kimball (1949a) suggested that most of the 
non-normal exautogamous clones were the result of the combined action 
of a number of mutant genes with individual effects too small to be 
detected. 

Powers and Shefner (1948) using X rays and Geckler (1950) using 
nitrogen mustard have both presented further e^'idence from breeding 
experiments for the genie or at least the micronuclear basis of the post- 
autogamous effect. However, Geckler (1950) reported on a number of 
findings which can be explained in terms of micronuclear inheritance only 
with great difficulty if at all. Kimball (1949b) reported one case of 
inheritance which did not conform to expectations. 

Another phenomenon, not at present explained, is that reported by 
Powers and Shefner (1950) for very high doses of X rays and by Powers 
and Raper (1950) for nitrogen mustard. In both cases, they found that 
death after autogamy rose to a maximum as the dose increased, then 
declined somewhat. With X rays, there was a secondary rise at very 
high doses. 

The reviewer believes that there is strong evidence that radiations and 
nitrogen mustard induce mutations in the micronuclei of P. aurclia and 
that these mutations express themselves in death and low rate of multi- 
plication of exautogamous clones. Ho\yever, there is rather convincing 
evidence that this is not the whole story and that other phenomena may 
also play a significant role. Further experiments to define this situation 
more thoroughly are needed. 

Most of the work with Paramecium has concerned itself more with the 



,'il I UADIATION liUJLOUY 

iKiturc of the iiiliciilcd chaiifJics which are prochiccd tliaii with the mech- 
anism !)>• which they are pnxhiced. 'I'he work of Powers (I'.)IS) with 
raihoactix-e isotojx's in the mechuin is an exception, lie lonnd that tor 
eijual activities, as measnred by an air ionization chamher and vibrating; 
reed electrometer, P^' was four to six times as effective in producing death 
after autogamy as a mixture of Sr«", Sr«", and ^''"'. It would Ix; expected 
tliat the phosphorus would be eoneentrated in the nucleus but not the 
strontium and yttrium. Rubin (1948) computed from Powers' data the 
expected increase of specific ionization due to the concentration of phos- 
phorus in the nucleus and came to the conclusion that it could not account 
for the total difference in effect which Powers found. He concluded that 
some other factor must be involved and was inclined to believe that it was 
the transmutation phenomenon, i.e., the result of the radioactive disin- 
tegration of phosphorus atoms incorporated in the molecules of the chro- 
mosomes. A number of approximations of necessity enter the calcula- 
tions so that it would seem well to withhold (inai judgement until further 
investigations of this matter have been made. 

In summary, nuclear mutations have been induced in Paramecium 
aurelia and are subject to ciuantitative study. In addition, several ill- 
defined inheritable changes have been found after irradiation of various 
protozoa. Further advances in understanding these changes must depend 
on obtaining them in a situation in which definitive genetic analysis is 
possible. 

MISCELLANEOUS EFFECTS 

Activation of Eggs. Loeb (1914) discovered that unfertilized eggs of 
Arhacia and Chartopierus could be stimulated to l^egin parthenogenetic 
development by exposure to ultraviolet from a ([uartz mercury vapor 
lamp. Giese (1949) has recently reviewed the subject but a brief dis- 
cussion of it seems desirable here. 

The activation of the sea urchin egg has been studied by several investi- 
gators. This egg is mature when shed and activation is indicated by 
membrane elevation and cleavage. The later cleavages in activated eggs 
may be quite irregular, with spindle abnormalities and fragments of 
chromatic material on the spindle (Nebel et al, 1937). Hollaender (1938) 
has shown that wave lengths of 2()50 A and longer have very little effect on 
the whole Arhacia egg. The curve of effectiveness rises sharply around 
2400 A and is still rising at the shortest wave length used, 22()0 A (Fig. 
8-0). This type of curve is a rather generalized one, resembling the 
absorption curves for certain proteins and for lipids. Giese (1949) 
believes that it may be the result of absorption in the lipids of the cell 
membrane. It would appear that the curve for different eggs may not be 
the same. Giese ( 1 938d) found no activation by 2537 A ultraviolet, in the 
doses used, of the eggs of the sea urchin Sfrongyloccntrotus, but he (1939c) 



PROTOZOA AND INVERTEBRATE EGGS 



3io 



lOOn 



readily activated eggs of the marine worm Urechis caupo with this same 
radiation. 

Harvey and HoUaender (1938) fractionated the Arbacia egg by ceiitrifu- 
gation into white (nucleated) and red (nonnucleated) halves and separated 
the latter into yolk and pigment (luarters. Activation was obtained with 
some differences in detail when either half or either of the two quarters 
were exposed to ultraviolet of 2480 A or below. Activation was also 
obtained for the red half and its two quarters with doses of the band of 
wave lengths 2650-3000 A, which were ineffective with the whole egg or 
the white half. It can be concluded 
that the nucleus does not play an im- 
portant role in activation by ultraviolet. 
While there seem to be differences in 
detail in the activation of different parts 
of the egg, it is not at all unlikely that 
changes in the surface of the eggs are 
involved. This is further indicated by 
the studies of Reed (1943) and Spikes 
(1944) which have shown localized effects 
on membrane formation as the result of 
unilateral irradiation of sea urchin eggs. 
Similarly, Tchakotine (1935a, b) has 
shown localized changes in the surface 
followed by activation phenomena as a 
result of localized irradiation of the 
Pholas egg. 

The investigations of Heilbrunn and 
Wilbur (1937) and Wilbur (1939) on the 
effects of calcium and magnesium on 
ultraviolet activation of the Nereis egg 
also suggest a surface phenomenon. 
Heilbrunn and Wilbur (1937) have shown 
that citrate inhibits the activation ; they 
suggest that this is due to removal of cal- 
cium from the cortex, so preventing 
Heilbrunn's views, is responsible for 




•— 



2200 



2400 
WAVE LENGTH, A 



2600 



Fig. 8-6. Action spectrum for 
activation of Arbacia eggs, from 
HoUaender (1938). 



the calcium release which, on 
the activation. Alsup (1941) 
found that photodynamic activation of Nereis eggs by visible light in 
the presence of eosin or rose bengal was likewise inhibited by 
citrate. Wilbur (1939) has shown that magnesium inhibits activa- 
tion by small doses of ultraviolet but the inhibition is overcome by 
larger doses. Calcium antagonized this action of magnesium. It was 
suggested that magnesium acts in the same manner as calcium but less 
efficiently. 

Excystment of Protozoa. The process of excystment in the ciliate pro- 



316 UADIATION HI()I,(»(;V 

tozoan Colpoda steinii is under excelleiil experimental control. Exposure 
to a special medium results in complete excystment, startinji; at alxjut 2 hr 
after exposure and endinji" in less than I hr from its inception. Taylor el 
al. (193()) huve shown tiiat a dose of 88, 100 i- of X rays fj;i\'(>n at the rate of 
1280 r/'sec within the first (iO min after exposure of the ciliates to the 
excystment medium increases the time to 50 per cent excystment to about 
420 mill. The same dose given 120 min after exposure to the excystment 
medium has very little effect. From the form of the curves of percentage 
excj'sted against time, it is concluded that, in the period between GO and 
120 min, there is a mixture of sensitive and resistant cysts and some in a 
transitional state between sensitive and resistant. The cysts in this 
transitional state are apparently moi'e easily prevented from excysting by 
the X rays than are cysts in the; other two states. 

Giese (1938a, 1941, 1945a), using the same techniques, has shown thai 
monochromatic ultraviolet also increases the time to excystment. The 
action spectrum has been determined for wave lengths from 2537 to 3()()() 
A. There is a small peak at 2804 A suggestive of the absorption spectrum 
of certain proteins. The dose of 2654 A ultraviolet to double excystment 
time is approximately ()00 ergs/mm-. 

MoiUiti) and Beha.'ior of Protozoa. There have been a number of obser- 
vations on the behavior and motility of protozoa during or immediately 
following irradiation, (liese and Leighton (1935a; Giese, 1938b, 1945a) 
have presented a series of quantitative observations on the effect of ultra- 
violet on a variety of ciliates. In particular, Giese (1938b) gives com- 
parative data for 50 per cent rotation on the long axis and 50 per cent 
immobilization for Tetrahymena glaucomiformis, Colpidium colpoda, 
Stylonychia curvata, Paramecium bursaria, P. aurdia, P. caudatiim, and 
P. multimicronucleatum , Blepharisma undulans, Spirostomum ambiguum, 
Bursaria truncateUa, and Fabrea salina. Giese (1945a), using monochro- 
matic ultraviolet, has shown that the action spectrum for immobilization 
and for ciliary reversal in Paramecium has a peak at 2804 A, suggesting 
that it is similar to the absorption spectra for certain proteins (Fig. 8-7). 
A variety of other studies on motility and behavior are summarized in 
Table 8-2. 

Immobilization has been observed often and in many cases is probably a 
sign of impending death. However, this is not necessarily so. The 
reviewer (unpublished) has observed complete immobilization of P. 
aurelia by 2250 A ultraviolet at a dose (1000 ergs/mm'- incident on the 
quartz container) which not only is not lethal but has only a very small 
effect on the time to the first division after irradiation. 

Wichterman (1948a, b) has reported in some detail the effect of X rays 
on the mating reaction in P. bursaria and P. calkinsi. Doses in the range 
between 100,000 and 700,000 r lessen or prexeiit the mating reaction and 
the pair formation which usually follows from it. Apparently, pair for- 



PROTOZOA AND INVERTEBRATE EGGS 



317 



rnation is somewhat more easily affected than is the mating reaction itself. 
At nonlethal doses, recovery from these effects seems to be possible. 
Paramecium calkinsi is affected by somewhat smaller doses than P. 
bursaria. 

Sensitization to Heat. Bo vie and Klein (1919) first reported that para- 
mecia could be made more sensitive to heat by exposure to ultraviolet. 
Giese and his coworkers have made a detailed investigation of this phe- 
nomenon. Giese and Grossman (1945a) may be consulted for reports of 
work with other organisms. 

The time to death at a single lethal temperature, 42°C, was used as a 
measure of the resistance to heat. The possibility that the minimum 
lethal temperature was changed was not tested. Giese and Grossman 

IOOt 



80H 

>• 

o 

2 60- 

UJ 



o 



40- 



20- 




2400 2600 2800 

WAVE LENGTH, a 



3000 



3200 



A CILIARY reversal"! 

# IMMOBILIZATION J STARVED 

O IMMOBILIZATION- FED 

Fig. 8-7. Action spectra for immobilization and ciliary reversal in Parawectuw, from 
Giese (1945a). 

(194oa) investigated the action spectrum for heat sensitization of Para- 
mecium. They found that 2483 A, the shortest wave length used, was 
much more effective than 2537 A or still longer wave lengths. There was 
a small secondary peak at 2804 A. The dose to reduce the time to death 
at 42°C to half its control value can be calculated to be about 400 ergs/mm- 
for 2483 A and about 1800 ergs/mm^ for 2804 A. 

They also found that recovery from the effect occurred. For all wave 
lengths other than 2654 A the course of recovery was much the same, and 
it was complete in about 4 days. Animals exposed to 2654 A recovered 
much more slowly and recovery was only about two-thirds complete by 4 
days. Giese and Grossman suggest that this means that more than one 
material is involved and that the slow recovery from 2654 A is due to a 
larger component of effect on nu(;leoproteins than at the other wave 
lengths. 

Giese and Grossman (1945b) have shown that visible light in conjunc- 
tion with a photodynamic dye can increase the sensitivity of Paramecium 



318 RADIATION BIOLOGY 

lo heat. Gipso (194r)a) has shown that viRil)l(' lijj;ht hy itself can do so 
Tor the ciliatc liU phitrismn, wliicli contnins u natural pliolodynjimic 
pifiiiKMit . 

Ciicsc ami Heath ( M> IS) dcnioiistrated that paramocia can be sensitized 
to heat by sublethal doses of X rays. They emphasize that dosage calcu- 
lations with the thill window 1ul)e employed are subject to much ((uestion 
l)ut give 50,000 r/min as an approximate rate. On this basis, it can be 
estimated from their data that do.ses between lOO.OOO and 300,000 r are 
necessary, depending on the nutiitive state of the animals, to reduce the 
time for death at 42°C" to half. Well-fed, rai)idly dividing animals are 
more sensitive than starved ones. This finding contrasts with Giese and 
Reed's finding (1940) that well-fed paramecia are more resistant to the 
division-retarding eifects of ultraviolet than are starved ones. Thus one 
cannot speak of a general resistance to radiation but only resistance to 
specific effects of specific radiations. Recovery from heat sensitization 
was shown to occur when the paramecia were fed after irradiation but 
not when they were starved. Giese and Heath (1948) conclude that 
recovery must involve the synthesis of new materials bj'' the cell. 

Giese and Grossman (1945b) .suggest that radiation partially denatures 
proteins. Exposure to heat is then supposed to complete the denatura- 
tion and so lead to death more rapidly than in animals not exposed to 
radiation. Giese (1947b) found evidence for sensitization of nucleo- 
proteins to heat by ultraviolet. Partially purified nucleoprotein from 
Strongylocentrotus sperm, dissolved in 2 M sodium chloride, forms threads 
when poured into dilute sodium chloride. Brief exposures to ultraviolet 
(mainly 2537 A) followed by a 10-min exposure to 80°G changed the 
luicleoprotein so that the threads did form. The reverse procedure, 
exposure to 80°C and then to ultraviolet, had no effect. 

Miscellaneous Microscopicalli/ Visible Changes. ^Microscopically visible 
changes in the nucleus of the cell are treated by Garlson (Ghap. 1 1 , volume 
1 of this series). Some of the more recent observations of various changes J 
in both the nucleus and cytoplasm in protozoa and invertebrate eggs are | 
summarized in Table 8-3. No attempt has been made to include papers 
in which microscopic observations were merely incidental to other work. 
As has been pointed out in a previous section, many of these changes may 
be those that occur in dying cells. 

Various Physiological, Biophysical, and Biochemical Effects. For the 
most part, the work on protozoa and invertebrate gametes has not been 
directed to a study of the enzyme systems affected by the radiation, the 
colloidal changes in protoplasm, the changes in permeability of mem- 
branes, and alterations in the chemical composition of the cell. Infor- 
mation of an indirect sort on some of these matters has been mentioned 
in preceding sections of this chapter but direct investigations are scarce 
and are listed briefly in Table 8-4 without discussion in the text. 



PROTOZOA AND INVERTEBRATE EGGS 3 ID 

Tablk 8-1. KiLLixn of Parasitic Protozoa by 1{ adiatiox 



Organism 


Radiation 


Criterion 


Dose 


Reference 


Tr[ipano^oma 


Soft X rays 


Decreased infective 


6,000 r 


Patel (1936) 


brucei 




power 








Ultra\- inlet 


Decreased infective 
power 


H.S.E. 


Patel (1936) 


Trypunosoina 


X ra3's 


Visible change 


100,000 r 


Halberstaedtcr 


gambiense 




Failure to infect 


12,000 r 


(1938) 


Triipanosoma 


X rays 


No visible change 


100,000 r 


Emmett (1950) 


cruzi 




Decreased infective 
power 


10,000 r 




Plasmodium 


Ultraviolet" 


Failure to infect 


Not given 


Russell ft al. 


(jallinaceum 








(1941) 


sporozoites 










I'lasmodium 


X rays 


No effect on infection 


150-700 r 


Zain (1943) 


gaUinaceum 










endothelial 










stages 










Plasmodium 










gaUinaceum 










sporozoites 


X rays 


Failure to infect 


8,000 r 


Bennisoii and 


trophozoites 


X rays 


Failure to infect 
Rate of development 
of the infection 


20,000 r 
4,000 to 
8,000 r 


Coatney (1945) 


Plasmodium 


X rays 


Failure to infect 


5,000 r 


Bennison and 


malaria 








Coatney (1945) 


trophozoites 










Plasmodium 


X rays 


Failure to infect 


16,000 r 


Rigdon and Rudi- 


lophurae 








sell (1945) 


trophozoites 










Eimeria tenella 


Ultraviolet 


No infection of chicks 


2 units 


Fish (1932) 


oocysts 




100 per cent mortal- 
ity in vitro 


1 unit 




Eimeria tenella 


X rays 


Failure to infect 


13,500 r 


Albanese and 


oocysts 




Effect on severity of 
infection 


9,000 r 


Smetana (1937) 


Eimeria tenella 


X raj'S 


Distinct effect on se- 
verity of infection 
Some infection 


9,000 r 
13,500 r 


Waxier (1941) 


Eimeria 


Ultraviolet 


Inhibition of in rilro 


Not given 


Litwer (1935a, b) 


perforans 




development of 






Eimeria 




oocysts 






stiedae 










A delina 


X rays 


Abnormal oocysts 


1,000 r 


Hauschka (1944) 


deronis 










Endamoeha 


Ultraviolet 


Decrease in luimber 


Not given 


Stoll el al. (1945) 


hisiolylicu 




excysting 






cysts 










Endamoeba 


X rays 


Partial inliihition of 


60,000 and 


Sadun et al. 


his(oh/ficu 




growth in vitro 


120,000 r 


(1950) 


trophozoites 




Infectivity unchanged 


120,000 r 





» Unless otherwise stated in this and other tables " Ultraviolet " moans the radiation 
from a quartz mercury arc presmnably witli major output at 2537 A. 



320 



RADIATION BIOLOGY 

Tabi.k 8-2. Motility and liKiiAVioK ok I'hotozoa 



Radiation 


Organism 


Effect 


Dose 


Reference 


X rays 


Paramecium 


Acceleration of 
movement 
followed by slow- 
ing, spinniufi, and 
iiiiin()l>ili/.ati()ii 


<5 X 10* r 


Dognon and Pif- 

fault (1931a) 


X rays 


Paramecium 


Irregular swint- 


1 to 6 X 10' r 


Back and Hal- 




caudatum 


niing, immobiliza- 
tion 




berstaedter 
(1945) 


X rays 


Paramecium 


Acceleration 


1 X 10'' r 


Wichterman 




bursaria 


Retardation 
Immobilization 


2 X 10'' r 
1 X 10'^ r 


(1948a) 


X rays 


Paramecium 
calkinsi 


Retardation 


4 X 10' r 


Wichterman 
(1948b) 


X rays 


Trypanosoma 
gambiense 


A few nonmotile 


2 X 10' r 


Halberstaedter 

(1938) 


X rays 


Pandorina 


Immobilization 


3 to () X 10' r 


Halberstaedter 
and Back (1942) 


Neutrons . . . 


Euglena 


Clear zone at top of 
culture (change in 
motility?) 


1250 n 


Jennings and 
Garner (1947) 


y (radium) . . 


Physarium 
polycephalum 


Plasmodium moves 

more slowly 
Plasmodium moves 

away from needle 


Not given 


Seifriz (1936) 


a (polonium) 


Polytoma 
uvella 


Immobilization 


Not given 


Holweck and 
Lacassagne 
(1931a, b) 


2804 A 


Paramecium 


50 per cent immo- 


1.1 X 10' 


Giese and Leigh- 


ultraviolet 


multimicro- 
nucleatum 


bilization 


ergs /mm ^ 


ton (1935a) 


2537 A 


Paramecium 


50 per cent rotation 


1.8 X 10' 


Giese and Leigh- 


ultraviolet 


multimicro- 
nucleatum 




ergs/mm^ 


ton (1935a) 


Intense 


Paramecium 


Avoiding reaction, 


Not given 


Rent.schler and 


flashes of 


muUimicro- 


retardation, rota- 




Giese (1941) 


ultraviolet 


nucleatum 


tion, immobiliza- 
tion 






Ultraviolet. . 


Paramecium 


Acceleration of con- 


Not given 


Roskin and Shl- 


' 


caudatum 


tracitile vacuoles, 
retardation of food 
vacuole formation 




shliaeva (1933) 


Ultraviolet. . 


Spiroiitomum 
ambiguum 


Various effects 


Not given 


Shirley and 
Finley (1949) 


Ultraviolet . . 


Amoeba proteus 


Various effects of 
several salts 


Not given 


Black (1936) 


Visible plus 


Trypanosoma 


Immobilization 


Not given 


Levaditi and 


I)hoto(ly- 


(jamhiense 






Prudhomme 


namic dye 


and T. evatisi 






(1945) 



PROTOZOA AND INVERTEBRATE EGGS 321 

Table 8-3. Microscopically Visible Changes Induced by Radiation 



Radiation 



•> (radium) 

7 (radium) 

X rays 

Ultraviolet 

Ultraviolet 

2537, 2654, 

2804, 3025 A 
2650, 2804 A 

Ultraviolet 

Ultraviolet 



Organism 



Ultraviolet, 
microbeam 

Ultraviolet, 
microbeam 



Ultraviolet, in- 
tense flashes 

Ultraviolet, 
intense 
flashes 



V'isible light 
plus photo- 
dynamic dye 

Visible light 
plus photo- 
dynamic dye 



Physarium 
Opalinid eiliates 
Unto tumidus eggs 

I'tiio tumidus eggs 

Amoeba proteus 

Paramecin m multimi- 

cronurleatum 
Paramecium aurelia 

Kahlia simplex 

Spirostomum 
ambiguum 



Amoeba proteus, 
Avioeba verrucosa, 
Paramecium caudalum 

Paramecium caudatum, 
Spirostomum 
avibigu um, A m ph ilep- 
tus claparedei 

Paramecium multim.i- 
cronuclealum 

Amoeba proteus, 
Amoeba dubia, 
Euglena, Volvox, 
Chilomonas, 
Stylonychia, 
Chilodonella, 
Coleps, Urocentrum, 
Paramecium bursaria, 
Epistylis, Stentor, 
Bursaria, Frontonia 

Amoeba dubia 



Effect 



Protoplasmic stream- 
ing, viscosity, distri- 
bution of granules 

Distribution of mito- 
chondria and vegeta- 
tive granules 

Changes in appearance 
of nuclei due to coagu- 
lation and permeabil- 
ity changes 

Coagulation within 
nucleus 

Form and general ap- 
pearance 

Vesiculation 

Formation of large, 
vacuolated bodies in 
the macronucleus 

Double animal forma- 
tion 

Fragmentation of 
macronucleus, 
various cytoplasmic 
changes 

Localized coagulation 
in cytoplasm 

Localized colloidal 
changes in macronu- 
cleus 

Change in shape, vesic- 
ulation 

Various changes, 
mainly fragmentation 



Reference 



Lytechinus eggs 



Blister formation, 
hyaline areas formed, 
pseudomombranes 
formed 

Variety of nuclear and 
cytoplasmic changes 



Seifriz (li)36) 
Horning (1937) 
Wottge (1939) 

Wels (1938) 
Black (1936) 

Giese and Leigh- 
ton (1935a) 

Kimball (1949c) 

Kimball and 
Gaither (1951) 

Horvath (1947) 

Shirley and 
Finley (1949) 



Tchakotine 
(1935c) 

Tchakotine 
(1936) 



Rentschler and 

Giese (1941) 
Harvey (1942) 



Hvman and How- 
land (1940) 



Tenncnt (1942) 



32: 



|{ SDI \ riON lUOhoCV 
TaBI-K 8-4. liloniKMICAI. and HiOIMIYSKAI, I'lKKKfTS 



JOllVct 


t 
Organism i 

liainiii ((uididu 


Hadiafion 

1 

and 7 


Heferencc 


Increased pcniH-ability at 


Simon (l<)3y) 


low tlo.sos 


I'KK 


(radium) 




Dccrcasoil poriiioahility at 








lii^li (loses 








Increased permeability of 


Unto tumid us egg 


X rays 


Wottge (1939) 


nuclear membiaiie 








("liaiine in pernieahility . . . . 


I'uianifcium 


Ultraviolet 


Roskin and 
Shi.shlmeva (1933) 


No detectable change in 


Strongiflocentroius 


lltraviolet 


Reed (1948) 


pertiieabilitj' at doses 


Pgg 


2483, 2537, 




wliich retard cleavage 




2654, 2804, 
3130 A 




No detectable change in 


Arbacia egg 


X rays 


Lucke et al. (1951) 


permeability at doses 




10,000 r 




which retard cleavage 








Changes in viscosity 


Amoeba proteus 


Photodynamic 


.\lsup (1942) 


1 and .4. duhia 


action 




No effect on viscosity at Arbacia eggs 


X rays 


W'illjur and 


doses which markedly re- 






Recknegel (1943) 


tard cleavage 








Changes in viscosity 


Spirostomum 


Ultraviolet 


Shirley and Finley 
(1949) 


No effect on respiration at 


Arbacia and 


X rays 


Chesley (1934) 


doses causing early death 


Chaetopterus 






eggs 






No effect on respiration .... Baniea Candida 


/3 and y 


Simon (1939) 




egg 


(radium) 




50 per cent inhibition of 


Arbacia sperm 


.\ rays 


Barron et al. 


respiration 




10,000 to 
20 , 000 r 


(1949a, b) 


Decreased assimilation of 


Paramecium 


Ultraviolet 


Roskin ^nd 


lipoid and glycogen 






Shishlmeva (1933) 


Changes in the amount of 


Bodo audatus 


y (radium) 


Lawrie and 


ammonia produced per 






Robertson (1935) 


cell 








Free fat formed in the cyto- 


Amoeba proteus 


ITltraviolet 


Heilbrunn and 


plasm 


and .4. dubia 




Daugherty (1938) 


Destruction of red pigment 


Blepharisma 


Visible light 


Giese (1938e); 
Giese and Zeuthen 
(1949) 


Change in the mineral con- 








stituents of protoplasm . . 


Paramecium 


X rays 


Berner (1942) 


Destruction of the enzymes 


Arbacia sperm 


X rays 


Barron el al. 


for oxidation of acetate 






(1949a, b) 


and succinate 








More rapid killing by sulf- 


Paramecium 


Visible light 


Calcutt (1950) 


hydryl inhibitors 









PROTOZOA AND INVERTEBRATE EGGS 323 

REFERENCES 

Albanese, A. A., and H. Smetana (1937) Studies on the effects of X rays on the 

pathogenicity of Eimeria tenella. Am. J. Hyg., 26: 27-36. 
Alpatov, W. W., and O. K. Nastiukova (1933) The influence of different (juantities 

of ultra-violet radiation on the division rate in Paramecium. Protoplasnia, 18: 

281-285. 

(1934a) Differences in susceptibility to ultraviolet radiation of Paramecium 

caudatum and P. birrsaria. Proc. Soc. Exptl. Biol. Med., 32: 99-101. 

(1934b) Susceptibility of infusoria to ultraviolet rays as related to the col- 
loidal properties of their protoplasm changed by means of different physico- 
chemical methods. Doklady Akad. Nauk S.S.S.R. 3: 395-600. (Not seen. 
Abstract in Biol. Abstracts, 11: 1419 seen.) 

(1934c) Influence of ultraviolet radiation on the division rate of P. caudatum 



in relation to temperature during and after irradiation. Doklady Akad. Nauk 
S.S.S.R., 4: 62-68. (Not seen. Abstract in Biol. Abstracts, 11: 1419 seen.) 
AIsup, F. W. (1941) Photodynamic action in the eggs of Nereis limbata. J. Cellular 
Comp. Physiol., 17: 117-130. 

(1942) The effects of light alone and photodynamic action on the relative 

viscosity of amoeba protoplasm. Physiol. ZooL, 15: 168-183. 

Back, A. (1939) Sur un type de lesions produites chez Paramecium caudatum par les 

rayons X. Compt. rend. soc. biol., 131: 1103-1106. 
Back, A., and L. Halberstaedter (1945) Influence of biological factors on the form of 

roentgen-ray survival curves. Experiments on Paramecium caudatum. Am. J. 

Roentgenol. Radium Therapy, 54: 290-295. 
Barron, E. S. G., B. Gasvoda, and V. Flood (1949a) Studies on the mechanism of 

action of ionizing radiations. IV. Effect of X-ray irradiation on the respiration 

of sea urchin sperm. Biol. Bull., 97: 44-50. 

(1949b) Studies on the mechanism of action of ionizing radiations. V. The 

effect of hydrogen peroxide and of X-ray irradiated sea water on the respiration 
of sea urchin sperm and eggs. Biol. Bull., 97: 51-56. 

Bennison, B. E., and G. R. Coatney (1945) Inactivation of malarial parasites by 
X rays. U.S. Public Health Service, Health Reports, 60: 127-132. 

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PROTOZOA AND INVKRTEHRATE EGGS 327 

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J. Cellular Comp. Physiol., 12: 263-271. 
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Spirostomum amhiguum. Trans. Am. Microscop. Soc, 68: 13()-153. 
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de I'oeuf de pholade, Barnea Candida. Arch. biol. Li6ge, 50: 95-203. 
Sonneborn, T. M. (1947) Recent advances in the genetics of Paramecium and 
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(1949) Ciliated protozoa: cytogenetics, genetics and evohition. Ann. Rev. 

Microbiol., 3: 56-80. 
Spencer, R. R., and D. Calnan (1945) Studies in species adaptation. III. Con- 
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sea urchin eggs. J. Exptl. Zool., 95: 89-103. 
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Stoll, A. M., P. A. Ward, and I). R. Mathieson (1945) The effect of ultraviolet 

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PROTOZOA AND INVERTEBRATE EGGS 331 

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Manuscript received by the editor, Xov. 29, 1950 



CHAPTER 9 

Radiation and Viruses* 

S. E. LURIA 

Department of Bacteriology, University of Illinois 
Urbana, Illinois 

Introduction. Effect of radiations on virus infectivity: Ionizing radiations — Ultra- 
violet radiation — Visible light. Differential effect of radiations on various properties of 
viruses: Nonlethal effects — Separation of properties of inactive virus particles. Irradiated 
virus in multiple and mixed infection: Interference phenomena — Reactivation phenomena. 
Intracellular irradiation of viruses: Irradiation of cells infected with exogenous viruses — 
Radiation and latent viruses. References. 

1. INTRODUCTION 

To few fields of biology have radiation studies contributed as much as 
they have to virology. The reasons are to be found in the properties of 
viruses and in the methodology of virus research. A virus can be defined 
as a submicroscopic entity capable of self-reproduction after exogenous 
infection of specific Uving cells (Luria, 1950). According to host, viruses 
may be classified as animal viruses, plant viruses, and bacterial viruses or 
bacteriophages. In their extracellular state, virus particles have charac- 
teristic sizes and chemical compositions, a common feature of which is the 
presence of protein and of nucleic acid of either the ribose or the deoxy- 
ribose type. Their small size, nucleoprotein content, and ability to 
reproduce inside cells make them useful models for the study of repro- 
duction of genetically specific biological units. At the same time they 
offer unique opportunities for observation both in the resting, extra- 
cellular state, in which they can be submitted to a variety of treatments, 
and in the reproductive, intracellular state, in which they behave as com- 
ponents of an integrated cellular system. Radiations, because of the 
discrete nature of their action, are more useful than chemicals in affecting 
free virus in such a way that, upon reintroduction into a host, the host- 
virus interaction will be abortive or variously modified; the results are 
very informative as to the biology of viruses. Moreover, it is possible by 

* This chapter has not been revised since being wyitteji in 1951. Vakiable discussion 
and bibliography of more recent work are given by Pollard (1953). 

333 



334 IJADIATION mOLOGY 

means of radiation to reach a virus within the host cell and to obtain 
information as to its intracellular properties. 

Althou}i;h viruses do not constitute a homogeneous group that may 
a piiori he expected to react to radiation in a uniform way, there does not 
appear to be any fundamental dilTerence in the he^havior of different 
\iruses toward ratliation, and most effects can he described under com- 
mon headings for all viruses. The major differences reflect the different 
purposes for which radiation experiments with different viruses have been 
performed. In this chapter various types of radiation effects and appli- 
cations of radiation analysis will be discussed, different viruses being used 
as examples rather than each virus being followed separately through the 
various approaches. It should be the goal of the radiobiologist to inter- 
pret the effects of radiation on \'iruses in terms of chemical alterations in 
nucleoproteins and other virus components. At this time, however, the 
radiation chemistry of large biological molecules is so poorly understood 
that a strictly chemical approach to the topic is precluded. 

2. EFFECT OF RADIATIONS ON VIRUS INFECTIVITY 

The most thoroughly investigated effect of radiation on viruses has been 
the loss of infectivity or "inactivation " of free virus particles when 
exposed to radiation. It must be recalled that "activity" of a virus can 
be defined as the ability to reproduce and to cause a detectable manifes- 
tation when introduced by a proper route into a suitable sensitive host. 
Quantitative studies are made possible by the relative accura(;y of the 
titration methods for viruses ; the amount of virus in different samples can 
be compared fairly accurately with a precision that may vary from 5 per 
cent in some instances to a factor of 2 or more in others. According to a 
majority of authors the results of virus titration give values proportional 
to the actual number of individual virus particles, each particle acting as 
one infectious unit with a probability that may be much lower than unity 
(Luria, 1940; Lauffer and Price, 1945). The results of titration can be 
used, however, to compare the active virus content of different samples 
(e.g., of an irradiated and a control suspension) even if infection requires 
the summation of the action of large numbers of virus particles rather 
than the reproduction of one successful particle, provided that a definite 
rule of proportionality exists between the amount of active virus intro- 
duced and the number or extent of the host manifestations. This is true, 
for example, for a method of titration that uses the incubation period or 
the time of death of an infected animal as related to the infecting dose 
(Bryan and Beard, 1939; CJard, 1943) rather than the counting of indi- 
vidual lesions or the dilution end point. 

One important caution for the radiobiologist is the control of the mode 
of testing virus activity. This has become particularly important since 



KAIMATION AND VIRUSES S.S.') 

the recognition that ba('teriophag;o inactivated l)y radiation can be reac- 
tivated in its host (Luria, 1947; Dulhecco, IQoOj; siniihir ])henomena may 
occur for other viruses. In the case of bacterioj^hases, because of the 
possil)iht3" of reactivation, an inactive virus particle must be defined as 
one that is unable to parasitize a host cell or that, upon parasitizing a 
bacterial cell under conditions where no reactivation occurs, fails to give 
rise to the production of active bacteriophage (see Sect. 3-2). 

A comparison of the titers of control and irradiated virus suspensions 
generally provides reproducible inactivation curves. In many cases the 
rate of inactivation of viruses by irradiation has been found to follow a 
simple exponential relation according to the equation 

N - iVoe-^-«, (9-1) 

where A^o = the titer in the unirradiated control, 

N = the titer in an irradiated sample, 

D = the dose of radiation, and 

k = Si constant, characteristic for a given virus, a given radiation, 
and sometimes for a given mode of titration. 
The dose for which N = A^oe~^ is often called the "inactivation dose" 
and designated as Di/e (Lea, 1946). 

2-1. IONIZING RADIATIONS 

X rays, y rays, a rays, electrons, neutrons, and deuterons have all been 
used to inactivate virus particles. It is now recognized that the inacti- 
vation of viruses by ionizing radiations may result from two major cate- 
gories of effects: indirect and direct. 

2- la. Indirect Effects. These effects are mediated by toxic substances 
produced by radiation in dilute aqueous solutions. The toxic products 
are responsible for most of the inactivation observed, provided that suffi- 
cient amounts of protective substances which compete for the toxic 
products are not present. The current theories on the nature and 
chemical properties of the toxic substances produced by ionizing radia- 
tions in water and on the mode of their action are discussed by Dale and 
by Barron (in Vol. I of this series). As far as viruses are concerned, the 
indirect effect of X rays was first recognized on papilloma virus (Friede- 
wald and Anderson, 1940, 1941), then on phage (Luria and Exner, 1941), 
and on plant viruses (Lea et ai, 1944). The effect manifests itself by a 
higher inactivation rate of viruses exposed to radiation in water or in 
saline solutions than when exposed either in crude suspensions containing 
large amounts of foreign substances or in the dry state. A variety' of 
substances can act as protective agents. Proteins such as gelatin and egg 
or serum albumin are effective. Several substances of small molecular 



33() UAOIATION mOLOOY 

woiglit, t'.f!;., tryptopliane, thiourea, and histidiiie, give complete protec- 
tion of hacteriophafj;e in \ery low concent rations (Latarjet and Ephrati, 
1948; Watson, 1952). Ordinary bacteriological media such as beef broth 
are excellent protecting metlia. 

(Concentrated \'irus suspensions in nonprotective media are inactivated 
more slowly than dilute suspensions both because of the presence of 
impurities and because the virus itself may act as a protective agent. In 
some cases the inactivation rate does not increase with dilution beyond a 
certain point; the diluting medium itself may contain a small amount of 
protective substances. In very dilute suspensions there is also the possi- 
bility that some of the toxic agents produced by the radiation, such as 
free H and OH radicals, may recombine before having an opportunity to 
meet a virus particle and react chemically with it (Lea, 194()). 

The rate of inactivation by the indirect effect of ionizing radiation 
varies from case to case. Deviations from the logarithmic relation Eq. 
(9-1), with the rate increasing with the dose, could be due to a need for 
cumulative damage of the virus (true multiple-hit effect), to a progressive 
accumidation of toxic products in the medium, or to destruction of pro- 
tective substances by radiation itself. With purified tobacco mosaic 
virus. Lea et al. (1944) reported logarithmic rates of inactivation by X 
rays even for dilute suspensions of purified virus, but their data are not 
very satisfactory. For bacteriophages (Alper, 1948; Watson, 1952) the 
inactivation rate increases with time of exposure. This was interpreted 
by Alper (1948) as indicating progressive accumulation of toxic products 
in the medium. Watson (1952), however, was able to show that the 
increasing rate of inactivation was due almost exclusively to accumulation 
of damage in the phage particles. A phage sample in buffer received a 
small dose of X rays; most inactivation was due to indirect effect. The 
phage was then diluted in a completely protecting medium and later was 
again diluted in buffer and exposed to the same dose of X rays. The 
second exposure was much more effective than the first, as expected from 
the previously determined survival curve, although the second medium 
had not been irradiated previously. 

Not all the indirect effect of X rays on viruses is exerted during actual 
exposure. There are also aftereffects of irradiation, mediated by rela- 
tively stable toxic products. If either water or buffer solutions are 
exposed to radiation, and virus is then introduced without appreciable 
amounts of protective substances, some inactivation will follow. In the 
case of bacteriophage (Watson, 1952) at least tw^o agents are invoh-ed, one 
short-lived, detectable only during actual irradiation, and the other a 
long-lived one, which is quite stable at 5°C and is slowly inactivated at 
room temperature. Many of the effects of the latter can be duplicated by 
peroxides. The different nature of the two agents is evident from the 
different properties of phage inactivated by one or the other of them (see 



RADIATION AND VIRUSES 337 

Sect. 3-2). Protection against both agents is generally afforded by the 
same substances. 

2-1 b. Direct Effect. The inactivation of viruses b}' the indirect effect 
of ionizing radiations is mediated by chemicals produced in the medium. 
Its rate is affected by the temperature and by the distribution of dose in 
time, as e.xpected from considerations of chemical kinetics. Because of 
its occurrence some questions have been raised as to the very existence of a 
direct effect exerted by the primary absorption of radiation energy within 
the physical domain of the virus particles. Yet such a direct effect is 
certainly present, and its analysis is possibly more revealing than that of 
the indirect effects, as far as the mechanism of biological effect of radia- 
tions is concerned. A direct effect of ionizing radiations is defined as a 
"nonprotectable" effect, i.e., an effect that cannot be eliminated by alter- 
ations of the medium. If the concentration of protective substances in 
the medium is increased beyond a certain level or if the virus is irradiated 
in the dry state, inactivation will proceed at a minimum rate, which 
cannot be further reduced (Luria and Exner, 1941 ; Lea, 1946). This 
residual inactivation is a function of only the total radiation dose and is 
not modified by changes in oxygen tension (Hewitt and Read, 1950), in 
temperature (Watson, 1950), or in the intensity of the radiation beam 
(Wollman et al., 1940; Lea, 1946), thus exhibiting all the characteristics of 
photochemical reactions. The distinction between direct and indirect 
effects has recently been emphasized, at least in the case of bacteriophage, 
by the finding that the biological properties of phage particles inactivated 
by X rays in the presence of an excess of protective substances differ in 
many respects from those of particles inactivated by various types of 
indirect effects (Watson, 1952; see Sect. 3-2).^ 

The direct effect of ionizing radiation on viruses has been analyzed 
repeatedly in relation to the mechanism of radiation action not only on 
viruses but on genetic units and cells in general (Lea, 1946). In all well- 
investigated cases, inactivation of viruses proceeds according to Eq. (9-1). 
This indicates that one radiation "hit" inactivates a virus particle, i.e., a 
particle is inactivated by one successful act of absorption, without sum- 
mation of individual effects. 

Thus virus inactivation is a good test for further analysis based on the 
hit theory, and it has been employed widely in testing the validity of the 

'Experiments by A. H. Doermann in 1951 (unpubli.shed) show that addition of 
cysteine or BAL to a suppo.sedly completely protecting medium such as nutrient broth 
can reduce the rate of bacteriophage inactivation by X rays by as much as a factor of 
2. This important discovery, if confirmed, might force a revision of the definition of 
direct effect. More likely, it may be an indication that the direct efTect, although 
direct in a geometric sense, i.e., exerted through acts of radiation absorption within the 
virus particle, is in part mediated through water tiound around or within the virus in 
a way that permits agents such as cysteine or BAL to interact with the oxidizing 
products of water decomposition. 



.338 |{.\l)l A riO.N HKtLOCJY 

so-called "target theory," accordiiifi; to which the effective hits are those 
that occur within a .specific physical domain which may coincide with all 
or parts of the l)iolo<i;ical ohjcci in\('.stifi;ated. Iiil'ormation concerning 
the geometry of this domain is sought in the following way. An ideal 
"target " or "sensitive xolunic" is delinetl by the value of the constant A; 
in Eq. (9-1): 

If D is measured in acts of absorption per unit volume, /,• will have the 
dimension.'-' of a \olume (sensitive volume); if D is measured in number of 
paths of ionizing particles (such as protons) crossing a unit area, then k 
will have the dimension of an area, the "sensitive cross section." Clearly, 
the target or sensitive volume thus defined is not a priori identifiable with 
a physical portion of the biological object. Two extreme po.ssibilities, 
and several intermediate ones, are conceivable: 

Hypothesis 1. The target corresponds to a real volume, within which 
each hit is effective, whereas all hits without are ineffective. 

Hypothesis 2. There is no such completely sensitive " real target " ; the 
probability that a hit in a given unit volume is effective is distributed o\'er 
a more or less large volume, which may even extend beyond the recognized 
boundaries of the organism. 

In the case of viruses the recognition of the direct effect and its distinc- 
tion from the indirect effects suggests that the probability that hits out- 
side the physical borders of the virus particles are effective is not appre- 
ciably different from zero. The effectiveness of a hit within the particle, 
however, may be lower than unity and may vary from point to point, with 
a distribution p{x,y,z) over the volume of the particle. If c is the \()lume 
of the particle, Eq. (9-1) becomes 

If p{x,y,z) = I* is constant, then 

Hypothesis 1 would divide a virus into two parts, one with P = 1 and 
the rest (if present) with P = 0. This point of view was vigorously 
defended by Lea (194G) who made extensive measurements of A* for dif- 
ferent biological effects of radiation and developed methods for analyzing 
the dependence of k on ionization density for various radiations. For the 
application of this type of analysis to viruses, the reader is referred to 
re\iews by Lea (19-1:()) and Bonet-Maury (1948). A brief summary will 
be sufficient here because the target theory is discussed by Fano (Vol. I of 
this series) and because, in the opinion of this writer, the information 
at)out \irus('s obtainable by this type of analysis is of limited \alue. 



RADIATION AND VIRUSES 330 

The first objective of the analysis has been to obtain vahies of k for dif- 
ferent viruses, to compare them with the physical dimensions of the virus 
particles, and to obtain estimates of P. A major difficulty is the unc^er- 
tainty as to what should be considered as the elementary act of absorp- 
tion, i.e., the "hit." For example, if the elementary act is considered to 
be the production of one single ionization, a different value is obtained for 
k than if the elementary act is taken to be the production of a cluster of 
ionizations supposedly occurring so closely together as to produce only one 
hit. Moreover, an appreciable fraction of the energy absorbed from 
ionizing radiation will be dissipated in the form of excitations without 
ionization. Ultraviolet irradiation studies (see Sect. 2-2) have shown, 
however, that an excitation has a very low probability of producing virus 
inactivation. The part of energy dissipated in this way probably makes a 
minor contribution to the biological effect of ionizing radiation. In the 
case of radiations that produce dense columns of ionizations, k could be 
measured as the cross section for collision between the target and the 
ionization column. There is some uncertainty as to the size of the latter 
and even as to its approximate reducibility to a cylinder. It should be 
emphasized that these uncertainties are due to the limited amount of 
information available concerning the distribution of the energy released 
by ionizing radiation in liquids. 

In spite of this, values of A- for inactivation of several viruses are avail- 
able for comparison with the known physical dimensions of the virus par- 
ticles. A representative group of such data is presented in Table 9-1, 
from which the following facts emerge: 

1. For a given virus, when the inactivation dose Dye for different radia- 
tion energies is measured in comparable units, based on ionizations per 
unit \'olume, there is a clear decrease of effectiveness per unit dose as the 
ionization density increases, as required by the target theory. Densely 
ionizing radiation should often produce more than one ionization (or 
cluster) within each target, with a resulting waste of ionizations. Lea 
(1946) proposed an elaborate method (the "associated volume method") 
to calculate the "true" size of the target (supposed to be spherical) from 
the dependence of k on ionization density. Target sizes obtained by this 
method are included in Table 9-1. For densely ionizing particles, Lea's 
method is approximately equivalent to measuring the cross section for 
collision between target and ionization column. 

2. There is a certain degree of parallelism between particle size and 
radiation sensitivity for different viruses and for a given radiation, at 
least qualitatively and with a few exceptions. This is particularly evi- 
dent for groups of agents presumablj^ similar in kind, such as bacterio- 
phages. This parallelism justifies the use of radiation data to estimate 
particle size by interpolation (WoUman and Lacassagne, 1940), a method 
that may still be of use when a virus cannot be purified enough for electron 



3 10 



RADIATION BIOLOGY 

Tahi.k '.1-1 













Diame- 














ter of 














spheri- 












Diame- 


cal 












ter of 


target 




\'iru8 


Radiation 


Inuctlvation 

dose* 

(Di/.). I 


Refer- 
encet 


spheri- 
cal 
target 
per ion 
pair, 
niM 


accord- 
ing to 
Lea's 

"associ- 
ated 

volume 

iiK'th- 

od,"t 

mil 


Particle diatiieter§ 

(or linear dimensions if 

not spherical), m^ 


BacteriophaRes: 














S13 


-) 


5.8 X 10' 


4 


12 


15 5 


16-20 (filtration, centrifu- 
Kation; shape unknown) 




X ray.i, 1.5 A 


9.9 X 105 


4 


10 


15 9 




a, 4 Mev 


3.5 X 10« 


4 


7 


16 3 




«X-174 


X rays, 0.9 A 


6.8 X 10* 


1 


12 


17 


15-20 (filtration, eentrifu- 




a. 6.5 Mev 


4.9 X 10« 


1 


6 


13 


eation; sliape unknown) 


C36 


y 


2.1 X 10' 


4 


18 


21 5 


42 (filtration; shape un- 
known) 




X rays, 1.5 A 


4.3 X 105 


4 


14 


22.3 




a, 4 Mev 


9.4 X 105 


4 


10 


33 




Tl (P28) 


X rays, <0.1 A 
Deutorons: 


9 X 10< 


5 


23 


30 


Head, .50; tail, 15 X 200 
(tadpole-shaped; elec- 




3 . 5 Mev 


3.2 X 105 


6 


16) 




tron inicroscope) 




2.5 Mev 


3.9 X 105 


6 


lol 


25-30 






1 5 Mev 


4.9 X 105 


6 


14j 






Staph. K . . . . 


y 


7.9 X 10* 


4 


24 


31 


60-100 (filtration) 




X rays, <0.1 A 


4.5 X 10^ 


5 


30 


40 






X rays, 1.5 A 


1.1 X 105 


4 


21 


40 






a, 4 Mev 


4.5 X 105 


4 


14 


50 




C16 


X rays, <0.1 A 


4 X 10' 


5 


30 


42 


Hea<i, 60 X 80; tail, 20 


(T2,T4,T6) 


X rays, 0.1 A 


3.5 X 104 


8 


31 


42 


X 120 (tadpole-shaped; 




X ravs, 0.15 A 


4 X 104 


9 


30 


40 


electron microscope) 




X rays, 0.7 A 


4.5 X 104 


9 


29 


58 






a, 6.5 Mev 


2.1 X 105 


9 


18 


70 




Vaccinia 


7 


8 X 104 


3 


24 


31 


210 X 260 (l.rick-shaped; 




X rays, 0.9 A 


3.5 X 105 


1 


22 


41 


electron niieroscope) 




X rays, 1.5 A 


1 X 105 


3 


18 


70 






a, 4 Mev 


2.1 X 105 


3 


16 


23 






a. 6.5 Mev 


3.5 X 104 


1 


32 


175 




Herpes 


a, 6.5 Mev 


3.5 X 104 


1 


32 


175 


265 X 300 (brick-shajied ; 
electron microscope) 














Foot and inouth 


X ray, 0.9 A 


2.8 X 105 


1 


17 


27 


1.5-30 (filtration, centrifu- 




a, 6.5 Mev 


1.3 X 10« 


1 


10 


23 


gation; shape unknown) 


Rabbit papil- 














loma 


X rays, 0.1 A 


4.4 X 104 


7 


29 


40 


66 (centrifugation) 


Tobacco 


7 


6.7 X 105 


2 


12 


14 


27.5 (electron microscope) 


necrosis 


X rays, 1.5 A 


9.4 X 105 


2 


10 


16 






X rays, 8.3 A 


5.15 X 10« 


2 


6 


12 




Tomato busliy 


7 


4.5 X 105 


2 


14 


17 


25.5 (electron microscope) 


stunt 


X rays, 1.5 A 


6.2 X 105 


2 


12 


19 


(37) (hydrated) 




X rays, 8.3 A 


3.1 X 10« 


2 


7 


14 






a, 4 Mev 


2.6 X lOe 


2 


8 


22 




Tobacco mosaic 


7 


3.7 X 105 


2 ' 


15 


18 


15 X 280 (rod-shaped; 




X rays, 1.5 A 


4.3 X 105 


2 


14 


22 


electron microscope) 




X rays, 8.3 A 


1.5 X 10» 


2 


9 


19 






a, 4 i\Iev 


1.9 X 10« 


2 


9 


26 





* When the inactivation dose was not given in roentgens by the authors, it was calculated on the 
assumijtion that 1 r corresponded to 1.7 X 10'- ion pairs per cubic centimeter for 7 and X rays and to 
1.9 X 10'2 for a rays and deuterons. 

t References: (1) Bonet-Maury, 1948. (2) Lea, 1946. (3) Lea and Salaman, 1942. (4) Lea and 
Salaman, 1946. (5) Luria and Exncr, 1941. (6) Pollard and Forro, 1949. (7) Syverton, Berry, and 
Warren, 1941. (8) Watson, 1950. (9) Wollman et al., 1940. 

J The values are either taken from Lea (1946) or read from Fig. 8 in Lea (1946). Since Lea assumed a 
value of 1.35 for the density of viruses, this value was adopted throughout, although for viruses irradi- 
ated in liquid a value of 1.13 would be a more likely estimate In no case would such a difference affect 
the calculated diameters, which at any rate arc simply rough estimates, by more than 10 per cent. 

§ The particle sizes ol)tained by filtration or centrifugation correspond to "wet" sizes; those by elec- 
tron microscopy are "drj-" sizes. 



RADIATION AND VIRUSES 341 

microscopy or ultraceiitrifugal analysis and when calibrated ultrafilters 
are unavailal)le. 

3. There is a progressively increasing discrepancy between particle 
volume and target volume, proceeding from small to large viruses, par- 
ticularly with sparsely ionizing radiation. Radiation sensitivity increases 
more slowly than virus size.- This is where the weakness of the target 
theory becomes evident. 

Lea (1946) favors the hypothesis that the target measures a true physi- 
cal volume within which each ionization is effective. The smallest viruses 
would be fully sensitive, whereas the particles of the larger viruses would 
contain both a radiation-sensitive (genetic) portion and a nonsensitive 
(nongenetic) portion. This analysis is carried further with the assump- 
tion that the sensitive portion always consists of one or more spheres. If 
the dependence on ionization density indicates that the effectiveness does 
not decrease fast enough with increasing density, it is postulated that the 
target consists of several spheres (a less wasteful arrangement for over- 
lapping ionizations). Moreover, each sphere is compared to a gene, 
whose lethal mutation results from a hit, and an estimate of the number of 
genes per virus particle is derived ; for example, vaccinia virus would con- 
tain about 100 genes, and a large phage would contain 10 or 20 (Lea and 
Salaman, 1946). This type of analysis hardly seems justified. 

In the first place, there are neither radiochemical nor genetic reasons to 
postulate a differentiation of biological materials into either indispensable 
and fully sensitive to radiation or fully dispensable. For viruses, it seems 
likely, in view of the complicated stages of interaction between virus and 
host, that many portions of the virus may be altered by radiation in such a 
way as to prevent reproduction. For example, surface groups may be 
involved in adsorption onto the host cells ; other groups may be operative 
in replication; and others in removing inhibitors. The probability of 
effective damage by one ionization (or cluster) may be different from one 
portion of a virus to another. For example, as mentioned later (see Sect. 
3-2), phage particles inactivated by X rays (direct effect) fall into two 
categories, some capable and some incapable of killing the bacterial host 
(Watson, 1950). The two types are probably damaged in different parts 
or in different ways. 

In the second place, even if there were a target within which an act of 
absorption was always effective, there would be no reason for its geometric 
interpretation as a sum of spheres. The observed dependence of radia- 

^ It has been stated that the discrepancies ahnost disappear for a particles (Bonet- 
Maury, 1947, 1948); this seems doubtful, however, a-ray data include disturbing 
contradictions such as a great disparity in sensitivity of vaccinia virus as measured by 
different authors (Lea and Salaman, 1942; Bonet-Maury, 1947) and a target size 
reported for poliomyelitis virus (Lansing strain) much larger than the generally 
accepted virus size (Bonet-Maury, 1948). 



."^12 UADiA ri(>.\ iii()L()(;v 

tion cfticieiicy on ionization density conid result from any goometric (lex i- 
atioii from the "single spherical target" model/' 

A study of the inactixat ion of a l)a('terioi)hage hy deutcron beams of 
diiferent energy (Pollard and Forro, 1!)4'J) sliowed a dependence of effect- 
iveness on the energy of the beam. This was interpreted at first as being 
due to a thlTerence in the effective diameter of the ionization column 
because of ultraviolet emission by excited atoms near the target but out- 
side it. This was clearly incompatible with the known low (juantum yield 
for ultraviolet inactivation of phage, and the deuteron data have been 
reinterpreted (Pollard, 1951) by a method substantially eciuivalent to the 
associated volume method of Lea (1946) and open to similar criticisms. 

In conclusion, it may be said that radiation studies on viruses based on 
the target theory have not yet provided any basic information on the 
nature and structure of viruses, especially because not enough attention 
has been paid to the interaction of an irradiated virus with its host. A 
virus particle is defined as inactive when it has become unable to produce 
active replicas of itself. This failure of reproduction may be caused either 
by inability of the virus to attach itself to a host or to a susceptible cell, by 
inability to penetrate and invade the host, or by inability to carry out any 
one of the probably numerous steps intervening between infection and 
production of new active virus in an infected cell.^ Different portions of a 
virus particle may be functioning in each of these processes. Chemical 
changes may be produced in a virus particle by radiation or other means 
without the particle registering as inactive, either because the damaged 
portion is not essential for reproduction or because that portion, although 
used in reproduction, may be replaceable by other portions. For example, 
suppose that a virus particle has a discrete number of different surface 
areas. A, B, C, . . . , any one of which can act to the exclusion of others 
as the "receptor" involved both in adsorption of the particle on a suscep- 
tible cell and in the following penetration, and suppose that, if receptor .1 
is utilized for absorption, the same receptor .4 will also control penetra- 
tion. If receptor A is damaged by a radiation hit in such a way that it 

" A similar criticism can be made of an attempt (Bonet-Maury, 1948) to interpret 
the supposedly lower sensitivity of vaccinia virus to X rays than to a particles (an 
unconfirmed observation; see Lea and Salaman, 1942) by assuming that the virus 
particle consists of an agglomeration of individual units, each of \vhi(4i nmst be inacti- 
vated for suppression of infectivity. The a rays supposedly would inactivate all the 
units in the agglomerate by energy spread. This hypothesi.s would require that each 
particle break apart into single units before infection ; otherwise, the X-ray inactivation 
curve would be of the multiple-hit type. There is no evidence for .such a structure of 
the vaccinia virus particle. 

* In the case of viruses acting on multicellular organisms, if activity is detected by 
appearance of multicellular foci of infection or of general reactions, a virus may be 
considered inactive if it is unable to reproduce sufficiently to overcome the host 
defen.ses. 



RADIATION AND VIRUSES 343 

can still function in adsorption but not in penetration, reproduction will 
1)6 blocked whenever receptor .1 is utilized, and the probability that 
inactivation results from such a hit is the inverse of the number of areas 
.1 , B, C, . . . present on the virus surface. Considerations of this type 
underline the naivete of the target theory in its narrow form, as applied to 
the analysis of virus inactivation. 

An interesting result has recently been obtained (Hershey et al., 1951) 
from a study of the spontaneous inactivation of bacteriophage that con- 
tains relatively large amounts of radioactive phosphorus. Bacteriophage 
particles containing up to 100 or more P^^ atoms (out of a total of approxi- 
mately 500,000 phosphorus atoms in nucleotides) show a definite insta- 
bility, with a one-hit type of activity decay. On the average, one particle 
is inactivated for every ten P^' disintegrations. The inactivation is 
apparently not due to the emission of /3 rays, but to the nuclear event 
itself. This result suggests either that only 10 per cent of the phosphorus 
atoms of a phage particle are necessary for infectivity, the others being 
dispensable, or that, when a phosphorus nucleus disintegrates, there is an 
average probability 0.10 that this change will result in inactivation. 

Some authors (see Riehl et al., 1941) have discussed the problem of how 
a hit in any one point of a large physical volume can produce inactivation 
of a virus (or mutation of a gene) and have speculated on the po.ssible 
need and mechanism for energy migration within a large biological mole- 
cule to a specific site of action. Such an approach has not led very far, 
however, since little is known about such energy-migration mechanisms. 
The need to invoke their intervention in virus inactivation is not apparent. 

2-2. ULTRAVIOLET RADIATION 

Viruses have generally been exposed to ultraviolet radiation either in 
stirred suspensions or in thin layers in order to avoid or equalize the 
screening effect of impurities. A continuous-flow technique has also been 
described (Levinson et al., 1944). The possibihty that in such experi- 
ments some indirect effects of radiations may be observed has often been 
neglected since the doses of ultraviolet radiation needed for inactivation of 
viruses do not seem to produce appreciable amounts of toxic substances in 
water. Toxic products might, however, originate from impurities. 

For most viruses the proportion of active virus has been reported to 
decrease exponentially with the dose according to Eq. (9-1), the total dose 
(intensity times time) being the relevant variable (Hollaender and 
Duggar, 193G; Price and Gowen, 1937; Taylor et al., 1941; Latarjet and 
Wahl, 1945; Oster and McLaren, 1950; Fluke and Pollard, 1949). One 
ciuantum is apparently the effective hit. Recent data on bacteriophage, 
for which the precise titration method makes it possible to obtain more 
accurate inactivation curves, indicate deviations from the simple loga- 
rithmic relation. Some phages (T2, T4, T6) exhibit a slow initial rate of 



344 RADIATION UIOLOGY 

iiiactivation for very siricall doses (about 10 crj^.s X mm-' for 2537 A), 
soon clian^iinjz; to a lotj;aritlimic rate as the dose increases (Beiizer cl al., 
1950). The reason for this heluvvior is obscure; it does not seem to be due 
to the presence of agf!;re}j;ates of \ irus particles. Other phages (e.g., Tl, 
T7) show an initial logarithmic rate with a br(!ak to a slower rate for sur- 
vivals lower than lO^-. The more resistant fraction of virus is not geneti- 
cally dilTerent; it is possible that it is combined with screening materials. 
Complications of this kind make calculations of iiiactivation rates and of 
(juantum yields somewhat (luestionablc. 

Action spectra have been reported for several viruses (liivers and ( lates, 
1028; Sturm et a/., 1932; dates, 1934; HoUaender and Duggar, 1936; 
IloUaender and Oliphant, 1944). Comparisons were generally made only 
for incident energies, however, by plotting the inverse of the incident dose 
re(iuired to produce a constant amount of inactivation versus the wave 
length. For most viruses the graph resembles the absorption curve of 
nucleic acids with a minimum at 2400 A, a maximum around 2000 A, and 
very low effectiveness beyond 3000 A. For some \'iruses, howe\'er, the 
maximum and minimum at 2()00 and 2400 A, respectively, are much le.ss 
pronounced than for other viru.ses. There is no clear correlation between 
total luicleic acid content and type of action spectrum since vaccinia virus 
and tobacco mosaic virus have approximately the same nucleic acid con- 
tent (in percentage of dry weight), yet give different action spectra. It 
has been pointed out that the viruses, whose action spectra are less similar 
to the absorption spectrum of nucleic acid, siipposedly contain the ribose 
instead of the deoxyribose type (HoUaender, 1946). 

Although these results indicate that, at least in most cases, a large pro- 
portion of the effective radiation is absorbed by the nucleic acid of the 
\irus particles, they do not indicate the relative effectiveness of quanta 
absorbed by different virus components. If the part of a radiation 
absorbed by luicleic acid and that absorbed by other components, e.g., 
proteins, were equally effective in producing inactivation, the greater 
absorption coefficient of the nucleic acids for most ultraviolet wave lengths 
would cause them to appear as the main contributors to the effective 
absorption whenever they are present in the amounts found in many 
viruses (5-40 per cent of the dry weight). 

More information could be gained from action-spectrum studies based 
on measurements not of incident radiation energy but of actual (juantum 
yield. Absorption measurements on purified virus preparations are easily 
feasible, yet surprisingly few data on quantum yield for virus inactivation 
have been reported. In most cases they are for one wa\(> length only, the 
2537 A line of mercury. One difficulty, of course, is that the actual virus 
content of a preparation, in terms of particles per milliliter, is .seldom 
accurately known. For tobacco mosaic virus and 2537 .\ the values of 
2.6 X 10-^ (Uber, 1941) and 4.3 X 10-'^ (Oster and McLaren. 1950) have 
been reported for the cinantum yield, the latter \alue being probably more 



RADIATION AND VIRUSES 345 

accurate. For phage T2, the quantum yield (2537 A) is about IQ-" 
(M. R. Zelle, personal communication), assuming a one-hit mechanism in 
spite of the deviation from the logarithmic inactivation rate. These 
(juantum yields are much lower than those reported for several chemical 
changes in simple organic compounds, including nucleic acid constituents 
(0.01-0.1). Most absorption takes place in the nucleic acid component of 
the virus. Unless absorption in this component happens to be much less 
effective than that in some other components (which is not supported by 
our knowledge of the action spectra), we are led to suppose that a virus 
can withstand an appreciable amount of chemical change in its nucleic 
acid moiety without being inactivated. This is in agreement with the 
results of experiments on phage inactivation following radioactive decay 
of its P^' atoms (Hershey et al, 1951; see Sect. 2-1), and the same con- 
siderations apply to both instances. 

The ultraviolet sensitivity of several viruses of a certain group, such as 
bacteriophages, roughly parallels the particle size when the doses are 
measured in incident energy (Luria and Dulbecco, 1949). This probably 
reflects in part the greater cross section of larger viruses and suggests that 
the quantum yields for inactivation may be of the same order. Bacterio- 
phages T2, T4, and T6 have equal size and morphology, yet T4 is twice as 
resistant to ultraviolet (2537 A) as T2 or TG. It is not yet clear whether 
this difference is due to a low^er nucleic acid content or to a lower quantum 
yield. The radiation resistance of T4 becomes associated with some of 
the distinctive characteristics of T2 or TO in type-hybrid phages produced 
by mixed infection (Luria, 1949). This makes it possible to investigate 
the determination of the ultraviolet sensitivity of a group of viruses by 
genetic means. 

2-3. VISIBLE LIGHT 

Wahl and collaborators (Wahl, 1946; Wahl and Latarjet, 1947) found 
that several bacteriophages are inactivated at an appreciable rate when 
exposed to visible light. The action spectrum has a maximum in the 
near-ultraviolet and violet regions and a limit of effectiveness in the green 
region of the spectrum. Yellow and red light are ineffective. This might 
indicate that the viruses contain a pigment with a maximum of absorption 
or of photochemical yield for the long ultraviolet radiation. It is 
unknown whether this pigment plays any role in the photoreactivation 
phenomenon (see Chap. 12 of this volume). It has also been suggested 
that the inactivation may be due to a photodynamic action mediated by 
components of the medium (Dulbecco, personal communication). 

3. DIFFERENTIAL EFFECT OF RADIATIONS ON VARIOUS PROPERTIES 

OF VIRUSES 

The loss of ability to reproduce is only one of the alterations that may 
be produced in a virus particle. Since inactivation results from suppres- 



346 i; \i)i A ri»).\ HioLOOY 

sion of any one of the steps needed for sueeossful infection and virus repro- 
duction, it is often more easily affected than any other recof^ni/ahle prop- 
erty of the virus particles. Changes produced by radiation, other than 
inactivation, may he even more interesting than inactivation itself since 
they may reveal new properties of the virus particles and their dependence 
on the integrity of specific virus functions. 

Only very large radiation doses cause actual disintegration of the par- 
ticles. In this section are considered, first, nonlethal efTects of radiation, 
i.e., changes recognizable in virus particles that survive irradiation; then a 
series of changes recognizable^ in iiiacti\-e particles. 

3-1. NONLETHAL EFFECTS 

Nonlethal effects are recognized as alterations in the properties of those 
virus particles that survive exposure to radiation; some alterations are 
nonhereditary, others are transmitted to the progeny. Among the former 
may be mentioned a delay in reproduction of bacterioptiage particles that 
survive ultraviolet irradiation, as evidenced by an increase in the latent 
period between infection of a bacterium and its lysis with liberation of new 
virus (Luria, 1944). The new virus gives a normal growth cycle; the 
reproductive delay, then, persists for only one cycle of intracellular repro- 
duction. Another nonlethal effect consists in a slower adsorption by 
bacteria of phage surviving exposure to X rays under conditions where 
indirect effects are prevalent (Watson, 1952) ; there is probably a surface 
alteration of the phage by toxic substances produced by X rays in the 
medium. 

A more important group of nonlethal effects of radiation on viruses is 
the induction of phenotypic mutations, a field as yet insufficiently investi- 
gated. Exposure of tobacco mosaic virus to X rays has been reported to 
produce mutations both from wild type to aucuba and back ((lowen, 
1941). The data indicate that the probability of inducing a mutation is 
about one one-thousandth that of inactivating a virus particle. A report 
is available on mutation induced in tobacco mosaic virus by irradiation of 
virus-infected leaves (Pfankuch el al., 1940). 

With bacteriophage T2, Latarjet (1949) has reported that, following 
ultraviolet irradiation of infected bacteria, there is an increase in the pro- 
portion of bacteria that liberate phage mutants T2h. 

3-2. SEPARATION OF PROPERTIES OF INACTIVE VIRUS PARTICLES 

The detection of the effect of radiations on different properties of viruses 
depends on the number of properties recognizable by the limited mode of 
analysis. With viruses such as bacteriophages and influenza viruses, 
several properties can be separated by increasing doses of radiation or by 
different types of radiations. Some groups of properties, however, are 
always lost simultaneously. When the properties studied represent 



RADIATION AND VIRUSES 347 

recognizable events in the interaction of a virus with the host cell, the 
simultaneous loss of two properties may be taken as an indication that the 
corresponding events result from the same step in interaction. Thus, 
successive steps in host-virus interaction can be traced by the analysis of 
the residual properties of virus particles exposed to different radiations 
under different conditions. As an example, this type of analysis as 
carried out for bacteriophages T2, T4, and T6 active on the common host 
Escherichia coli B will be described. 

The major phases of interaction between these viruses and their 
common host are fairly well known (for reviews, see Delbriick, 1942; 
Cohen, 1949; Luria, 1950; Benzer et al., 1950). One or more active par- 
ticles of bacteriophage become adsorbed by the susceptible bacterium; the 
adsorption is irreversible under the usual environmental conditions. 
Reproduction of the bacterial cell is stopped, and there is complete sup- 
pression of the synthesis of the specific components of bacteria, in par- 
ticular, of bacterial enzymes. There occurs a quick and profound change 
in the cytologically recognizable nuclear apparatus of the bacterial cell, 
which in fixed preparations appears to be disrupted and is later replaced 
by a fine, granular material giving the cytochemical reactions of deoxy- 
ribonucleate and probably representing the new virus. All synthetic 
processes in the infected cell are directed toward the synthesis of bacterio- 
phage components — phage protein and phage nucleic acid — through the 
activity of preexisting bacterial enzymes. After a rather precisely 
defined latent period, during which the synthesis of phage components is 
followed by the appearance of large numbers of new phage particles, the 
bacterial cell is lysed and releases the new phage into the medium. 

The outcome of the infection also depends on the number and genetic 
constitution of the infecting particles. If too many particles are present, 
there may occur a "lysis from without," apparently resulting from a 
massive damage to the bacterial surface. This type of lysis takes place 
without phage reproduction and without disruption of the bacterial 
nuclei. The T-even bacteriophages, in their wild types, also exhibit a 
phenomenon of "lysis inhibition," i.e., a delay in lysis if two or more par- 
ticles infect a bacterium at an interval of several minutes. 

Let us see what happens if active bacteriophage particles are replaced 
by particles inactivated by exposure to radiation. 

Phage particles inactivated by moderate doses of ultraviolet radiation 
(N/No > e~^^, by extrapolation), if tested under conditions where no 
reactivation occurs (see Sect. 4-2), are still capable of being adsorbed by 
bacteria and of killing the bacterial cell (Luria and Delbriick, 1942). 
Bacterial nuclei are disrupted and bacterial syntheses are suppressed, but 
no synthesis of phage components takes place. Lysis and liberation of 
active bacteriophage are absent; even if the infected cells arc artificially 
l)roken, no active bacteriophage is extracted (Luria and Human, 1950). 



348 HAUIATION HIOLOGY 

Thus ultraviolet irradiation separates the early phases of infection from 
the later ones; it provides evidence that bacterial killinji; occurs througli 
the disrupting; action of the infectin}^ pha}>;e and does not re(iuire its rei)ro- 
(hiction. With other ultraviolet-inactivated phages (e.g., Tl and T7j 
bacterial infection is followed by an increase of material that reacts cyto- 
ehemically like deoxyribonucleic acid, but no active phage can be 
recovered from the bacteria. 

For X-ray inactivation, it is necessary to distinguish between direct and 
indirect effects (see Sect. 2-1). Bacteriophage particles inactivated by 
the direct effects of X rays (Watson, 1950) are normally adsorbed by host 
bacteria, but their bacteria-killing ability is often lost. The fraction of 
"killing" particles diminishes logarithmically with the X-ray dose, with a 
slope approximately one-third the slope of the inactivation curve. The 
killing particles affect the bacteria in the same way as does ultraviolet- 
inactivated phage. All adsorbable phage particles, whether killers or not , 
retain both the "lysis-inhibiting" property and the ability to produce 
"lysis from without." These effects, then, require only the changes 
brought about by phage adsorption, without further intromission of the 
virus particle into the economy of the host cell. Thus the comparison 
between active phage particles and particles inactivated by ultraviolet 
and by X rays permits the distinguishing, in the early preproductive 
phases of host-virus interaction, of two stages — one of "adsorption" and 
one of "invasion." The latter involves the disruption of that part of the 
bacterial machinery that impresses on the newly synthesized material the 
specificity of bacterial protoplasm. Interference phenomena (see Sect. 
4-1) reciuire particles capable of invasion; they are produced only by 
particles that can kill bacteria. 

Phage particles inactivated by the indirect effect of X rays (exposure in 
the absence of protective substances; Watson, 1952) exhibit a greatly 
reduced rate of adsorption onto the host cells, which hinders the analysis 
of those phage properties that manifest themselves in later stages of the 
host-virus interaction. This suppression of adsorption results only from 
exposure to the short-lived toxic agent present during actual irradiation in 
water. Phage particles inactivated by introduction into a freshly irradi- 
ated medium give a completely different picture; they are readily adsorbed 
and retain their killing ability, with all the properties that attend this. 
No interpretation in chemical terms of the effects of indirect irradiation 
on various phage properties is available; differential effects of chemical 
poisons on different parts of virus particles are clearly to be expected. 

(lenerally, inactive virus particles that are still adsorbable by the bac- 
terial cells are not physically disintegrated and can still be recognized, e.g., 
in electron micrographs. Very large doses of ultraviolet radiation disrupt 
the complex morphological structure of some bacteriophages. Upon dis- 
ruption, some of the large coli bacteriophages release part of their nucleic 



RADIATION AND VIRUSES 349 

acid (Dulbecco, 1950) and also liberate an agent, smaller than the virus 
particles and separable by differential centrifiigation, which produces lysis 
of the susceptible bacteria (Anderson, 1945). This lytic agent may or 
may not be implicated in the normal lysis of bacteria infected with active 
phage; the possibility of liberating active principles from virus particles by 
means of radiation is, at any rate, suggestive of a new approach to virus 
research. 

The situation described for the phages of the T group is by no means 
unicjue; at least for influenza viruses, similar observations have been made 
(Henle and Henle, 1947). Exposure to ultraviolet for progressively 
longer periods of time eliminates, one after the other, all the properties 
of the virus that can be studied. Reproductive ability disappears first, 
followed by toxicity, which, according to Schlesinger (1950a), is a mani- 
festation of an abortive infection in cells incapable of supporting full 
reproduction of the virus. The ability of the virus to interfere with the 
reproduction of another virus disappears next, followed by the immu- 
nizing capacity for a susceptible host (which may have to do with both 
antigenicit}^ and interfering ability). Hemagglutination — that is, the 
ability to agglutinate red blood cells — is much more resistant and dis- 
appears only after doses of radiation which probably disrupt the virus 
particle. Complement-fixing antigens, mainly present in crude virus 
preparations in the form of small "soluble" antigens, are greatly resistant 
to irradiation. It is interesting that hemagglutination and complement 
fixation should be the two most resistant properties of the influenza virus 
since both of them can be found separated from virus activity in the 
course of normal growth (Hoyle, 1948; Henle and Henle, 1949) and may 
be in the form of immature elements of greater ultraviolet resistance. 

It may be noted that, to an inactive particle of influenza virus, radia- 
tion can leave both the ability to agglutinate red blood cells and the 
ability to be eluted from them enzymically, whereas heat, for example, 
preserves the ability to agglutinate red blood cells but suppresses the 
enzymatic elution. 

The separation of infectivity from the antigenic properties of a virus by 
radiation is of fairly general observation. It has been proved for phages, 
for plant viruses (which retain enough of their integrity to form the same 
crystals or paracrystals as their active counterparts; see Bawden, 1950), 
and for a series of animal viruses. The persistence of serological proper- 
ties, however, may be limited to the effect of ultraviolet or of X rays 
acting directly. In the case of papilloma virus, the indirect effect of X 
rays gives a closer parallelism between the destruction of infectivity and 
that of complement fixation than does the direct effect of X rays (Friede- 
wald and Anderson, 1941). 

Because of the persistence of its antibody-stimulating ability, virus 
which has been inactivated by radiation, in spite of some observation to 



350 RADIATION HIOLOOY 

the contrary, is now coiisidcred a rathor Kood .source of vacciiie.s (Web.ster 
and Ca.sals, 1942; Leviiusoii el *//., I'.tll; Milzer et at., 1941; Mil/er and 
Levinsoii, \\)V.)). As is the rule with inactive viruses, hirf^c; amounts of 
irradiated virus must be used in vaccination .since there is no increase in 
antigen l)y niultipHcation of virus in the host. It is po.ssihle that a certain 
role in the immunity phenomena oh.served with inactive virus vaccines 
may be played by the interference phenomena discus.sed in the next 
section. 

4. IRRADIATED VIRUS IN MULTIPLE AND MIXED INFECTION 
l-l. INTERFEREXCE PHENOMENA 

Under interference phenomena is included a complex jjjroup of phe- 
nomena involving an alteration in the growth or manifestations of a virus 
due to the presence in the same hc^st of more virus of the same or anotiier 
type. The virus particles do not interact among them.selves in vitro, and 
the interference phenomena are strictly cellular. Only with bacterio- 
phage, however, have interference phenomena been analyzed at the 
cellular level (Dell)rack, 1950). 

Mixed infection of a common host with two unrelated bacteriophages 
results in mutual exclusion, only one virus type reproducing in any one 
given cell. The excluded virus may exert a depressor effect on the yield 
of \vinning virus. These exclusion phenomena are not exerted at adsorp- 
tion but take place intraceUularly. Related viruses give incomplete 
exclusion, which becomes less and less evident as the viruses become more 
closely related; particles of two virus strains differing by one mutation 
only do not exclude one another. Whenever exclusion fails, the total 
yield of virus per cell is lower than the sum of the yields that each virus 
would produce by it.self; the two viruses share the maximum potential 
yield per cell. 

With irradiated phages the following rule is fairly well established: 
Whenever a phage particle, after exposure to ultraviolet or X rays, can 
still invade and kill the cell, it retains the exclusion power it had when 
active; particles that are adsorbed but do not kill the host do not produce 
exclusion (Luria and Delbruck, 1942; Watson, 1950). It is not known 
whether an ultraviolet- or an X-ray-inactivated virus particle, if excluded, 
can still exert a depressor effect on the yield of an active, heterologous, 
excluding virus. Irradiated interfering phage excludes homologous 
active phage if it reaches the bacterium several minutes earlier; otherwise, 
exclusion fails, and the yield of active phage is normal (Luria and Dul- 
becco, 1949). 

It is evident then that the interfering ability of inactivated phages is 
related to their ability to kill the bacterial host. If, as seems likely, the 
latter process results from the virus taking oxor and redirecting the syn- 



RADIATION AND VIRUSES 351 

thetic machinery of the host, interference is probably also a manifestation 
of the appropriation by one virus of the directive pattern of specificity to 
the exclusion of another virus. It is interesting in this connection that 
"lysogenic" bacteria, which carry a phage in a form that does not inter- 
fere with bacterial life, can be infected and lysed by other, unrelated 
phages and can liberate them normally. 

In the case of animal and plant viruses, interference phenomena have 
generally been studied only in their mass manifestations when a tissue or 
a whole organism is exposed to two viruses in succession or simultaneously. 
It is difficult therefore to interpret interference in terms of cellular events. 
For animal viruses, interference phenomena have been discussed by Henle 
(1950) and Schlesinger (1950b). Interference may occur between related 
or unrelated viruses, but not all unrelated viruses interfere with one 
another, and two viruses can often be shown to multiply in the same cell. 
Whenever there is interference between two active viruses, it is also 
observed with virus . ^activated by ultraviolet radiation ; other radiations 
have hardly been studied in this connection. With influenza viruses in 
the allantoic cavity of the chick embryo, it has been shown (Henle and 
Henle, 1943, 1945; Ziegler et al., 1944) that a large amount of a virus, e.g., 
influenza type A, after inactivation by ultra\iolet radiation can prevent 
reproduction either of homologous or of heterologous active virus, e.g., 
influenza type B. This interference was at first attributed to suppression 
of virus adsorption because the irradiated virus destroyed the virus recep- 
tors on the allantoic cells, 't is now known, however, that interference 
may occur with amounts of virus that do not prevent adsorption and also 
by introduction of the interfering virus after the first virus has been 
adsorbed (Henle, 1950). In the case of bacteriophage, as well as influenza 
virus, interference probably takes place at the level of the reproductive 
process. That a blockade of the synthv^^c machinery is involved, rather 
than a competition for building blocks, is suggested by the fact the 
inactive virus, although unable tc reproduce, retains the interfering ability. 

4-2. REACTFx vTION PHENOME A 

Reactivation phenomena hi. oeen reported only with bacteriophages, 
but the possibility of their occurrence in other viruses should be explored. 
Bacteriophages exposed to radiation give different activity titers accord- 
ing to the conditions of titration. Two factors have been found relevant : 
The number of irradiated particles absorbed per bacterium (Luria, 1947) 
and the exposure of the infected bacteria to light of certain wave lengths 
(Dulbecco, 1950). Xo reactivation effect has been observed following 
treatments of irradiated phage before adsorption to the host bacterium. 
Reactivation of phages by light ("photoreactivation") is discussed in 
detail in Chap. 12 of this volume. 

4-2a. MuUiplirify Reactivation. In phage titration the phage must be 



352 



RADIATION UIOI.OOY 



mixed witli scnsitiNc bacteria. Kor irradiated phase tlie residual titer is 
minimum when the phaf^e is exposed to such an excess of bacteria that the 
f2;reat majority of tlie infected l)acteria receive only one phage particle. 
I'nder these conditions, it is possible to distinguish a fraction of "residual 
active particles" and one of "inactive particles." The usual inactivation 
curves are obtained in this manner. For some phages and for some types 
of radiation, there is an apparent increase in activity under conditions of 
"multiple infection" of bacteria with phage. This multiplicity reacti- 
vation has been observed with the coli phages Tl, T2, T4, Tf), and T6 
after ultra\iolet irradiation (Luria and Dull)ecco, 1949) and with T2, T4, 
and T() also after exposure to X rays (Watson, 1950). To participate in 
reactivation, an irradiated particle must retain its ability to kill the host 
(see Sect. 3-2). 

Table 9-2. The Basic Observation in Multiplicity Reactivation of 

Bacteriophage 

(Modified from Luria and Dulbecco, 1949, Table 2.) 

Phage: T6r, 1.5 X 10'" units/ml exposed for 20 sec to ultraviolet germicidal lamp, 

General Electric Company, 15 watts, at 50 cm; 7 ergs/mm^/sec 
Bacteria: E. coli B, 2 X 10« colls/ml = B 
Platings: 0.05 ml of phage dilution plus 0.2 ml of B per plate 









Total dilution 










of original 


Plaque 


Mix- 




Dilution of phage 


phage in 


count 


ture 


Procedure 


when first mixed 


suspension 


(sum of 


No. 




with B 


from which 

samples were 

plated 


two 
plates) 


1 


O.I ml T6r -^ 0.9 ml B; kept 10 
min at 37°C; diluted 1 to 10^ 
0.05 ml plated 


1/10 


1/10^ 


1318 


2 


0.1 ml (T(ir 1/10) -^ 0.9 ml B; 
kept 10 min at 37°C; diluted 
1 to 102, 0.05 ml plated 


1/10* 


1/10^ 


474 


3 


0.1 ml (Tfir 1/10')-^ 0.9 ml B; 
kept 10 min at 37°C; diluted 
1 to 10, 0.05 ml plated 


1/10^ 


1/10^ 


250 


4 


0.05 ml (T6r 1/10^) plated 


<1/10* (on plate) 


I/IO^ 


57 



The basic observation is illustrated in Table 9-2 and consists in the fact 
that the same amount of irradiated phage gives a higher activity titer 
(number of lytic areas or "placiues" on a solid layer of sensitive bacteria) 
if the bacteria have been allowed to adsorb the phage from a more concen- 
trated phage .suspension. The effect is not caused by exposure of the 
infected bacteria to some factor other than phage present in crude concen- 
trated phage preparations since it occurs equally well with purified phage. 



RADIATION AND VIRUSES 353 

It must be remembered that, in the type of titration employed, only bac- 
teria which, after receiving phage particles, liberate active phage are 
measured. Only the.se bacteria are lysed; infected bacteria that fail to 
liberate active phage die unlysed. 

If the number of bacteria that liberate active phage is determined and 
the number of residual active phage particles is subtracted from it, the 
number of bacteria in which inactive phage was reactivated is obtained. 
This is never higher than the number of cells that receive two or more 
inactive particles. For small doses of ultraviolet radiations and for high 
multiplicities of infection, the two become approximately equal. Thus, 
reactivation is due to intracellular interaction between phage particles 
which, if adsorbed on separate bacteria, would have registered as inactive. 

The interpretation of the mechanism of "multiplicity reactivation" is 
at this time obscure. The theory originally proposed for its interpreta- 
tion (Luria, 1947; Luria and Dulbecco, 1949) is undergoing revisions. 
When the phenomenon was first recognized, it was quickly discovered 
that reactivation occurs not only among particles of the same phage 
but also among particles of two related phages. More especially, it occurs 
among particles of phages T2, T4, and T6. These exhibit the remarkable 
phenomenon of genetic recombination, in which mixed infection with two 
different phages results in the production of "hybrid forms," deriving 
some of their properties from one phage, some from the other (Delbriick 
and Bailey, 1946; Hershey and Rotman, 1948, 1949). 

This observation suggested a similarity of mechanism between recom- 
bination and reactivation, and the hypothesis was formulated that ultra- 
violet irradiation produced, by discrete hits, a damage localized in discrete 
gene-like individual "units" in each phage particle and that reactivation 
resulted from cooperation among the infecting particles. This coopera- 
tion was supposed to involve the same (unknown) mechanism as that 
involved in genetic recombination. 

The requirement for reactivation in a given bacterium was then postu- 
lated to be the possession by the infecting particles as a group of at least 
one set of undamaged units. This led to the expression, for the maximum 
frequency of production of active phage, 



00 

I 



x''e 



J., 11 - (1 - e-"-)']' 



where x = average number of inactive particles per bacterium, 
r = average number of hits per particle, 
k = an integer number, and 

n = number of the hypothetical units per particle (assumed in first 
approximation to have equal ultraviolet sensitivity). 



354 HADIATIO.N UIOLOGY 

The denominator in Fa\. (9-2) is the fraction of l)acteria receiving two or 
more particles; the luimerator is the prol)ahility that the group of k par- 
ticles infecting a given i)acterium contains one full set of active units. It 
is possible to determine x and r experimentally. A comparison of ij with 
the experimental frecjuency w of active phage production gave, at first, 
results compatible, with some limitations, with E(\. (9-2). The analysis 
was therefore pushed further along these lines, and estimates were given 
for the values of n for different phages. 

The tendency of the experimental ratios w/y toward unity for small 
values of r (low doses) and high values of .r (high multiplicities) suggested 
furthermore that any mechanism of recombination, if responsible for reac- 
tivation, should be an exceedingly efficient one in order to allow an essen- 
tially full utilization of needed units derived from many different phage 
particles in the formation of active phage. This led to the "gene-pool" 
hypothesis, according to which each unit reproduced independently of the 
others, and the resulting new units reassembled to form the new particles. 
This hypothesis could explain a number of features of the phage repro- 
duction process. 

Additional evidence, however, has forced revision of one basic assump- 
tion of the theory that multiplicity reactivation is due solely to a highly 
efficient mechanism of genetic recombination. According to theory, 
the minimum requirement should be the integrity of at least one full 
set of units in the infecting particles. If the frequency of reactivation is 
plotted against the dose of ultraviolet received by the particles, for high 
doses the curves should tend to an ultimate slope equal to the slope of the 
inactivation cur\-e for the free phage since both these slopes represent the 
probability of persistence of one full complement of active units (Dulbecco, 
1952). Analytically, it is easily seen that, for very large values of r, 
Eq. (9-2) tends to the form 




Dulbecco (1952), having by a special procedure obtained data on the 
frequency of recombination at very high radiation doses, found that for 
phage T2 the curves for w versus dose reach their ultimate slope much 
sooner than expected and that this slope is not the same as that of the 
inactivation curve of the single particles but only about one-fifth of it (see 
Fig. 9-1). This result indicates that the simple theory is inadequate. 

The situation may be summarized as follows: jMultiplicity reactivation 
represents the result of a cooperation among inactive phage particles in 
producing active phage. In this cooperation each particle contributes in 
a more than additive measure. For high ultraviolet doses, for example, 
bacteria with three particles have a probability of reactivation several 



UADIATION AND VIRUSES 355 

times greater than that of bacteria with two particles. Dulbecco's work 
on photoreactivation of phage (1951, unpiibhshed results) suggests that 
some phages (the T2, T4, TG group, in particular) may receive two types 
of ultraviolet damage, one photoreactivable by a one-quantum process, 
the other by a multiple-hit process. Multiplicity reactivation can over- 
come the effects of both types of damage. It may involve some kind of 




140 



280 420 560 

ULTRAVIOLET OOSE.Sec 



Fig. 9-1. Survival of phage T2r and its multiplicity reactivation after ultraviolet 
irradiation. Exposure was at 80 cm distance from a 15-watt germicidal lamp 
(General P^lectric Company). Broken line, free phage survival; solid line, reactiva- 
tion frequency (fraction of mvdtiple-infected bacteria that liberate active phage). 
The figures given for each solid-line curve indicate the average multiplicity of infec- 
tion in the whole population. (Modified frotn Dulbecco, 1952.) 



very efficient cooperation at the physiological level, together with a 
mechanism of genetic recombination of a more orthodox nature than the 
one postulated by the "gene-pool" theory. 

For phages inactivated by X rays, multiplicity reactivation is very 
slight, with a frecjuency much lower than with ultraviolet-irradiated 
phage (Watson, 1950). It has been suggested that it may occur only for 
that fraction of particles that are inactivated by acts of X-ray adsorption 
which resemble ultraviolet (juanta in the extent of damage they produce, 
and possibly in the amount of energy released. Interestingly enough, an 



350 UADIATION lUOLOGY 

ap|)iv('iahk' amount of multiplicity reactivation was found' with phage 
inactivated by the aftcretTect of X rays (see Sect. 2-1 a). 

6. INTRACELLULAR IRRADIATION OF VIRUSES 

5-1. IHHADIATION OF CELL8 INFECTED WITH EXOGENOUS VIRUSES 

Irradiation of cells durinjj; infection with \iruses may be of use in the 
study of \irus reproduction. This approach has as yet been limited to 
bacteriophage, but it could be applied to other viruses, particularly in 
tissue cultures. With bacteriophage the basic experimental procedure 
(Anderson, 1944; Luria and Latarjet, 1947) consists in infecting a bacterial 
culture with virus, taking samples at intervals during the period that pre- 
cedes lysis, and exposing them rapidly to various doses of ratliation. The 
irradiated infected bacteria are then tested immediately for their ability to 
liberate phage. This ability can be suppressed by either ultraviolet or 
X rays; and, if the fraction of bacteria that liberate phage is plotted 
versus dose of radiation, "suppression curves" are obtained. The sup- 
pression effect is exerted on the intracellular bacteriophage rather than on 
the bacterial host. This is shown by the following observations: 

1. Active phage can reproduce normally in bacteria exposed to ultra- 
violet radiation shortly before infection (Anderson, 1948). 

2. If infected bacteria are irradiated immediately after infection, the 
rate of suppression of phage liberation as a function of radiation dose is 
similar to the rate of inactivation of free virus. 

3. In multiple infection the suppression cur\e immediately after infec- 
tion is of the multiple-hit type and closely resembles the curves obtained 
for active phage production in multiple infection with irradiated bacterio- 
phage (see Sect. 4-2a). 

As the time after infection increases, the suppression curves change in a 
manner characteristic for the phage. The simplest case is that of phage 
T7 (Benzer, 1952; see Fig. 9-2). After infection there is no change in 
ultraviolet sensitivity for 3 or 4 min, but then the suppression curve 
becomes of the multiple-hit type without any change in the final slope of 
the curve; this seems to indicate a simple mechanism of multiplication of 
virus elements having the same sensitivity as the free virus. 

With phage T2 (Luria and Latarjet, 1947; Benzer, 1952) the first 
change in bacteria infected with one T2 particle and exposed to ultra- 
violet (2537 A) is an increase in ultraviolet resistance without appreciable 
change in the shape of the curve. Several minutes later the inactivation 
curve changes to a complex type, suggesting an effect on numerous 
objects within each cell. In the latest stages of infection, radiation 
sensitivity again increases. The results suggest that phage T2 must 
perform an early function easily blocked by ultraviolet damage and that 
the radiation sensitivity increases as this early phase is passed. 



RADIATION AND VIRUSES 



357 



Phage T2 has similarly been investigated with X rays (Latarjet, 1948). 
The picture is simpler than with ultraviolet since the suppression curve 
remains constant for several minutes, then becomes of a multiple-hit type 
(with lesser ultimate slope than for free phage), and finally becomes a 
multiple-hit curve with ultimate slope similar to that of free phage. 



> 

o 

z 
llJ 

o 

UJ 

a: 



a. u 

UJ 



< 
q: 



> 
> 

a. 

(ft 




05 - 



001 



0005 



100 200 

ULTRAVIOLET DOSE, Sec 



300 



Fig. 9-2. Intracellular irradiation of phage T7. Bacteria were washed in buffer, 
infected with phage T7 (single infection), and then placed in a nutrient medium. 
Samples were taken at intervals, chilled, exposed to ultraviolet radiation, and then 
plated to determine the number of bacteria that still liberate phage. The suppression 
curves are compared with the inactivation curve for free phage. Exposure conditions 
were the same as for Fig. 9-1. The time given for each curve is the time between 
addition of nutrient and chilling previous to irradiation ("growth timeT). {Benzer, 
1952.) 



Thus this type of analysis, although quite incomplete, suggests that a 
small phage such as T7 reproduces by multiplication of uniform elements, 
whereas T2 undergoes a complex series of changes, including a process of 
multiplication, which only in the latest phases leads to the presence of 
mature fully sensitive phage particles in the infected cells. This con- 
clusion is in accord with all our information on the reproduction of these 
viruses. 

The intracellular irradiation procedure allows the determination of the 



358 i{ \i)i AiioN ni(ti.(»r.v 

stage of iiif(>(t ion from the shape and slope of the suppression curve. This 
can he utiHzed to identify tiic stages at which a certain Ircatment stops 
virus reprochu'tion. For example, on exposure of l)acteria infected with 
phage T2 to a temperature of 45°C, the changes in ultraviolet sensitivity 
lake place normally for the first 7 min, after which no further change 
occurs, as though at this time a temp(M-ature-sensitive reaction entered tlie 
picture (Beii/cer, 19o(), unpublished data). 

Although no "cure" for phage-infected bacteria has been ol)tained in 
this work, an extension of these studies to other viruses might produce 
results of some therapeutic value in \ irus infection. Even if the cells 
already infected could not be saved by radiation, suppression of their 
ability to liberate virus might prevent the spreading of infection. The 
value of such a procedure would depend on the relative sensitivities of the 
infected cells and of the normal tissues. Experiments on rabbit papil- 
loma have shown that growth of the papillomas can be suppressed by 
doses of X rays much smaller than those necessary to inactivate the \^irus 
in vitro (Syverton, Berry, and Warren, 1941; Syverton, Harvey et al., 
1941). Actually, virus can be recovered in undiminished amounts from 
the irradiated papillomas (Friedewald and Anderson, 1943). Here the 
radiation probably acts on the host cell, rather than directly on the intra- 
cellular virus, in the same way as therapeutic doses of X rays affect 
bacterial infections bj' acting on the tissues of the host. 

5-2. RADIATION AND LATENT VIRUSES 

An interesting possibility is that of affecting, by means of radiation, 
viruses which may be present in the latent state, i.e., viruses which do not 
manifest themselves and which behave up to a certain point like normal 
cell components. The distinction between latent viruses and cell com- 
ponents is not always easy with the available knowledge and in some cases 
may actually be academic. Any self-reproducing element of the proto- 
plasm of a cell might conceivably become a virus if by some evolutionary 
accident it should ac(iuire the ability to enter other cells and there repro- 
duce its own kind. Such an origin of viruses has been suggested, but the 
question will remain academic until more definite knowledge is obtained in 
regard to the occurrence and properties of self-reproducing units (other 
than the nuclear genes) in most types of cells. Should such units be more 
widespread than they appear to be, their origin in the process of cell evolu- 
tion would still be unknown. It is known that a virus may enter a cell 
and reproduce while the cell goes through several cell generations, often 
without causing recognizable cell disturi)ances. Such a virus behaves at 
least for some time as a cell component. This type of symbiosis often 
prolongs itself for many cell generations, and in multicellular organisms 
some viruses are transmitted through the gametes from generation to 
generation. The recognition of the virus depends then only on indirect 



RADIATION AND VIRUSES 359 

tests, either the inoculation of tissue extracts in a virus-susceptible host or 
the search for antivirus antibody in the latently infected organisms. The 
distinction between virus and cell component is thus (luite difficult. The 
information relevant to this area of biology caiuiot be discussed here. In 
connection with the possil)ility of affecting latent viruses or self-repro- 
ducing cell components within cells as a means of analyzing the relation 
between these entities and the cell as a whole, some radiation results 
are pertinent. 

5-2a. Irradiation of Lysogenic Bacteria. Lysogenic bacteria carry one 
or more bacteriophages in a latent form without recognizable manifes- 
tations. In these bacteria the phage is apparently present as immature 
virus or "prophage." Occasionally a lysogenic cell is lysed and liberates 
a cluster of mature phage particles, whose presence can be recognized if a 
susceptible strain of bacteria that responds to phage infection by lysis is 
available (Lwoff and Gutmann, 1950) . In the course of attempts to define 
the conditions that lead to the occasional maturation of virus in the lyso- 
genic bacteria, it has been discovered (Lwoff et al., 1950) that ultraviolet 
irradiation produces in some lysogenic strains a massive lysis accompanied 
by liberation of mature phage. This suggests that something in the lyso- 
genic bacteria prevents the maturation of prophage into bacteriophage, 
thus preserving the symbiotic relation, and that ultraviolet, by removing 
the inhibition, releases the maturation process. 

5-2b. Irradiation and Cytoplasmic Factors. Another pertinent obser- 
vation concerns the destruction by radiation of the cytoplasmic factor 
"kappa" in Paramecium aurelia. Some strains of this organism produce 
a poison (paramecin) which is lethal for individuals of other strains. The 
production of the poison is always associated with the presence in the 
cytoplasm of the killer animals of peculiar Feulgen-positive particles, 0.3- 
0.8 M in size, which are the material carriers of a genetically recognizable 
self -reproducing mutable factor (kappa). The continuous production of 
kappa depends both on the presence of preexistent kappa and on the 
proper genetic background. Kappa has been transmitted from one indi- 
vidual to another by "infection " with cell extracts, thus resembling a virus 
or a rickettsia (Sonneborn, 1949). 

X rays (Freer, 1950), as well as ultraviolet (Sonneborn, personal com- 
munication) and nitrogen mustards (Geckler, 1949), eliminate the killer 
factor from the protoplasm at a rate that suggests a one-hit inactivation 
process with an inactivation dose of approximately 4000 r. This dose is 
comparable to the doses required for sterilization of bacteria which are 
somewhat larger in size than the kappa particles. This result suggests 
interesting applications of radiation analysis to the study of cytoplasmic 
inheritance and encourages speculation on the possible use of selective 
effects of radiation on cytoplasmic elements in modifying de\-elopment 
and differentiation (which have been suggested to be controlled by cyto- 



.3()() IIAIMATIOX MIOLOflY 

pliismic dotormiiiants of heredity) and in altering the neoplastic properties 
of tumors. 

It may he of interest to mention that another self-reproducinj^ mutahle 
cytoplasmic factor, the virus-like "genoide" for carbon dioxide sensitivity 
in Drusophila (L'lleritier, 1949), which can he transmitted from fly to fly 
by cell-free extracts, is inactivated in the extracts by X rays. The inacti- 
vation dose is around 10'' r, similar to that for medium-sized viruses 
(L'Heritier and Plus, 1950). 

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(194(i) Kxperinients on the inactivation of hacteriophaKc hy radiations and 

their Ixarin;! on tlie nature of l)a(t(riophaKe. I'roc. Roy. Soc. London, lil33: 
4:i4 444. 

Lea, D. K., K. .M. Smith, H. liulmcs, ami U. .Mari<ham (1944) Direct and indirect 
actions of radiations on viruses and enzymes. Parasitology, 36: 110-118. 

Levinson, S. ()., \. Milzer, H. J. Shaughnessy, J. L. Neal, and F. Oppenheimer (1944) 
Production of i)()(cnt inactivated vaccines with idtraviolet irradiation. IL .\n 
al)l)rc\iated preliminary rcj)ort on .sterilization of l)actcria and immunization with 
rabies and St. Louis encephalitis vaccines. J. Am. Med. Assoc, 125: 531-532. 

L'Heritier, P. (1949) Genoide sensibilisant la Dronophilf a I'anhydride carl)oni(iue. 
In, rnit(!''s biologiques donees de continuitc gencticiuc. (Vntre National de la 
Recherche Scientili(iue, Paris. Pp. 113 122. 

L'Heritier. P., and .\. Plus (1950) Inactivation par les rayons .\ du virus responsable 
de la .sensibilite au CO.. chez la Drosophile. Compt. rend. .soc. bio!., 231 : 192 194. 

Luria, S. Iv (1940) Methodes statist iques appliquees a I'etude du mode d'action des 
ultravirus. Ann. inst. Pasteur, 64: 415-438. 

(1944) A growth-delaying effect of ultraviolet radiation on bacterial viruses. 

Proc. Natl. Acad. Sci. U.S., 30: 393-397. 

(1947) Reactivation of irradiated bacteriophage by tramsfer of self-repro- 
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(1949) Type hybrid bacteriophages. Rec. Genetics Soc. .Vm., IS: 102 (also 

Genetics, 35: 122, 1950). 

(1950) Bacteriophage: an essay on virus reproduction. Science, 111: 507- 



511. 

Luria, S. E., and M. Delbriick (1942) Interference between inactivated bacterial 
virus and active bacterial virus of the .same strain and of a different strain. 
Arch. Biochem., 1: 207-218. 

Luria, S. E., and R. Dulbecco (1949) Genetic recombinations leading to production 
of active bacteriophage from ultraviolet inactivated bacteriophage particles. 
Genetics, 34: 93-125. 

Luria, S. E., and F. M. Exner (1941) The inactivation of bacteriophages by x-rays. 
Influence of the medium. Proc. Xatl. Acad. Sci. U.S., 27: 370-375. 

Luria, S. E., and M. L. Human (1950) Chromatin staining of bacteria during bac- 
teriophage infection. J. Bacteriol., 59: 551 560. 

Ltiria, S. E., and R. Latarjet (1947) Ultraviolet irradiation of l)acteriopliage during 
intracellular growth. J. Bacteriol., 53: 149-163. 

Lwoff, A., and A. Gutmann (1950) Recherches sur un Bacillus vUgatherinm lysog^ne. 
.\nn. inst. Pasteur, 78: 711-739. 

Lwoff, A., L. Siminovitch, N. Kjeldgaard, S. Rapkine, E. Ritz, and .V. Gutmann 
(1950) Induction de la production de bacteriophages chez im<' i)a(t( rie lysogene. 
.\mi. inst. Pasteiir, 79: 815 859. 

.Milzer, .\., and S. O. Levinson (1949) .Vctive immunization of mice with ultra- 
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Milzer, .\., V. ()j)])eMlieimer, and S. (). Levinson (1944) Production of potent 
inactivated vaccines with ultraviolet irradiation. III. .\n abbreviated pre- 



RADIATION AND VIRUSES 363 

liminary report on a complotoly inactivatod poliomyelitis vafcinc (Lansing strain 

virus) in mice. J. Am. Med. .V.ssoc, 125: 704 705. 
Oster, G., and A. D. McLaren (1950) The ultraviolet li^l't ami photosynthesized 

inactivation of tobacco mosaic virus. J. Gen. Physiol., '.i'A: 215-228. 
Pfankuch, K., G. A. Kausche, and II. Stubbe (1940) liber die Entstehung, die 

biologische und physikalisch-chemische Charakterisierung von KiHitgen- und 

7-Strahlen induzierte "Mutationen" des Tabakmosaikvirusproteins. Biochem. 

Z., 304: 238-258. 
Pollard, E. C. (1951) Ionizing radiation as a test of molecular organization. .Am. 

Scientist, 39: 99-109. 

(1953) The physics of viruses. Academic Press, New York. 

Pollard, E. C, and F. Forro, Jr. (1949) Examination of the target theory by deuteron 

bombardment of T-1 phage. Science, 109: 374-375. 
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Inactivation by ultra-violet light. Phytopathology, 27: 267-282. 
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Sonneborn, T. M. (1949) Beyond the gene. Am. Scientist, 37: 33-59. 

Sturm, E., F. L. Gates, and J. B. Murphy (1932) Properties of the causative agent 
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Syverton, J. T., G. P. Berry, and S. L. Warren (1941) The roentgen radiation of 
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Syverton, J. T., R. A. Harvey, G. P. Berry, and S. L. Warren (1941) The roentgen 
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Taylor, A. R., D. G. Sharp, D. Beard, H. Finkelstein, and J. W. Beard (1941) Influ- 
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Watson, J. D. (1950) The properties of X-ray-inactivated bacteriophage. I. 
Inactivation l)y direct effect. J. Bacteriol., 60: 697 717. 

(1952) The properties of X-ray-inactivated bacteriophage. II. Inactivation 

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J. Pub. Health, 32: 268-270. 



304 HADlVnoX UIDI.OOY 

W oilman, E., F. Ilolwock, iind S. I.uria (li>40) KlTcct of radiations on bacteriophage 
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Wolliiian, 10., anil A. Lai-assanno (liMO) Evaluation des dimensions des bacterio- 
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Ziegler, J. E., Jr., (!. I. Lavin, and F. L. Horsfall, Jr. (1944) Interforcnce between 
the inlluciiza viriises. II. The effect of virus rendered non-infective by ultra- 
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Manuscript received hii thr ((lilnr Apr. 20, 1951 



CHAPTER 10 

Effects of Radiation on Bacteria 

M. R. Zelle 

Cornell University 
Ithaca, New York 

Alexander Hollaender^ 

Biology Division, Oak Ridge National Laboratory 
Oak Ridge, Tennessee 

Bactericidal effects of radiation. High-energy radiations: General results of quatdita- 
tive investigations. Factors influencing sensitivity to ionizing radiations: Ultravioiet 
radiation — Extreme ultraviolet radiation — Near- ultraviolet and short-visible radiation. 
Physiological properties of bacteria following irradiation. Sublethal effects of radiation. 
Bacterial genetics: Radiation-induced mutations in bacteria — Mechanism of radiation 
effects. 

One of the first laboratory observations of the effects of radiations on bac- 
teria was published by Downes and Blunt in 1877. Since that date a volu- 
minous literature has accumulated as a result of the wide interest in the 
effects of radiation on bacteria per se and also because of the relative ease 
with which quantitative studies of the biological effects of radiation can 
be made with bacteria. 

Despite the numerous investigations during the past seventy-five years, 
understanding of the effects of radiation on bacteria is still only fragmen- 
tary. Furthermore, the advances since the 1936 predecessors of these 
volumes, especially during the past decade, have been rapid in comparison 
to the advances in earlier work. For example, almost all knowledge of 
radiation-induced mutations in bacteria has been gained since that time. 
Consequently, in the limited space available, no attempt will be made to 
review completely the earlier literature. Rather, only those earlier con- 
tributions considered to be especially significant will be included with corre- 
spondingly greater emphasis on recent research. The rather numerous 
reviews concerned with various aspects of the biological effects of radia- 
tion combine to give a complete coverage of the development of the field 

' ^\'o^k performed iiiuier Contract Xo. W-7405-eng-26 for the Atomic Energy 
Commission. 

365 



;^()() UADIATION lilOl.OGY 

(Dufijrar. \\yM\\ (liese, 1945, H)47. lUoO; K\\\s rl ai. 1041; Latarjet, 1046; 
Lea, 1017; Loofbourow, 1048; Mitchell, 1051; Rahn, 1045). 



BACTERICIDAL EFFECTS OF RADIATION 

When bacteria are exposed to radiation, in either the high-ener{j;y or the 
ultraviolet range, the most prominent clTcct is the apparent killing of a 
percentage of the cells, the fraction killed being a function of the absorbed 
energy. The usual criterion of survival is the ability of the bacteria to 
form a colony visible to the eye when incubated following plating on ordi- 
nary culture media. This arbitrarily adopted measure of the bactericidal 
effects of radiation, although convenient for (|uantitative studies, is influ- 
enced by a variety of experimental conditions. Ilollaender (1043) 
showed that prolonged exposure to saline, following long-ultraviolet or 
short-visible irradiation, reduced the fraction of colony-forming organisms. 
Roberts and Aldous (1940) made careful studies of various experimental 
conditions both before and after ultraviolet irradiation which affect the 
survival of Escherichia coli, strain B. TIkmt results will be considered in 
more detail later, but for this strain, a hundredfold variation in survival 
could be produced by changing the postirradiation treatment. Further- 
more, the shape and slope of the survival curve were markedly influenced 
by different conditions. Strain B/r, a radiation-resistant mutant deri\'ed 
from strain B (Witkin, 1946, 1947), did not show similar variation in 
survival when subjected to the same experimental treatments. Kelner 
(1949a, b) observed that exposing bacteria to visible light following 
exposure to ultraviolet significantly increased sur\'ival (photoreacti- 
vation). Anderson (1949, 1951b) and Stein and Meutzner (1950) have 
shown that increasing the temperature of incubation increases the sur- 
vival of ultraviolet-irradiated E. coli B. Survival following X irradiation 
was shown by Hollaender, Stapleton, and Martin (1951) to be influenced 
by the oxygen concentration of the medium at the time of irradiation. 
Stapleton et al. (1953) have discovered that incubation at suboptimal 
temperatures markedly increases the survival of bacteria exposed to X 
rays, and Stapleton (1952) has found marked differences in the radio- 
sensitivity of E. coli cells at different stages of the growth cycle. 

These findings are mentioned to emphasize the multiplicity of factors 
which influence the quantitative results obtained in studies of the bac- 
tericidal effects of radiation. Consequently, in order to obtain reproduc- 
il)le results, it is necessary that these variables be adequately controlled. 
Furthermore, it is impossible to determine how much of the variation in 
the results obtained bj' difl'erent investigators is attributable to differences 
in their experimental techniques. This is especially true in regard to 
many of the earlier studies. 

Bactericidal effect, lethal effect, killing, and inactivation are used 



EFFECTS OF RADIATION OX BACTKHIA 367 

synonymously to indicate the failure of the cells to form a colony visible 
to the naked eye when plated under the particular conditions of the 
experiment under discussion. 



HIGH-ENERGY RADIATIONS 

GENERAL RESULTS OF QUANTITATIVE INVESTIGATIONS 

Minch in 1896 was apparently the first to attempt to study the bac- 
tericidal effects of X rays. His results were essentially negative as were 
those of man}' other investigators during the next thirty years. Low 
intensities of X rays and rather insensitive bacteriological techniques 
seem to be the chief reasons for the conflicting and often negative results 
of different investigators during this period. Duggar (1936) briefly dis- 
cusses these early X-ray results, and the bibliographies given by him and 
by Pugsley et al. (1935) form a helpful guide to the early literature. 

Diu'ing this period, however, it was convincingly demonstrated by a 
number of investigators that ionizing radiations do exert a marked bac- 
tericidal effect. Green (1904)^ employing semiquantitative bacterio- 
logical techniques, studied the bactericidal effects of radium /3 rays on 23 
species of bacteria including five spore formers. In his experiments all 
species were killed by the /3 rays, and the spore formers were found to be 
considerably more resistant than the vegetative forms. One of the out- 
standing contributions during this period was that of Chambers and Russ 
(1912). These workers studied the effects of radium emanation, pri- 
marily /3 rays, on distilled water suspensions of Staphylococcus aureus, 
Escherichia coli, Bacillus pyocyaneus, and Bacillus anthracis. A pro- 
nounced bactericidal action was observed with all species; anthrax spores 
were observed to be the most resistant to radiation. Quantitative esti- 
mates of surviving organisms made by plate counts of an irradiated S. 
aureus suspension, when plotted semilogarithmically, gave rise to a 
straight line. This is the first exponential survival curve reported for 
bacteria subjected to radiation. Furthermore, these workers observed 
motile cells of B. pyocyaneus in irradiated suspensions in which no colony- 
forming organisms were present. Similar observations of inactivated but 
motile cells have been made by Bruynoghe and Mund (1925). 

Following application of the target theory by Crowther (1924, 1926) 
to inhibition of mitosis in tissue culture cells observed by Strangeways and 
Oakley (1923) and to his own data on killing of Colpidium colpoda, a num- 
ber of investigators applied similar analyses to the bactericidal effects of 
ionizing radiations. Holweck (1929) and Lacassagne (1929) irradiated 
"pyocyanique S" with soft X rays of 4 and 8.3 A wave lengths. They 
observed exponential killing with 4 A X rays, but a multihit or sigmoidal 
type of survival vxivxe was obtained with 8.3 A. However, Lea, Haines, 
and Coulson (unpublished, see Lea, 1947) observed exponential sur- 



3(1S It \i)i A iio.v iii()L(jr.Y 

vival curves \vh(Mi icpcMliiifj; this work with the same strain and wave 
Iciijitlis. 

Wyckort" (lU:i()a. 1)) and WyckolT and Rivers (llKiO), in a series of care- 
tul stiuHes, more firmly estal)Msiied the occui'rence of exponential survival 
curves followiufi; exposure to ionizing radiations. Wyckoff and Rivers 
( 1930) studied the bactericidal effects of 15o-kv (8 rays on E. coli, Salmo- 
nella h/pliimurium, and Staphylococcus aureus cells seeded on the surface 
of agar plates. Exponential survival curves were obtained for all except 
S. aureus. By allowing E. coli to divide before irradiation, they showed 
that clumping of the cells w'as probably the reason that exponential sur- 
vival curves were not obtained for S. aureus. They conclude that a single 
electron is sufficient to inactivate a cell of these species. Similar results 
(Wyckoff, 1930a) wxre obtained in studies with E. coli and *S. tijphimurium 
using copper-K X rays and the soft general radiation from a tungsten 
tube operated at 12 kv. Later, Wyckoff (l!)30b) studied the killing of 
E. coli with X rays of wave lengths varying from 0.5 to 4 A. Exponential 
killing was observed at all wave lengths. Wyckoff interpreted his results 
to indicate that a single quantum of X rays was sufficient to kill the bac- 
teria. His estimated values of the sensitive volume of the organisms 
decreased with increasing wave lengths as a result of the greater incident 
energy required for inactivation at the longer w^ave lengths. 

Similar results have been obtained by other workers with various bac- 
terial species subjected to various ionizing radiations. Hercik (1933, 
1934b) observed exponential sur^•ival curves wuth Serratia marcescens 
irradiated with a particles emitted by polonium. Pugsley et al. (1935) 
observed exponential killing for E. coli irradiated wdth 40-kvp X rays but 
obtained sigmoidal curves for Sarcina lutea. A correction applied for the 
degree of clumping as determined by microscopic examination of the 
irradiated suspension resulted in an exponential survival curve. Lorenz 
and Henshaw (1941) made extensive tests of the bactericidal effects of 
200-kvp X rays on Achromobacter fischeri. Statistical analj'sis showed no 
systematic deviation from an exponential survival curve. During the 
past decade, in which there has been almost a routine use of radiation 
for the induction of mutations in microorganisms, numerous investigators 
have observed exponential survival curves (e.g., Lincoln and Gowen, 
1942; Demerec and Latarjet, 1946; Witkin, 1947; Roepke and Mercer, 
1947; Anderson, 1951a). Frara et al. (1950) report exponential survival 
curves for six species following irradiation with 50-kvp X rays. A typical 
exponential survival curve with 5 per cent confidence limits of the plotted 
points is shown in Fig. 10-1 (Stapleton, unpublished data). 

Not all investigators have observed exponential survival curves, how- 
ever. Observation by Holweck (1929) and Lacassagne (1929) of sig- 
moidal survival curves with 8.3 A X rays has already been mentioned. 
Glaus (1933) observed sigmoidal sur\i\al curves for E. coli following 



EFFECTS OF RADIATION ON BACTERIA 



369 



100 



X irradiation in the presence of heavy metal ions. The sigmoidal curves 
may have been due to the short wave lengths of the secondary radiations 
which were considered to be the main cause of inactivation. Luria 
(1939), employing the same bacteriological techniques in both cases, 
observed exponential survival curves with polonium a particles and a 
two-hit sigmoidal curve with 0.7 A X rays. Microscopic examination of 
the irradiated cells of E. colt revealed that, although some of the cells were 
killed immediately and did not divide or grow, others continued to grow 
without dividing and ultimately 
developed into long filamentous 
forms. These filamentous forms 
either divided a few times and 
then died or else recovered and 
proceeded to develop normal col- 
onies. Exposure to both a and 
X radiation caused filamentous 
forms, but the proportion was 
higher with X rays. Luria points 
out that death by several mecha- 
nisms is incompatible with the 
simple mathematical formulation 
of the target theory. 

Similar results with Aspergillus 
terreus spores have been reported 
by Stapleton, Hollaender, and 
Martin (1952) and Zirkle et al. 
(1952) who report sigmoidal sur- 
\ival curves with hard X rays and 
exponential curves with densely 
ionizing protons and a particles. 
Since in both cases air-dried 
spores were irradiated, there is no 
possibility that prodviction of a 
toxic substance in the irradiated suspending medium could be responsible 
for the sigmoidal curves. 

Stapleton (1952) made the important observation that the form of the 
survival curve obtained after X irradiation (250 kvp) of E. coli B/r cells 
depends on the stage of the growth cycle of the culture. Cells from fully 
grown cultures in the stationary phase yield exponential survival curves 
(Fig. 10-1). However, when cells in the lag phase were exposed to X rays, 
sigmoidal survival curves were obtained, the deviation from exponential 
killing increasing to a maximum at the end of the lag phase. Interpreta- 
tion of the sigmoidal cur\es on the multitarget theory (Atwood and Nor- 
man, 1949) shows an increase from one to about eight targets during the 




001 



40 

DOSE, kr 



Fig. 10-1. Exponential survival curve with 
5 per cent confidence limits for each point; 
E. coli B/r resting cells irradiated in air- 
saturated buffer with 250-kvp X rays. 
(Stapleton, unpuhlished data.) 



;^7() RADIATION HIOLOGY 

lufj; phase. Cytolofiical studies show a hifi;h correspondeiu'e between the 
iiuniher of ol^servahle nuclear l)odies and the numher of t argots estimated 
for cells in different f^rowth jihases. The survival curves become expo- 
nential and the target iniinber ix'comes 1 as growth proceeds through the 
logarithmic phase and the stationary phase is approached. Stapleton 
made the further interesting observation that stationary-phase cells sub- 
jected to 7 rays from a Co^" source exhibit a "two-hit " killing curve as 
compared to an exponential curve for X rays. 

Lea et al. (193()), in addition to discussing the principal theories of the 
mechanism of the bactericidal action of ionizing radiations, present 
important (luantitative data on survival curves of irradiated bacteria. 
They studied primarily the survival of Bacillus mesentericus spores and, 
less extensively, the survival of E. colt and S. aureus. The radiations 
employed were a particles emitted by polonium and /3 rays produced by 
radon disintegration. Careful energy measurements were made and the 
geometrical conditions were controlled. The spores and bacteria were 
irradiated in dried gelatin films of approximately l-yu thickness. The 
fraction of organisms surviving was found to be a diminishing exponential 
function of the time of exposure, or dose, for all organisms with both 
radiations. The target areas and mean lethal doses (MLD) were com- 
puted for each organism for both a and 13 rays. 

The studies with B. mesentericus spores and E. colt were extended to 
include radium y rays (Lea et al., 1937), neutrons, hard X rays of an effec- 
tive wave length of 0.15 A, and soft X rays of L5, 4.1, and 8.3 A (Lea 
et al., 1941). The organisms were irradiated in aqueous suspensions; 
suspending the cells in previously irradiated distilled water given com- 
parable exposures was shown to have no effect. Again, exponential 
survival curves were observed in all cases. 

Spear (1944), utilizing the same bacterial strains employed by Lea and 
his associates, presents the most extensive data on the bactericidal effects 
of neutrons. The neutron source was a beryllium target bombarded by 
8-Mev deuterons accelerated in a 37-in. cyclotron. Acjueous suspensions 
were exposed, and the dose was measured with a Victoreen dosimeter 
calibrated in roentgens. The sur\i\'al cur\'e did not depart systemati- 
cally from an exponential curve. The ratio of the T-ray dose in roentgen 
units to the neutron dose in n units refjuired to produce 50 per cent 
lethality was 3.2 for E. coli and 5.3 for B. mesentericus spores. Because 
of the lack of absolute dosimetry with neutrons, Spear presents a curve 
(Fig. 10-2) showing a systematic decrease in the MLD ratio of B. mesen- 
tericus spores to E. coli cells as the ionization density increases. This 
curve includes all the data of Lea and his associates and shows that the 
neutron data fit into the general relation. 

No effect of varying temperature between 2.5° and 3()°C at the time of 
irradiation was observed by Hercik (1934a) in studies employing a par- 



EFFECTS OF RADIATION ON BACTERIA 



371 



tides and Serratia marcescens. Lea et at. (1936) found the rate of iuacti- 
vation of B. mesentericus spores by a and particles to be independent of 
temperature between -20° and +50°C. No influence of temperature 
was noted on the rate of inactivation of *S. aureus. These results differ 
markedly from the high temperature coefficients experienced with chemi- 
cal disinfectants. Lea e( al. (1937) showed that varying the temperature 
from 0° 37°C had no influence on the survival of E. colt exposed to y rays. 
Lea et al. (1936) observed that a sixfold variation in intensity of a-par- 
ticle flux had no effect on the proportion of organisms surviving a given 
dose. This was confirmed by experiments in which a given dose was 
administered in a number of fractions, no effect on survival being observed. 



30- 



_i 
o 
o 

CO 

o 

ir 
uj 20 



(ij 

5 



o 

I 



BETA RAYS 
Oc-GAMMA RAYS 

SOFT X RAYS I 5 A 
O' \ O-SOFT X RAYS 83 a 



O-NEUTRONS 



(- 



10- 




ALPHARAYS 



1^34 
IONS PER cm(xlO^) IN BIOLOGICAL MATERIAL 

Fig. 10-2. Curve showing ratio of MLD mesentericus: MLD coli plotted against ion 
density for a number of different radiations. {Adapted from Spear, 1944.) 



Lea et al. (1941) reported further data on the lack of an intensity effect 
of a particles and 8.3 A X rays. A seventy-fivefold variation in intensity 
was employed in the latter case. Extremely high intensities of /3 par- 
ticles have been shown by Huber (1951) to be bactericidally effective. 

The observation of filamentous forms in bacterial cultures following 
ultraviolet irradiation was reported by Gates (1933) who concluded that 
the mechanism of cell division was more sensitive to radiation than the 
processes of growth. Similar observations have been reported by several 
investigators (Spencer, 1935; Luria, 1939; Witkin, 1947; Rolierts and 
Aldous, 1949). Lea et al. (1937) observed such filamentous forms follow- 
ing continuous y irradiation of growing E. coli cultures. Subsequent 
experiments indicated that the filamentous cells reacted to y rays in the 
same manner as the normal cells, i.e., they were equally sensitive to 
radiation and were inactivated in an exponential manner. 

Careful studies of the effect of ionization density on the biological effec- 



372 u\i)i\ri()N iii()L()(!Y 

li\ciu>.s.s of poloiiuim (I particles liaxc l)i'cii report (id by Zirkle (11)10) who 
obtained dilTereiit ioni/atioii densities b}- utilizing dilTerent portions of 
the path lengtii. An increase in elTectivcness with increasing ionization 
density was obserxed with Aspenjillns nujer spores whereas, for inactiva- 
tioii of E. coll, an inverse iclation was observed. Little influence of 
ionization ciensity on sur\ i\al of yeast cells was noted. 

In Table 10-1 are assembled the available data on the relation of ioni- 
zation density and the bactericidal effect of radiations (see also Fano, 
Chap. 1, and Zirkle, Chap, (i, \'olume I of this series). The estimates 
made from Spear (1044) and Zirkle (1040) are only approximate but 
serve to indicate the general relations. It will be observed that the MLD 
(37 per cent survival dose) for E. coli increases as the iotiization density of 
the radiation increases. On the contrary, the MLD for B. mesentericus 
and for .1. nufcr spores decreases with increasing ionization density. 
This difference is as yet unexplained, and it may indicate that different 
mechanisms are involved in the lethal effects of irradiation for the two-cell 
forms. The increased efficiency of more densely ionizing radiations in 
killing ,4. terrcus spores is confirmed by Stapleton, Hollaender, and Martin 
(lOo'i) who add the further interesting observation that more densely 
ionizing a particles are less efficient than hard X rays in inducing 
mutations. 

Summarizing, three general conclusions seem warranted: (1) Both 
exponential and sigmoidal survival curves may be observed, depending on 
the bacterial strain, the technique of irradiation, the characteristics of the 
radiation, and the stage of growth of the irradiated cells. (2) The sur\iv- 
ing fraction for a gi\'en dose of radiation is independent, within limits, of 
the intensity of the incident radiation or of the fractionation of the dose. 
(3) For vegetative cells the bactericidal effectiveness of a given dose 
decreases with increasing ionization density; the opposite seems to be 
true for spores. 

The interpretation to be placed on these general results is not clear at 
this time. Lea et at. (1036) and Lea (1947) have discussed at length the 
various interpretations proposed for the observed survival curv^es. 
Interpretation in accordance with the target theory seems the most 
plausible since the first-order kinetics are a natural consequence of the 
theory. Similaily, the sigmoidal survival curves are also easily accounted 
for on the basis of either multiple hits re(iuired in one target or single hits 
in multiple targets. Atwood and Norman (1940) discuss this latter 
interpretation. Lea (1947) especially has been a strong proponent of the 
target theory. Il(> interprets the results to indicate that a single ioniza- 
tion is sufficient to inactivate a bacterial cell. He has developed the 
hypothesis, first suggested by Rahn (1020, 1030), that the bactericidal 
effects are due to lethal mutations induced by the radiation. Following 
elaborate analysis of the results obtained with E. coli exposed to radiations 



EFFECTS OF RADIATION ON BACTERIA 



373 



Table 10-1. Mean Lethal Doses" of Various Rai)l\tions on 

Various Bacteria 



Reference 



Radiation 



MLD 



E. coll (doses in 10' r or 10' n) 



Wyckoff (H)30b) 


X rays, 


A: 






0.56 




4.2 




0.71 




4.6 




1.5 




4.3 




2.3 




6.7 




4.0 




8.4 


Zirkle (1940) 


X rays 


0.3 A 


3.9 




a Particles, Mev: 






~5 




5.7 




~2 




6.4 


heaetal. (1941) 


^ rays 




4 




7 rays 




5.2 




X rays, 


A: 






0.15 




6.0 




1.5 




6.5 




8.3 




7.5 




Neutrons 


7.1 (4.1)'> 




OL Partic 


iles 


24 



B. mesentericus spores (doses in 


10^ 


r^ 




LeaeiaL (1941) 


^ rays 
7 rays 
X rays, A: 

1.5 

4.1 

8.3 
Neutrons 
a Particles 






1.1 
1.3 

1.3 

1.1 
1.5 

0.61'- 
0.26 





A. niger spores" (doses in 10^ r) 




Zirkle (1940) 


a Particles, 


Mev: 








~4 




11.0 






~3 




3.6 






~2 




4.7 






~1.5 




3.9 






~1 




3.2 



" MLD, 37 per cent survival dose. 

* Estimated from Spear (1944). 

« Spore germination, not colony growth, wasmeasured: estimated from Zirkle (1940). 



874 UADIATION ItlOI.OOY 

of (lilTcrcMit ionization (ioiisitics, Leu (11)47) coMcludos ih.il the hiictcricidal 
cITccts can be accounted for on the hypothesis of lethal niiitalions induced 
aiuoiifi; 2')() ^enes liavinj; an avera}j;e diameter of 12 ni^u. 

An iucreasinii; amount of evidence has l)een accumuhiting to indicate; 
that a major porti(in of tlie effects of ionizing radiations may he iiuhrect. 
Conse(iuently there is consi(hMal)le doubt that the target theory, without 
modification, may Ix' Nalidly ajjplied to interpretation of those bac- 
tericidal phenomena in which indirect actions aic known to be involved. 
Further discussion is gix en in a later section. 



FACTORS INFLUENCING SENSITIVITY TO IONIZING RADIATIONS 

Few comparisons have been made of the X-ray sensitivity of spores and 
the parent vegetative forms. In general, spores have been found to l^e 
more resistant (Green, 1904; Chambers and Russ, 1912; Baker, 1935; 
Lea et al., 193G, 1941). The greater resistance to radiation of spores may 
be partially due to the lower water content (Stapleton and Hollaender, 
1952). Of equal interest is the generality of the inverse relation of sensi- 
tivity of spores and vegetative cells to radiations of different ionization 
densities (Table 10-1). 

The relative sensitivity of E. coli B/r cells to 250-kvp X rays at different 
stages of the growth cycle has been studied by Stapleton (1952), as dis- 
cussed earlier. There is a sharp decrease in sensitivity during the lag phase, 
followed by a marked increase in sensitivity during the logarithmic phase. 
The maximum sensitivity is reached at the end of the logarithmic phase, 
and as the stationary phase progresses, the sensitivity gradually declines 
to the initial level. 

Table 10-2. Mean Lethal Doses" of Escherichia coli Strains Irradiated 

DURixr, the Stationary Phase in Air (Incubatkd at 37°C and 

Irradiated with 250-kvp X Rays) 

(PVoin Stapleton, pensonal (.'ommunication.) 

Strain .MLD, (r) X 10' 

B 3.5 

B/r 6.2 

Tennessee 6.0 

86G 6.2 

Gratia 6.5 

n-52 6.5 

• Crook 8.0 

Texas 10.0 

"• MLD, 37 per cent survivaL 

The relation of genetic constitution to radiation resistance wall be dis- 
cussed later. That strains within a species maj^ vary widely in sensitivity 
to radiation is showai in Table 10-2, in which the MLD \alues for eight 
E. coli strains exposed to 250-kvp X rays are showai (Stapleton, personal 



EFFECTS OF RADIATION ON BACTERIA 375 

communication). Although strain B/r shows about the median resist- 
ance in this group, strain B is significantly more sensitive than any of the 
others. 

Relation of Oxygen Concentration to X-ray Effects. The current interest 
in the relation of oxygen tension to the effects of X rays on living cells 
stems largely from the work of Thoday and Read (1947j, although earlier 
investigators had made similar ol)servations. The relation of oxygen 
concentration to cytogenetic effects is discussed by Giles (Chap. 10, 
volume I of this series) along with possible interpretations to be placed 
upon the observations. 

A similar relation between oxygen concentration and bactericidal effec- 
tiveness of X rays has been observed for E. coli B/r by Hollaender and 
coworkers (Hollaender, Stapleton, and Martin, 1951; Hollaender, Baker, 
and Anderson, 1951: Hollaender, Stapleton, and Burnett, 1951; Hol- 
laender and Stapleton, 1953). This same group has obtained interesting 
results on the closely allied problem of chemical protection against X rays. 

Except when studying temperature effects, all irradiations were per- 
formed at 2°C with washed cells suspended in M/lb phosphate buffer. 
Reduction of oxygen tension was accomplished by partial evacuation fol- 
lowed by saturation with nitrogen, helium, hydrogen, or carbon dioxide. 
The particular gas used to replace oxygen was of no importance. 

Figures 10-3 and 4 from Hollaender, Stapleton, and Martin (1951) show 
that lowering the oxygen tension changes not only the slope of the sur- 
vival curve but also the shape, although in a later publication (Burnett 
et al., 1951) exponential survival curves are shown for both oxygen-satu- 
rated and nitrogen-saturated suspensions as well as for suspensions con- 
taining 0.04 M concentrations of sodium hydrosulfite (Na2S204), British 
anti-Lewisite (BAL), and ethanol. Equally apparent in Figs. 10-3 and 4 is 
the lower sensitivity of cells grown in glucose broth and of cells grown 
anaerobically, in contrast to the sensitivity of aerobically grown cells. 
Although the ultimate slope of the nitrogen-saturation survival curves is 
less steep than the slope of those for oxygen saturation, cells grown in 
glucose broth or nutrient broth, either aerobically or anaerobically, 
exhibit parallel survival curves once the threshold dosage is exceeded. 
The threshold dose is a function of the method of culturing. 

Hollaender, Stapleton, and Martin (1951) observed that cells irradiated 
in oxygen-saturated suspensions were more sensitive at 2°C, whereas cells 
irradiated in the absence of oxygen were more sensitive at 37°C. As dis- 
cussed earlier, Hercik (1934a) and Lea et al. (1936) failed to detect an 
effect of temperature. The apparent disagree