Inhibition and Destruction of the Microbial Cell

817 Pages·1971·13.016 MB·English

by  W. Hugo (Auth.)

INHIBITION and
DESTRUCTION of the
MICROBIAL CELL
EDITED BY
W. B. HUGO
Department of Pharmacy,
University of Nottingham, Nottingham, England
ACADEMIC PRESS
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Copyright © 1971 by ACADEMIC PRESS INC. (LONDON) LTD
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FROME
Contributors
A. c. ΒAIRD-PARKER Unilever Research, Golworth/Welwyn Laboratory,
Sharnbrook, Bedford, England
M. R. w. BROWN Department of Pharmacy, University of Aston,
Birmingham, England
B. CROSHAW Boots Pure Drug Co. Ltd., Research Department, Biology
Division, Nottingham, England
p. F. D'ARCY Riker Laboratories, Loughborough, Leicestershire, England
Η. M. DARLOW Microbiological Research Establishment, Porton Down,
Salisbury, Wiltshire, England
J. H. s. FOSTER School of Pharmacy, Bath University of Technology,
Claverton Down, Bath, England
J. A. FAREWELL Department of Pharmacy, Bath University of Technology,
Claverton Down, Bath, England
M. FRIER Pharmaceutical Development Department, Boots Pure Drug
Co., Ltd., Beeston, Nottingham, England
s. A. GOLDBLITH Department of Nutrition and Food Science, Massa-
chusetts Institute of Technology, Cambridge, Massachusetts, U.S.A.
p-o. HAGEN Oak Ridge Associated Universities, Oak Ridge, Tennessee,
U.S.A.1
w. A. HAMILTON Department of Biological Chemistry, University of
Aberdeen, Aberdeen, Scotland
R. κ. HOFFMAN Decontamination Section, Physical Defence Division,
Department of the Army, Fort Detrick, Frederick, Maryland, U.S.A.
w. B. HUGO Department of Pharmacy, University of Nottingham,
Nottingham, England
D. j. KUSHNER Department of Biology, University of Ottawa, Ottawa,
Canada
w. H. LEE School of Veterinary Medicine, Department of Epidemiology
and Preventive Medicine, University of California, Davis, California,
U.S.A.
A. R. LONGWORTH Imperial Chemical Industries Ltd., Pharmaceuticals
Division, Macclesfield, Cheshire
j. MELLiNG Microbial Research Establishment, Porton Down, Salisbury,
Wiltshire, England
1 Present address : Department of Experimental Surgery, Duke University,
Durham, North Carolina, U.S.A.
ν
vi CONTRIBUTORS
Ε. j. MORRIS Microbiological Research Establishment, Porton Down,
Salisbury, Wiltshire, England
H. p. RIEMANN S'chool of Veterinary Medicine, Department of Εepidem-
iology and Preventive Medicine, University of California, Davis,
California, U.S.A.
A. D. RUSSELL Welsh School of Pharmacy, University of Wales Institute
of Science and Technology, Cardiff, Wales
j. R. TRUEMAN Lever Brothers and Associates Ltd., Product Develop-
ment Division, Port Sunlight, Wirral, Cheshire, England
Preface
When approached by Academic Press to edit a book on the general
area of disinfection I felt at first that such a topic was well served.
However, no book was available that dealt in detail with the inhibition
and destruction of groups of micro-organisms, although the information
must be available either scattered in the literature or, for some groups,
in occasional review articles. From this realization the concept of the
book crystallized—namely, that the treatment of the inhibition and des-
truction of the microbial cell should be written from two points of view :
the agent, the traditional approach, and the target (the microbial cell),
which is a novel approach. Consequently, six chapters comprising 306
pages are devoted to weaponary and eight chapters, 445 pages, to
targets.
Inevitably, such an approach leads to a certain amount of duplica-
tion, but I believe that, in the form in which this book appears, only
advantage can result from this.
Moreover contributors have confirmed that my original concept
regarding targets was justified and I heard many reports concerning
the diversity of literature sources which finally gave rise to their
contributions.
I was also fortunate in being able to include a chapter on another
important unreviewed area in the general field of this work, namely the
effect of the cultural prehistory of microorganisms upon their response
to inhibition and destruction (Chapter 14).
It was not my intention to include antibiotics and sulphonamides in
this volume, but the author of Chapter 12 felt he could not treat his
subject adequately if these were omitted, and he further considered,
rightly in my opinion, that while excellent data were available on their
antimicrobial action there was a need for collated information on their
action on yeasts and moulds.
This book will be of prime interest to graduate research workers and
scientists in pure and applied microbiology, while it is hoped that
undergraduates reading specialized courses at honours level in micro-
biology or applied courses in food science and agriculture will be able
to refer to the book profitably. It will also be of interest to the medical
profession, especially those involved in public health and pathology,
and to scientists in the pharmaceutical industry.
vii
viii PREFACE
Finally, I would like to thank the staff of Academic Press for un-
failing help and courtesy during the preparation of the volume.
I am very greatly indebted to the contributors and record in this
preface my grateful thanks for their efforts, cooperation and expertise
in bringing the information relating to their topic to its final form.
Some have undertaken commissions at short notice and I am fully
aware of the time they have spent on their work.
W. B. HUGO November 1970
Chapter I
Inhibition and Destruction of Microorganisms
by Heat
M. R. W. BROWN AND JACK MELLING
Department of Pharmacy, University of Aston in Birmingham,
Birmingham, England and Microbiological Research Establishment,
Porton Down, Salisbury, Wiltshire, England
I. INTRODUCTION . . . . . . . . .. 1
II. GROWTH TEMPERATURE RELATIONSHIPS . . . .. 3
A. Species and strain variation . . . . . .. 3
B. Growth medium composition . . . . .. 4
C. Biochemical basis of temperature relationships . .. 5
III. LETHAL TEMPERATURE RELATIONSHIPS . . . . .. 8
A. Lowered temperatures . . . . . . .. 8
B. Elevated temperatures . . . . . .. 9
REFERENCES . . . . . . . . . .. 33
I. Introduction
It is proposed to consider the inhibitory and lethal effects of heat
on vegetative bacterial cells. The inhibitory effects of heat on spores
will only be referred to briefly to provide appropriate illustrative
examples. They are considered elsewhere in this book (page 451).
The potential range of responses of a bacterium to any particular
temperature is determined genetically. Nevertheless, the past and
present environment play a profound role in modifying the phenotypic
expression of the genetic determinants. Consequently (cf. page 703),
quantitative comparisons between different cell populations of their
response to heat are extremely difficult. We define heat resistance in
terms of the maximum heat treatment during which the bacterium
retains viability. Valid comparison of resistances can be made only if
environmental conditions before, during and after treatment are con-
trolled and the validity of the comparison not stretched too far from
the confines of those conditions.
ID M 0—Β
2 M. R. W. BROWN AND JACK MELLING
The introduction of a toxic chemical agent into the environment
of a bacterium and thence into the cell itself makes a radical, qualita-
tive change in the system. This is not to say that the disruption to the
cell quantitatively may not be slight. Furthermore, the bacterium may
possess a mechanism of excluding or inactivating the toxic chemical.
The situation with thermal energy is quite different. For all practical
purposes thermal energy is always present and the capacity of a bac-
terium to insulate itself from changes in the temperature of its environ-
ment is negligible. For these reasons the questions asked when inves-
tigating the mechanism of the inhibition and destruction of bacteria by
heat will include somewhat different ones to those asked about the
mechanism of action of a toxic chemical. Addition of a chemical agent
causes both a qualitative and a quantitative change in a system. Heat
treatment, no matter how severe, is only a quantitative effect. The
turnover of complex molecules is such as to enable the thermodynamic
balance sheet to give an ultimate loss of free energy. Thermal energy
is an integral part of the entire complex system. In other words,
thermal energy not only inevitably affects the cell as a whole but also
every molecule and every reaction in it.
For these reasons we have been unable to separate a consideration
of the effects of heat on bacterial growth from that of lethal effects.
It would not be correct to say that dead bacteria provide no information,
but living ones may certainly be more informative. Furthermore, as
a generalization, psychrophiles are inhibited from growth at tempera-
tures appropriate for mesophiles and these latter are inhibited from
growth by temperatures suitable for thermophilic bacteria. Brock (1967)
quotes examples of bacteria growing in pools at boiling point (about
92 °C) at Yellowstone National Park. From this point of view interest
in the effects of heat on growth and on inhibition would seem to
merge.
The bulk of this chapter is appropriately devoted to the inhibitory
effects on bacteria of elevating the temperature. Nevertheless, the in-
hibitory and lethal effects of temperature decreases are briefly con-
sidered because they are effects of thermal energy.
Because of the universal effects of thermal energy on biochemical
reactions the Arrhenius relationship has been used as a quantitative
expression relating temperature and rates of biological reactions in
general.
The empirical Arrhenius equation is given by
log k = G
6 2-303.fi Τ
where k is the reaction velocity, C is a constant, R is the gas constant,
1. INHIBITION AND DESTRUCTION OF MICROORGANISMS BY HEAT 3
Τ the absolute temperature and Ε is the activation energy for the
reaction. This important parameter may be obtained by plotting log k
versus l/T which is linear within the limits of applicability of the
Arrhenius equation.
A more fundamental treatment of this idea depends upon the appli-
cation of the theory of absolute reaction rates (Eyring, 1935) to bio-
chemistry (Johnson et al., 1954). When a reaction, biological or other-
wise, conforms to the Arrhenius relationship it enables the investigator
to predict events, and thus exercise control. It is thus possible some-
times to use elevated temperatures to study reactions which occur at
an inconveniently slow rate at lower temperatures (Brown and Meiling,
1967).
There is an enormous literature related to heat resistance of micro-
organisms and no attempt has been made to compile a comprehensive
review. Rather, in the context of this book, we wish to consider recent
work on the biochemical basis both of inhibition of growth and on
loss of viability resulting from thermal energy.
We have found the following reviews helpful: Ingraham (1962), the
Symposium on Molecular Mechanisms of Temperature Adaption (Ed.
Prosser, 1967), Symposium on Growth of Microorganisms at Extremes
of Temperature (1968) and several chapters in the text Thermobio-
logy (Ed. Rose, 1967).
II. Growth Temperature Relationships
A. SPECIES AND STRAIN VARIATION
Bacterial growth has been found to occur at temperatures ranging
from about 0° to 90 °C, with different species and strains having dif-
ferent maximum and minimum growth temperatures within this range.
Organisms have been divided into psychrophiles, mesophiles and ther-
mophiles according to the range of temperatures over which they will
grow. Although rigid differentiation has not been possible, owing to the
occurrence of borderline cases and the influence of environment, never-
theless examination of the characteristics of members of the three
groups may begin to give some indication of the way in which they are
able to cope with their different environments.
An Arrhenius plot of log bacterial growth rate versus the reciprocal
of incubation temperature (° Abs.), when linear, gives a value analo-
gous to the activation energy and is the temperature characteristic of
growth (μ). Recent evidence (Hannus and Morita, 1968) indicates that
μ values may be the property of a particular species or of the growth
medium, but not of the temperature range of growth.
Ingraham (1962), in a useful review of this subject, considered that
4 M. R. W. BROWN AND JACK MELLING
the ability of a population to adapt to growth at different temperatures,
or the selection of mutants having this ability, was likely to provide
information regarding the means by which organisms are able to grow
at different temperatures. However, it appears that the temperature
range for most organisms is not readily altered (Farrell and Rose, 1967 ;
Stanier, 1942). This suggests that considerable differences in the genetic
make-up of the various organisms may be involved. There is some
evidence that transfer of genetic material may convert mesophilic into
thermophilic types (Sie et al., 1961; McDonald and Matney, 1963).
Observations on some mesophilic bacteria (Mefferd and Campbell, 1952 ;
Allen, 1953) have indicated the presence of about one thermophilic
variant in 10 6 cells of the original population. Thus, in the absence of
evidence of any mutagenic action of heat, it appears that these thermo-
philes may have arisen by spontaneous mutation.
B. GROWTH MEDIUM COMPOSITION
There is now considerable evidence that the temperature at which
growth of microorganisms can take place is affected by the composition
of the growth medium. An early report is that of Mitchel and Houlahan
(1946) who isolated a mutant of Neurospora which required riboflavin
for growth at temperatures above 28°C. Begue and Lichstein (1959)
observed that several strains of Saccharomyces cerevisiae were unable
to grow at 38 °C in a chemically defined medium which was adequate
for growth at 30 °C. The organism grew well at both temperatures in a
complex medium and subsequent investigation indicated that addition
of calcium pantothenate enabled growth to occur in the chemically
defined medium at 38°C. It appeared that synthesis of pantothenic acid
by these organisms at 38°C was somehow prevented. Maas and Davis
(1952) obtained a mutant strain of Escherichia coli which required
pantothenic acid for growth above 30° and they concluded that the
mutation resulted in production of an altered heat-labile enzyme in-
volved in pantothenic acid synthesis.
The examples quoted above have all indicated increased nutritional
requirements as the temperature increased. Campbell and Williams
(1953) divided the thermophilic organisms which they were studying
into three groups, based on their nutritional requirements at different
temperatures. The first group showed no difference in requirements
regardless of incubation temperature over the range 36°C to 55°C.
Group two had increased requirements as the incubation temperature
was increased, but the third group exhibited increased nutritional re-
quirements as the temperature was decreased. The authors suggested
that these results could be explained on the basis that in the first
group the gene responsible for synthesis of some enzyme was either