Table Of ContentObservations relatives à la Note précédente de M. Daclaux ; par M. PASTEUR.
« Je prends la liberté, en présentant cette Note de M. Duclaux, de lui
suggérer l'idée d'un travail auquel le préparent non seulement celui que
je dépose, en son nom, à l'Académie, mais les travaux non moins distin
gués qu'il a déjà produits sur le rôle des microbes dans la digestion.
» Souvent, dans nos causeries du laboratoire, depuis bien des années,
j'ai parlé, aux jeunes savants qui m'entouraient, de l'intérêt qu'il y au
rait à nourrir un jeune animal (lapin, cobaye, chien, poulet), dès sa
naissance, avec des matières nutritives pures. Par cette dernière expression,
j'entends désigner des produits alimentaires qu'on priverait artificiellement
et complètement des microbes communs.
» Sans vouloir rien affirmer, je ne cache pas que j'entreprendrais cette
étude, si j'en avais le temps, avec la pensée préconçue que la \ie, dans
ces conditions, deviendrait impossible.
)> Si ces genres de travaux se simplifiaient par leur développement même,
on pourrait peut-être tenter l'étude de la digestion par l'addition systé
matique, aux matières nutritives pures dont je parle, de tel ou tel microbe
simple ou de microbes divers associés bien déterminés,
» L'œuf de poule se prêterait sans difficulté sérieuse à cette nature
d'expériences. Privé extérieurement au préalable de toute poussière vi
vante au moment où le petit poulet va sortir, mis aussitôt après dans
un espace sans germes quelconques de microbes, espace où se renouvel
lerait un air pur, on fournirait facilement du dehors au jeune poulet des
aliments purs (eau, lait, grains).
» Que le résultat soit positif et confirme la vue préconçue que je mets
en avant ou qu'il soit négatif et même en sens inverse, c'est-à-dire, que la
vie soit plus facile et plus active, il y aurait un grand intérêt à tenter l'ex
périence. *
COMPT. REND. FRENCH ACAD. SCI., 100:68 (1885)
GERMFREE LIFE
AND
GNOTOBIOLOGY
by
THOMAS D. LUCKEY
Department of Biochemistry
University of Missouri School of Medicine
Columbia, Missouri
ACADEMIC PRESS, New York and London
1963
COPYRIGHT© 1963, BY ACADEMIC PRESS INC.
ALL RIGHTS RESERVED.
NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM,
BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT
WRITTEN PERMISSION FROM THE PUBLISHERS.
ACADEMIC PRESS INC.
Ill Fifth Avenue, New York 3, New York
United Kingdom Edition published by
ACADEMIC PRESS INC. (LONDON) LTD.
Berkeley Square House, London W.l
LIBRARY OF CONGRESS CATALOG CARD NUMBER: 63-13105
PRINTED IN THE UNITED STATES OF AMERICA
Dedicated to the scientists and friends of science who helped
establish germfree research as a new biological tool.
PREFACE
During the past decade the use of the techniques of germfree research
has increased to such an extent that now is the appropriate time for
the appearance of this comprehensive review of the whole field of
germfree research and gnotobiology. The last detailed review, written
in German, is that of Glimstedt (1936). Several brief reviews which
have appeared since then are mentioned in Chapter 2; the most
recent of these is that of Mickelsen (1962).
This volume treats the theory and general aspects of germfree
research and provides a summary of germfree work in all phyla, thus
presenting an over-all picture of the subject. The chapters on methods
and nutrition are sufficiently detailed to serve as guides for experi
mental work. Germfree vertebrates are characterized from available
data on morphology, biochemistry, nutrition, serology, and physiology.
The final chapter reviews exploratory research concerned with the
inoculation of germfree animals with known kinds of microorganisms.
The phylogenetic approach has been used in order to give perspec
tive to the work with any given species, to emphasize areas needing
further research, and to provide a broad enough survey to facilitate
evaluation of gnotobiotics by workers in peripheral fields. Certain
aspects have been discussed within more than one context in the
book in order to develop the subject fully enough so that the reader
will not be burdened by the need for undue cross-reference to other
sections.
New terms have been introduced to describe new concepts and to
provide precise communication. For the convenience of the reader
there is a glossary in the appendix section. Also included in the
appendix is a chronology which gives some idea of the extent to which
various people, laboratories, and nations have been involved in germ
free research.
The extensive bibliography will serve as a guide to the literature
in the field. Where a specific reference is not given for data in the
text the implication is that the source can be found in references
listed under the authors name or under Reyniers et al. (with the
author as a collaborator); in some cases data are from the author's
unpublished work.
vii
Preface viii
This volume has been planned for those who are engaged in germ
free research, those just entering the field of gnotobiology, those in
related fields who need to incorporate the concepts of gnotobiology
into their own thinking and practice, and who wish to apply the
techniques in their own work, and those who are interested in the
progress of science in our time. The monograph will also provide a
background for many practical aspects: the maintenance and supply
of specific pathogen-free and gnotobiotic laboratory animals, tech
niques in experimental surgery, modification of hospital procedures,
the developing industry of raising disease-free pigs, and biological
problems in space exploration.
Personal conversations or correspondence with most of the current
investigators in germfree research have enriched this manuscript.
The trust evidenced in their helpful responses has had a sobering
and humbling influence.
T. D. LUCKEY
April, 1963
Acknowledgments
The unpublished work was done with financial aid of the Office of Naval
Research at the University of Notre Dame, and the National Science Foundation
at the University of Missouri. Collaborators at the University of Notre Dame were
J. A. Reyniers, R. F. Ervin, M. Wagner, Dr. H. A. Gordon, R. Meeks, Dr. T.
Mende, J. Pleasants, and B. A. Teah. The technical assistance of Mrs. Akrevy
Mauer, Mrs. Margaret Beaver, Mrs. Lou McAllister, Mrs. Nancy Conbere,
A. Lorance, and D. Donalek is gratefully acknowledged. Dr. H. T. Miller has
been of special assistance in the work with the germfree room at the University
of Missouri and also in the preparation of this manuscript. A special contribution
to the work described in this monograph was the provision of five germfree mice,
by the University of Notre Dame through Dr. R. E. Thorsen, F. X. Bradley, and
Dr. H. A. Gordon.
Credit or reference is given with each photograph excepting those by the
author. Acknowledgment is given for permission to duplicate material from the
publishers of the following journals: Annals of the New York Academy of
Sciences, Journal of Dental Research, Journal of Nutrition, Lobund Reports,
Texas Reports of Biology and Medicine, and The Journal of Experimental
Medicine.
Special thanks are expressed to those who translated literature: Drs. G. J.
Mannering, S. Landry, H. A. Gordon, H. T. Miller, Fun Sun Chu, and Mrs. Leida
Piip. Drs. M. Miyakawa and J. Tanami introduced me to the Japanese research
and literature. Dr. J. J. Landry was helpful in allowing use of material on
sterile surgery and pig operations. I am especially indebted to Dr. Victor Cabelli
for critical reading of the manuscript, to Morris Wagner for reviewing the
methods on testing for sterility, and to Mrs. Pauline Luckey for final proof
reading. While all have helped make this document presentable, the author
assumes full responsibility for errors and omissions.
ix
CHAPTER 1
Theory and General Aspects
of Germfree Life and Gnotobiology
Biological Isolation as the Basis of Germfree Life
Complete isolation, a state which has more philosophical than prac
tical importance, is not easily attained. A small fraction of the air we
breathe was in the lungs of Christ, Mohammed, and Buddha. The
complete biological isolation of any but the first living organism is
denied by the, ontogenetic law. No organism is entirely alone; the
phylogeny of the individual transcends species barriers. Living cells
are born of other living organisms. Plants use carbon dioxide from
animals to build their substance; and animals must rely upon the
photosynthetic activity of plants for fuel and specific organic nutrients
without which animals would soon perish. Autotrophic bacteria and
some plants can survive on inorganic substances, but even they use
biologically formed organic molecules on most occasions.
Since experimentation with complete biological isolation is not
practical with present day limitations, the most strict definition of pure
culture may be ignored. More pertinent is a consideration of a usable
definition of germfree with the concomitant development of a detec
tion system for any and all living contaminants and the relegation of
viruses to an acceptable and practical category for future experi
mental work. Classically, germfree animals, sterile plants, and axenic
invertebrates are each defined as a single species isolated from all
other demonstrable living microorganisms. Theoretically, germfree
research is an extension of the pure culture concept of bacteriology.
This concept began in the early days of taxonomy and may be traced
back to Aristotle. It gathered strength from the theory of the immuta
bility of species, was tarnished by the theory of evolution, and finally
was crystallized by the work of Koch (and Lister) who isolated single
microbial cells to give assurance of a pure culture according to the
ι
1. Theory and General Aspects 2
cell theory of biology. The weakness of the identification of biological
isolation procedures and of germfree research with the pure culture
concept is that inherent in the cell theory of life itself.
As Hughes ( 1959 ) points out, the cell is a convenient mechanical
unit for the basis of life but not the ultimate one. The individuality of
the cell is less fundamental than is usually portrayed. Consider cells
which harbor intracellular parasites, cells such as striated muscle with
more than one nucleus, bacteria without well defined nuclei, or living
organisms without cell walls such as the slime mold Physarum
polycephalum. Corliss (1957) briefly reviewed the complexity of
protozoa whether considered as cells or individuals. Blood has many
characteristics of living tissue without being strictly cellular. Mature
red blood cells are cellular and respire, but the absence of nuclear
material precludes them from being called living. Viruses obviously
do not fit into the cell theory; they are analogous to only one
part of a cell—a fragment of a chromosome. If such a contaminant
is part of the genetic apparatus, Reyniers (1959c) suggests it may
be eliminated by following appropriate breeding patterns or by
finding a strain of the same species which does not carry the contami
nant. The occult or nonsymptomatic virus becomes of real importance
in the definition of the individuality of the cell or the organism. Is it
really a stranger when it has occupied a regular place in the chromo
somal pattern? If viruses are considered to be living and if a regular
place in a chromosome is occupied by viral nucleic acids, no organism
can be completely gnotobiotic. Infectious nucleic acids must be
considered in similar concept. Practically, the definitions of germfree
and gnotobiosis must include the term "detectable living forms."
Dmochowski (1961) emphasizes the fact that living organisms
should be defined neither in terms of cell morphology nor of cellular
constituents. The cell is a useful biological compartment which allows
concentration of bioconstituents for the reactions of growth, organiza
tion, and reproduction characterizing living organisms. The cell is a
characteristic unit of living organisms, but this unit is of little im
portance as a characteristic of life. An organism has the same number
and kinds of cells immediately after death as it had as a living
organism; placement of the cells is similar; form is little changed;
and most of the bioconstituents are present in concentrations char
acteristic of life. Only the reactions are different; the energy equi
libria have shifted. Life must ultimately be defined only as it can
be understood, in biochemical and biophysical concepts. The detec-
3 Biological Isolation as Basis of Germfree Life
tion of unwanted living organisms must ultimately be based upon
life as a process characterized by acceptable morphologic, chem
ical, and physiological criteria. The importance of this view to
the theory of germfree research will become apparent in the testing
for contaminants. This view suggests that the ultimate test for living
organisms cannot rest entirely upon the presence of cells or other
morphologic characteristics, but it must include the biochemical and
thermodynamic characteristics of life.
Of the many possible approaches to define life, a descriptive defi
nition in physical and chemical concepts might be the most useful for
the detection of unwanted living forms which may or may not be
known forms. Life is the consummate activity of specially organized
systems (usually cellular organisms) in which material is processed
and energy is continually expended for: (1) synthesis of more
system(s) through growth and reproduction; (2) increased assurance
of survival by variation via adaptation and mutation; and (3) main
tenance of the effectiveness and internal integrity of the system(s) to
prevent disability, disorder, and death. The advantage of using such a
definition is that it will not only fit the classical living systems for
which tests have been routine, but it also asks for new and improved
methods for examination and suggests that there are broader views
of life than is usually seen in laboratory testing. For example, the
synthesis of more system(s), or the increase of total mass through
growth and reproduction, includes the nutritive requirements and the
"physiology" of microorganisms usually considered in differentiation
between one species and another. Respiration and metabolism are
needed for each item considered, but they are more easily examined
by activities associated with the first point. The second point empha
sizes that growth and reproduction alone are not enough for a general
criterion of life; mutation is a means for variation on a grand scale
and adaptation allows for variation by the individual. Both are essen
tial for the continuity and survival of life. The third point, the con
tinuous victory of life over randomness, could be explored as a means
for detecting life in an unknown area, or for finding new systems
which actually are living according to the general definition but would
not otherwise be recognized as living. Captured matter and energy
are incorporated into the metastable complex required for the equi
librium and further development of delicate catalytic and coupled
reactions without net energy loss to increased randomness. It is sug
gested that cybernetic mechanisms of life will differ in complexity if
