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Microbes and Infection 5 (2003) 553560

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Review: on the shoulders of giants

Louis Pasteur (18221895)


Guy Bordenave 1
Unit dimmunophysiologie et Parasitisme Intracellulaire, Institut Pasteur, 25, rue du Dr.-Roux, 75724 Paris cedex 15, France

Abstract
In Louis Pasteurs scientific career it is striking to note the exponential character of the research he introduced in all the fields he opened up.
He offered fabulous opportunities to stereochemistry. He is acknowledged as one of the founders of microbiology. He established the
possibility of anaerobic life. He pointed the way to epidemiology, public health, and the bacteriologic fight. He struggled against the idea of
spontaneous generation of life. He irrevocably substantiated the microbial theory of infectious diseases. He demonstrated that bacterial
virulence could be attenuated, he evidenced immunity and generalised the vaccination principle. He also was an incomparable experimenter.
2003 ditions scientifiques et mdicales Elsevier SAS. All rights reserved.
Keywords: Molecular dissymmetry; Microbiology; Aerobic and anaerobic lives; Rebuttal of spontaneous generation of life; Immunity; Vaccines; Microbes and
infectious diseases; History of science

1. Introduction
What more can be said about this exceptional man who
has already had so much written about him2 [1]? It is true that
every generation has a duty to remember the immense contribution made by some of our predecessors. Pasteurs patriotism, for example, has been widely discussed and evidence of
the influence of his fathera former soldier of the First
Empire who later became a tanner craftsmanis sought in
vain within this patriotism. His mother appeared to have a
heightened sense of the relativity of human concerns as,
shortly before her death in 1848, she wrote the following
words to him: whatever happens, never let yourself get
down. Little is of great importance in life [2]. The human
species is made in such a way that it is easily fascinated by
everything that relates to the major work of so-called eminent
people. We can therefore ask ourselves if, in this case, it was
the conservatism origins of that characterised Pasteur and,
under Napoleon III, inspired him to talk about the exaltation
created by a great reign, while Victor Hugo was himself in
exile. However, it is difficult to assess whether such reasoning is really worthwhile. The most sensible approach would
E-mail address: gmilon@pasteur.fr (G. Bordenave).
1
G. Bordenave is a retired Institute Pasteur scientist. For correspondence
and reprints please contact G. Milon at the given address.
2

Pasteurs correspondence and other documents: Annick Perrot, Conservateur, Muse Pasteur, 25-28, rue du docteur Roux, 75524 Paris cedex 15;
E-mail: aperrot@pasteur.fr.
2003 ditions scientifiques et mdicales Elsevier SAS. All rights reserved.
DOI: 1 0 . 1 0 1 6 / S 1 2 8 6 - 4 5 7 9 ( 0 3 ) 0 0 0 7 5 - 3

probably be to try and limit ourselves to an impersonal


description of his scientific work. But is this really possible
given that he was such an exceptional being? How can we
possibly define a scientific career in relation to such work? In
any case, this account could only ever approach such a
subject with great humility.
Dole, a small sub-prefecture in the Jura region, can be
proud, and rightfully so, of having witnessed, in 1822, the
birth of the one who, indisputably, belongs to that category
of children whose genius immortalises the memory of
civilisations.

2. Molecular dissymmetry
It was in 1844, at the age of 22, while he was studying
chemistry at the Ecole Normale Suprieure in Paris, that
Pasteur made one of his first, key discoveries. It was already
known that quartz crystals were able to deviate polarised
light either to the right or to the left. This property was
directly linked to their crystalline configuration: some facets
of the crystalsthe hemihedral facetsare inclined in relation to the edges which support them, sometimes in one
direction and sometimes in the other. Biot, an expert in
crystallography and optical phenomena, noticed that tartaric
acid deviated polarised light both in its liquid state and
crystalline form. Two crystalline forms of tartaric acid were
identified: tartaric acid and paratartaric acid. Mitscherlich,
another reputable chemist, had shown that these two forms as

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G. Bordenave / Microbes and Infection 5 (2003) 553560

Today, we are more aware of the importance, in pharmacology for example, and even in perfumery, of the two enantiomer forms (the mirror image of each other) of a same
substanceone can be active, whereas the other remains
inactive [1,4].
3. Fermentations and microbiology

Fig. 1. Facets of the double sodium ammonium paratartrate crystals: some


are inclined to the right and others to the left. Photo courtesy of Institut
Pasteur.

well as those of their respective saltstartrates and


paratartrateswere identical in all respects except in their
ability to deviate polarised light. The tartrates deviated light
to the right while the paratartrates remained inactive. Pasteur
noticed small facets similar to those of quartz crystals on the
tartaric acid and diverse tartrate crystals (Fig. 1). He also
noticed that all the facets were orientated to the right in the
tartrate crystals, while in the double sodium ammonium
paratartrate crystals, as in those studied by Mitscherlich,
some were inclined to the right and others to the left. He
divided up the two categories of crystals with tweezers and
used them to make separate solutions. Both solutions deviated polarised lightone to the right and the other to the left.
Mixed in equal quantities, they formed an optically inactive
solution. The concept of molecular dissymmetry was born
from the interpretation of this observation. The age of stereochemistry was beginning. In comparison to Biots often repeated exclamation: My dear child, I loved science so much
during my life it makes my heart flutter (Biot who imposed
draconian measures on the experiment), Pasteurs reaction is
moving in other ways. So violent was the shock, that he
rushed out of the laboratory and, when he came across a
chemistry technician, he grabbed him and cried out: Ive
just made an important discovery... Im so happy Im trembling all over, I cant even look through the polarimeter.
What an extraordinary moment of elation! What researcher,
even when his find is more modest, has not experienced such
emotion? This is a joy dominating over all others, an inexpressible and absolute joy! Pasteur was truly won over and
this impetuous whirlwind was going to take over his whole
life.
Pasteurs choice of science was significant, as during his
adolescence he also showed an undeniable talent for painting, proof of which is shown in the pastel pictures he left [3].
Pasteur was convinced that molecular dissymmetry was one
of the most fundamental characteristics of living organisms.

After being a professor in Dijon and then Strasbourg,


another challenge awaited Pasteur in Lille. Here, he was
drawn to study the problems that industrialists in the region
were having with their sugar factories or breweries. He was
faced with the problem of fermentation and, from this confrontation a new discipline would be born: microbiology. The
scientists who prevailed in this field at the time were Berzlius, Liebig, Helmholtz and Berthelot. Using all the influence
that their wide fame gave them, they fought against what was
known as the vitalist theory, i.e. the involvement of living
organisms in fermentations. They attributed this involvement
to chemical agents, which they referred to as ferments and
which they considered independent of the vital processes.
Pasteur studied ethanolic, lactic, butyric and acetic fermentations. He began by explaining lactic fermentation because, of all fermentations, it is chemically the most simple.
On fission of the glucose molecule, lactic fermentation only
consists of two lactic acid molecules. He isolated the lactic
bacillus from the greyish deposit which accompanies the
souring of milk and he cultivated it in a clear, nutrient broth.
Once a sufficient quantity had been produced, the population
of microscopic living organisms (which were all alike) ensured that lactic acid was formed from glucose.
As for ethanolic fermentation, he began by showing that
glucose was not only transformed into carbonic gas and
ethanolsomething that had been accepted since
Lavoisierbut that other products were also found as a result
of this fermentation such as glycerine, succinic acid and amyl
alcohol. Above all, he showed that the yeast, which was very
different from the bacillus involved in lactic fermentation,
induced ethanolic fermentation in a liquid which only contained glucose and mineral salts. There was total parallelism
between fermentation and yeast multiplication.
The agent responsible for butyric fermentation appeared
in the form of small rods, which were capable of undulatory
vibrations and could therefore move. Whilst observing a drop
of liquid undergoing butyric fermentation between a slide
and a coverglass, he noticed that the bacilli at the edge of the
coverglass were losing their mobility while those in the
middle retained it. This situation was the reverse of what he
was used to recording with other microorganisms, as they did
the opposite and gathered on the edges where there was more
oxygen. He was convinced that life without oxygen was
possible as butyric fermentation could be stopped by blowing
air into the liquid where this fermentation was occurring. He
came up with the terms aerobic and anaerobic to refer,
respectively, to life in the presence of oxygen and life without
it. Pasteur still examined acetic fermentation and here he also

G. Bordenave / Microbes and Infection 5 (2003) 553560

introduced the vitalist theory even though it was known that


vinegar was the result of a simple chemical reaction: the
oxidation of ethanol in acetic acid. He demonstrated that the
transformation of wine into vinegar depended on the activity
of a bacterium whose population formed a thin layer, which
floated on the surface of liquids. It is this bacterium which
incorporates oxygen from the air into the ethanol [1,5].
In 1897, 2 years after Pasteurs death, Bchner managed
to extract a soluble fraction from yeast, which was able to
trigger ethanolic fermentation in the absence of living cells.
It seemed that the catalytic theory of Liebig and Berzlius,
etc. was basically correct. The two great trends of scientific
thought during this periodthe physiological and chemical
theories of fermentation and metabolismwere going to be
reconciled. Microscopic living organisms produce
fermentsenzymes, which act either inside or outside the
microbial cell. Enzymes can carry out chemical transformations in the absence of the living organisms which produced
them but, under ordinary conditions, these transformations
give the living organisms energy and basic elements which
are essential to their metabolism. Pasteur had never ruled out
this possibility as, for example, in 1878 he wrote I should
not be surprised to see the yeast cells produce a soluble,
alcoholic ferment... soluble ferments have only yet been
produced by a vital function. In any case, the products of
ethanolic, lactic, butyric and acetic fermentations were
linked to bacterial life.
4. Rebuttal of the idea of the spontaneous generation
of life
Having studied microbial life, Pasteur found himself
drawn into the controversy surrounding the idea of the spontaneous generation of life. He joined this debate on the origin
of life in 1859, the year Darwins The Origin of Species
was published. Here, he fully demonstrated his talent as an
exceptional bench scientist. From the concept of specificity,
born out of the study of fermentations, was derived that of
hereditary characters which, in turn, depended on normal
generations. He naturally admitted that it was possible that,
somewhere in the universe, life could be reproduced but it
was necessary to check the assertions of those who claimed
to have witnessed the birth of life. In 1859, Pouchet, Director
of the Natural History Museum in Rouen, claimed that he
could bring about spontaneous generation whenever he
wished. His demonstration consisted of taking a flask of
boiling water, sealing it tightly and then plunging it upside
down into a bath of mercury. Once the water had cooled
down, the flask was opened in the mercury and half a litre of
pre-sterilised oxygen as well as a little hay infusion were
introduced. A microbial population regularly developed after
a few days. Pasteur demonstrated that by eliminating the
mercury from the experiment, he avoided contaminating the
liquids and the air. He then conducted those famous experiments in his swan-neck flasks (Fig. 2). Having introduced a
culture broth into a flask, he heated the neck in a flame and

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Fig. 2. Louis Pasteur observing a culture broth sterilised in straight and


swan-neck flasks. Painting Robert Thom. Photo courtesy of Institut Pasteur.

then bent it into the shape of an S (hence the name swanneck). Vapour from the boiling broth expelled the air via the
opening in the s-shaped tube. The flask cooled down and the
outside air entered slowly, without having been heated or
filtered, but having been sterilised by the humidity of the tube
which retained the germs which were unable to get past the
bend in the swan-neck. As the s-shaped tube remained open,
the air on the inside and outside of the flask communicated
freely but the broth remained sterile indefinitely. The lack of
air was therefore not responsible for the fact that life did not
appear. Moreover, the broth retained the elements that were
necessary for the development of life as, when the neck of the
flask was broken, microbial life appeared quickly.
It was with straight-neck flasks that Pasteur discovered the
uneven distribution of microorganisms in the atmosphere.
Flasks containing a nutrient broth underwent sterilisation by
boiling, which also helped to expel the air due to the stream
of water vapour. The flasks, which were closed using a flame
while the water vapour was still coming out, were almost
empty of air, and the nutrient broth remained sterile as long
as the flasks were sealed. Pasteur opened these flasks in many
different places and took extreme care not to contaminate
them. By breaking the neck of the flasks, air from the outside
was allowed in. The flasks were then re-sealed and put into an
incubator. Some of these flasks remained sterile while in
others, microbial life appeared. The evidence of the uneven
distribution of microorganisms in the atmosphere was proven
when it was noticed that the number of flasks with a contaminated content was higher at low altitudes, near inhabited
areas and cultivated lands than in the high mountains or
places where the air remained still for a long time. This also
proved, once again, that non-heated air could only trigger
microbial growth if it contained living germs likely to culture
the nutrient broth [1,5]. These demonstrations led to aseptic
manipulation, sterilisation and autoclaving techniques, and
the Englishman, Tyndall, played a key role in founding them.
It was at this time that scientists also became aware that if air
contained microorganisms, then water and solids must also
contain them and maybe even greater numbers of them.

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G. Bordenave / Microbes and Infection 5 (2003) 553560

After the death of Claude Bernard, a renowned physiologist, notes were published relating to some experiments that
he conducted on the fermentation of grapes. In particular, he
stated that, contrary to Pasteurs opinion, fermentation was
possible independently of living processes and he seemed to
implicitly suggest that yeast could be the consequence of
fermentation rather than its cause. Pasteur detected there a
disguised resurgence of the idea of the spontaneous generation of life and he devised, by way of response, a fabulous
experiment. He owned a small vineyard in the district of
Arbois. Using glass, he isolated a section of the vines at a
time when the yeast germs were not yet deposited on the
grapes. It was impossible to obtain ethanolic fermentation
with ripe raisins from the protected bunches, but the opposite
was true of the ripe raisins from the exposed bunches. If the
protected bunches were placed on vine-stocks left in the open
air, fermentation was easily obtained with their ripe grapes.
Nothing had been discovered about the conditions in
which life came about. All that was shown was that microbial
life did not occur in a properly sterilised nutrient medium,
which was protected from outside contamination. Furthermore here is what Pasteur wrote on this subject: Ive
searched for spontaneous generation in vain for twenty years.
No, I dont think its impossible but what leads you to believe
that it is the origin of life?You put matter before life so matter
has therefore existed since the beginning. How do you know
that the incessant progress of science will not force scientists
in a century, a thousand or two thousand years time... to
assert that life, and not matter, has existed since the beginning. You go from matter to life because your current knowledge... does not allow you to understand things differently.
Who can assure me that in ten thousand years time we will
believe it impossible not to go from life to matter...?[6].

5. Wine, beer and silk worm diseases


It is Pasteur who rightly received the credit for having
discovered that ethanolic fermentation diseases were caused
by microorganisms which entered into competition with
yeast. He went to great lengths to prevent microorganisms
from developing in finished products, i.e. after the involvement of yeast. He tried several antiseptics and then used heat
as a sterilisation agent. Very wisely, he knew how to take
advantage of the vulnerability of microorganisms by combining the slight acidity of wine with a moderate rise in temperature (55 C), over a short period of time (and better still
without oxygen), to obtain partial, but generally sufficient,
sterilisation. This principle would later be applied to a very
large number of perishable liquid foodstuffs (wine, beer,
vinegar, milk, etc.) or solids, and would lead to what is now
known throughout the world as pasteurisation. His work on
the role of microorganisms in fermentations and then in wine
and beer diseases, would gradually lead him to the microbial
theory of infectious diseases and give this remarkable unity
and outstanding direction to his scientific career [1,7].

Fig. 3. Silk worm on mulberry-tree leaves. Drawing by Lackerbauer, published in Maladie des vers soie, Paris, 1870. Photo courtesy of Institut
Pasteur.

It was then that he returned to Paris, to the cole Normale


Suprieure, and was called upon by Dumas, one of his former
masters/matres to study the silkworm disease which was
causing devastation in the sericulture industry in the south of
France (Fig. 3). When suffering from the disease, the worms
weaving the cocoons from which the silk was extracted
stopped growing at very different stages. They showed small
marks, which resembled specks of pepper on their
skinhence the name pebrine, which was given to the
disease. Pasteur noticed that only butterflies from sick worms
showed the characteristic corpuscles, which were easily detected under the microscope. He set up a method of selection
based on this observation to sort eggs from healthy butterflies, which could be kept for reproduction, and eggs produced by affected butterflies that had to be destroyed. The
healthy eggs made healthy worms, which wove cocoons with
healthy chrysalises. Today, it is known that pebrine is caused
by a protozoan parasite. At the time it appeared that the
problem was more complicated than first thought because at
least two simultaneous diseases were present: pebrine and
flacherie. Flacherie was only really identified when it persisted on its ownpebrine having been eliminated from the
breeding of healthy worms. The general view today is that
flacherie is above all caused by opportunist infections which
accompany a benign viral attack. These varied infections
resulted in diverse and disturbing forms of the disease. Pasteur acquired a certain understanding of flacherie and devel-

G. Bordenave / Microbes and Infection 5 (2003) 553560

oped appropriate techniques for keeping it under control.


One of his recommendations was not to retain eggs from
worms which appeared languid during the stage preceding
the weaving of the cocoon or from those which died in large
numbers at this time. It was a contagious disease problem and
it was necessary to keep healthy worms in non-infected
premises using non-infected material. From this approach
emerged the principleelementary in infectious diseases but
not yet implementedof separating the healthy from the
affected [1,8]. (This principle was applied to humans by
Emile Roux and his colleagues for the first time ever at the
Pasteur Hospital). Pasteur learnt much from this work and
was now in a better position to tackle the problem of infectious diseases in superior vertebrates.
The period when the laboratory moved to the Cevennes
has been described as a probably happy time in Pasteurs life,
although it is easy to imagine that the word happiness, in
the most general sense of the term, did not feature frequently
in the vocabulary of a man who was so intellectually and
morally demanding. He called on his whole family to help
with his silk worm research. To people who clung to traditional practices and who protested at the difficulty of new
methods suggested, Pasteur would reply that he knew of a
child (his daughter Marie-Louise, then aged ten) who excelled in the art of telling healthy butterflies from sick ones.
This period spent in the countryside must have made a
wonderful holiday for this young town girl! His wife, Marie,
was also mentioned. She took notes and wrote up the reports
of his experiments, which he dictated. She inquired into
problems, followed the progress attained and also probably
made suggestions, because in this field and in such an exciting atmosphere, it is difficult to remain passive or indifferent.
The image of the two of them in the calm surroundings of this
provincial environment (rather than in a relationship that
revolved around domination of one by the other as has been
too often suggested) paints a peaceful picture. It is satisfying
to imagine the hours of deep communion and the oases of
serenity and intimacy known to this household where science
seemed to take centre stage.
6. Chicken cholera, sheep anthrax, swine erysipelas
and the birth of immunity
In 1879, Pasteur succeeded in cultivating the bacillus
involved in chicken cholera and showed how the disease
could be triggered by injecting a pure suspension of this
bacillus. With this material, Pasteur demonstrated that, unlike chickens and rabbits, adult guinea pigs resisted the infection, developing only a local abscess, which was nevertheless
the local outbreak site of the same bacilli, whose full virulence subsisted in chickens and rabbits. This concept of a
healthy host opened the way for investigation into the wider
problem of epidemiology. It is said that the fundamental
observation was discovered by chanceif this was not so it
must be said that Pasteurs foresight was all the more
astonishingbut, whatever the case, the main point was that

557

the issue could no longer be avoided. Pasteurs observations


highlighted immunity and led to a general understanding of
the vaccination principles. His research was interrupted for a
while and the suspensions containing the chicken cholera
bacillus aged in the laboratory. When he resumed his research, it became apparent that the chickens injected with the
bacillus taken from these old suspensions not only survived,
but went on to resist a second injection of a fresh suspension
of the virulent bacilli which resulted in the rapid death of
untreated chickens. Pasteur understood that the chickens had
been immunised and that the bacilli responsible for causing
the disease could be attenuated. Attenuation was achieved by
exposing the culture to the air for some time periods (and at a
suitable temperature), as the same culture, which was attenuated when exposed to the air, retained all its virulence when
enclosed in an airtight container. Virulence was therefore not
a permanent characteristic but rather an unstable property
that could be lost and even regained without affecting any of
the other bacillus characteristics. Pasteur established a connection between this observation and the one made at the end
of the 18th century by Jenner, an English country doctor, who
succeeded in protecting humans against smallpox by inoculating them with the substance contained in cowpox pustules. Cowpox or vaccinia was a similar disease found in
cows but which was harmless in humans. At that point, it was
the only application known to man. In order to combine the
two discoveries, the name vaccination was given to the
phenomenon brought up to date by the chicken cholera experiments. Pasteur then became aware of the existence of a
general rule, which he had already suspected: the possibility
to make an animal less susceptible to the virulent form of a
microorganism by putting it in contact with the attenuated
form. This research would now have an impact on the wider
issue of attenuating virulence in germs.
While Pasteur was studying the problem of sheep anthrax,
the disease was causing devastation on sheep, goat and cow
farms. Koch, a young German doctor, had already won recognition through his work on the sheep anthrax bacillus. He
proved, among other things, that the anthrax bacillus actually
caused the disease and he also ascertained the complete life
cycle of this bacillus: immobile rods which develop into long
filaments and then into round granulesthe spores (or the
resistant form) which, in the right conditions, changed back
into rods. It was at the beginning of his research into the
anthrax bacillus that Pasteur discovered large amounts of
another bacillus in the tissues of some of the animals infected
by the disease. He named this second bacillus (which is rare
in blood) septic vibrio. He showed that it was a strict
anaerobic bacteria, which was responsible for causing both
abnormal death and erratic results in animals which had
purposely been contaminated with anthrax, using the blood
from animals that had died from the disease. The vibrio
septic bacteria killed the animals before the anthrax bacillus
had a chance to grow. Pasteur also used the anthrax bacillus
to prove that the presence of a pathogen in an organism did
not necessarily mean that the disease was manifested itself,

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G. Bordenave / Microbes and Infection 5 (2003) 553560

and that the environment could be crucial in influencing its


development. Although chickens appeared to resist anthrax,
when their usual temperature of 42 C was lowered to 38 C,
they could be made almost as susceptible to the disease as
rabbits or guinea pigs. The anthrax bacillus turned out to be
extremely sensitive to temperatures of around 45 C, which
explains why it did not multiply or did not multiply well in
chickens. If a chicken, which had been purposely infected
with anthrax by artificially lowering its temperature, then had
its usual temperature raised back to 42 C, it was restored
back to health. Pasteur also concluded that anthrax bacillus
spores could survive over long periods of time in the earth
around where the animal that had died from the disease was
buried, and that worms in this earth were involved in bringing
the spores back up to the surface. He injected earth taken
from the worms intestines into guinea pigs, infecting them
with anthrax. Pasteur pointed out that stubble left behind
after harvest could injure the animal and thus provide the
infection with a means of entry. This research into the etiology of anthrax has been widely acknowledged as exemplary
work. The main incentive behind it was, of course, to develop
a vaccine. However, the task turned out to be more difficult
than for chicken cholera. At the most usual temperatures, the
bacillus is present in its rod form (i.e. sensitive to virulence
attenuation) but the spore form of the bacillus (i.e. resistant to
attenuation process) is also detected. The problem was how
to keep the bacillus alive while at the same time preventing
the spores from developing. After much trial and error, the
objective was achieved by storing the cultures at 42-43 C
inside shallow containers with oxygen present. Eight days in
these conditions resulted in producing bacilli that were harmless to guinea-pigs, rabbits and sheep. Before reaching this
final state, the bacilli went through various stages of attenuation in which they could be maintained. Pasteur discovered
that it was best to vaccinate against anthrax in two steps: an
initial inoculation using bacilli from a culture with very weak
virulence followed by a second inoculation, 12 d later, using
bacilli from a more virulent culture. This process protected
the guinea-pigs, rabbits and sheep against the most virulent
form of the bacillus. This led to a public experimenta
memorable event that took place at Pouilly-le-Fort in the
Spring of 1881, in the presence of a crowd of journalists and
inquisitive on-lookers. The challenge was to vaccinate 24
sheep, one goat and six cows and then to inject them with the
virulent strain of the anthrax bacillus. At the same time, a
similar group of unvaccinated, control animals was also injected with the same anthrax bacillus strain. The experiment
was a complete successonly the vaccinated animals survived.
Evidently, critics protested at the time against the principle of vaccination. Some of these criticisms came from
Koch, the famous German microbiologist. He criticised Pasteur, who did not use his solid culture medium method, for
working with impure bacillus cultures. Koch remained unfamiliar with this emerging new disciplineimmunity
perhaps because he had apparently failed in his own attempt

to protect against the tuberculosis bacillus. He remained


relatively sceptical to the possibility of attenuating pathogen
virulence, even though his own work on the anthrax bacillus
and his important discoveries (the tuberculosis bacillus and
the human cholera vibrio) contributed a great deal to establishing the microbial theory of infectious diseases. Most of
the ill feeling between Koch and Pasteur stemmed from the
extreme sensitivity of scientists to anything related to their
own personal work, to say nothing of the severe FrancoGerman bitterness that existed at the time. It would appear
that a scientists sensitivity could be as acute as his contribution to progress was crucial.
The German school of medical bacteriology attracted considerable international interest. It discovered various bacteriological agents of infectious diseases. It had an advantage
over the French school becauseso it is claimedit was
larger and better organised. However, through its research
and particularly through Pasteurs contribution, the French
school can be credited with the discovery of streptococcus
(1879) (which was causing damage in maternity wards under
the name of puerperal fever), staphylococcus (1878), pneumococcus (1881) and septic vibrio (1877). This is what Roux
said on the subject: A small, round organism, made up of
clusters, that cultivates easily in nutrient broth, can be found
in the pus of warm abscesses and furuncles. It can also be
found in infectious osteomyelitis in children. Pasteur affirmed that osteomyelitis and furuncle are two forms of the
same disease and that osteomyelitis was really a furuncle of
the bone. This assertion caused much amusement among
surgeons in 1878. Pasteur never gave botanical names to the
microbes he discovered, but named them according to their
shape or culture. Thus, for him, the furuncle microbe was the
grain cluster microbe and the puerperal fever microbe was
the grain chaplet microbe. These same discoveries are
better known in the world of bacteriology as Staphylococcus
and Streptococcus pyogenes [9]. And they also enabled
certain people to claim them as their own discoveries. The
discovery of the plague bacillus by the pasteurien, Yersin,
could also be added to this never-ending list.
As far as research into erysipelas or swine fever was
concerned, the problem was of a different nature. If the swine
fever bacillus is injected from rabbit to rabbit, the bacillus
acclimatises to the rabbit. All the rabbits end up dying after a
few days, whereas initially the rabbits always got sick but did
not die with the same frequency. If pigs are then injected with
the blood from this group of rabbits, the virulence for pigs
gradually decreases over several successive rabbit inoculations and this injection no longer causes the pigs to die but
just makes them sick. Once they recover from the disease, the
pigs are immunised.
This must have been enormously encouraging for everyone who worked alongside Pasteur in this amazing development, i.e. the application of essential principles: the use of
attenuated, living bacilli in relation to a recognition system
that was gradually being discovered and which ensured that

G. Bordenave / Microbes and Infection 5 (2003) 553560

559

the superior organism being studied was protected in some


way. Two fundamental approaches, centred around a concern
for public health, immediately set the Pasteur school apart
from other bacteriology schoolsfirstly, research into microbial agents that cause infectious diseases and secondly,
using this research (with the aim of attenuating virulence as
soon as the disease agent is exposed) to prepare a vaccine for
prophylactic use and afterwards, a serum for use as a cure. As
far as virulence attenuation procedures for microorganisms
were concerned, initial attempts dramatically showed how
diverse they could be, as no less than three different attenuation methods were designed under Pasteurs supervision for
the first three bacterial vaccines. One of the main reservations
at the time was based on the fact that people remained
reluctant to the idea that these tiny and apparently insignificant beingsbacillicould have such a devastating effect
on superior organisms [1,10].
7. Human vaccination against rabies
Without a doubt, these experiments required an enormous
amount of intellectual courage, of courage in every sense of
the word. There has been much debate about the decision to
carry out research into rabies. Clearly it was Pasteurs choice.
We can assume that the determining factor was the fact that
rabies was a disease common to humans and animals and that
it was possible to experiment on animals. The least that can
be said is that the experiments involved several rather startling activities. Firstly, they involved a virus that could not be
detected under an optical microscope. Secondly, the disease
had a long incubation periodover a month from the moment of contamination to the appearance of the first symptoms. This period was used to try and obtain resistance by
vaccination, even after the infectious bite. The first positive
result was the discovery that the virulence was based in the
nervous system and that the best inoculation route was via
dura mater after trephination. By regularly transferring contaminated spinal cord via this route, from rabbit to rabbit (a
process requiring around fifty passages), a fixed virus was
obtained which had a regular incubation period of 7 d. The
second positive result was that viral virulence could be attenuated and extinguished by exposing the spinal cord from
contaminated rabbits to dry, sterile air (Fig. 4). It was not
denied that this could have been due to a reduced amount of
rabies virus rather than a reduction in virulence as, unlike
bacteria, it was impossible to quantify the virus.
It is easy to imagine the moral dilemma that Pasteur must
have faced when 9-year-old Meister came to him on 6th July
1885 from an Alsatian village where he had been bitten on
the 4th July. Imagine what a request for help from Alsace
must have felt like in France in 1885. The child was suffering
from 14 wounds and his death appeared unavoidable. The
decision was made to apply the treatment that had been
continually successful in dogs. Roux was working on the
same problem and we can credit him for his idea of gradually
drying out rabbit spinal cord in air inside sterile flasks.

Fig. 4. A painting by Edelfelt showing Louis Pasteur inspecting a rabbit


spinal cord drying out in a sterile flask. Photo courtesy of Institut Pasteur.

Pasteur adapted this idea by adding potassium particles to


speed up the dehydration process. Roux believed that the
animal experiments had not been perfected enough for use on
humans. He left the laboratory to mark his disapproval and
pointedly got back to his seat when the diatribe against
Pasteur reached its paroxysm. The method involved injecting
subcutaneously suspensions of spinal cord via the peritoneal
region. First non-virulent spinal cord was used, then more
and more recent spinal cord until finally an extremely virulent spinal cord was administered. The treatment began on
6th July 1885 and continued until the 16th. This is Pasteurs
brief account: For each of the different spinal cords used, we
also inoculated two new rabbits by trephination in order to
monitor the virulence of the spinal cords.... Spinal cords
dated from the 6th, 7th, 8th, 9th and 10th July were not
virulent as they did not cause rabies in the rabbits. All the
spinal cords from 11th, 12th, 13th, 14th, 15th and 16th July
were virulent and the virulent matter became progressively
stronger and rabies broke out in the rabbits.... Joseph Meister
therefore avoided not only the rabies that his bite wounds
may have otherwise caused but also the rabies that I inoculated him with..., a more virulent form of rabies than the kind
found in stray dogs... [1,10]. This description, which remained true for all subsequent individuals treated for the
disease, meant that there was no basis for the allegations
accusing the treatment of being ineffective. Strangely
enough, however, the same allegations still exist today.

560

G. Bordenave / Microbes and Infection 5 (2003) 553560

8. By way of conclusion
What more can be said about this scientific career when all
attempts at describing it are feeble in comparison. The most
striking element is the exponential nature of every area of
Pasteurs research and the fact thatdue to a lack of time
owing to the choices madehe was not able to investigate
further, or perfect every area of his work. He opened up
fantastic opportunities for stereochemistry. He is recognised
as one of the founders of microbiology. He discovered that
life without oxygen is possible. His observation that chicken
cholera bacillus, deadly for this species of animal yet harmless in guinea-pigs (which could carry the bacillus without
becoming sick), provided a foundation for epidemiology.
Lister, the English surgeon, revered as the founder of antiseptic techniques, always remained grateful to Pasteur for his
intellectual contribution to medicine. Pasteur did not lay
down any specific rules concerning hygiene but we can
consider that he left it up to others to enforce such rules after
he had brought the problem to light. He noticed that microorganisms in soil could affect the anthrax bacillus to such an
extent that it no longer spread the disease. He realised that
this discovery could lead to therapeutic applications. He
played his part in the bacteriological struggle, offering, albeit
unsuccessfully, to provide the Australian Government,
whose territory was infested with rabbits, with the chicken
cholera bacillus as he knew that rabbits were sensitive to the
bacillus and that it could destroy them. He identified the
principle of immunity and appreciated that all animals were
indelibly marked when they came into contact with a bacillus. He also discovered that it was possible to attenuate the
virulence of such bacilli. Even if we only consider that he
showed medicine the microbial approach to infectious
diseasesan approach marked by rigorous experimentshe
would have been one of the very few who, during their
lifetime, must have noticed the enormous repercussions their
scientific work had on the improvement of the human condition. As well as being enormously generous in his contributions towards public health, Pasteur also gave rise to a long
tradition of scientists who were uninterested in money. We
have heard that he invested all the money from registered
patents for the pasteurisation of beer, vinegar and wine back
into the public domain, and he did not benefit personally
from any of the industrial tools developed either. After the
Pouilly-le-Fort experiment, he allocated to his laboratory the
earnings from sales of sheep anthrax vaccinations in France,
reserving only the proceeds from foreign markets for himself
and his closest partners. He must have felt intensely nostalgic
at not being able to travel to distant lands and study infectious
diseases peculiar to different regions and contribute to their
eradication. One of his major concerns was to send some of
the most eminent students from his school instead. In this
way Instituts Pasteur developed and prospered, and they still
form a unique network in the world today. Some of them
operate in the same way as the Parisian model, which Pasteur
founded and devised policies for.

His life is a constant example of relentless demands of


originality and quality, a permanent invitation to anxiety and
cross-examination, a glorification of willpower. Pasteurs
fascinating self-assurance encourages us to ask ourselves
what state of perfect equilibrium he must have reached and
whether or not he belonged to those Plato enthusiasts for
whom to discover means to remember. Better than anyone
else, he demonstrated the presence of that strange energy that
exhorts humans to surpass themselves and prevents them
from sinking into indifference. Finally, he wished to see
Bossuets philosophical principle engraved on the front wall
of all laboratories: the most unsettling thing for the spirit is
to believe in things because we want to believe in them ....
On his death at Villeneuve-lEtang, in September 1895, he
left his successors vast areas of research to investigateall
the different future prospects that he had laboured so hard at
creating.
It seems appropriate to apply to Pasteur those verses that
Malraux borrowed from Hugo for the epigraph of his book on
de Gaulle [11].
Oh! What a terrible sound from the dusk
Oak trees felled for Hercules stake!

Acknowledgements
This article was translated from the French by Victoria
Skewes. My warmest thanks go to Genevive Milon and
David Ojcius for their help and encouragement during the
writing of this article. I also wish to thank Chantal Brul,
Sylvie Delassus and the Publications Service of the Institut
Pasteur for their assistance.

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