| We need no reminder that
the foundations of our knowledge of health and disease were constructed
by scientific giants who worked decades, even centuries, ago. It is with
tributes such as the one today to Louis Pasteur that we pay homage to these
great minds -- to acknowledge their achievements and our indebtedness to
them which we can never repay.
With certainty, one hallmark
of Pasteur's research was not only the importance of his individual discoveries,
but the overwhelming breadth of his accomplishment. Pasteur's long time
collaborator, Emile Duclaux, wrote, "A mind ... of a scientific man is
a bird on the wing; we see it only when it alights or when it takes flight.
... We may by watching closely keep it in view, and point out just where
it touches the earth. But why does it alight here and not there? Why has
it taken this direction and not that in its flight toward new discoveries?"
Pasteur, himself, provided
us with an answer: He believed that his research was "enchained" to an
inescapable, forward moving logic. As we review today Pasteur's scientific
discoveries we shall see the truth of this statement: how one discovery,
one concept, led almost "inescapably" to another.
Education and
Growing Up
Pasteur was born in Ole
and grew up in the nearby town of Arbois, the only son of a poorly educated
tanner, Jean Pasteur. Louis was not an outstanding student during his years
of elementary education, preferring fishing and drawing to other subjects.
In fact, young Louis drawings suggested that he could easily have become
a superior portrait Artist. His later drawings of friends done at college
were so professional that Pasteur was listed in at least two compendia
of XIX C. artists.
The Senior Pasteur, however,
did not see his son ending up as an Artist, and Louis, himself, was showing
increasing interest in chemistry and other scientific subjects. The highest
wish Father Pasteur had for his son was that he complete his education
in the local schools and become a professor in the college at Arbois. However
the headmaster of the college recognized that Louis could do much better
and convinced father and son that Louis should try for the Ecole Normale
Sup rieure in Paris. This most prestigious French University was founded
specifically to train outstanding students for University careers in science
and letters. And it was here that Pasteur entered and began his long journey
of scientific discovery.
Crystallography
It may surprise some to
learn that Pasteur, the father of microbiology and immunology, was a chemist
who launched his memorable scientific career by studying the shapes of
organic crystals. Pasteur was 26 years old, working for his doctorate in
chemistry in the laboratory of Antoine Balard. Crystallography was just
emerging as a branch of chemistry. His project was to crystallize a number
of different compounds. Happily he started working with tartaric acid.
Crystals of this organic acid are present in large amounts in the sediments
of fermenting wine. Often one also found in the sediments in the wine barrels
crystals of a second acid called paratartaric acid or "racemic acid". A
few years earlier, the chemical compositions of these two acids, tartaric
and paratartaric, had been determined. They were identical. But in solution
there was a striking difference. Whereas tartaric acid rotated a beam of
polarized light passing through it to the right, paratartaric acid did
not rotate the light. This puzzled the young Pasteur. How could this be?
Pasteur refused to accept
the notion that two compounds that had the same chemical composition yet
acted so differently in respect to rotation of light could be identical.
He was convinced that the internal structure of the two compounds must
be different and this difference would show itself in the crystal form.
The experts in this field had looked examined tartrate and paratartrate
crystals but never saw a difference, perhaps because, as Duclaux thought,
they believed that no difference could exist. Pasteur believed that there
were differences and indeed found them!
Upon intense examination
beneath his microscope, he saw that every crystal of pure tartaric acid
looked like every other one. When he examined the paratartrate crystals,
on the other hand, he saw two types of crystals, nearly identical but not
quite! One type was the mirror image the other -- the way the right hand
mirrors the left hand. This was the difference he was looking for!
Pasteur then performed
one of the simplest and yet most elegant experiments in the annals of chemistry.
With a dissecting needle and his microscope, he separated the left and
right crystal shapes from each other to form two piles of crystals. He
then showed that in solution one form rotated light to the left, the other
to the right. This simple experiment proved that the organic molecules
with the same chemical composition can exist in space in unique stereospecific
forms. And with this work did Pasteur launch the new science of stereochemistry.
To Pasteur this discovery
had a deeper meaning. He proposed that asymmetrical molecules were indicative
of living processes. In the broadest sense, he was correct. We know today
that all of the proteins of higher animals are made up of only those amino
acids that exist in the left-hand form. The mirror image right-hand amino
acids are not used by human or animal cells. Likewise, our cells burn only
the right-handed form of sugar, not the left-handed form that can be made
in the test tube. It was the discovery of asymmetry of organic molecules
that provided Pasteur with the "inescapable forward moving logic" that
enchained him as he began his studies on alcoholic fermentation.
Alcoholic Fermentation
Pasteur served on the
faculty of science of Dijon for a brief period and then was transferred
to Strasbourg University where he continued his studies on molecular asymmetry.
In Strasbourg, Pasteur had the immense good fortune to meet and marry the
University Rector's daughter Marie Laurent, who was to be his devoted wife,
mother and scientific helpmate through the remainder of his life.
In 1854 Pasteur was appointed
Dean and professor of chemistry at the Faculty of Sciences in Lille, France.
Lille was an industrial town with a number of distilleries and factories.
The Minister of Public Instruction was not completely sold on "science
for science's sake". He reminded university faculty that (and here I quote
the Minister's words) "whilst keeping up with scientific theory, you should,
in order to produce useful and far reaching results, appropriate to yourselves
the special applications suitable to the real wants of the surrounding
country."
Pasteur, in contrast to
other faculty, needed no prodding. He enjoyed taking his students on tours
of the factories and was quick to advise the managers that he was available
to help solve their problems. In the summer of 1856, M. Bigot, father of
one of his students in chemistry, called upon Pasteur to help him overcome
difficulties he was having manufacturing alcohol by fermentation of beetroot.
Often, instead of alcohol, Bigot's fermentations yielded lactic acid.
To better appreciate the
discoveries to follow, we should understand what was believed at that time
about alcoholic fermentation. Chemistry was emerging as a true science,
freed from the pseudoscience of the alchemist. The mysterious chemical
processes of living animals were slowly being unraveled in strictly chemical
terms. Lavoisier had shown that chemical combustion in living animals was
quantitatively identical to that occurring in a furnace. Lavoisier also
showed that sugar, the starting product of fermentation, could be broken
down to alcohol, CO2 and H2O by simply dropping a sugar solution on heated
platinum. Woehler startled the scientific world by sythesizing the organic
compound urea, showing for the first time that organic compounds, believed
up to then as capable of synthesis only by living animals could be made
in a test tube. And due, in no small part to Pasteur's work on crystals,
internal structure and analysis of complex organic compounds was becoming
routine.
In this light, fermentation
leading to production of wine, beer and vinegar was believed to be a straightforward
chemical breakdown of sugar to the desired molecules. The chemical experts
of the day proclaimed that the breakdown of sugar into alcohol during fermentation
of sugar to wine and beer was due to the presence of inherent unstabilizing
vibrations. One could transfer these unstabilizing vibrations from a vat
of finished wine to new grape pressings to start fermentation anew.
Yeast cells were found
in the fermenting vats of wine, and were recognized as being live organisms,
but they were believed simply to be either a product of fermentation or
catalytic agents that provided useful ingredients for fermentation to proceed.
Those few biologists who earlier concluded that yeast was the cause of,
and not the product of, fermentation were ridiculed by the scientific experts:
The deep conviction of the scientific establishment was that chemistry
had come too far to allow a vitalistic life force theory to challenge pure
chemical explanations of molecular reaction. To attribute such chemical
changes to mysterious life forces would represent a major backward step
in science!.
Unfortunately, the "scientific
establishment" was not providing much help to the brewers of wine, beer
and vinegar. These manufacturers were plagued by serious economic problems
related to their fermentations. Yields of alcohol might suddenly fall off;
wine might unexpectedly grow ropy or sour or turn to vinegar; vinegar,
when desired, might not be formed and lactic acid might appear in its place;
the quality and taste of beer might unexpectedly change making quality
control a nightmare! All too often the producers would be forced to throw
out the resultant batches, start anew, and sadly have no better luck!
Into M. Bigot's factory,
microscope in hand, came Pasteur. He quickly found three clues that allowed
him to solve the puzzle of alcoholic fermentation. First, when alcohol
was produced normally, the yeast cells were plump and budding. But when
lactic acid would form instead of alcohol, small rod like microbes were
always mixed with the yeast cells. Second, analysis of the batches of alcohol
showed that amyl alcohol and other complex organic compounds were being
formed during the fermentation. This could not be explained by the simple
catalytic breakdown of sugar shown by Lavoisier. Some additional processes
must be involved. Third, and this may have been the critical clue to Pasteur,
some of these compounds rotated light, that is they were asymmetric. As
we said earlier, Pasteur suspected that only living cells produced asymmetrical
compounds. He concluded and was able to prove that living cells, the yeast,
were responsible for forming alcohol from sugar, and that contaminating
microorganisms turned the fermentations sour!
Over the next several
years Pasteur identified and isolated the specific microorganisms responsible
for normal and abnormal fermentations in production of wine, beer, vinegar.
He showed that if he heated wine, beer, milk to moderately high temperatures
for a few minutes, he could kill living microorganism and thereby sterilize
(pasteurize), the batches and prevent their degradation. If pure cultures
of microbes and yeasts were added to sterile mashes uniform, predictable
fermentations would follow.
Spontaneous Generation
In the midst of the great
excitement and controversy created by Pasteur's research on fermentation,
a debate was ongoing in the scientific world on the theory of "spontaneous
generation". The idea that beetles, eels, maggots and now microbes could
arise spontaneously' from putrefying matter was speculated on from Greek
and Roman times. And in the 1860's spontaneous generation was still a subject
of debate in the exalted French Academy of Sciences. Against the advice
of his colleagues, who saw dabbling in this field as thankless and unrewarding,
Pasteur entered the fray. Based on his work on fermentation it seemed obvious
to him that the sources of yeasts and other microorganisms that were found
during fermentation and putrefaction entered from the outside, for example,
on the dust of the air. Pasteur conducted a series of ingenious experiments
that destroyed every argument supporting "spontaneous generation". He showed
that the skin of grapes towards the beginning of grape harvest was the
source of the yeast. Drawing grape juice from under the skin with sterile
needles gave juice that would not ferment. Covering the grape arbors with
fine cloth or wrapping the grapes with cotton to keep off contaminating
dust, gave grapes that would not produce wine. In order to show that dust
of the air was the carrier of contamination, he allowed air collected at
different altitudes, from sea level to mountain tops, to enter sterilized
vessels containing fermentable solutions. The higher the altitude the less
the dust in the air and the fewer flasks showed growth.
The experimental design
that clinched the argument was the use of the swan-neck flask. In this
experiment, fermentable juice was placed in a flask and after sterilization
the neck was heated and drawn out as a thin tube taking a gentle downward
then upward arc -- resembling the neck of a swan. The end of neck was then
sealed. As long as it was sealed, the contents remained unchanged. If the
the flask was opened by nipping off the end of the neck, air entered but
dust was trapped on the wet walls of the neck. Under this condition, the
fluid would remain forever sterile, showing that air alone could not trigger
growth of microorganisms. If, however, the flask was tipped to allow the
sterile liquid to touch the contaminated walls and this liquid was then
returned to the broth, growth of microorganisms immediately began.
In the words of Pasteur
"Never will the doctrine of spontaneous generation recover from the mortal
blow of this simple experiment. No, there is now no circumstance known
in which it can be affirmed that microscopic beings came into the world
without germs, without parents similar to themselves."
Silk Worms
As if Pasteur was not
busy enough with his studies on fermentation and spontaneous generation,
hE was asked by the Department of Agriculture to head a commission to see
what could be learned about a devastating disease of silkworks that was
destroying the French silk industry. Even though Pasteur knew nothing of
silkworms and had no idea that they suffered from disease, his research
on silkworms forged another link in his "inevitable" chain of discovery.
Now there were at least
two different types of silkworm diseases that Pasteur came to grips with:
Pebrine, in which black spots and corpuscles are generally, but not always,
present on the worm. In such cases the worms often die within the cocoons
. In the second type of disease, flacherie, the worms exhibit no corpuscles
or spots but fail to spin cocoons. Pasteur suspected, but was not sure,
that pebrine corpuscles were associated with the failure of the worms.
Nonetheless, by examining the worms under the microscope he was able to
identify those free of pebrine and used only their eggs for breeding. Next
he excluded from breeding eggs from worms with flacherie whom he identified
by their sluggish behavior in climbing leaves when about to construct cocoons.
He instructed the silkworm farmers on these methods of selection and how
to use the microscope to detect sickness in the worms. Soon the silk industry
in France, Italy and other European countries returned to health.
Pasteur considered these
studies important landmarks in his investigations on infection and infectious
disease. As he expanded his research, he found that healthy worms became
infected when allowed to nest on leaves used by infected worms. He also
noted that the susceptibility of the worms varied widely, some worms dying
shortly after infection, some weeks later, some not at all. He determined
that temperature, humidity, ventilation, quality of the food, sanitation
and adequate separation of the broods of newly hatched worms each played
a role in susceptibility to the disease. So here from Pasteur's research
we see the emergence of his future concepts of the influence of environment
on contagion.
Germ Theory of
Disease
The crowning achievements
of Pasteur's career were development of the germ theory of disease and
the use of vaccines to prevent these diseases. Pasteur's studies on contamination
of wine and beer by airborne yeast clearly stimulated certain investigators
to recognize that these "diseases" were due to entry of foreign microorganisms.
Lister in England was so impressed by Pasteur's work that he began to systematically
sterilize his instruments, bandages and sprayed phenol solutions in his
operatories thus reducing infections following surgery to incredibly low
numbers.
By 1875 many physicians
recognized that some diseases were accompanied by specific microorganisms,
but the body of medical opinion was unwilling to concede that important
diseases --cholera, diptheria, scarlet fever, childbirth fever, syphilis,
smallpox - could ever be caused by these agents. To give you an idea of
the magnitude of the problem, according to Pasteur's biographer son-in-law
Vallery-Radot between April 1 and May 10, 1856, in the Paris Maternity
Hospital there were 64 fatalities due to childbirth fever out of 347 confinements.
The hospital was closed and the patients were transferred to a different
hospital. Sadly, the contagion followed these women and nearly all of them
died!
As Pasteur wandered through
hospital wards he became increasingly aware that infection was spread by
physicians and hospital attendants from sick to healthy patients. Pasteur
impressed on his physician colleagues that avoidance of microbes meant
avoidance of infection. In a famous speech before the august Academy of
Medicine in Paris he stated, "This water, this sponge, this lint with which
you wash or cover a wound, may deposit germs which have the power of multiplying
rapidly within the tissue....If I had the honor of being a surgeon....not
only would I use none but perfectly clean instruments, but I would clean
my hands with the greatest care...I would us only lint, bandages and sponges
previously exposed to a temperature of 1300 to 1500 degrees. Slowly, but
surely, through the preachings of Pasteur, Lister and other physicians
antiseptic medicine and surgery became the rule.
Anthrax
At this time, anthrax,
a fatal disease of sheep and cattle, was decimating the sheep industry
and the economy of France. Important strides in identifying the causative
agent of anthrax had been made by the time Pasteur entered the arena. The
great German physician/scientist Robert Koch, isolated the anthrax bacillus,
previously identified by the French physician Davain, from infected spleens
and showed that under resting conditions the bacillus formed long-lived
spores.
Definitive proof was still
lacking that the cultured bacillus, itself, and not something carried along
in Koch's culture medium was responsible to giving injected animals anthrax.
Pasteur provided this proof. As described by Dubos, Pasteur placed one
drop of blood from a sheep dying of anthrax into 50 ml of sterile culture,
grew up the bacterium, and then repeated this process 100 times. This represented
a huge dilution of the original culture so that not a single molecule of
the original culture remained in the final culture. Yet, the last culture
was as active as the first in producing anthrax. As only the bacillus,
itself, by growing up each time in the new culture, could escape dilution,
it proved beyond all doubt that the anthrax bacillus and nothing else could
be responsible for the disease. Thus was the germ theory of disease firmly
established!
But how did the disease
spread? Why was one field deadly to sheep, another harmless? Here Pasteur's
studies on silkworm contagion provided the clue. During one of Pasteur's
excursions to a field where sheep were grazing he noted that the ground
in one part of the field was differently colored than the rest. There it
was that the farmer had buried some sheep dead of anthrax. The color of
the soil was due to earth worm casts. He realized that earth worms were
feeding on the carcasses of the buried sheep and bringing the anthrax spores
to the surface where other sheep could graze on the contaminated soil.
Although containment of the animals on uncontaminated fields would help
control the spread of anthrax, more was needed.
Interestingly, Pasteur's
studies on chicken cholera going on at this time provided the breakthrough
that led to development of specific vaccines to fight disease. Cholera
was a serious problem for farmers. Chicken cholera would spread through
a barnyard rapidly and wipe out the entire flock in as little as 3 days.
Spread could be by contaminated food or animal excrements. Pasteur had
identified the cholera bacillus and was growing it in pure culture. When
injected, chicken invariably died in 48 hours.
Then luck intervened.
During the heat of the summer, Pasteur returned to Paris leaving the cholera
cultures used for infection stored on the shelves of the Arbois laboratory.
Upon return, Pasteur's collaborators were disappointed to find that these
stored cultures no longer killed injected chickens, nor even made them
sick. The group set to work to make new cultures of the bacillus and tested
these batches on new birds and those healthy previously treated birds.
The results were astonishing: The previously injected birds were unaffected
by the bacillus, while the new birds all died. When Pasteur saw these results
he immediately realized that in a sense he was repeating the studies of
Jenner 80 years earlier who had conferred on humans immunity to smallpox
by vaccinating individuals with a mild form of cowpox. Pasteur then reproducibly
manufactured attenuated cultures of chicken cholera vaccines and could
routinely prevent cholera in the vaccinated chickens.
If attenuated cholera
bacillus could render chickens resistant to the disease, would not an attenuated
anthrax bacillus render sheep immune to anthrax? By various techniques
involving oxidation and aging, anthrax vaccines indeed prevented anthrax
in laboratory trials. Pasteur's reports on preventing sheep anthrax were
so exciting to some and unbelievable to many, that he was challenged by
the well-known veterinarian Rossignol to conduct a carefully controlled
public test of his anthrax vaccine. This was to take place at Pouilly le
Fort, a farm in the town of Melun south of Paris. Twenty-five sheep were
to be controls, the other twenty-five were to be vaccinated by Pasteur
and then all animals would receive a lethal dose of anthrax. All of the
control sheep must die and the vaccinated sheep must live. When Pasteur's
colleagues learned that he had agreed to the test they were concerned.
The challenge was severe and there was no room for error. The vaccines
were still in the developmental stage. "What succeeded with 14 sheep in
our laboratory will succeed with 50 at Melun", said Pasteur.
The publicity was intense.
A reporter from the London Times sent back daily dispatches. Newspapers
in France followed the events with daily bulletins. There were crowds of
onlookers, farmers, engineers, veterinarians, physicians, scientists and
a carnival atmosphere. Would Pasteur's claims of vaccination hold up? Even
Pasteur was privately concerned that he had acted impetuously in accepting
the challenge. Happily, the trial was a complete success -- indeed, a triumph!
Two days after final inoculation (May 5, 1882), every one of 25 control
sheep was dead and every one of the 25 vaccinated sheep was alive and healthy.
The fame of Pasteur and these experiments spread throughout France, Europe
and beyond. It was, says Duclaux, "the anthrax vaccine that spread through
the public mind faith in the science of microbes". Within 10 years a total
of 3.5 M sheep and a half M cattle had been vaccinated with a mortality
of less than 1%. The immediate savings to the French economy were enormous,
at least 7 M francs, estimated to be enough to cover the reparations that
France was required to pay to Prussia for the loss of the Franco-Prussian
War in 1880.
Supported by the successes
with anthrax and fowl cholera diseases, Pasteur identified and isolated
over the next 2-3 years the microbes for many other diseases including
swine erysipelas, childbirth fever and pneumonia.
Rabies
The final and certainly
most famous success of Pasteur's research was the development of a vaccine
against rabies or hydrophobia as it is also known. The disease has always
had a hold on the public imagination and has been looked upon with horror.
It evokes visions of "raging victims, bound and howling, or asphyxiated
between two mattresses" (Duclaux). The treatments applied to victims were
as horrible as the supposed symptoms: this included cauterizing the bite
wounds with a red-hot poker. Actually very few persons die in any year
from being bitten by a rabid dog or wolf. The symptoms of the disease are
variable: onset may take weeks to months to develop if they develop at
all. Nonetheless, Pasteur and his colleague Roux realized that conquest
of rabies would be recognized as a great achievement to the world of science
and to the public at large.
Pasteur and Roux initially
attempted to transfer infection by injecting healthy dogs with saliva from
rabid animals. The results were variable and unpredictable. Later, recognizing
that the active agent was in the spinal cord and brain, and because they
were unable to detect a specific rabic microorganism, Pasteur and Roux
applied extracts of rabid spinal cord directly to the brain of dogs. With
this technique they could reproducibly produce rabies in the test animals
in a few days.
The goal was next to
develop a vaccine that would provide protection to the subject before the
rabic agent moved from the bite site to the spinal cord to the brain. This
was achieved by injecting into test animals suspensions of spinal cord
of rabid rabbits that were attenuated in strength by air drying over a
12-day period in the now-famous Roux Bottle. A strip of spinal cord was
suspended from a hanger in the center of the bottle containing a hole at
the top of the bottle and one on the lower side. Air entered from the bottom
opening, passed over a drying agent and exited from the top. The longer
the cord was dried, the less potent was the tissue in producing rabies.
The treatment plan used
to develop immunity to rabies was to inject under skin of a dog the least
potent preparation of minced spinal cord, followed every day for the next
12 days with a stronger and stronger extract. At the end of this time,
the animal was completely resistant to bites of rabid dogs and failed to
develop rabies if the most potent extracts were applied directly to the
brain.
Following confirmation
of his reports in 1885 that he had made dogs refractory to rabies by vaccination,
Pasteur received wide acclaim and much favorable publicity. But why not
use the vaccine on humans? Frankly, Pasteur was terribly afraid of things
going wrong and he was particularly uneasy about being unable to isolate
the rabic substance. And so he continued to insist that many years of additional
research was necessary before the treatment could be tried on humans.
But the press of events
made him act sooner. On July 6, 1886, 9 year old Joseph Meister and his
mother appeared at Pasteur's laboratory. Two days earlier the young boy
had been bitten repeatedly by a rabid dog. He was so badly mauled that
he could hardly walk. His mother appealed to Pasteur to treat her son.
At the time Pasteur had treated about 40 dogs, most of whom resisted rabies.
Could he risk treating this youth who faced certain death? Pasteur, after
consultation with physician colleagues, and much trepidation treated the
youth. Despite Pasteur's fears, Meister made a perfect recovery and remained
in fine health for the remainder of his life.
A few months later a second
victim turned up. He was a young shepherd also bitten by a mad dog. Following
reports of his successful treatments, the wild acclaim for Pasteur knew
no bounds! Victims of dog and wolf bites from France, Russia, the United
States poured into his laboratory for treatment. The newspapers and public
followed these treatments and cures with intense interest. Pasteur became
a hero and a legend. The Pasteur Institute funded by public and governmental
subscriptions was built in Paris initially to treat victims of rabies who
were coming to Pasteur's laboratory in increasing numbers. Later, Pasteur
Institutes were built, including 3 in the United States, to deal with human
rabies and other diseases.
Rabies was the last major
research of the master scientist. His health was failing and a paralysis
of his left side from a serious stroke he suffered in his 46th year made
his working in the laboratory increasingly difficult. Pasteur died in 1895
after suffering additional strokes. He was buried, a national hero, by
the French Government. His funeral was attended by thousands of people.
His remains, initially interred in the Cathedral of Notre Dame, was transferred
to a permanent crypt in the Pasteur Institute, Paris.
In a tragic footnote to
history, Joseph Meister, the first person publicly to receive the rabies
vaccine, returned to the Pasteur Institute as an employee where he served
for many years as Gatekeeper. In 1940, 45 years after his treatment for
rabies that made medical history, he was ordered by the German occupiers
of Paris to open Pasteur's crypt. Rather than comply, Joseph Meister committed
suicide! |