The Life of Sir Alexander
Chapter VIII - First
hope: the Lysozyme
... sometimes a man whose intelligence is arrested by things
which do not strike others, who, knowing how to use his eyes, looks hard at
what the others do not see.leriche
Never neglect any appearance or any happening which seems to
be out of the ordinary: more often than not it is a false alarm, but itmaybe
an important truth.Fleming
1922', writes Dr Allison, 'I went to St Mary's to work inIthe
Inoculation Department with Fleming. From the veryJLfirst
he started to pull my leg about my excessive tidiness. Each evening I put my
"bench" in order and threw away anything I had no further use for. Fleming
told me that I was a great deal too careful. He, for his part, kept his
cultures sometimes for two or three weeks and, before finally getting rid of
them, looked very carefully to see whether by chance any unexpected or
interesting phenomenon had appeared. The sequel was to prove how right he
was and that, if he had been as neat as I am, he would probably have found
out nothing new.
'About a month or two after I had started working with him,
he was busy one evening cleaning up several Petri dishes which had been
lying on the bench for perhaps ten days or a fortnight. As he took up one of
the dishes in his hand, he looked at it for a long time, showed it to me,
and said: "This is interesting." I had a good look at it. It was covered
with large yellow colonies which appeared to me to be obvious contaminants.
But the remarkable fact was that there was a wide area in which there were
no organisms; and another, farther on, in which the organisms had become
translucent and glassy. Beyond that, again, were organisms which were in a
transitional stage of degradation, between the very glassy ones and those
which were fully developed with their normal pigment.
'Fleming explained that this particular dish was one to which
he had added a little of his own nasal mucus, when he had
happened to have a cold. This mucus was in the middle of the
zone containing no colony. The idea at once occurred to him that there must
be something in the mucus which dissolved or killed the microbes in its
immediate neighbourhood, and then became diffused in such a way that it
progressively contaminated the already developed colonies. "Now, that really
is interesting," he said again. "We must go into it more thoroughly.5'
His first care was to pick off the organism and to stain it by gram. He
found it was a large gram-positive coccus, not a pathogen, and not one of
the known saprophytic organisms commonly met with, but obviously a
contaminating organism which was more likely to have been in the atmosphere
of the laboratory and may, of course, have blown in through the window, from
the dust and air of Praed Street.
'The next step in the investigation was to try again the use
of nasal mucus, and he tested some for its effect on this large
gram-positive coccus, not on a plate but in a tube. He made a culture of the
organism in broth and added nasal mucus to it. To his great surprise and
mine, the opaque suspension of organisms became, in the space of a few
minutes, completely clear — "clear as gin," he said at the time. Immediately
afterwards, and in the same conditions, he tried the effect of tears. A
single drop of tear-fluid dissolved the organisms in probably less than five
seconds. It was astonishing and thrilling.
the next five weeks, my tears and his were our main supply of material for
experiment. Many were the lemons we had to buy to produce all those tears!
We used to cut a small piece of lemon-peel and squeeze it into our eyes,
looking into the mirror of the microscope. Then, with a Pasteur pipette, the
point of which had been rounded in a flame, we collected the tears which we
proceeded to put into a test-tube. In this way I often collected as much as
J-J c.c. of tears for our experiments.'
Visitors, men and women alike, were put under contribution in
this matter of tears. TheSt
Mary's Hospital Gazettepublished
a drawing which depicted children coming, for a few pennies, to the
laboratory, where one attendant was administering a beating, while another
collected their tears in a receptacle on which was written the word
'Antiseptics'. Even the laboratory attendants were condemned to undergo the
'ordeal by lemon', but they got threepence each time. They kept regular
accounts and were paid for all their tears at the end of the month. Once,
when Fleming noticed that the eyes of one of these men were very red, he
remarked: Tf you cry often enough, you'll soon be able to retire!'
These experiments had proved that tears contain some
substance which can dissolve certain microbes with surprising speed. Tt was
possessed5, said Fleming, 'of extraordinary power. Up till then I
used to wonder at the much slower action of the antiserum which, when added
to an infected broth warmed in an incubator or in the water bath, takes some
considerable time to dissolve the microbes, and then only incompletely. But
when I studied this new substance, I put into a test-tube a thick, milky
suspension of bacteria, added a drop of tear, and held the tube for a few
seconds in the palm of my hand. The contents became perfectly clear. I had
never seen anything like it.'
The phenomenon was indeed very impressive, and Fleming was
the first person to observe it. The double piece of luck had been
miraculous, for the mysterious substance had been brought in contact with
the one microbe which was most sensitive to its action. All the same, though
its power of dissolving (and so, killing) had been demonstrated in a more
spectacular fashion in the case of the yellow 'coccus', which was
inoffensive, the substance also dissolved, though more feebly, other
microbes, some of which were pathogenic. In a series of experiments, Fleming
showed that it had the properties of an enzyme (natural ferment).
What should this substance be called? As usual, the question
was debated in the library, round the tea-table. Wright, as we have seen,
delighted in constructing words from Greek roots. Since the new substance
was a species of enzyme, its name must end with the syllable 'zyme'; and
since it dissolved, or 'lysed', certain microbes, it was agreed to given it
the name of 'lysozyme'. As to the microbe so easily 'lysed', Wright named it
— from 'lysis'(dissolution)
show): in other words — the organism which makes it possible to show, or
note, a power to dissolve.
Fleming continued tenaciously with his investigation of the
lysozyme. Since he had made the initial discovery, an idea had been taking
form in his mind and becoming more and more insistent. How did it happen
that a natural secretion of the body should possess such great strength as a
bactericide? Obviously because it had a protective effect on exposed
surfaces. This was a necessary provision of nature since, did it not exist,
the human species would have died out long ago, or would never have
developed at all, seeing that, from the moment of birth, human bodies are in
contact with the innumerable germs which air, earth and water contain. At
every moment of our lives microbes are being deposited on the surface of the
skin, and are penetrating into the nose, the mouth and the alimentary canal.
Many of these microbes are harmless, some are even useful and, for example,
facilitate digestion. The organism tolerates them, but resists any attempt
they may make to get beyond the mucous membrane or to multiply too rapidly.
The blood and its army of phagocytes provide one part of this
system of natural defences. But there are certain sensitive and fragile
areas, such as the conjunctiva of the eye, the membrane of the nose, and the
mucous membrane of the respiratory channels, which are exposed to airborne
microbes, and do not have the advantage of an abundant blood-flow. These
parts of the body cannot be left without protection. It looked as though
lysozyme might be one of the body's natural defences and, if the hypothesis
could be verified, it seemed probable that this substance, or other
substances of the same type, would be found distributed all over any animal
body — whether of a man, a bird or a fish — and that this peculiarity would
be present, too, in the vegetable world.
Fleming, therefore, organized a series of experiments with
the object of showing that lysozyme would be found in other secretions and
even in tissues. He discovered that a nail-paring, a scrap of skin, a drop
of saliva or a few hairs, when introduced into a test-tube, exercised the
same miraculous solvent action. He got into the habit, when speaking to his
students about natural defences, of asking them to take a cutting from the
edge of one of their finger-nails and place it in a microbial suspension.
The instantaneous effect amazed them — 'especially as they had recently come
from the hands of a physiologist who had taught them that fingernails
consist of inert tissue.' Meanwhile, as he went on with the researches, he
was finding more and more lysozyme everywhere: in the secretions of the
buccal mucus; in the sperm of all animals; in the spawn of the pike; in a
woman's milk; in the tip of a stalk; in leaves.
All the growing things in the garden were tested. Tulips and
buttercups, nettles and peonies, were found to contain lysozyme. The turnip
had an unusually large amount. But the richest store was egg-white. Fleming
demonstrated that egg-white, when diluted in sixty million times as much
water, was still capable of dissolving some microbes. The egg, therefore,
possesses considerable power as a bactericide, and it needs to, for the
white, and even the yolk, of an egg provide a marvellous culture medium for
microbes. The shell of an egg is not impervious to them: consequently, if
eggs can remain sterile'for several days in a dairyman's shop-window, where
they are exposed to the attack of every kind of germ, the reason must be
that they have some form of natural protection. 'It looks,5said
Fleming to his colleague, Ridley, 'as though the surfaces most exposed to
infection are also the best protected. For instance, the slime secreted by
an earth-worm is a highly potent bactericide.' He found lysozyme in the
blood, especially inside the leucocytes and in the fibrin of clots. 'Would
not this be,' he asked, 'a protective mechanism for open wounds, which
rapidly become covered with a layer of fibrin and leucocytes, both of which
are rich in lysozyme?'
Yes, lysozyme really did seem to be the body's natural
antiseptic, the cells' first line of defence against microbic invasion.
Fleming had every right to be proud of his work. He had discovered a new and
very important aspect of those natural defences of the human body to which
he had devoted so much study, in the worship of which he had been brought up
by Wright. Not so very long ago, Metchnikoff had demonstrated the fact that
certain special cells, the phagocytes, opposed the invasion of microbes.
Fleming had found that these cells contained lysozyme. Was it not possible,
therefore, to conclude that lysozyme was one of the weapons employed by the
leucocytes in their battle against the microbes?
As to the skin and the mucous membranes, Metchnikoff had
thought that they were protected only by mechanical means. 'Nature', he had
said, 'does not use antiseptics to protect them. The fluids which bathe the
surface of the mouth and other mucous membranes are not bactericidal, or
only very imperfectly so.
Nature removes from the mucous membranes and the skin
quantities of microbes by epithelial desquamation,1and
these are then expelled by the liquid secretions. Nature has chosen this
mechanical procedure, just as the surgeon replaces the antisepsis of the
mouth with a lavage of salt water.5In
1921 most bacteriologists held this opinion.
Fleming had just proved that Metchnikoff's argument must, on
this point, be modified. 'From the aforementioned experiments5,
he said, 'it is clear that these secretions, and the greater part of the
tissues, have, in a very high degree, the power to destroy microbes.5This
discovery was one of capital importance. But Fleming never used the word
'discovery5. It was one of those 'big words5which
he disliked. He always said 'my observation5. But, whether
discovery or observation, this one gave him more satisfaction than any
other. So great was his secret elation that in the first paper which he
wrote on lysozyme he, as a rule so prudent, so reserved — he, who either
from temperamental shyness, or in reaction from Wright's passion for vast
abstractions, would never permit himself to talk of anything but facts —
opened the flood-gates of his caution to a tide of wonderful hypotheses.
Not only was this discovery of his tremendous in its own
right; it also brought to a head ideas which he had been pondering for a
very long time. Somewhat later, in one of his rare moods of expansiveness,
he said to Ridley: 'When I was a young doctor in the514-'18
war, the Old Man was very much concerned with the power of the blood to kill
bacteria by means of its own leucocytes and serums. But I realized that
every living thing must,in
all its parts,have
an effective defence-mechanism; otherwise, no living organism could continue
to exist. The bacteria would invade and destroy it.' Ridley adds: 'He left
me in no doubt that he had disclosed to me in that simple sentence "every
living thing must in all its parts be protected" something fundamental in
his thinking. This, I believe, was the star that guided him all through his
Against what microbes was lysozyme effective? Fleming devised
an ingenious method by which to arrive at an answer to this question. He
hollowed out in a gelatinous substance contained in a Petri dish either a
hole or a gutter, in wjiich he placed some agar impregnated with lysozyme.
Next, he 'planted5certain
microbes, some in streaks perpendicular to the gutter, others in lines
forming the radii of a circumference of which the hole was the centre. Some
of the microbes developed right up to the gutter or the hole. They were
obviously insensitive to lysozyme. Others stopped short at a greater or less
distance, and this distance marked the measure of their sensitiveness.
Unfortunately, lysozyme, which was so powerful against the
inoffensive microbes, turned out to have a much weaker effect upon the
dangerous germs, the pathogenic ones. Nothing, thought Fleming, could be
more understandable. For what were the pathogenic germs if not those which
could penetrate the defences of the organism, establish themselves and cause
infection? Now, had they been as sensitive to the action of lysozyme as, for
instance, the yellow 'coccus'(lysodeikticus),
they would have been destroyed by the defenders, they would have been unable
to establish themselves and do harm, and this in itself would be contrary to
their own definition.
Does not all the difference between 'pathogenic'and
precisely there? he reflected. Certain microbes can infect certain varieties
of animal and not others; certain tissues, and not others. Is the solution
to the problem of predilection to be found in a difference of the quantity
or the quality of lysozyme in these animals or tissues? Starting from this
hypothesis, Fleming conceived one of those experiments which were always so
simple, but never failed to go straight to the heart of the problem.
He tried the effect of human tears on three groups of germs.
The first was composed of one hundred and four inoffensive species, found in
the air of the laboratory. The second contained eight germs, pathogenic to
some animals, but not to human beings. The third was made up of germs which
were pathogenic to human beings. The results were exactly what he expected
them to be. The lysozyme exercised a very powerful action on seventy-five
per cent of the first group, and on seven species (out of eight) of the
second. Its action was weak in the third group, though not completely
absent. Consequently, if the amount of lysozyme in the organism were
increased, it might be possible in that way to stop the development of
certain dangerous microbes. There was material here for investigation.
Fleming asked Dr Allison to join him in a programme of
research along these lines. But before making further experiments, he read a
paper on his discovery and on the conclusions he had drawn from it, in
December 1921 to the Medical Research Club,a
of respectable age (it had been founded in 1891) which was both exclusive
and influential. The reception accorded to the paper was cold beyond belief.
Not a single question was asked and no discussion followed the reading. Only
utterly worthless papers were treated in this manner. Sir Henry Dale, who
was among those present, has written: I very well remember his interesting
paper, and the way in which we all of us said: "Charming, wasn't it? Just
the sort of naturalist's observation Flemingwouldmake"
' — and that was all.
This icy reception of so original a study hurt Fleming's
feelings, for beneath his impenetrable mask he was extremely sensitive. But
it did not stop him. He prepared another paper on the same subject which
Wright presented to the Royal Society in February 1922.1But,
once again, it did not receive the attention it deserved. Fleming without
being unduly upset continued with Allison's help to work on the substance,
in the importance of which, despite the indifference of his peers, he
persisted in believing. Between 1922 and 1927, they published a further five
brilliant papers on lysozyme. They made an attempt to extract it in its pure
state, but neither of the two men was a chemist (Fleming used to say that he
would fail in an examination in elementary chemistry), and in the
laboratories of the St Mary's Research Service there was no chemist or
biochemist to be found. They could not isolate lysozyme, though they noted
that alcohol could precipitate it, without destroying it.
Having observed that lysozyme found in egg-white was a
hundred times more concentrated than that found in tears, they used it for
their experiments and established conclusively that the substance, at a
concentration double that to be found in tears, had a bactericidal action on
almost all the pathogenic germs and, in particular, on the streptococci, the
staphylococci, the meningococci, and the bacillus of diphtheria. They even
tried the effect of egg-white, administered by mouth, on the streptococci of
the intestine. Having made certain that lysozyme was not destroyed by the
gastric juices, they chose a patient who had an abnormal quantity of
streptococci in his intestine and made him swallow the white of four eggs
every day. The streptococci returned to normal. Encouraged by this temporary
success, they prescribed egg-white for several patients presenting the same
anomaly, who complained of fatigue and 'migraine'. They obtained a change
for the better in the symptoms. With prudence and honesty they concluded
that: 'This may, of course, have been merely a psychological effect, or it
may have been due to a temporary action of the lysozyme on the
Fleming, all this while, was going on with his general study
of antiseptics. The purpose of it was the same: to conquer the infections.
In 1923, the combined efforts of several research-workers in the lab.
produced a new and effective technique for this type of investigation.
Elliott Storer, who had thought it out, called it the slide-cell (a slide
divided into cells) method, but the slides prepared by him gave
disappointing results. Wright, who realized the value of this technique,
perfected it, and Dyson added a further improvement. It had everything in it
to please Fleming: it required skill in its manipulation; it cost nothing;
and it could be worked with small quantities — a great advantage where human
blood was concerned.
The slide-cell consisted of two slides of glass separated by
five strips of Vaselined paper, placed at regular intervals at right-angles
to the longest axis of the slides. The space between the slides was thus
divided into four equal compartments, each one of which could contain a
small quantity of blood. (Fleming had noticed that the paper on which a
certain medical journal was printed had the ideal thickness required for the
strips. When he was describing the method in his lectures, he would say,
with perfect gravity, and much to the surprise of his students: 'For the
Vaselined strips you should use theJournal
The small compartments were then filled with defibrinated
blood infected with the microbes to be studied, sealed at the two open ends
with paraffin, after which the whole slide-cell was placed in the incubator.
The microbes multiplied in colonies which, in this thin layer of blood, were
easy to count. For instance, it was possible to observe that, if about one
hundred staphylococci were put into a compartment containing normal blood,
the leucocytes killed, on an average, ninety-eight per cent of them, so that
only two of the colonies developed.
Fleming thought that this was an ideal technique for making a
definitive study of the action of the antiseptics on the leucocytes. In the
compartments of the slide-cell he mixed blood with more and more
concentrated solutions of the antiseptic which he wanted to study. He
noticed that the antiseptic killed the leucocytes at concentrations far
below those required to kill the bacteria and so there were concentrations
in which all the leucocytes, in other words all the defenders, were killed,
staphylococci flourished: a hundred microbial colonies were counted in each
compartment instead of only twowithoutantiseptics.
His conclusion was as follows: 'These experiments show that there is little
hope that any of the antiseptics in common use could be successfully
introduced into the blood stream to destroy the circulating bacteria in
cases of septicaemia.'1By
this beautiful and simple experiment, he had proved irrefutably that the
antiseptics then in use destroyed the leucocytes in much weaker solutions
than those which would have enabled them to act upon the microbes.
On the contrary, when the slide-cell was used to study the
action of egg-white on the phagocytes, Fleming and Allison observed that
'whereas egg-white, in marked contrast to the chemical antiseptics, has no
destructive effects on the leucocytes, it has considerable inhibitory or
lethal effect on some of the bacteria'. They made the experiment of giving
intravenous injections of an egg-white solution to a rabbit, and then
measuring the bactericidal power of its blood. There were no unfortunate
consequences. The anti-bacterial power was markedly enhanced. 'And it is
possible', wrote Fleming,'that
in cases of generalized infection with a microbe susceptible to the
bacteriolytic action of egg-white ... the intravenous injection of a
solution of egg-white might be beneficial.. .'This
was an important conclusion, for, with it, Fleming, the victorious adversary
of antiseptics, was affirming that he had no prejudice against chemotherapy,
provided the product employed did not destroy the natural defences of the
But in order to make a series of intravenous injections
without danger, it would have been necessary to rid the lysozyme of
egg-white. Fleming and Allison, as we have seen, had attempted, without
success, to extract lysozyme in its pure state. In 1926, a young doctor
named Ridley came to do research work in Wright's laboratory. He was not a
professional chemist, but he knew a great deal more about chemistry than did
his colleagues. Fleming asked him to extract lysozyme in its pure state.
Ridley tried, but unsuccessfully. Fleming was greatly disappointed. Tt is a
pity,' he told Ridley, 'because if we had this substance pure, it ought to
be possible to maintain in the body a concentration which would kill certain
Later, as we shall see, a biochemist did manage to purify and
Fleming was an obstinate man. He continued to make a studyin
the action of other products upon the bactericidal power of the blood. He
wanted, for instance, to measure the action of salt. He found that every
saline concentration which departed from that normally found in the human
body weakened the phagocytes.
What would be the effectin
In order to find out, he gave an intravenous injection of hypertonic salt
(that is, a solution of greater concentration than the normal concentration
in the organism) to a rabbit. The first injection was too strong. The rabbit
had convulsions and for a few seconds seemed to be on the point of dying.
Two minutes later, however, it had got over the shock. Fleming examined its
blood. At first, and for as long as the concentration of salt in the
animal's blood remained above the normal, the result was identical with that
The bactericidal action of the blood was diminished. But, to his great
astonishment, Fleming discovered that, after two hours, the concentration of
salt in the blood having returned to normal, the bactericidal power of the
blood was greatly enhanced, and this lasted for several hours.
Having perfected the experiment in such a way as to give a
quantity of salt so little above the normal that it caused the animal no
distress, Fleming tried his hypertonic salt on a patient. An intravenous
injection produced an increase of the bactericidal power without causing the
He made further experiments on patients whenever his medical
colleagues allowed him to do so. But, generally speaking, he was given only
desperate cases, and even those very rarely. One or two other doctors made
similar experiments and observed that the results were good. But they did
not continue. Fleming was very fond of this little discovery and always
regretted that it had been more or less ignored. He could not understand why
greater advantage was not taken of a treatment which was wholly inoffensive
and was probably more effective than the therapeutic vaccines. His sixth
paper on lysozyme was written in 1927. It deals with an important
phenomenon. By exposing microbes to increasing concentrations of lysozyme
and picking up the few survivors, he had managed to create 'strains5of
the famous yellow coccus, or of the faecal coccus, eighty times more
resistant than had been originally the case. Had these microbes, in
developing greater resistance to lysozyme, also become more resistant to the
action of the blood? Experiment showed that they had. Why? As we have seen,
Fleming had found lysozyme in the phagocytes. Since the increase in
resistance to lysozyme keptpari
resistance to phagocytosis, did it not look as though the action of the
phagocytes was, in part, due — as he had already thought it was — to the
lysozyme which they contained?
As in his first paper, he here found himself faced by
problems of the greatest importance. The pathogenic germs are enemies
dangerous to man, because they force the natural defences. Gould it be that
lysozyme had in primeval times been an all-powerful weapon supplied to
primitive man by nature to protect him againstallgerms?
Might not the pathogenic germs be, in fact, descendants of the few germs
which, having resisted lysozyme, had acquired an ever-increasing power of
resistance, until they had become capable of overcoming nature's other
defences? If that were so, could one not, by selection, transform an
inoffensive into a virulent germ? This was the subject of his sixth paper.
Why was it that this series of superb studies, which had
opened up new and vast horizons, attracted so little attention from British
scientists? Was it due to the fact that Wright at that time was 'slowing
down', and that the enemies of his school were now inclined to accept with a
considerable degree of suspicion the work carried out in his laboratory?
Fleming, loyal as ever, said that the fault was his, and that he should have
presented his findings in the matter of lysozyme to an audience, not of
doctors, but of physiologists, who would, most certainly, have been
interested. However that may be, the indifference of his colleagues to work
which, in spite of his modesty, he knew to be remarkable, made him more
silent and reserved than ever, but also stronger. His judgment was not
influenced by that of others. His momentum had by no means come to a halt.
All through his life he never abandoned the search for a substance which
would kill the microbes without weakening the phagocytes. He had looked for
it, with his master, in the vaccines. He had hoped that he had found it in
lysozyme, a substance which could have reconciled the physiological and the
antiseptic schools, because it was an antiseptic enlisted in the service of
the body's natural defenders. Being a tenacious scientist who was sure of
his facts, he still looked forward to a future in whichhislysozyme
would play an important part.
Nor was he wrong. Even today much work is being done on
lysozyme. It interests bacteriologists because it dissolves the mucins with
which the microbes are coyered; industrialists because it protects
foodstuffs from infection (the Russians use it for preserving caviare);
doctors, because, when added to cow's milk, it reproduces the component
structure of human milk, and also because they use it in the treatment of
eye and intestinal affections. All this has come about because an attentive
observer, when about to throw away a contaminated culture, looked at it
carefully, and said:cThis
is interesting.' The discovery which was received with icy silence in London
in 1921, has in the space of thirty years become the subject of two thousand
papers. Alexander Fleming always said: 'We shall hear more about lysozyme
one day.' Who knows?
In the laboratory, those who held aloof from cliques realized
its value. Martley, a charming bearded Irishman and one of nature's
gentlemen, said to Pryce in 1927: 'Fleming's the really intelligent chap ...
If the Old Man had been the author of those experiments with lysozyme and
other things, what a sensation there'd have been!At
the first International Congress of Microbiology, held in'93'?
Jules Bordet, a Belgian scientist, a former pupil of Pasteur and President
of the Congress, spoke in his opening address of Fleming's work on lysozyme
in the highest terms. Fleming, completely taken by surprise, was
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