Significant Scots - Sir Alexander Fleming

Fortune favours the prepared mind, pasteur

All the same, the spores didn't just stand up on the agar and say 'I produce an antibiotic, you know.' Fleming

The mysterious mould from Praed Street produced a substance which stopped the growth of certain pathogenic microbes. The first point to get clear was: 'Have other moulds the same power?' Fleming's friends remember the time when he would stare at every mouldy surface, his eyes glinting with curiosity, and be for ever asking whether they hadn't got any rotting old shoes to give him. The sculptor Jennings, a member of the Chelsea Arts Club, recollects how on one occasion Fleming suddenly addressed a general question to the artists clustered round him: Tf any of you chaps has got a pair of mouldy old shoes, Fd very much like to have 'em.' When somebody asked what for, he said: 'Oh, for something I'm doing at the lab.'

Experiment showed that the other moulds which he tested did not produce an antibacterial substance. His penicillium, therefore, was more than ever worth looking into. What he needed now, in order to get on with his research-work, was a great quantity of 'mould juice'.

For some time a young assistant, Stuart Craddock, had been working with him. Fleming had asked him to help with his study of mercuric chloride and to see whether, by injecting it in very small doses, it might be possible, not to kill, but to inhibit, the microbe, and so facilitate the work of the phagocytes. Tf he told me once, he told me a hundred times', says Craddock, 'that the only usable antiseptic would be one which would arrest the growth of the microbes without destroying the tissues. On the day when such a substance should be discovered, he said, the whole treatment of the infections would be transformed.' That was the leit-motif of his scientific life.

Very soon Fleming told Craddock to abandon mercuric chloride at once and devote his attention to the production of mould juice. They began by cultivating the penicillium in a meat-broth at a temperature of just under ioo° Fahrenheit. Then, the mycologist La Touche said that the penicillium would be happier at about 69°. A large black incubator was installed in the room where Craddock worked. The latter planted the penicillium in a number of flat bottles of the kind used for preparing vaccines, and left them in the incubator for a week. In this way he obtained from two to three hundred cubic centimetres a day of the mysterious substance, and this he put through a '^Seitz⁵ filter with the aid of a bicycle pump — a somewhat primitive method.

Fleming studied the cultures in order to determine on what day of growth, at what temperature, in what nutritive medium, he would get the greatest yield of the active principle. The methods he had perfected in the old lysozyme days enabled him to measure the antibacterial strength and to standardize the concentration of the cultures. He observed that if the broth was left at laboratory temperature, its antibacterial strength rapidly diminished. This meant that the marvellous substance showed a disquieting degree of instability. He discovered that it became more stable if the alkaline reaction of the broth (pHg) were changed to a neutral reaction (pH6.8).

At last he was able to submit his 'juice⁵ to the test which no antiseptic had so far passed successfully — that of toxicity. To his great — and silent—joy, he observed that 'the toxicity to animals of powerfully antibacterial mould broth filtrates appeared to be very low. Twenty c.c. injected intravenously into a rabbit were not more toxic than the same quantity of broth. Half a c.c. injected intraperitonically into a mouse weighing about 20g. induced no toxic symptoms. Constant irrigation of large infected surfaces in a man was not accompanied by any toxic symptoms, while irrigation of the human conjunctiva every hour for a day had no irritant effect. In vitro, penicillin which completely inhibited the growth of staphylococci in a dilution of 1 in 600, did not interfere with the leucocytic function to a greater extent than did ordinary broth.'

It was all becoming tremendously interesting. 'There', says Craddock, 'was the antiseptic of his dreams, a substance which, even in diluted form, remained bactericidal, bacteriostatic and bacteriolytic, without producing any harmful action on the blood.' Craddock just then was suffering from an infected antrum. Fleming washed out the sinus with penicillin-broth. In his laboratory notes we read: 'January gth, 1929: mould filtrate antiseptic power on Craddock's antrum. Swab from antrum on blood-agar: 100 staphylococci with myriads of Pfeiffer around. Then 1 c.c. mould filtrate put into right antrum. Swab three hours after on blood-agar. One colony of staphylococci and a few colonies of Pfeiffer. In films as many bacteria seen after as before but mostly phagocytosed.'

Thus, even when immensely diluted, the substance killed nearly all the staphylococci. That it would have no action on the Pfeiffer bacillus, Fleming had expected, since it was one of the microbes which had shown resistance in the early experiments. The result of this first and modest therapeutic attempt with raw penicillin on a human being was not too bad.

Craddock also tried to cultivate penicillin in milk. After a week the milk had curdled and the 'juice' had turned into something resembling Stilton cheese, which Craddock and another patient ate, with results that were neither harmful nor beneficial. Fleming had asked his colleagues at St Mary's to let him try his filtrate on infected wounds. One of the cases on which the 'juice' was tried was that of a woman. Coming out of Paddington Station she had slipped and fallen under a motor-bus. She had been taken to St Mary's with a terrible open wound in her leg. An amputation was performed, but she developed septicaemia and it was quite certain that she would die. Fleming, when his opinion was asked, judged the case to be desperate. Then he added: 'Something very odd has happened in my lab. At this very moment I have got a culture of a mould which destroys staphylococci.' He tried soaking a dressing in the 'juice' and applying it to the surface of the amputation. He had not much hope that this application would do any good. The concentration was too weak, and the damage too generalized. The effect was nil.

But he remained just as much convinced as ever of the importance of his discovery. Sir Alexander McCall tells how, one day in 1928, 'Alec and Mrs Fleming spent a Sunday with us. Alec brought a glass slab from his pocket, showed it to my wife, and expressed the opinion that from this slab things would come which would create world-wide interest. My wife, just to pull Alec's leg, said that it was "only a dirty slab".'

About this time it occurred to Fleming that the substance discharged by the mould into the culture broth deserved a name. The one he gave it, 'penicillin', is, as he said later, a word of 'perfectly orthodox formation'. 'Penicillin' comes from 'penicillium', as 'digitalin' from 'digitalis'. Since he had not isolated the active antibacterial principle, he continued to apply the name to the raw filtrate, but his conversation, as well as his papers, leaves no doubt that what interested him was the antibacterial substance contained in the filtrate.

What he wanted now was to extract this active principle. As has been already pointed out, he was not himself a chemist, and there was neither a chemist nor a biochemist on Wright's staff. In one of his moments of paradox, Wright had said: 'There is not enough of the humanist in chemists to make them suitable colleagues.' There is no reason whatever why a biochemist should not be an excellent humanist, but the fact remains that chemistry was not represented in the laboratory, unless we accept as a chemist the young doctor, Frederick Ridley, who, though an amateur, had proved to be skilful up to a point. In 1926, Fleming, having noticed his competence, had asked him to purify lysozyme. Now he begged him once again to make, in association with Craddock, an attempt to extract the antibacterial principle of penicillin.

'So long', says Craddock, 'as penicillin was mixed with the broth, it was obvious to all of us that it could not be used for injections until it had been freed from foreign proteins' (a series of protein injections would have caused anaphylactic accidents). It was essential that extraction and concentration should be attempted before any serious therapeutic use of the substance could be made. 'I have always thought', Craddock continues, 'that the end aimed at in extracting and purifying penicillin was to make it suitable for purposes of injection. When Fleming had started me working on mercuric chloride, he said that it might be possible to use it intravenously in doses sufficiently massive to inhibit bacterial growth without killing the patient, and I am sure he had the same thing in mind with regard to penicillin, provided we could extract it from the broth as a stable and pure substance.

In this way it came about that two young men who had only just finished their medical studies, set out to find the solution of a chemical problem which proved to be an extraordinarily difficult one. The astonishing thing is that, though they did not know it, they came within measurable distance of success. 'Ridley⁵, says Craddock, 'had sound and pretty advanced ideas about chemistry, but when it came to methods of extraction, we were driven back on to books. We read up a description of the classic method: using acetone, ether or alcohol as solvent, and evaporating the broth at a fairly low temperature, because we knew that great heat would destroy the substance; working in a vacuum. We knew very little when we began. We knew just a little bit more when we had finished: we learned as we went along.'

They worked in a narrow sort of passage, which contained a sink, where in the old days the nurses had washed out bed-pans, filled hot-water bottles and kept specimens of urine. It dated from the time before the laboratory had been installed in this wing of the building. They chose it because there were running water available and a water pump. They had to construct their own apparatus from what odds and ends they could find. They evaporated the broth by vacuum, because they could not use heat for fear of the penicillin vanishing. After evaporation there remained at the bottom of the bottle a brown, syrupy mass in which the strength of the penicillin was about ten to perhaps fifty times greater than that measured in the broth. But this 'melted toffee' could not be used. Their aim was to obtain pure penicillin in the form of crystals.

'We were full of hope when we started', says Craddock, 'but, as we went on, week after week after week, we could get nothing but this glutinous mass which, quite apart from anything else, would not keep. The concentrated product retained its power for about a week, but after a fortnight it became inert.' Later on (when the brilliant work of Chain had made possible the extraction of pure penicillin) they realized how close they had been to success. 'We could not know at the time that we had only one more hurdle to cross. We had been so often discouraged. We thought we had got the Thing. We put it in the refrigerator, only to find, after a week, that it had begun to vanish. Had an experienced chemist come on the scene, I think we could have got across that last hurdle. Then we could have published our results. But the expert did not materialize.' And so it was that the attempts at extraction were abandoned.

There were also personal reasons why the two young doctors should give up. Craddock had just got married and was about to go to a better paid post at the Wellcome Research Laboratories. Ridley, who was suffering from boils, had tried various vaccines in vain and had become discouraged. He abandoned the problem of penicillin in favour of a cruise which he hoped might cure him. An ironical feature of his case is that if he had succeeded in the business of extraction, penicillin would have put an end to his boils! When he returned, he gave the whole of his time to ophthalmology, in which he later specialized. It was, after all, but natural that both men should be out of love with the research-work they had been doing. Chemistry was not their speciality. They had made an immense effort and worked for several weeks, only in the end to find themselves with a 'batch' which had vanished almost at once.

Fleming had not taken an active part in their labours. T am a bacteriologist,' he had said, ^cnot a chemist.' He had asked his two 'amateur experts' to take over that side of the work and had waited, full of hope, for their results. Meanwhile he had prepared a paper on penicillin which he read on February 13th, 1929, to the Medical Research Club. Sir Henry Dale, who was then its chairman, remembers the reactions of the audience. They were more or less the same as they had been in the case of lysozyme. 'Oh, yes, we said, Fleming does observe that sort of nice thing.' It is certainly true that he never knew how to present his findings in the best light. 'He was very shy, and excessively modest, in his presentation, he gave it in a half-hearted sort of way, shrugging his shoulders as though he were deprecating the importance of what he said... All the same the elegance and beauty of his observations made a great impression.' That impression may have been real enough, but nothing in the strangely superior and sullen attitude of his listeners gave any indication of it.

When a paper has been found interesting, it is always followed by questions, and the greater the interest aroused, the more numerous are the questions. The reader stands at his desk, waiting for them. If none comes that period of waiting in the silent room is a terrible ordeal. Fleming experienced it when he spoke of penicillin, as he had done, formerly, when he spoke of lysozyme. Not a single question was asked, whereas, the next paper: 'On the Nature of the Lesion in Generalized Vaccinia⁵provoked a lengthy discussion. The icy reaction to something which he knew to be of capital importance appalled him. In 1952, when he was at the summit of his fame, he was still talking about 'that frightful moment^'. But in 1929 he gave no sign of disappointment. He knew the value of what he had done and that knowledge gave him strength and made it possible for him to remain unshaken in his belief.

He had now to prepare for publication in the British Journal of Experimental Pathology a report on penicillin. This first paper is a triumph of clarity, sobriety and precision. In a few pages it covers all the facts. It does justice to the efforts made by Ridley to purify the substance. It shows that penicillin, since it can be dissolved in pure alcohol, is neither an enzyme nor a protein. It speaks of the innocuousness of the substance when injected into the blood stream and says that it is more effective than any other antiseptic and can be used in the treatment of infected surfaces. It states that he is engaged in studying its value in the treatment of pyogenic infections. In the final summing-up, it recapitulates all these points and, in particular, the following: '(1) A certain type of penicillium produces, in culture, a powerful antibacterial substance ... (7) Penicillin is non-toxic to animals, even in massive doses, and is non-irritant ... (8) It is suggested that it may be an efficient antiseptic for application to, or injection into, areas infected with penicillin-sensitive microbes.'

This conclusion was the cause of the first, and perhaps the only, quarrel between Fleming and his master, Wright. When the latter read the paper, prior to authorizing its publication (his imprimatur was customary in the Department), he demanded the suppression of paragraph 8. Had he n&t said a hundred times that the natural defences of the body alone were effective? Had he not established, in conjunction with Fleming himself, the fact that antiseptics were the enemy? But Fleming, the ever cautious Fleming, who never used a word without weighing it well, and who, as the greatest possible compliment to another bacteriologist, Jules Bordet, was to say: 'Marvellous theories are sometimes promulgated — not always with sufficient scientific backing. Young Bordet set to work, not to invent theories, but to bring facts to light...' —Fleming stuck to his guns, and paragraph 8 appeared, together with the rest of the paper, in June 1929.

While waiting for the doctors and surgeons of the hospital to provide him with patients on whom to test his penicillin (tests, the results of which he published in 1931-2), he finished his article on the staphylococci for A System of Bacteriology. A little later, he returned to the subject in connection with what was known as 'the Bundaberg catastrophe', when, in 1929, at Bundaberg, Queensland (Australia), a number of children had been inoculated against diphtheria, and twelve of them had died within thirty-four hours. The vaccine had been contaminated by a very virulent staphylococcus.

In the meantime, one of the best chemists in England, Professor Harold Raistrick, who taught biochemistry at the School of Tropical Medicine and Hygiene, had developed an interest in the products of moulds in general and especially in penicillin. A bacteriologist, Lovell, and a young chemist,- Clutterbuck, joined forces with him. They obtained strains both from Fleming himself and from the Lister Institute. This team succeeded in cultivating penicillium, not, this time, in a broth, but in a synthetic medium containing some salts and a little glucose. Clutterbuck, Raistrick's assistant, studied the filtrate from the biochemical point of view; Lovell from the bacteriological.

Raistrick succeeded in isolating the yellow pigment which gave the juice its colour, and showed that it did not contain the antibacterial substance. The objective in view was, of course, to isolate this substance itself. Raistrick managed to extract it in ether and hoped that, by evaporating the ether, he would obtain penicillin in its pure state. In the course of this operation, however, the pemcillin, fugitive as ever, vanished. The activity of the filtrate, if it was kept, diminished in strength from day to day and very soon disappeared entirely.

In all research there is a human element. Raistrick wanted to continue his investigation of penicillin, but the mycologist of the team was killed in an accident. Clutterbuck, too, died while still quite young. Then the bacteriologist, Lovell, left the School to enter the Royal Veterinary College. 'But I did not go', he writes, 'until October 1933 and, so far as I was concerned, work on penicillin had stopped well before that date, though why, I do not know. I had intended to test penicillin by injecting it into the peritoneal cavity of mice infected by pneumococci. Having observed the astonishing activity of the substance on pneumococci in vitro, I wondered whether it would be equally effective in vivo. I was stimulated by some work done by Dubos on this, but my investigations remained in the planning stage, and never got farther.'

'During all the time I was working on this subject', continues Professor Lovell, 'Fleming was very much interested in what we were doing, and gave us all the help he could. I constantly rang him up about the difficulties we were experiencing over the mutations which occurred with certain strains of penicillium. He was always ready to co-operate. He told me of the incorporation of a malt substance which he had obtained from the pharmacy at St Mary's, and I realized that he was treating the subject more as an artist than a chemist. It mattered little to him what the composition of the product was, so long as it gave good results. That was all he wanted to know. He offered to send me some.

'I think that our main contribution had been to show that the mould could be cultivated in a synthetic medium; that it was possible to keep it longer when the pH had been brought over on to the acid side, and that we could remove the penicillin by extraction with ether. It was a great misfortune that Clutterbuck died while still a young man. I feel quite sure that, had he lived, it would not have been long before he would have realized that by switching the pH over to the alkaline side he would have been able to recover the penicillin which was apparently lost when we treated the filtrate with ether — as became obvious when Chain took over from that point and succeeded in concentrating penicillin, which was the starting point of the work which he and Florey did.'

It is only fair to admit that Raistrick and his assistants achieved useful results and were moving forward in the right direction. It is not surprising that they should have been brought up short, as Craddock and Ridley had been before them, by the baffling instability of the substance. ^cWe had realized⁵, says Raistrick, 'that the effectiveness of penicillin was no less destroyed in an alkaline than in an acid medium, and that, when extracted with ether, it vanished. Such a thing had never happened to a chemist. It seemed incredible. Faced by difficulties of this kind, we had to abandon the work, and pass on to other experiments.'

Those who take a harsh view of the discontinuance of these attempts for the purification of penicillin forget that similar breaks are always occurring, either because the results obtained are disappointing, or because of the convergence of fortuitous circumstances. In the case of penicillin, all these factors played a part. The substance was more than usually unstable and, on two occasions, the investigating teams, which deserved to succeed, were dispersed by illness and death. Luck, bad as well as good, is always present in research. The man who is forced to stop short on the very threshold of discovery can have a clear conscience, provided he feels sure that he has done everything which was (for him) humanly possible. This was so with Raistrick and Lovell. T am only too glad', writes the latter, 'that I was able to contribute even a small amount towards the use of penicillin, and to the good it has done.'

The scientific research-worker finds his satisfaction in the knowledge that he has played his part in a great common task, without being influenced by either personal ambition or jealousy. 'No research is ever quite complete. It is the glory of a good bit of work that it opens the way for still better and thus rapidly leads to its own eclipse. The object of research is the advancement, not of the investigator, but of knowledge.'¹

Meanwhile, Fleming was going ahead with his experimental local applications of penicillin at the hospital. The results were encouraging though not miraculous because, owing to its instability, penicillin had a way of giving out just when its use would have been most rewarding. 'I am convinced', said Fleming, 'that, before it can be used on a large scale, it must be concentrated.'

Speaking at the Royal Dental Hospital in 1931, he reaffirmed his faith in the substance and, a year later in the Journal of Pathology and Bacteriology published the results of his experiments on infected wounds. He had been bitterly disappointed by the ill-success of the chemists. It had never occurred to him that the extraction of a substance could present so many difficulties, and he had felt certain that, after the work done by Raistrick, the substance would at last be available for use in its pure state. All through the years to come, he obstinately retained a secret tenderness for 'his baby'. There is much evidence to show that, in spite of his habitual reserve, he frequently spoke of penicillin, and never despaired of one day seeing it purified.

Dr A. Compton, for a long time Director of the Laboratories of the Egyptian Department of Public Health, describes how in the summer of 1933 he paid a visit to Fleming who gave him a bottle containing a culture of penicillium notatum with a request that he should try it on his patients when he returned to Alexandria. But Compton at that time was hoping for great things from another bactericidal principle which he had himself discovered, with the result that the bottle remained in a corner of the laboratory at Alexandria and was never used. Fortune was not favourable.

Dr Rogers (who now works in Birmingham) was a student at St Mary's round about 1932 or 1933. Just before a shooting-match between the London hospitals in which he was due to figure, he was laid low with an attack of pneumococcal conjunctivitis. 'You'll be all right by Saturday,' Fleming told him, after treating his eye with a yellow liquid and remarking that, in any case, it couldn't do him any harm. On the day of the match, Rogers found that he was cured. Whether this cure was the result of penicillin, he never knew.

To Lord Iveagh, his neighbour in the country, who bred cows and was consequently brought face to face with the problem of mastitis, a streptococcal infection, Fleming spoke of a fungus which could arrest the development of certain microbes. 'Who knows?' said he. 'One of these days, perhaps, you'll be able to put it in the animals' feed, and be rid of your mastitis trouble for good and all.'

In 1934 Fleming took on as an assistant Dr Holt, a biochemist, for the purpose of preparing antitoxins. He went through the now classic experiments for his benefit: action of penicillin on a mixture of blood and microbes — microbes killed, leucocytes intact — which was the reverse of what happened with the known antiseptics. ^cHe was well aware', says Holt, 'of the therapeutic potentialities of penicillin, and was extremely keen that it should be purified, because, he said, it was "the only product capable of killing microbes with a high degree of resistance, such as the staphylococci, without injury to the white corpuscles" ...'

Holt was struck by the spectacular nature of these experiments and agreed to make an attempt at purification. He reached the same point as Raistrick had done, but could go no farther. He succeeded in passing penicillin into an acetate solution, but it disappeared with great suddenness. After numerous failures he gave up. Fleming was once again disappointed, 'but', says Holt, 'to those of us who lived with him in the lab. he said over and over again that penicillin had great potential therapeutic value. He continued to hope that some day somebody would come along and solve the chemical problem, and that he would then be able to make the appropriate clinical tests.'

In 1935, he copied into his diary — where he was in the habit of recording quotations to which he attached great importance — under the date December 20th, Friday, Ember Day, the following passage from a speech delivered by Lister in 1898 on the occasion of his having the Freedom of the City of Edinburgh presented to him: 'I must confess that highly, and very highly, as I esteem the honours which have been conferred upon me, I regard all worldly distinctions as nothing in comparison with the hope that I may have been the means of reducing, in however small a degree, the sum-total of human misery.' Such was his secret ambition. One day, it was going to be satisfied beyond his wildest dreams.