Serendipity and the Discovery of Penicillin

Guest Article by Prof. K.J. Rao

Solid State and Structural Chemistry Unit

Indian Institute of Science

Bengaluru, 560012

Serendipity is discovering something pleasant and useful quite unexpectedly. In this sense discovery of penicillin was a perfect example of serendipity. Let us see how. First the interesting story of penicillin.

Alexander Fleming was already a famous scientist in 1928, working at St. Mary’s Hospital, Paddington, London and had just become a full professor. In 1921 he had discovered Lysozyme*, a vital protective enzyme, present in our tears, saliva nasal mucosa, breast milk and even in blood. It kills bacteria by ‘lysing’ them or breaking open the microbial cell after attaching to the peptidoglycan of the bacterial cell wall. But lysozyme was not effective in killing more virulent and disease-causing bacteria. That is why Fleming turned his attention towards discovering more powerful antimicrobial molecules. Growing bacterial colony of streptococci and staphylococci was integral to his research. Therefore, his laboratory was always filled with a number of Petri dishes with colonies of bacteria grown in them for his experiments. It was rumored that Fleming was often a bit untidy in maintaining his biological samples and the labware (see below the photograph of Fleming in his laboratory).

*Lysozyme is famous for a couple of other reasons. It is the second protein and the first enzyme whose structure was determined using X-rays (C. C. F. Blake, D. F. Koenig, G. A. Mair, A. C. T. North, D. C. Phillips & V. R. Sarma, Structure of Hen Egg-White Lysozyme: A Three-dimensional Fourier Synthesis at 2 Å Resolution. Nature 206, 757–761 (1965)). Lysozyme was also the first enzyme which was fully sequenced. It is a single polypeptide with 129 amino acids and folded into a globular structure.It contains all twenty common amino acids.

In 1928 August, Fleming was growing bacterial cultures of staphylococci in his lab and he left for a holiday in his home town, Loch field near Ayrshire in Scotland where he was born in 1881. When he returned to his lab on the 28th of September, 1928, he found bacterial cultures fully grown in all his Petri dishes except in one of them. His trained eye noticed that there was growth of a fungus with a bluish green tinge in that Petri dish and the growth of bacteria – staphylococci – was restricted to a zone away from the fungus. In his mind Fleming concluded that the fungus has some chemical principle that killed the bacteria and hence the zone of no-growth. Otherwise, why wouldn’t the bacteria grow? This Fungus had gotten into the Petri dish because it was kept carelessly uncovered and the breeze from the open window brought the fungus inside the laboratory. Being untidy pays - sometimes!

Fig 1. Alexander Fleming studies mold cultures in his lab at the Wright Fleming Institute in London.

Now Fleming tried culturing of bacteria in the presence of fungus in Petri dishes. This was to ascertain his conjecture that the fungus contains a killer chemical. He repeated the experiments and confirmed he was correct. He had an intelligent assistant, Robert Hare and the latter discovered that the fungus grew better at optimum temperatures. Fleming named the bacteria killing chemical in the fungus, Penicillin and the mold - the fungus - Penicillium Notatum. It is the same as Penicillium Chrysogenum. The fungus was then squeezed and the juice was shown to have the antibacterial property. Fleming thought that the Penicillium mold can be harnessed to fight infectious diseases. But Fleming was not a chemist by training. He was a physician, pharmacologist and medical microbiologist.

No wonder, he didn’t proceed to isolate the active principle in the fungus and characterize it more fully. But he did one good thing; sent samples of the mold (fungus) to any number of other scientists, (including a German scientist, Dr. Schmidt) who expressed interest in this discovery. Fleming also published his findings in the British Journal of Experimental Pathology in 1929. For almost a decade there wasn’t much happening with this discovery.

Fig 2. Penicillin kills gram-positive bacteria very well. On the plate P. chrysogenum (a descendent of Fleming's original strain) was inoculated into the center of a lawn of Staphylococcus aureus. Note the clear zone of inhibition of growth of S. aureus

Dr. Howard Florey was a professor of pathology and director of Sir William Dunn School of Pathology at Oxford University. He was interested in investigating how bacteria and fungi kill each other. The 1929 paper of Fleming referred to above caught his attention. He also had the sample of Fleming’s mold which Florey’s predecessor, Dr. George Dreyer had obtained from Fleming.

Florey was a go getter. He had the reputation for skillfully squeezing research grants from the tight-fisted bureaucrats. He was also respected for successfully managing intelligent-but-impossible colleagues in his research team. One such gifted but sensitive colleague was Dr. Ernst Chain, a Jew from Germany. Without any delay Florey and Chain started work on Penicillium molds from where Fleming had left. Another biochemist, Dr. Norman Heatley, joined the team. Heatley was an expert at growing fungus. He was reputed for having grown the mold in practically any and every container in the laboratory including bedpans.

Their first experiments were on mice, 50 of them. They infected all the mice with streptococci and treated half the infected mice with the penicillium extract from the molds. Their happiness knew no bounds when they discovered that all of the treated mice lived and all of the untreated ones died (who has the time to mourn the dead mice!). They were convinced that “penicillin kills bacteria”. Now Florey and his colleagues had to try their experiments on humans.

Quite unexpectedly, during the lunch time gossip at the high table** at Oxford, Florey heard about a patient at Radcliff Infirmary suffering from severe infection (sepsis). The patient got wounded while pruning plants in a rose garden. Doctors at Radcliffe had almost given up on this patient, Albert Alexander, a policeman. Florey got permission to try his purified penicillin juice on Alexander. Upon treatment with the penicillin juice Alexander showed signs of rapid recovery but the penicillin juice got exhausted. Alexander died for want of more penicillin. It would take 2000 liters of the penicillin juice to save one patient. Even Heatley the wizard mold grower found it challenging.

**The High table is a table for the use of fellows (members of the Senior Common Room) and their guests in large dining halls, where the students eat in the main space of the hall at the same time. They remain the norm at Oxford, Cambridge, Dublin and Durham universities, where the university is organized into colleges. Wikipedia.

The task now was to find a better way to grow more fungus, isolate penicillin and convince the public that penicillin cures otherwise fatal diseases.

Florey and Heatley decided to reach the other side of the Atlantic in search of support for their work. They left for US in mid-1941 along with a sample of their P. Chrysogenum fungus. Apparently, they were afraid that they may lose the vials containing the mold during their travel and therefore they smeared the inside of their coats with the mold in order to secure its safe transport. In the US they tried to convince several pharmaceutical companies to take up manufacture of penicillin. But their proposal was cold-shouldered. Companies didn’t find the prospect of money in it. It was largely because the yields of penicillin were very low, 4Units/ml of the purified fluid from the fungus; 1Unit is ~0.6 micrograms. Florey approached the US National Academy of Sciences and they directed him to a fungus expert, Dr. Charles Thom, who in turn sent the duo – Florey and Chain - to the Fermentation Division of the Northern Regional Research Laboratory at Peoria in Illinois. Success of their mission at last began at Peoria.

Scientists at NRRL scoured a number of P.Chrysogenum samples to find if particular molds gave better yields of penicillin. They rummaged through samples of fungi obtained from near and far (including a fungus sample from Bombay, now Mumbai) and employed several assistants for the job. One such assistant, Mary Hunt (later popularly known as Moldy Mary) brought a ripe cantaloupe from a nearby market which was covered all over with a mold of a golden hue. It turned out to be just a golden chance. This culture yielded 250 units /ml of penicillin. Upon tweaking it with mutagenetic (X-ray) irradiation, they could increase the concentration of penicillin to 2500 units/ml. It resulted in a bonanza for penicillin. We may note that Industrial strains today are reported to yield up to 50,000 units/ml. In the meantime, many University labs such as those at Stanford, Minnesota and Carnegie got involved in this research for two reasons; war time obligations and scientific novelty of the therapeutic namely penicillin.

Fig 3. Penicillium mold on a fruit

And that was it, Penicillin became available in quantities for treatment of all sorts of infections. In 1942 March, Anne Miller at the New Haven Hospital in Connecticut became the first patient to be successfully treated for infection arising after a miscarriage. In UK, Fleming cured Harry Lambert of a fatal infection, streptococcal meningitis, by spinally administering penicillin, in August 1942.

Recall, it was war time – World War II. Soldiers were dying more from infections than injuries. Penicillin was in great demand by the allies. The two governments, of UK and US came together to sponsor and support manufacturing penicillin. US didn’t have enough penicillin to treat even one patient in 1941 and at the end of 1942 it had barely enough to treat about 100 patients. But by the end of 1943 the entire demand for penicillin by the warring forces on their side could be met by the allies. We may note that while in World War I, 18% of all deaths were due to bacterial pneumonia, it was just less than 1% in World War II, thanks to penicillin.

During this time efforts were on in other European countries to make penicillin. Large parts of Europe were under Nazi occupation including northern France, Netherlands and Belgium. German intelligence learnt about the penicillin story. Dr. Schmidt in Germany tried to get the P. Chrysogenum from Pasteur Institute in France and the latter sent them the mold that didn’t yield penicillin. The story was roughly the same with Netherlands. Penicillin was not being made on large scale anywhere in Europe

However, a pharmaceutical company known as NG&SF in Netherlands, acquired the complete know how to manufacture penicillin from a Jewish scientist, Andries Querido, from Switzerland. But to manufacture penicillin they needed to hide their operations from a German guard posted by Nazis at their facility. After learning about the guard’s weakness for a particular brand of liquor they ensured that the guard received an uninterrupted supply of good quality gin. From there on manufacture of penicillin under the name, Bacinol, went on in Netherlands unnoticed by the Nazis.

Thus, the first antibiotic, penicillin, arrived as a savior of mankind, considered by many as the most important drug of the last century. Fleming himself had rightly exclaimed,

When I woke up just after dawn on September 28, 1928, I certainly didn’t plan to revolutionize all medicine by discovering the world’s first antibiotic, or bacteria killer. But I guess that was exactly what I did.”

Fleming was elected a Fellow of the Royal Society in 1943 and knighted in 1944. He was named in the list 100 Most Important People of the 20th century by Time magazine and as the the Third Greatest Scot by the Scottish Television. This great scientist, Sir Alexander Fleming, had the following to say in his Nobel Lecture:

We all know that chance, fortune, fate or destiny - call it what you will-- has played a considerable part in many of the great discoveries in science. We do not know how many, for all scientists who have hit on something new have not disclosed exactly how it happened. We do know, though, that in many cases it was a chance observation, which took them into a track which eventually led to a real advance in knowledge or practice. This is especially true of the biological sciences for there we are dealing with living mechanisms about which there are enormous gaps in our knowledge.

– a glorious testament to Fleming’s humility and a sobering thought for young scientists.

Chemical and crystal Structures of penicillin were determined later. The chemical structure of penicillin (C16H18N2O4S) was first proposed by Edward Abraham at Oxford in 1942. Dorothy Hodgkin determined the correct chemical structure of penicillin using X-ray crystallography, again at Oxford in 1945. It was also established that there are different forms of penicillin compounds all of which shared the same structural component called β-lactam (see below).

Fig 3 & 4. Structure of Penicillin

There was no dearth of high drama when it came to rewards and recognitions related to penicillin. When Harry Lambert was cured of meningitis by the use of penicillin, an influential newspaper, The Times, interviewed Fleming and wrote a big article on the development but didn’t even mention the names of Fleming or Florey.

That upset Fleming’s professor, Sir Almroth Wright, who wrote to The Times highlighting Fleming’s work and stating that Fleming deserved a “Laurel wreath” for the scientific contribution. This naturally infuriated Florey and his team but they chose to remain silent. Fleming himself was very modest about his contributions referring to the public admiration of his work as the “Fleming Myth”. He was also utterly human so much so that he refused to file a patent on penicillin. When he heard about American companies getting patents on penicillin, he was infuriated and said; “I found penicillin and have given it free for the benefit of humanity. Why should it become a profit-making monopoly of manufacturers in another country? “

“The Nobel Prize in Physiology or Medicine 1945 was awarded jointly to Sir Alexander Fleming, Ernst Boris Chain and Sir Howard Walter Florey”, "For the discovery of penicillin and its curative effect in various infectious diseases." What about Professor Heatley, without whose contribution penicillin would not be there. It appears that Sir Henry Harris once said, “without Fleming no Chain; without Chain, no Florey; without Florey, no Heatley, and without Heatley, no penicillin”. How true it was.

In 1990, after 45 years of Nobel recognition to his collaborators, this oversight of the Nobel Committee was sought to be assuaged by the Oxford University; they awarded Professor Norman Heatley the first ever Honorary Doctorate of Medicine in its 800-year history.

Let us now get back to serendipity.

When he returned to his laboratory after vacation, Fleming was only expecting healthy growth of staphylococci in his Petri dish, not the landing of a mold that generated penicillin and partly killed his bacteria. That was the serendipitous finding. Let us not forget the keen intellect of Fleming. His was a prepared mind that the luck favored.

There was another serendipitous discovery that got partially eclipsed in this whole affair. And that was Mary Hunt’s bringing to the lab that heavenly cantaloupe covered all over in golden mold. It yielded a lot more penicillin which instantly raised hopes of all around Florey and Heatley. The project would get halted otherwise.

That is why discovery of penicillin was an excellent example of serendipity.

There are plenty of serendipitous discoveries in science. Let us get to review some of the famous ones in coming months. If you want see the sources of this essay, go through:

1. Macfarlane G. Alexander Fleming: the man and the myth. Cambridge (MA): Harvard University Press; 1984.

2. Gaynes R. Paul Ehrlich and the magic bullet. In: Germ theory: medical pioneers in infectious diseases. Washington (DC): American Society for Microbiology Press; 2011. p. 250.

3. Robert Gains, Emerg. Infect Dis. 2017 May; 23(5): 849–853.