By March 1940 the Oxford team had sufficient impure penicillin to commence testing whether it was toxic. Over the next two months, Florey and Jennings conducted a series of experiments on rats, mice, rabbits and cats in which penicillin was administered in various ways. Their results showed that penicillin was destroyed in the stomach, but that all forms of injection were effective, as indicated by assay of the blood. It was found that penicillin was largely and rapidly excreted unchanged in their urine. [80] They found no evidence of toxicity in any of their animals. Had they tested against guinea pigs research might have halted at this point, for penicillin is toxic to guinea pigs. [81] In 1895, Vincenzo Tiberio, an Italian physician at the University of Naples, published research on moulds initially found in a water well in Arzano; from his observations, he concluded that these moulds contained soluble substances having antibacterial action. [17] Two years later, Ernest Duchesne at École du Service de Santé Militaire in Lyon independently discovered the healing properties of a P. glaucum mould, even curing infected guinea pigs of typhoid. He published his results in a dissertation in 1897. [18] Duchesne was using a discovery made earlier by Arab stable boys, who used moulds to cure sores on horses. He did not claim that the mould contained any antibacterial substance, only that the mould somehow protected the animals. Penicillin does not cure typhoid and so it remains unknown which substance might have been responsible. A Pasteur Institute scientist, Costa Rican Clodomiro Picado Twight, similarly recorded the antibiotic effect of Penicillium in 1923. In these early stages of penicillin research, most species of Penicillium were non-specifically referred to as P. glaucum, so that it is impossible to know the exact species and that it was really penicillin that prevented bacterial growth. [10]

Sheehan, John C.; Henery-Logan, Kenneth R. (5 March 1957). "The Total Synthesis of Penicillin V". Journal of the American Chemical Society. 79 (5): 1262–1263. doi: 10.1021/ja01562a063. ISSN 0002-7863. Sheehan, John C.; Henery-Logan, Kenneth R. (1962). "The Total and Partial General Syntheses of the Penicillins". Journal of the American Chemical Society. 84 (15): 2983–2990. doi: 10.1021/ja00874a029. ISSN 0002-7863.Efforts were made to coax the mould into producing more penicillin. Heatley tried adding various substances to the medium, including sugars, salts, malts, alcohol and even marmite, without success. [63] At the suggestion of Paul Fildes, he tried adding brewing yeast. This did not improve the yield either, but it did cut the incubation time by a third. [59] The team also discovered that if the penicillin-bearing fluid was removed and replaced by fresh fluid, a second batch of penicillin could be prepared, [59] but this practice was discontinued after eighteen months due to the danger of contamination. The mould had to be grown under sterile conditions. [64] Abraham and Chain discovered that some airborne bacteria produced penicillinase, an enzyme that destroys penicillin. [65] It was not known why the mould produced penicillin, as the bacteria penicillin kills are no threat to the mould; it was conjectured that it was a byproduct of metabolic processes for other purposes. [64]

Ethel and Howard Florey published the results of clinical trials of penicillin in The Lancet on 27 March 1943, reporting the treatment of 187 cases of sepsis with penicillin. [98] It was upon this medical evidence that the British War Cabinet set up the Penicillin Committee on 5 April 1943. The committee consisted of Cecil Weir, Director General of Equipment, as chairman; Alexander Fleming; Howard Florey; V. D. Allison, another one of Fleming's former research students; Sir Percival Hartley, the head of the MRC; and representatives from pharmaceutical companies. [99] This led to the mass production of penicillin by the next year. [100] Deep submergence for industrial production [ edit ] Laurence, William L. (6 May 1941). " 'Giant' Germicide Yielded by Mold; New Non-Toxic Drug Said to be the Most Powerful Germ Killer Ever Discovered". The New York Times . Retrieved 13 February 2023. a b Houbraken, J.; Frisvad, J. C.; Samson, R. A. (June 2011). "Fleming's penicillin producing strain is not Penicillium chrysogenum but P. rubens". IMA Fungus. 2 (1): 87–95. doi: 10.5598/imafungus.2011.02.01.12. PMC 3317369. PMID 22679592. For the effect on the cultures of staphylococci that Fleming observed, the mould had to be growing before the bacteria began to grow, because penicillin is only effective on bacteria when they are reproducing. Fortuitously, the temperature in the laboratory during that August was optimum first for the growth of the mould, below 20°C, and later in the month for the bacteria, when it reached 25°C. Had Fleming not left the cultures on his laboratory bench and put them in an incubator, the phenomenon would not have occurred. [41]Heatley developed a penicillin assay using agar nutrient plates in which bacteria were seeded. Short glass cylinders containing the penicillin-bearing fluid to be tested were then placed on them and incubated for 12 to 16 hours at 37°C. By then the fluid would have disappeared and the cylinder surrounded by a bacteria-free ring. The diameter of the ring indicated the strength of the penicillin. [67] An Oxford unit was defined as the purity required to produce a 25mm bacteria-free ring. [57] It was an arbitrary measurement, as the chemistry was not yet known; the first research was conducted with solutions containing four or five Oxford units per milligram. Later, when highly pure penicillin became available, it was found to have 2,000 Oxford units per milligram. [72] Yet in testing the impure substance, they found it effective against bacteria even at concentrations of one part per million. Penicillin was at least twenty times as active as the most powerful sulfonamide. [68] The Oxford unit turned out to be very small; treating a single case required about a million units. [73] By the mid-1950s, there were reports in the United States that milk was not curdling to make cheese. The FDA found that the milk was contaminated with penicillin, which was killing the bacteria required for cheesemaking. In 1963 the World Health Organization reported high levels of penicillin in milk worldwide. People who were allergic to penicillin could now get a reaction from drinking milk. [232] A committee chaired by Lord Netherthorpe was established in the UK in 1960 to inquire into the use of antibiotics in animal feed. In 1962, the committee recommended that restrictions on the use of antibiotics in animals be relaxed. It contended that the benefits were substantial and that even if bacteria became resistant, new antibiotics would soon be developed, and there was no evidence that bacterial resistance in animals impacted human health. [233] [234] Arseculeratne, S. N.; Arseculeratne, G. (May 2017). "A re-appraisal of the conventional history of antibiosis and Penicillin". Mycoses. 60 (5): 343–347. doi: 10.1111/myc.12599. PMID 28144986. S2CID 21424547. Penicillin patents became a matter of concern and conflict. Chain had wanted to apply for a patent but Florey had objected, arguing that penicillin should benefit all. [183] Florey sought the advice of Sir Henry Dale, the chairman of the Wellcome Trust and a member of the Scientific Advisory Panel to the British Cabinet, and John William Trevan, the director of the Wellcome Trust Research Laboratory. On 26 and 27 March 1941, Dale and Trevan met at Oxford University's Sir William Dunn School of Pathology to discuss the issue. Dale advised that patenting penicillin would be unethical. Undeterred, Chain approached Sir Edward Mellanby, then Secretary of the Medical Research Council, who also objected on ethical grounds. As Chain later admitted, he had "many bitter fights" with Mellanby, [184] [185] but Mellanby's decision was accepted as final. [186]