Editing Howard Florey (section)

=== Development ===
In the course of his work on lysozyme, Chain read papers on the enzyme in the ''British Journal of Experimental Pathology'' by [[Alexander Fleming]] in volumes 3 and 8, and by Florey in volume 11. While doing so he came across Fleming's paper discussing the antibacterial effects of ''[[Penicillium notatum]]'' mould in volume 10. The erroneous impression given by Fleming that penicillin was a [[Bactericide|bactericidal]] enzyme led Chain to consider that it would be similar to lysozyme.{{sfn|Macfarlane|1979|pp=281–283}} Money was short at the time; the office had an overdraft of £500 ({{Inflation|UK|500|1935|fmt=eq|r=-3|cursign=£}}) and Florey had to forbid the purchase of any further equipment. Chain and Florey decided to create a large research project on antibacterial substances produced by micro-organisms that could attract long-term funding.{{sfn|Chain|1971|p=297}} Three sources were initially chosen for investigation: ''[[Bacillus subtilis]]'', ''[[Trueperella pyogenes]]'' and [[penicillin]] mould.{{sfn|Macfarlane|1979|p=285}} Florey later said: 
{{blockquote|People sometimes think that I and the others worked on penicillin because we were interested in suffering humanity. I don't think it ever crossed our minds about suffering humanity. This was an interesting scientific exercise, and because it was of some use in medicine is very gratifying, but this was not the reason that we started working on it.<ref>{{Cite web |url=http://www.asap.unimelb.edu.au/bsparcs/exhib/nobel/florey.htm |title=Howard Walter Florey |last1=Sutherland |first1=Denise |last2=Tenkate |first2=Elissa |date=1998-02-19 |work=Australian Nobel Laureates |publisher=[[University of Melbourne]] |access-date=21 February 2019 |archive-date=24 March 2019 |archive-url=https://web.archive.org/web/20190324145611/http://www.asap.unimelb.edu.au/bsparcs/exhib/nobel/florey.htm |url-status=live }}</ref>}}

Florey approached the MRC for funding in September 1939, shortly after the outbreak of the [[Second World War]], and Mellanby authorised the project, allocating £250 ({{Inflation|UK|250|1939|fmt=eq|cursign=£}}) to launch the project, with £300 for salaries and £100 for expenses per annum for three years.{{sfn|Macfarlane|1979|pp=300–303}} Florey felt that more would be required. On 1 November 1939, Henry M. "Dusty" Miller, Jr, from the Natural Sciences Division of the Rockefeller Foundation dropped by to discuss funding Heatley's position. Heatley had fallen out with Chain, and had accepted a new position at the [[Carlsberg Laboratory]] in [[Copenhagen]] on a Rockefeller Fellowship, but due to the outbreak of the [[Second World War]], he had decided to remain at Oxford. Miller arranged for Heatley to retain his fellowship. Instead of working for Chain, Heatley would report directly to Florey as his personal research assistant.{{sfn|Macfarlane|1979|pp=300–303}}{{sfn|Mason|2022|pp=119–121}}

[[File:Penicillin development (43818514272).jpg|thumb|left|[[Blue plaque]] commemorating the isolation and purification of penicillin at the [[Sir William Dunn School of Pathology]] in Oxford]]
As a bacteriologist, Miller was enthusiastic about the antibacterial project; he encouraged Florey to apply for a grant from the Rockefeller Foundation, and recommended to his headquarters that the request for financial support be given serious consideration.{{sfn|Mason|2022|pp=119–121}}{{sfn|Jonas|1989|pp=267–268}} "The work proposed", Florey wrote in his application letter, "in addition to its theoretical importance, may have practical value for therapeutic purposes."{{sfn|Jonas|1989|p=269}} Florey's application was approved, with the Rockefeller Foundation allocating US$5,000 (£1,250) per annum for five years.{{sfn|Macfarlane|1979|pp=300–303}}{{sfn|Mason|2022|pp=119–121}}

Florey's team already had a sample of penicillin mould; Dreyer had been given a sample of the mould in 1930 for his work on [[bacteriophage]]s. He had lost interest in penicillin when he discovered that it was not a bacteriophage, but Campbell-Renton had continued to cultivate it.{{sfn|Hobby|1985|pp=64–65}}{{sfn|Wilson|1976|p=156}} The team developed techniques for growing the mould on a surface of liquid [[Czapek-Dox medium]]. Most laboratory containers did not provide a large, flat area, so glass bottles laid on their sides were used.{{sfn|Macfarlane|1979|pp=306–307}} Later, specially-made containers were fabricated.{{sfn|Macfarlane|1979|p=316}} As the laboratory gradually became a penicillin factory, Florey hired six women to perform the cultivation and extraction work.{{sfn|Macfarlane|1979|p=325}} It had to be carried out under sterile conditions;{{sfn|Macfarlane|1979|p=325}} Abraham and Chain discovered that some airborne bacteria produced [[penicillinase]], an enzyme that destroys penicillin.<ref>{{cite journal | title=An Enzyme from Bacteria Able to Destroy Penicillin |last1=Abraham |first1=E. P. |author-link=Edward Abraham |last2=Chain |first2=E. |author-link2=Ernst Chain |journal=[[Nature (journal)|Nature]] |issn=0028-0836 |year=1940 | volume=46 |page=837 |doi=10.1038/146837a0 |issue=3713 |bibcode=1940Natur.146..837A |s2cid=4070796 |doi-access=free }}</ref>

Heatley and Chain tackled the problem of how penicillin could be extracted from the mould. The liquid was filtered through parachute silk to remove the [[mycelium]], [[spore]]s and other solid debris.{{sfn|Macfarlane|1979|pp=305–308}} The [[pH]] was lowered by the addition of [[phosphoric acid]] and the liquid was then cooled.<ref>{{cite journal|last1=Abraham |first1=E. P. |author-link1=Edward Abraham |last2=Chain |first2=E. |author-link2=Ernst Chain |last3=Fletcher |first3=C. M. |last4=Florey |first4=H. W. |author-link4=Howard Florey |last5=Gardner |first5=A. D. |author-link5=Arthur Duncan Gardner |last6=Heatley |first6=N. G. |author-link6=Norman Heatley |last7=Jennings |first7=M. A. |author-link7=Margaret Jennings (scientist) |date=16 August 1941 |title=Further Observations on Penicillin |journal=[[The Lancet]] |issn=0031-6970 |volume=238 |issue=6,155 |pages=177–189 |doi=10.1016/S0140-6736(00)72122-2 |pmid=1541313}}</ref> In this form the penicillin could be drawn off by a solvent. Initially [[diethyl ether]] was used,{{sfn|Macfarlane|1979|pp=305–308}}{{sfn|Mason|2022|pp=122–123}}  but it is highly inflammable.<ref>{{cite web |title=Research School of Chemistry Safety Regulations |date=July 2011 |publisher=Australian National University |url=http://rsc.anu.edu.au/~perri/RSC_Web/RSC%20Safety%20Regulations%20July%202011.pdf |access-date=7 July 2023 |archive-date=12 July 2012 |archive-url=https://web.archive.org/web/20120712175122/http://rsc.anu.edu.au/~perri/RSC_Web/RSC%20Safety%20Regulations%20July%202011.pdf |url-status=live }}</ref> At Chain's suggestion, they tried the much less flammable [[amyl acetate]], and found that it also worked. Penicillin-bearing solvent was easily separated, but now they encountered the problem that had stymied earlier attempts: recovering the penicillin from the solvent. Heatley reasoned that if the penicillin could pass from water to solvent when the solution was [[acid]]ic, maybe it would pass back again if the solution was [[alkaline]]. Florey told him to give it a try. This method, which Heatley called "reverse extraction", was found to work.{{sfn|Macfarlane|1979|pp=305–308}}{{sfn|Williams|1984|pp=69–70}}{{sfn|Mason|2022|pp=122–123}} Chain hit upon the idea of [[freeze drying]] to enable the water to be removed without damaging the penicillin. The team had thus developed a complete process for growing, extracting and purifying penicillin, resulting in a dry, brown powder.{{sfn|Macfarlane|1979|pp=305–308}}{{sfn|Wilson|1976|pp=158–159}} By early 1942, they could prepare a highly purified compound,<ref>{{Cite journal |last1=Abraham |first1=E. P. |last2=Chain |first2=E. |date=1942 |title=Purification of Penicillin |journal=[[Nature (journal)|Nature]] |issn=0028-0836 |volume=149 |issue=3,777 |pages=328 |doi=10.1038/149328b0 |bibcode=1942Natur.149..328A |s2cid=4122059|doi-access=free }}</ref> and had proposed the chemical formula.<ref>{{Cite journal |last1=Abraham |first1=E. P. |author-link=Edward Abraham |last2=Baker |first2=W. |last3=Chain |first3=E. |author-link3=Ernst Chain |last4=Florey |first4=H. W. |author-link4=Howard Florey |last5=Holiday |first5=E. R. |last6=Robinson |first6=R. |date=1942 |title=Nitrogenous Character of Penicillin |journal=[[Nature (journal)|Nature]] |issn=0028-0836 |volume=149 |issue=3,778 |page=356 |doi=10.1038/149356a0 |bibcode=1942Natur.149..356A |s2cid=4055617|doi-access=free }}</ref> Heatley developed an assay method. An Oxford unit was defined as the purity required to produce a 25&nbsp;mm bacteria-free ring.{{sfn|Sheehan|1982|p=30}} It was an arbitrary measurement, as the chemistry of penicillin was not yet known; the first research was conducted with solutions containing four or five Oxford units per milligram. Later, highly pure penicillin became available with 2,000 Oxford units per milligram.{{sfn|Williams|1984|p=99}}
[[File:Penicillin Past, Present and Future- the Development and Production of Penicillin, England, 1943 D16965.jpg|thumb|right|A laboratory worker sprays a solution containing penicillin mould into flasks of [[corn steep liquor]] to encourage further penicillin growth.]]

The team discovered that ''Penicillium'' extract killed several types of bacteria. Gardner and Orr-Ewing tested it against [[gonococcus|gonococci]] (against which it was most effective), ''[[meningococcus|meningococci]]'', ''[[streptococcus|streptococci]]'', ''[[staphylococcus|staphylococci]]'', [[anthrax bacteria]], ''[[Actinomyces]]'' and the organisms that caused [[tetanus]] and [[gangrene]]. Florey and Jennings experimented on rats, mice, rabbits and cats in which penicillin was administered in various ways,{{sfn|Macfarlane|1979|pp=308–312}} and found no evidence of toxicity.{{sfn|Sheehan|1982|p=32}} On 25 May 1940, Florey injected eight mice with a virulent strain of ''streptococcus'', and then four of them with penicillin. A day later all four of the untreated mice were dead, but all of the treated ones were still alive, although one died two days later.{{sfn|Macfarlane|1979|pp=313–316}} Over the following weeks Jennings and Florey repeated the experiment with ever-larger batches of mice, and with different bacteria. They found that penicillin was also effective against ''staphylococci'' and the bacteria that cause gangrene.{{sfn|Macfarlane|1979|pp=319–320}} They published their findings in ''[[The Lancet]]'' on 24 August 1940.<ref>{{Cite journal |last1=Chain |first1=E. |author-link1=Ernst Chain |last2=Florey |first2=H. W. |author-link2=Howard Florey |last3=Adelaide |first3=M. B. |last4=Gardner |first4=A. D. |author-link4=Arthur Duncan Gardner |last5=Heatley |first5=N. G. |author-link5=Norman Heatley |last6=Jennings |first6=M. A. |author-link6=Margaret Jennings (scientist) |last7=Orr-Ewing |first7=J. |last8=Sanders |first8=A. G. |date=1940 |title=Penicillin as a Chemotherapeutic agent |url=https://pubmed.ncbi.nlm.nih.gov/8403666 |journal=[[The Lancet]] |issn=0140-6736 |volume=236 |issue=6104 |pages=226–228 |doi=10.1016/S0140-6736(01)08728-1 |pmid=8403666 |access-date=8 February 2023 |archive-date=31 October 2021 |archive-url=https://web.archive.org/web/20211031222612/https://pubmed.ncbi.nlm.nih.gov/8403666/ |url-status=live }}</ref> Florey reminded his staff that as promising as their results were, a man weighed 3,000 times as much as a mouse.{{sfn|Mason|2022|p=152}}

In February 1941, Florey and Chain treated their first patient, [[Albert Alexander (police officer)|Albert Alexander]], who had had a small sore at the corner of his mouth, which then spread, leading to a severe facial infection involving ''streptococci'' and ''staphylococci''. His whole face, eyes and scalp were swollen to the extent that he had had an eye removed to relieve the pain. Within a day of being given penicillin, he started to recover. However, the researchers did not have enough penicillin to help him to a full recovery, and he relapsed and died. Because of this experience and the difficulty in producing sufficient penicillin, Florey switched his focus to children, who could be treated with smaller quantities of penicillin.{{sfn|Macfarlane|1979|pp=330–331}}

Florey expected that penicillin would be hailed as a breakthrough, but he was disappointed; his results aroused little interest. He spent the next two years attempting to generate interest in what he believed to be the most important medical discovery of the century.{{sfn|Macfarlane|1979|p=322}} He was elected a [[Fellow of the Royal Society]] in March 1941,{{sfn|Williams|1984|p=128}}<ref>{{cite news |url=http://nla.gov.au/nla.news-article74465805 |title=Out Among the People |newspaper=[[The Advertiser (Adelaide)]] |location=South Australia |date=5 June 1941 |access-date=9 February 2023 |page=15 |via=National Library of Australia |archive-date=25 October 2023 |archive-url=https://web.archive.org/web/20231025104213/https://trove.nla.gov.au/newspaper/article/74465805 |url-status=live }}</ref> but his work with penicillin played little part in this.{{sfn|Macfarlane|1979|p=346}}