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Fleming was born on 6 August 1881 at Lochfield, a farm near Darvel in Ayrshire, Scotland. He was the third of the four children of Hugh Fleming (1816-1888) from his second marriage to Grace Stirling Morton (1848-1928), the daughter of a neighbouring farmer. Hugh Fleming had four surviving children from his first marriage. He was 59 at the time of his second marriage, and died when Alexander (known as Alec) was seven.

Fleming went to Loudoun Moor School and Darvel School, and earned a two-year scholarship to Kilmarnock Academy before moving to London where he attended the Royal Polytechnic Institution.[4] After working in a shipping office for four years, the twenty-year-old Fleming inherited some money from an uncle, John Fleming. His elder brother, Tom, was already a physician and suggested to his younger sibling that he follow the same career, and so in 1903, the younger Alexander enrolled at St Mary's Hospital Medical School in Paddington. He qualified MBBS from the school with distinction in 1906.

By chance, however, he had been a member of the rifle club (he had been an active member of the Volunteer Force since 1900). The captain of the club, wishing to retain Fleming in the team suggested that he join the research department at St Mary's, where he became assistant bacteriologist to Sir Almroth Wright, a pioneer in vaccine therapy and immunology. He gained a BSc with Gold Medal in 1908, and became a lecturer at St Mary's until 1914. On 23 December 1915, Fleming married a trained nurse, Sarah Marion McElroy of Killala, County Mayo, Ireland.

Fleming served throughout World War I as a captain in the Royal Army Medical Corps, and was Mentioned in Dispatches. He and many of his colleagues worked in battlefield hospitals at the Western Front in France. In 1918 he returned to St Mary's Hospital, where he was elected Professor of Bacteriology of the University of London in 1928.

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