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Slotin was the first of three children born to Israel and Sonia Slotin, Yiddish-speaking refugees who had fled the pogroms of Russia to Winnipeg, Manitoba. He grew up in the North End neighborhood of Winnipeg, an area with a large concentration of Eastern European immigrants. From his early days at Machray Elementary School through his teenage years at St. John's High School, Slotin was academically exceptional. His younger brother, Sam, later remarked that his brother "had an extreme intensity that enabled him to study long hours."

On 21 May 1946, with seven colleagues watching, Slotin performed an experiment that involved the creation of one of the first steps of a fission reaction by placing two half-spheres of beryllium (a neutron reflector) around a 3.5-inch-diameter (89 mm) plutonium core. The experiment used the same 6.2-kilogram (13.7 lb) plutonium core that had irradiated Harry Daghlian, later called the "demon core" for its role in the two accidents. Slotin grasped the upper 228.6 mm (9-inch) beryllium hemisphere with his left hand through a thumb hole at the top while he maintained the separation of the half-spheres using the blade of a screwdriver with his right hand, having removed the shims normally used. Using a screwdriver was not a normal part of the experimental protocol. At 3:20 p.m., the screwdriver slipped and the upper beryllium hemisphere fell, causing a "prompt critical" reaction and a burst of hard radiation. At the time, the scientists in the room observed the blue glow of air ionization and felt a heat wave. Slotin experienced a sour taste in his mouth and an intense burning sensation in his left hand. He jerked his left hand upward, lifting the upper beryllium hemisphere and dropping it to the floor, ending the reaction. However, he had already been exposed to a lethal dose of neutron radiation. At the time of the accident, dosimetry badges were in a locked box about 100 feet (30 m) from the accident. Realizing that no one in the room had their film badges on, "immediately after the accident Dr. Slotin asked to have the badges taken from the lead box and placed on the critical assembly". This peculiar response was attributed to "vertigo" and was of no value for determining the actual doses received by the men in the room. Others in the room at the time included Raemer E. Schreiber, Alvin Cushman Graves, Stanley Allan Kline, Marion Edward Cieslicki, Dwight Smith Young, Theodore P. Perlman, and Pvt. Patrick J. Cleary. As soon as Slotin left the building, he vomited, a common reaction from exposure to extremely intense ionizing radiation. Slotin's colleagues rushed him to the hospital, but the radiation damage was irreversible. By 25 May 1946, four of the eight men exposed during the incident had been discharged from hospital. The Army doctor responsible for the hospital, Captain Paul Hageman, said that Slotin's, Graves', Kline's and Young's "immediate condition is satisfactory."
Louis Slotin