how we counted human chromosomes

The Mechanism

*Joe Hin Tjio* (born Pekalongan, Dutch East Indies, *2 November 1919*; died Gaithersburg, Maryland, *27 November 2001*, aged 82 — an ethnically Chinese Indonesian who had spent the Second World War in a Japanese internment camp on Java, escaped to mainland Asia after the war, and emigrated to Europe in 1947) had been working since 1948 as a plant cytologist at the *Estación Experimental de Aula Dei*, the rural Spanish agricultural research station outside Zaragoza. Each summer from 1948 onwards he travelled to the *Institute of Genetics of the University of Lund*, in southern Sweden, to spend the European summer working in the laboratory of the Swedish geneticist *Albert Levan* (born Gothenburg, *8 March 1905*; died Lund, *28 March 1998*, aged 93), the institute's professor of cytology. Through the late 1940s and early 1950s Levan's laboratory had been a centre of *plant cytogenetics* — the microscopic counting and morphological analysis of chromosomes in plant tissues, principally *Allium* (onions) — and had produced a series of methodological refinements that would matter to what came next: the *hypotonic-shock* pretreatment of cells, which makes chromosomes spread out on the microscope slide instead of bunching into an unreadable knot; the use of *colchicine*, an alkaloid extracted from the autumn crocus, to arrest dividing cells at the metaphase stage where the chromosomes are maximally condensed and individually visible; and the use of *tissue culture* to obtain a uniform population of actively dividing cells from a single starting tissue sample. None of these techniques had been applied to *human* tissue. The dominant view of the human diploid number, since *Theophilus Shickel Painter's* 1921 *Science* abstract and 1923 *Journal of Experimental Zoology* paper based on testicular biopsies of *three institutionalised mental patients* who had been castrated for medical reasons (a procedure considered routine at the time for treatment of "Negro lunatics" in the Texas state hospital where Painter had been working), was that humans had *48 chromosomes* — 24 pairs. Painter had himself acknowledged in 1923 that *"the number is either 46 or 48 ... I feel that the diploid number 48 is correct"* — the count of 48 was reported as definite in the published abstract but as preferred-over-46 in the full paper. The 48-chromosome consensus was reinforced by *Theophilus Painter and Henry Eldridge Crampton 1929*, by *Cyril Dean Darlington 1932*, by *Jack Schultz 1936*, and by every karyotyping textbook from 1923 to 1955. Histological textbooks reproduced Painter's photographs in support. *Sajiro Makino and S. Sasaki 1953*, in a *Cytologia* paper using Japanese tissues, reported a count of 48. In late 1955 Tjio, in Spain, applied for and received a *Spanish state visiting fellowship* to work in Lund for the late autumn and winter of 1955-56, with the explicit goal of *applying Levan's plant-cytogenetic methods to human tissue*. The starting material was *cultured fibroblast tissue from four 12-week-old human embryonic lungs*, supplied to Levan's laboratory by the Swedish gynaecologist *Rune Grubb* of the Department of Obstetrics and Gynaecology, Lund University Hospital, from legally terminated pregnancies. Tjio cultured the fibroblasts in a modified Hanks' balanced salt solution, applied a 30-minute *colchicine* arrest of metaphase, performed the *hypotonic-shock* pretreatment with 0.95% sodium citrate, fixed in *acetic-methanol* (3:1), squashed onto glass microscope slides, and stained with *orcein*. The improvement in chromosome separation over Painter's testicular-tissue squashes was visually obvious immediately — Tjio's metaphase plates showed *individually-resolvable, well-separated, condensed chromosomes* without the clumping that had made nineteenth- and early-twentieth-century counts so difficult. On the *evening of 22 December 1955*, working in the basement laboratory of the Institute of Genetics on Sölvegatan in Lund, Tjio examined fixed metaphase spreads from the four fetal lung cultures under a Leitz monocular microscope, took photographs through the microscope objective (using a 35-mm camera he had personally built, modelled on the darkroom-camera his father — a portrait photographer in colonial Java — had taught him to assemble), developed the photographs the same night in the laboratory darkroom, and counted the chromosomes on the enlarged prints under a desk lamp. The count in every cell was *46*. The result was so unexpected that Tjio's initial response was to assume the four embryos he had been given were chromosomally abnormal — that he had received a non-representative sample of pathological tissue. He counted *261 cells* across the four cultures over the next month; *242 cells* showed exactly 46 chromosomes, *7 cells* showed 47, *7 cells* showed 45, and the remaining 5 cells were unclear because of overlap. The modal number was 46, beyond any doubt. The paper was submitted to *Hereditas*, the journal of the Mendelian Society of Lund, on *26 January 1956*, listing *Tjio and Levan* as authors. Tjio insisted on first authorship — a contentious arrangement, given that the work was done in Levan's laboratory using Levan's methods, but Tjio's photographs were the proof. The paper, titled *"The Chromosome Number of Man,"* appeared in *Hereditas* vol. 42, pp. 1-6, in April 1956. Its opening paragraph was characteristically restrained: *"In 1921 Painter reported, in two short communications, the somatic chromosome number of man to be 48 ... we have, however, been led to suspect that the chromosome number 48 is incorrect."* The body of the paper presented sample photographs of metaphase spreads with the 46 chromosomes individually circled and identified by relative length and centromere position. The community response was immediate and almost entirely accepting. *Charles Ford and John Hamerton*, at the Medical Research Council's *Radiobiology Research Unit* at Harwell in England, had been independently working on chromosome counts in *human testicular biopsies* — using methods adapted from Tjio and Levan's earlier work in plant cytology — and confirmed the count of 46 in adult human male meiotic preparations within weeks. Their paper, *"The Chromosomes of Man,"* appeared in *Nature* on 26 May 1956, two months after Tjio and Levan. Within three years, the new field of *human cytogenetics* had identified the chromosomal basis of *Down syndrome* (Lejeune, Gautier, and Turpin, *Comptes Rendus de l'Académie des Sciences* Paris, 26 January 1959 — an extra copy of chromosome 21, the *trisomy 21* now known to be the cause of Down syndrome); the chromosomal basis of *Klinefelter syndrome* (Jacobs and Strong, *Nature*, 4 April 1959 — an XXY rather than XY sex-chromosome complement); and the chromosomal basis of *Turner syndrome* (Ford, Jones, Polani, de Almeida, and Briggs, *The Lancet*, 4 April 1959 — a single X, denoted XO). Within the decade the entire field of medical genetics would be redrawn around the human karyotype. Tjio remained at Aula Dei until 1959 and then joined the *National Institutes of Health* in Bethesda, Maryland, where he ran a cytogenetics laboratory in the National Institute of Arthritis and Metabolic Diseases until his retirement in 1992. Albert Levan continued as professor of cytology in Lund until 1971, was elected to the *Royal Swedish Academy of Sciences*, and never received the Nobel Prize that many of his colleagues thought the chromosome work deserved. The *original Tjio-Levan 1956 photographic plates* are held in the *Lund University archives* in the Institute of Genetics building on Sölvegatan, where the basement laboratory itself was preserved as a historical workspace until the building's late-1990s renovation. Painter's original 1923 count of 48, by contrast, has had a quieter afterlife. The reason the wrong number persisted for thirty-three years was not because Painter was incompetent — his sample preparation was simply not good enough, and the chromosomes in his squashes overlapped sufficiently that any of several counts in the 46-49 range could be supported by the same photographs. Once Tjio and Levan's improved hypotonic-shock-and-colchicine method made individual chromosomes routinely resolvable, the textbook number changed in a single publishing cycle.