Human chromosomes / E.H.R. Ford.

  • Ford, E. H. R. (Edward Hugh Rawlinson)
Date:
1973
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Human chromosomes / E.H.R. Ford. Source: Wellcome Collection.

    104/408 (page 84)
    84 HUMAN CHROMOSOMES However the technical problems have been overcome, as outlined in Chapter 1, by the use of colchicine (or other antimitotic substances) to arrest cells at metaphase and so accumulate dividing cells; and by treating cells with hypotonic solutions in order to swell them up before squashing. The essential stages in obtaining preparations of mammalian chromosomes are thus as follows: (a) The cells are usually obtained in suspension; either because they have been cultured in suspension (as in blood cultures) or by suspending them in a physiological fluid. Fibroblast cultures, which grow in flat sheets, can be suspended by treatment with trypsin to separate them from each other (though this is not essential); marrow or testicular cells can be suspended by breaking up the tissue with a pipette in a physiological solution (or in some other way). (b) The suspended cells are exposed to colchicine for a certain period. (c) The cells are centrifuged, the colchicine solution removed by pipetting off the supernatant fluid, and a hypotonic solution is added and allowed to remain in contact with the cells for a certain, rather precise period. (d) The hypotonic solution is removed by centrifugation and removal of supernatant fluid, and the cells are fixed by addition of a fixative which will not harden them too much for subsequent flattening to be achieved. (e) The cells are flattened on to a slide, either by squashing or by an air drying technique (see p. 87). (f) The preparation is stained, and, if necessary, mounted. There are a number of possible variations on each of these stages; those which give the best results for a particular tissue can be ascertained by trial and error. Probably nearly every worker and laboratory uses slightly different methods, and there is a certain mystique involved. However the method of any experienced worker, in his own hands (though not always in the hands of others), will usually give excellent results. The following comments are offered from the author's personal experience. B. Colchicine Pretreatment The alkaloid colchicine (prepared from the autumn crocus) is a very potent substance, which has long been used for the empirical treatment of gout. Very low concentrations are adequate to inhibit mitosis. Its synthetic analogue, colcemid (CIBA), deacetylmethyl colchicine, is commonly used instead of the natural alkaloid. It is