10. b. SMEAR METHOD FOR 

 CHROMOSOME PREPARATIONS* 



Very recently it has become possible to prepare and examine chromosomal complexes of 

 various animals, including man, by the smear method. Entire chromosomal configura- 

 tions from somatic or germinal cells may be studied without the tedium of sectioning and 

 reconstituting. 



All the methods now used are based upon two rather simple innovations. These include 

 the treatment of dividing cells with hypotonic media prior to fixation, and then flattening 

 the cells prior to examination. In this manner the chronnosomes are spread with mini- 

 mum overlapping, all within a single optical plane, allowing photographic karyotype 

 analysis and recording. The chromosomes are cut out of an enlarged print of a metaphase 

 cell and paired, on the basis of relative lengths and the position of the centromere. This 

 shows the karyotype of the individual, and genetic variations become evident. The photo- 

 graphs are generally taken under oil immersion or phase microscopy, at approximately 

 1100 X or even less. 



1. MITOTIC CHROMOSOMES : The source of rapidly dividing cells may be the embryo, 

 malignant tissue, testis, or other tissues that may be growing or regenerating. 

 Tissue cultures afford an excellent source, and in these bone marrow, solid tissues, 

 or blood cells may be used and studied. 



a. Blood Cell Cultures : A 10 ml heparinized blood sample is centrifuged at 300 rpm 

 for 2 minutes and the plasma, which contains the white blood cells, is pipetted 

 off and placed in a sterile culture bottle. Tissue culture medium 199 is added, 

 making up about 60-80 percent of the final volume. The cell concentration should 

 be 1, 000-2, 000 cells/mm . Penicillin and streptomycin are also added, plus a 

 mitogenic agent, Phytohemagglutinin. After incubation for 3 days at 37°C. , 

 Colchicine (2 x 10" molar final concentration) is added for 1 hour. The cells are 

 spun down in a centrifuge (300 rpm for 3 minutes) and the medium is replaced 

 with 1% sodium citrate solution. After 2-4 minutes this is removed (after cen- 

 trifugation) and the acetic alcohol added. Several changes of this fixative may be 

 used, after which a drop of the cell suspension can be placed on a slide and al- 

 lowed to air dry. This preparation can be stained with acetic orcein and is then 

 ready for examination. Alternatively, the acetic alcohol can be replaced with 45% 

 acetic acid and a small drop of this cell suspension squashed beneath a siliconized 

 coverslip. This slide can be floated off at the time of staining with acetic orcein 

 and made permanent, or the stain can be applied by capillary action and the 

 chromosomes examined in the wet preparation, (see Hungerford et al, 1959). 



b. Bone Marrow : A bone marrow sample may be incubated for 6-24 hours in a 

 medium made up of glucose-saline and human AB serum. The treatment of. the 

 cells at the end of the period of incubation is the same as described above for 

 blood cell samples, (see Ford, Jacobs, Lajtha 1958). The quality of the prepara- 

 tion can usually be enhanced by separating the marrow cells from the large num- 

 ber of erythrocytes present in marrow samples, by using TC-199 instead of 

 glucose-saline, and by using the patient's own plasma in place of human AB serum. 



Bone marrow can also be cultured by a plasma clot technique such as is described 

 on the following page for solid tissues. 



* This section is written with the generous aid of Dr. O. J. Miller, who also provided the excellent photographs of human chro- 

 mosomes. 



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