CYTOGENETIC ANALYSIS 475 



reported by Rothfels and Siminovitch. 1077 Tjio and Puck 1322 noted a final concentra- 

 tion of 0.3-0.5 y/ml. to be effective for cells of human, opossum, and Chinese hamster 

 in tissue culture. The length of colchicine treatment in vitro will vary considerably 

 from one cell type to another, depending on the generation time of the average cell. 

 Nevertheless, the minimum time for colchicine should be 90 minutes and the maximum 

 14-18 hours for cells having generation times of 14-24 hours. 



An interesting application of Vincaleukoblastine (VLB), an alkaloid of Vinca 

 rosea, as a mitotic arresting agent similar to Colcemid, is reported by Palmer. 989 

 Exceedingly low dosages of VLB, in vivo or in vitro, lead to mitotic arrest in a number of 

 species and strains of cells. The J-96, human, monocytic-leukemia cell is readily 

 arrested in metaphase at the low dosage rates of 0.01 and 0.001 gamma. Its value 

 may be applied whenever a cell undergoes a rate of mitosis slower than desired. 



Preparations of bone marrow using the technique of Ford and Hamerton 386 are 

 most adequate for the majority of species. Recently, the additional step of air drying 

 such preparations has eliminated the need for squashing delicate bone-marrow cells. 

 Bender has followed, essentially, Ford and Hamerton's procedures up through fixation 

 for 30 minutes. After centrifuging and resuspending in fresh fixative, small drops of 

 material are placed on clean, wet slides. As the drop spreads out and the preparation 

 dries, the cells flatten quite well, as noted in figure 54a-d. After drying completely, 

 several drops of aceto-orcein are added, followed by dehydration (3 : 1 and 95 per cent 

 alcohol) and mounting in Euparal. For similar preparations of embryonic cells of the 

 mouse, 0.2 mg. of heparin is added to 100 ml. of the citrate solution. This modifica- 

 tion is apparently detrimental for bone-marrow cells, but most helpful for embryos. 

 In the latter case, pregnant females are treated with colchicine (25 x 10 ~ 6 M/10 g. 

 body weight for 2-6 hours) . After sacrifice, the liver and spleen of the embryo are 

 dissected and placed in citrate solution. By means of aspirating with a 26-gauge 

 needle, a fairly uniform suspension of intact cells is obtained. Thereafter, the suspended 

 cells are treated exactly like bone-marrow cells (figure 54a) . 



The simplest procedure for softening surgically removed tissues for squashing is 

 that modified further by Makino and Sasaki 842 in which they employ distilled water, 

 followed by fixation with 50 per cent acetic acid. The absence of ethyl alcohol, as found 

 in Carnoy's 3 : 1 fixative, assures softness during subsequent but brief storage. With 

 tissue cultures, these same workers, using the method described by Awa et al., 41 trypsi- 

 nized and centrifuged cells previously treated with colchicine and attained hypo- 

 tonicity by adding an equal volume of tap or distilled water for 5-10 minutes. Direct 

 fixation and staining was accomplished with acetic dahlia (0.75 gm. of dahlia violet in 

 30 per cent acetic acid) . Examples of their observations are given in figures 55, 57, 

 and 61. Ohno, Kaplan, and Kinosita 964 ' 966 applied distilled water to seminiferous 

 tubules for the purpose of rendering spermatogonial chromosomes more favorable for 

 the squash technique. Pachytene bivalents, however, were badly affected by this 

 treatment and, to enhance pachytene morphology, storage of freshly removed semi- 

 niferous tubules, for two hours in the refrigerator, was found satisfactory (figure 59). 



