CYTOGENETIC ANALYSIS 



477 



Fetal chromosomes of the mouse were thoroughly disrupted after five minutes' pre- 

 treatment with distilled water. 615 Yerganian 1460 noted that 3 : 1 fixed and stored 

 human pachytene chromosomes remained satisfactory for many months, even after 

 detrimental effects of a lengthy necroscopy. Figure 60 illustrates Hauschka's un- 

 published method of pretreating testicular fragments in cold 50 per cent hypotonic 

 Earle's solution. A useful variant of a pretreatment procedure is one employed by 

 Tanaka and Kano. 1310 Following extirpation of the regenerated liver, small bits of 

 tissue were placed in tap water adjusted to a pH of 7.4 for about 18 minutes. The 

 water and tissue were heated to about 38° C. for 2-3 minutes before adding, in equal 

 parts, a 20 per cent solution of Sudan black in propionic acid. Squashes are made 



Fig. 59. Late diplotene bivalents of 



house mouse, Mus musculus. 



Fig. 60. First meiotic metaphase of 



MALE HOUSE MOUSE, MuS UlUSCuluS. 



Nineteen autosomal pairs and a densely 

 staining, X-Y pair showing end-to-end 

 association (no chiasma is formed) . 900 X. 

 Technique described by Ohno, Kaplan, 

 and Kinosita. 964 (Photograph provided 

 by S. Ohno.) 



Note end-to-end association of XY bi- 

 valent at top of plate. Acetic orcein squash 

 made after 20minutes treatment of testicular 

 (semininiferous tubules) fragments in cold 

 50% hypotonic Earle's solution. 1100 X 

 (Photograph by T. S. Hauschka.) 



after one hour of storage in the latter solution. Tonomura and Yerganian 1324 treated 

 regenerating liver by what is now considered the conventional scheme outlined herein 

 for tissue culture. Softening was regained by transferring the tissue (stored in 3: 1 

 solutions) into dilute, nonmordanted acetocarmine for several hours or overnight prior 

 to squashing. This procedure helped to alleviate scattering of chromosomes in other- 

 wise brittle hepatic cells that result after too lengthy a storage period in 3: 1 solution. 



The use of trypsin ( 1 per cent) to soften ovarian tissues for the release and squash- 

 ing of oocytes has been found to be most appropriate by Ohno, Kaplan, and Kinosita. 965 

 They generally employed 1- to 3-day-old females for pachytene-diplotene configurations. 



Pretreatment with hot water is not applicable for cells in tissue culture. Glass- 

 attached and suspended cultures may be handled in the manner described by Nowell 948 

 and Moorhead et a/. 893 The latter procedure is most effective in spreading colchicine- 

 resistant, Syrian hamster chromosomes (figure 56). 



It appears that excessive hydration increases fuzziness of metaphase chromosomes. 

 To offset this undesirable feature, the hypotonic pretreatment period has been reduced 

 to five minutes when handling tissue cultures. 1077 The slow addition (drop by drop) 



