CYTOGENETIC ANALYSIS 487 



fluid containing several million cells and restoppered with the cotton plug. The am- 

 poules are sealed some 20 minutes later with the aid of a small propane torch and placed 

 in a small rack or container on the top shelf of an upright deep freezer at —4° C. for 

 several hours. Thereafter, the vials are stored directly at —79° C. in a cabinet 

 designed by Stulberg et a/. 1301 Practically all types of Chinese-hamster cells (over 

 200 genetic clones) are favorably preserved when prepared in this simple manner. 

 Maximum periods of storage and percentages of successful regrowth have yet to be 

 determined. It appears, however, that other laboratories have had equivalent 

 success without the necessity of following the more elaborate approach of slow freezing 

 as described by Hauschka et al. 53i Both approaches have been employed with tissues 

 of the Chinese hamster, and time will prove which is the better method. In this 

 manner, genetic continuity is assured and restoration is greatly facilitated in the event 

 of sudden, undesirable shifts in the karyotype or outright losses due to accidents. 



Wet-squash preparations of colchicine-arrested metaphases generally exhibit 

 minimal overlapping of chromosomal arms. Some care, of course, must be taken to 

 assure cytoplasmic integrity as contrasted to the safety of air-drying procedures. 

 Nevertheless, various strains respond differently in regard to this vital feature. 



Squashing flattens cells more effectively than drying in air, particularly when 

 cellular membranes are exceedingly elastic or excessive clumping of chromosomes 

 results as a response to certain treatments. In the event air-dried cells on a cover glass 

 are preferred, the procedures outlined by Rothfels and Siminovitch 1077 and Tjio and 

 Puck 1322 are readily combined to handle cells cultured in these vessels, along with a 

 saving of several ml. of medium per trial. 



The use of ferric acetate as a mordant for carmine stains will be discussed in detail. 

 This adaptation, stemming from Belling's classical developments in plant cytology, has 

 been found useful by the author for both phase-contrast and bright-field microscopy 

 of mammalian chromosomes. Chromosomes stain intensely, along with minimal 

 uptake of the dye in the cytoplasm. 



Preliminary preparations 



Several ounces of cover glasses (0 thickness, 10 x 45 mm., or less) are placed in a 

 beaker containing 100 ml. of a 1 per cent solution of 7 X detergent and heated for a 

 period of about one-half hour. The cover glasses are rinsed thoroughly with running 

 tap water for several hours, with frequent agitation. Then they are rinsed and 

 agitated thoroughly with distilled water and stored overnight in it. After draining the 

 distilled water, 70-85 per cent ethyl alcohol is added and the jar or container covered 

 tightly until needed. When cover glasses are placed in culture vessels, they are 

 removed individually from the alcohol with the aid of forceps and wiped dry with lens 

 paper to prevent fingertip smudges along the edges which are excellent adjuncts for 

 cell proliferation. The cover glass is placed in the culture vessel and stoppered loosely 

 for autoclaving. (Caution: The rubber-lined bakelite cap should be tightened only 

 after there has been sufficient cooling following the autoclaving.) 



