488 GENETICS OF SOMATIC CELLS 



Preparation of iron mordant 



Place 6 to 10 razor blades (Gillette blue blades) in a loosely stoppered Erlenmeyer 

 flask and add 100 ml. of 45 per cent acetic acid. Agitate occasionally during a three 

 to six week period of brewing. When the solution is a deep amber color, filter re- 

 peatedly several times and store overnight. In the event of further precipitation, add 

 25-50 ml. of 45 per cent acetic acid and *efilter. Store without precipitation at room 

 temperature. A second batch of mordant should be left to age for several months ; a 

 gradual drying-out of the acetic acid and eventual erosion of the razor blades will 

 occur. By simply adding 50 ml. of 45 per cent acetic acid to the crumbled mass and 

 filtering, a very effective and concentrated mordant is made available immediately 

 upon filtration. The original corroded mass is retained for future needs. Several 

 hundred milliliters of mordant can easily be obtained in the manner described. 



Culture vessels are seeded with appropriate numbers of trypsinized cells (approxi- 

 mately 50-100,000 maximum) and kept either in a CO s incubator (loosely stoppered 

 bakelite cap) or a conventional incubator (tightly stoppered) depending on cellular 

 performance. When cells enter the log phase of growth some 48 hours later, colchicine 

 pretreatment, with the addition of 0.5-1.0 y/ml. of medium, is generally initiated. 

 Stock colchicine is prepared by dissolving 0.5 mg. of colchicine in 100 ml. of complete 

 medium to provide a concentrate of 5 y/ml. Five- to ten-ml. batches of filtered solu- 

 tion are stored at —4° C. until needed. Thereafter, prolonged storage at +4° C. is 

 adequate and 0.1-0.2 ml. of this stock colchicine solution is added to provide a final 

 concentration of 0.5-1.0 y/ml. in the supporting medium. 



The culture may be incubated for an additional two hours, or to a maximum 

 period of 15 hours, or overnight, depending on the experimental requirements and 

 generation time of the tissue employed. For example, some Chinese-hamster deriva- 

 tives have 10- to 12-hour generation times and one must act accordingly to minimize 

 the frequency of endoreduplications and polyploidy that can arise following prolonged 

 colchicinization. 



To reduce the degree of fuzziness displayed by the pellicle of chromosomes as a 

 result of hypotonic pretreatment, the time of exposure has been reduced from the 

 conventional period of 20 minutes to only 5 minutes. Hypotonicity is achieved by 

 adding one-half ml. (10 drops) of tap water to the supporting medium in the vessel. 

 After 5 minutes, 0.5-1.0 ml. of Carnoy's fixative (3:1) is added to the hypotonic 

 medium. Thus, the volume of solution in the vessel is approximately doubled at this 

 point. For best results, the culture tube should be stored at room temperature for at 

 least 18 hours to encourage some degree of hardening of the cellular membrane for 

 better squashes. In the event air-dried specimens are desired, several rinses with 50 

 per cent (one part 3 : 1 fixative and one part water) may be added to the schedule prior 

 to air drying. Media containing horse serum will generally exhibit a mucoidal 

 precipitate and should, therefore, be thoroughly rinsed and replaced with 50 per cent 

 3 : 1 fixative for delayed wet squashing. Fixed media containing fetal calf and other 

 kinds of sera are unaffected by acetic acid and remain clear for many months. 



