4-92 GENETICS OF SOMATIC CELLS 



morphology of attached and spread cells. However, the length of time required to 

 conduct the staining, along with the many solutions needed for the schedule, tend to 

 make such preparations a major undertaking. The procedure outlined above permits 

 rapid and better visualization than staining with hematoxylin and eosin. The 3:1 

 fixative yields nuclear details with distinct clarity. Chromocentral masses, nucleoli, 

 sex chromatin (in man), and cytoplasmic vacuoles and metabolic by-products are 

 noticeably enhanced when using phase microscopy. 



Nuclear and cytoplasmic features serve as phenotypic expressions among a large 

 number of Chinese-hamster clones. Variations or deviations from classic diploidy, loss 

 of sex heterochromatin, monosomaty for distinct autosomes and the like, reflect in the 

 appearance of nuclei of 3:l-fixed cultures. The clonal cell can be utilized to record 

 its specific phenotype-karyotype complex most conveniently with the above schedules 

 for chromosomes and general morphology. 



Routine for tissue culture 



Schedules can readily dominate the attention of a cytologist beyond the desired 

 limit. To help minimize routines, this laboratory changes the medium in flasks only 

 twice weekly in place of the three changes customarily recommended. Also, in the 

 process of changing the medium, only half the volume is replaced in large farming 

 bottles, thereby conserving better than 50 per cent of the expense and labor con- 

 ventionally assigned to the preparation of media. Literally thousands of cultures 

 have been handled this way most favorably, and none have displayed deleterious 

 effects. The media of culture tubes are never changed and thereby retain the initial 

 1-1 -j ml. of medium provided at the time of seeding. Stock colchicine is added to the 

 partly spent medium at the prescribed time, or at the start of various pretreatments. 

 This may occur as late as three days after initiating the tube culture. Chinese 

 hamster cells proliferate rapidly in neutral or slightly acid media. In the absence of a 

 change of medium, acidity still favors rapid metabolism and proliferation. Con- 

 servation of media is also extended to routines of tending to petri dishes placed in the 

 C0 2 incubator for cloning trials (figure 66). In these instances, the medium is never 

 changed during a 7- to 10-day period of incubation. 



Every effort is made to insure sterility of the media and other culture solutions, to 

 control the temperature of the incubators, and to regulate the amount of C0 2 being 

 delivered to the incubators. Adequate pretesting of media for sterility with surplus 

 cultures, and with thioglycollate (Difco Manual) is one of the basic steps to help retain 

 genetic continuity of unique sublines. 



Freezing and storage of pretested media is also an essential precaution, particularly 

 if minimal inoculations of cells are to be done. Such procedures appear to be un- 

 necessary or disregarded in laboratories where detection of genetic variations is of 

 secondary importance. Researchers should be aware of the cytologic progression and 

 cellular contaminations that have occurred among the various strains, 1076 despite 

 efforts to characterize given types at various repositories. 206 



