CYTOGENETIC ANALYSIS 485 



squashes as a means of determining which approach provides the better spread of 

 chromosomes for individual needs. Delaying wet squashes for some 48-72 hours 

 following fixation will help to harden glass-adhered mitoses for improved and intact 

 squashing onto siliconed slides. 



The procedure of Conger and Fairchild 221 for permanent preparations has 

 become universally adopted with slight modifications, such as the use of liquid nitrogen 

 for quicker freezing in place of dry ice (C0 2 ). 



PROCEDURES in vitro FOR CHINESE-HAMSTER CULTURES 



A culture vessel has been designed by the author to serve in a number of capacities 

 in current techniques of tissue culture (figures 67, 68, and Addendum) . The neck of the 

 vessel may be bent slightly to insure against accidental spillage and contamination 

 while the cap is left loosely turned when placed in a C0 2 incubator. The neck of 

 the tube is reduced, as contrasted to the lengthier version of the original Leighton tube, 

 to facilitate removal of the cover glass as well as to perform limited clonal isolation 

 procedures. 



The use of ferric hydrate or acetate mordant is relatively new for tissue cultures of 

 mammalian cells, even though it has been extensively incorporated in procedures for 

 the staining of plant chromosomes for well over thirty years. The use of razor blades 

 and repeated filtration of the resulting saturated solution of ferric acetate has eliminated 

 the adverse precipitation experienced in the past, particularly when staining botanic 

 specimens. To reiterate, acetocarmine is preferred as a universal stain because it 

 requires less care and rarely precipitates in the bottle after many months of exposure 

 at room temperature. It stains the inner components of the chromosome, leaving the 

 matrix clear. In contrast, aceto-orcein requires daily attention to remove trouble- 

 some precipitation, and it stains the entire thickness of the chromosome. Con- 

 sequently, the former stain leaves the chromatids slender and distinct whereas the 

 latter stain results in a thicker and more densely stained structure. Acetocarmine 

 assists in revealing structural entities, such as secondary constrictions and hetero- 

 chromatic segments that are consistently noted as being part of the more slender 

 portions of chromatin, especially along the length of the sex chromosomes (figures. 63 

 64, and 65). 



The use of petri dishes for the cultivation of cells onto flying cover glasses in a 

 C0 2 environment is, comparatively speaking, less efficient with regard to utilization of 

 medium and incubator space. When several cover glasses are placed in petri dishes, 

 they generally overlap or are readily disturbed while the contents are viewed with an 

 inverted microscope. In addition, an average of 5 ml. of medium is required for 

 each 60-mm. petri dish. This same amount of medium, when used with the culture 

 vessel described herein, will yield three times more surface. 



In the event excessive cytoplasmic staining is experienced, propionic acid may be 

 substituted in place of acetic acid for the preparation of stains. The chromosomes 



