486 GENETICS OF SOMATIC CELLS 



appear slimmer (less or no matrical staining) than that resulting from acetocarmine. 

 The most intense staining results when acetocarmine and aceto-orcein are mixed (1:1) 

 and further amplified with the aid of the mordanting procedure. Since aging 

 improves stains, it is best to make large batches and store for several years, if this is 

 practicable. 



The tissue-culture preparations described below may be dried in air after being 

 fixed primarily for wet squashing. This practice provides some versatility when 

 appropriate attention cannot be given at the time of fixation to follow the precise, 

 step-by-step air-drying procedures described by Rothfels and Siminovitch 1077 and Tjio 

 and Puck. 1322 Air drying of wet-stored cultures is satisfactory only when the cellular 

 population is minimal and the cytoplasm well spread. Following satisfactory wet 

 squashes, duplicate fixations may be rinsed with fresh fixative prior to air drying and 

 storing in a slide box in which the wooden length of vertical slots has been reassembled 

 to accommodate the length of the cover glass. When excellent observations are in the 

 making and duplicate preparations are available, it is best to proceed to complete the 

 slide making. On many occasions, the second observation provides equal or better 

 squashes with the help of features noted in the initial preparation. 



In addition to providing adhering cells for a variety of wet and dry squashing 

 procedures for chromosomal preparations, the cover glass provides cells for general 

 study of clonal (phenotypic) features or responses to agents as well as intraclonal and 

 interclonal variation of chromosomal complements. The culture vessel encourages 

 isolation of clones by means of directing a simple, orally manipulated (latex tube) 

 micropipette while viewing the proceedings under the low power of an inverted micro- 

 scope. The cover glass can be transferred, if desired, to another vessel for pretreatments 

 and the like, so that genetic continuity can be maintained by repopulation of the area 

 barred by removal of the cover glass. Actively proliferating cells along the periphery 

 of the vessel tend to migrate centrally after removal of the cover glass. By so doing, a 

 larger sample of genetically related sublines may be retained each generation without 

 the need for preparing and maintaining an equal number of farming bottles (without 

 cover slips) for replicate samplings and general continuity of the stock. The size of the 

 cover glass can vary, depending on the needs of the experiment and the ability of the 

 strain in question to repopulate and subculture readily following minimal inoculations 

 of cells. In general, 10 x 45 mm., 0-thickness cover glasses are employed with this 

 vessel for purposes of sampling and maintaining sublines with minimal handling. 

 Periodically, auxiliary subcultures are grown in large farming bottles (200-ml. serum 

 bottles, or milk-dilution bottles) for purposes of freezing viable cells, according to the 

 procedures described by Stulberg et al. 1301 and Hauschka et a/. 534 



In our hands, cells grown in 200-ml., serum, farming bottles are prepared for 

 freezing by simply draining off the medium and scraping with a rubber policeman. 

 One ml. of complete medium, adjusted to pH 7.0 and containing 10 per cent 

 glycerol (Merck) is added to the scrapings gathered at the base of the flask. With the 

 aid of a Pasteur pipette two 1.5-ml., Wheaton freezing ampoules are filled with the 



