CYTOGENETIC ANALYSIS 501 



present in the majority of tissues available today. On the other hand, colchicine alone 

 or in conjunction with reduced hypotonicity (5 minutes or less), is most adequate for 

 wet squashes when chromosomes are few and cytoplasm is abundant. Examples of 

 the latter, such as the numerous lines from the Chinese hamster, do not have the 

 fuzziness that water-pretreated chromosomes generally display. Clean lines de- 

 lineating the chromosomal contour favor exact identification of chromosomal type and 

 component parts, particularly centromeres, secondary constrictions, and sites of fusion 

 involving euchromatin-heterochromatin. 



The need for colchicine is also reduced when cellular generation times are rapid, 

 and fixation or slight hypotonicity is applied during the log phase of growth. Famili- 

 arity with the cellular type allows the reduction of pretreatments to a minimum, a 

 procedure which promotes the clarity of details. Overspiralization from colchicine 

 pretreatment is helpful when chromosomes are to be counted. However, colchicine 

 reduces the opportunity to identify distinct chromosome types that resemble one 

 another very closely, as in the case of the mouse and the 6-12 group of the human 

 karyotype. There is a real need for methods which will reveal the minute structure of 

 the individual chromosomes (satellites and the like) in attempts to associate their 

 appearance with metabolic and other disorders. 1323 Other aspects to consider are the 

 nature of very short arms, the components of the centromere, appearance of secondary 

 constrictions following chemical pretreatments administered at nontoxic levels, and the 

 need to start correlating component parts of mitotic chromosomes with meiotic bi- 

 valents. 



It is recommended that the degree of hypotonicity be varied when attempting new 

 trials with wet squashes. Hypotonicity appears to be most helpful when preparing 

 solid tissues of normal and malignant derivation for squashing. Nevertheless, by means 

 of colchicine or Colcemid pretreatment of the intact animal, and by using minimal 

 hypotonicity and delaying squashing at least overnight, an adequate start can be made 

 in obtaining satisfactory preparations. It must be remembered that extensive hypo- 

 tonicity is not always necessary and will only lead to chromatid separation and fuzzy 

 outlines. Storage in the fixed state, that is, 3 : 1 fixative added to the natural exudate 

 of the peritoneum or to the culture medium, results in the retention of sufficient 

 softness in practically all tissues encountered to date. 



The choice of stain is a matter of the individual's preference. Propionocarmine 

 or acetocarmine are considered better (by the author) than aceto-orcein for chromo- 

 somes in vitro. One can rely on the availability of clean stain at all times without 

 troublesome precipitates. Since time is an important factor, and one need not refilter 

 the stain, as is required with the use of aceto-orcein, related stains may be preferred 

 when they are equally applicable. The use of iron mordant in conjunction with 

 carmine may make up for the intensity of staining that has become associated with 

 aceto-orcein. 



The procedures of drying in air are not yet accepted by all investigators. For best 

 results, the log phase of growth provides sufficient numbers of mitoses in a sparse to 



