CYTOGENETIC ANALYSIS 479 



the mouse. 781 The simplest procedure used in the writer's laboratory is illustrated by 

 Kato's photograph (figure 62) of an Ehrlich tumor cell pretreated with colchicine in situ 

 at the rate of 25 x 10~ 6 M/10 g. of body weight for six hours. Following removal of 

 the exudate from the peritoneal cavity, tap water (50 per cent by volume) is added as 

 the hypotonic agent for a period of 10 minutes. Finally, fixative is added (three parts 

 95 per cent ethyl alcohol: 1 part glacial acetic acid) at 50 per cent volume of hypotonic 

 exudate, and stored until needed. Fixed cells settle to the bottom of the vial and can 

 readily be pipetted or raised by means of an eyedropper. A drop of fixed cells is 

 placed on a siliconed slide and a drop of acetocarmine is added. Conventional 

 squashing procedures follow, depending on the manner of observation, that is, phase 

 or bright-field microscopy. 



Other pretreatments. — A number of simple practices have resulted in synchronizing 

 mitoses in populations otherwise randomly dividing. For instance, Wildy and 

 Newton 1389 subjected the HeLa cell to cold pretreatment (1 hour at 4° F.) and noted 

 numerous synchronous divisions, some 18 hours later. Such a step would be useful 

 for the evaluation of somatic pairing of homologues of noncolchicinized metaphases and 

 for increasing the incidence of anaphases for purposes of evaluating the action of 

 radiation and drugs. More recently, Nowell 948 noted the stimulatory effect of 

 Bactophytohemagglutinin (Difco Laboratories) on mitoses in leucocytes of human 

 peripheral blood cultures. It can be readily ascertained that increasing the serum 

 concentration (1-5 per cent) of spent basal media to the normal range (10-25 per 

 cent) may also serve to synchronize mitoses. 



A masked secondary constriction on chromosome III of the Chinese hamster 

 suddenly becomes visible following treatment with hyaluronidase for two hours. 1459 

 This enzyme has been quite effective for spreading human chromosomes in bone- 

 marrow aspirates. 855 The alkylating agent, triethylenemelamine (TEM), promotes 

 elongation and separation of chromatids within cells that are readily damaged by the 

 agent (Yerganian, unpublished data). Minimal concentrations of the drug in the 

 range of 1 y/ml. of medium, or less, may readily lead to matrical anomalies that affect 

 the spiralization diameter of major coils. 



Ribonuclease has been utilized by Ohno et al. 96i as an agent to reduce the amount 

 of RNA that masks the allocyclic sex bivalent of the rat and mouse during meiotic 

 prophase. The end-to-end association of the X and Y chromosomes was readily seen 

 after 15 minutes' incubation in 0.1 per cent solution of Armour ribonuclease in physio- 

 logic saline (figure 59). The same authors 965 noted the immediate dispersion of 

 oocytes of newborn rats during a 10-minute period of incubation. Hyaluronidase is 

 reported to be quite effective in dispersing chromosomes and cells of bone marrow. 

 Marshall et a/. 855 placed human bone marrow in a solution containing 15 units of 

 hyaluronidase (Wydase, or Diffusin) per 0.1 ml. of distilled water for a period of one 

 hour. Without squashing, Wright and Giemsa smears were prepared routinely. 

 Marshall (personal communication) states that she and her colleagues "have no support- 

 ing evidence for the hypothesis that bone-marrow cells contain 46 chromosomes. In 



