CHROMOSOMES 



66 



CHROMOTROPE 2R 



of the best fixatives for small mam- 

 malian tissues is that given by LaCour, 

 L. F., Proc. Roy. Soc. Edin. B., 1944, 

 62, 73-85. It is a mi.xture of methyl 

 alcohol (15 parts), 5% acetic acid (5 

 parts), formalin (1 part), and water 

 (5 parts). The proportions may need 

 to be varied according to the material. 

 For a study of the morphology of mouse 

 chromosomes the liver tissue of a new- 

 born mouse is particularly good. 



Chromosome structure. The nuclear 

 cycle, whether in plants or animals, is an 

 alternation between two stable states — 

 spiralization and despiralization. The 

 metaphase chromosomes usually repre- 

 sent a state of maximum spiralization 

 and the chromosomes in the resting 

 nucleus, the maximum of despiraliza- 

 tion or minimum of spiralization. For 

 demonstration of the spiral structure 

 of the chromosomes and unravelling of 

 the coils, fixation should be preceded 

 by some form of pretreatment. Differ- 

 ent methods such as hydration, dessi- 

 cation, exposure to acid vapors or sub- 

 jection to dilute solutions of alkalis, 

 treatment with dilute solutions of salts 

 of strong alkalis and weak acids such 

 as KCN, NaCN, in fact anything that 

 tends to change the pH, have been em- 

 ployed to bring out the real structure 

 (Nebel, B. R., Zeitschr. Zellf. u. Mikr. 

 Anat., 1932, 16, 251-284; Kuwada, Y. 

 and Nakamura, T., Cytologia, 1934, 5, 

 (2), 244-247; Sax, K., and Humphrey, 

 L. M., Bot. Gaz., 1934, 96, 353-362; 

 Huskins, C. L., and Smith, 8. G., Ann. 

 Bot., 1935, 49, 119-150; LaCour, L. F., 

 Stain Tech., 1935, 10, 57-60; Oura, G., 

 Zeit. f. Wiss. Mikr., 1936, 53, 36-37; 

 Kuwada, Y., Shinke, N., and Oura, G., 

 Zeit. f. Wiss. Mikr., 1938,55, 8-16; Cole- 

 man, L. C, and Hillary, B. B., Am. J. 

 Bot., 1941, 28, 464-469; Gopal-Ayen- 

 gar, A. R., Genetics, 1942; Coleman, 

 L. C, Genetics, 1943, 28, 2-8; Ris, IL, 

 Biol. Bull., 1945, 3, 242-257). Some of 

 the best results have been obtained by 

 treatment with K or NaCN 21-^ to 21-5 

 mol. solutions for periods varjdng with 

 the material. 



Stain with aceto-carmine, acetic 

 orcein, acetic lacmoid or Feulgen. If 

 Feulgen is used a counter stain with 

 fast green in acetic acid may be used 

 if desired. The cells are squashed on 

 the slide after staining. The amount 

 of pressure needed is determined by 

 experience. The following schedules 

 of treatment for mouse cliromosomes 

 may be applied mutatis mutandis in the 

 study of chromosomes from other 

 tissues. 



Fix pieces of liver from a newborn 

 mouse in a mixture of methyl alcohol - 

 formalin-acetic acid of LaCour for 15 



min. Wash in 70% ale. Transfer 

 small piece of material on to a slide and 

 add first drop of acetocarnnno or acetic 

 orcein and then coverslip. Gently tap 

 with the rubber tipped end of a pencil 

 until the cells are loosened up and ai'e 

 more or less one layer in thickness. 

 Squeeze out gently the excess of stain. 

 Apply pressure on the coverslip with 

 thumb or by carefully rolling a round 

 edged pencil over it, taking care to see 

 that the coverslip does not slide during 

 the process. The amount of pressure 

 needed is judged by experience. If air 

 bubbles get in add a drop or two of the 

 stain at the edge of coverslip and repeat 

 the process if necessary. Seal edge of 

 coverslip with beeswax and vaseline. 

 If it is desired to make slides permanent 

 follow McClintock's method (McClin- 

 tock, B., Stain Tech., 1929, 4, 53-56). 



For Feulgen-squash preparations fix 

 material as in the preceding outline. 

 Wash in water thoroughly. Hydrolyse 

 in N.HCl at 60°C. for 6-8 min. Trans- 

 fer to Leuco-basic fuchsin for 20 min. 

 to I hr. Pour off stain and add SO2 — 

 water and allow it to remain for 3 min. 

 Change 2-3 times. Place a small piece 

 of material in a drop of 45% acetic acid 

 on a slide and add a coverslip. Gently 

 tap and squeeze out excess of stain as 

 described above. Flatten out the cells 

 by applying pressure with thumb or by 

 rolling a round edged pencil over the 

 coverslip. Transfer slide into large 

 petri dish containing dioxan until cover- 

 slip floats off. The cells will adhere 

 either to the coverslip or slide. Use 

 dioxan balsam as mounting medium. 



Chlorazol black E -|- acetocarmine 

 (Nebel, B. R., Stain Techn., 1940, 15, 

 69-72). Fixation in cold Flemming's 

 fluid plus urea (Hance, R. T., Anat. 

 Rec, 1917, 12, 371-382). Microincin- 

 eration of (Barigozzi, CL, Bull. d'Hist. 

 Appl., 1938, 15, 213-219). Method of 

 localization of genes by experimental 

 deletions, distribution of protein and 

 nucleic acid, classification, etc. (Pain- 

 ter, T. S., J. Roy. Micr. Soc, 1940, 60, 

 161-176). Feulgen stain for chromo- 

 somes (Mensinkai, S. W., J. Roy Micr. 

 Soc, 1939, 59, 82-112). Aceticorcein 

 is advocated as a new stain-fi.xative for 

 chromosomes (LaCour, L., Stain 

 Techn., 1941, 16, 1G9-174). Demonstra- 

 tion of alkaline phosphatase in chromo- 

 somes (Krugelis, E. J., J. Cell. & Comp. 

 Physiol., 1942, 19, 376-379). 

 Chromotrope 2R (CI, 29) — acid phlo.xine 

 GR, chromotrope blue 2R, fast fuchsin 

 G,XLcarmoisine6R — An acid mono-azo 

 dye employed by Lendrum, A. C, J. 

 Path. & Bact., 1935, 40, 415-416 in a 

 study of breast carcinoma and skin 

 lesions as counterstain for celestin blue. 



