The Effect of HCI Hydrolysis on the Retention of Thymidine in DNA*

Allium roots grown in C14-thymidine and H3-thymidine media were treated with N hydrochloric acid at 60°C. as in standard Feulgen hydrolysis. The retention of the radioactive thymidine in DNA as a function of hydrolysis time was studied autoradiographically. No significant loss of label was detected until hydrolysis was extended beyond the optimal time for Feulgen staining. The data are consistent with the assumption that there is no significant loss of DNA during normal Feulgen hydrolysis in the material used. Recently it has been suggested (Woods, 1957) that acid hydrolysis optimum for Feulgen staining of cellular deoxyribose nucleic acid (DNA) results in the loss of nearly two-thirds of the DNA. For this study, anthers of Lilium Iongiflorum ("Croft") were frozen and dehydrated according to the method of Woods and Pollister (1955), fixed in hot 75 per cent alcohol, and hydrolyzed with 10 per cent perchloric acid at 20°C. for periods of time ranging from 15 minutes to 144 hours. The liberation of thymine into the hydrolysates at these time periods was taken as the criterion of D N A loss. The implications of this suggestion to general quanti tat ive cytochemistry of D N A are sufficiently important to warrant reinvestigation of the problem under the standard conditions of the Feulgen procedure. Such a study is reported in this paper. Allium root tips were fixed in alcoholacetic acid mixture, and hydrolysis was carried out with hydrochloric acid. Changes in D N A content of the root tips as a function of hydrolysis time were followed by the use of radioactive isotopes. Material and Methods Allium sp. set bulbs, placed on vials of distilled water, were allowed to germinate roots during 4 days. Two bulbs, each with twenty to thirty roots, were then placed on separate vials with their roots in contact with White's inorganic medium. One of the vials contained * This investigation was supported by a research grant (C-3276) from the National Cancer Institute, United States Public Health Service. approximately 0.24 microcuries Ct4-thymidine per milliliter of White's medium, and the other contained approximately 0.32 microcuries of 55 per cent radiopure H3-thymidine (Schwarz Laboratories, Inc., Mount Vernon, New York) per milliliter of solution. Roots were grown in these media for 72 hours, rinsed with distilled water, fixed for 2 hours in 3 parts ethanol: 1 part glacial acetic acid, and hydrolyzed for lengths of time varying from 0 minutes to 20 minutes in 1 N HCI at 60°C. Squashes in 45 per cent acetic acid were made on microscope slides treated with egg albumen. The tissue on the slides was quick-frozen in liquid air, the coverslips removed, and the slides placed in absolute alcohol. The slides were passed through increasing dilutions of alcohol and finally rinsed three times in distilled water. The tissue was covered with stripping film (Kodak plate "autoradiographic" stripping film Kodak, Ltd., London) after the method of Pelc (see Doniach and Pelc, 1950). Films were exposed for 11 days in the dark under refrigeration (5-7°C), developed with Kodak D-19b, and fixed with Kodak F 5. All autoradiographic slides were examined with a Leitz ultropak objective lens fitted with a phase ring, enabling location of cells by phase contrast microscopy and counting of emulsion silver grains by reflected light. The number of silver grains reduced over a prophase or metaphase figure was counted at X 460 magnification. When the number of grains above one nucleus was counted two or three times, the reproducibility of counts was found to be good (6 per cent error or less). The numbers given in Tables 11 and I l i represent averages where more than one count was made. There was considerable variability in the number of silver grains above nuclei of identical stage and treatment. The exposure of the roots to the radioactive media for as long a period as 72 hours had been an at-