The Chemical Determination of Deoxyribonucleic Acid in Tissue Cultures

The protein content and dry weight of tissue fragments may vary considerably without any real change in cell number. In view of the rather constant amount of deoxyribonucleic acid per cell found for any given species (1) it has been suggested that it should be used for the measurement of cell number (2). In tissue culture studies where it has been used in this way micro modifications of the procedure of Schmidt and Thannhauser (3, 4) have been employed for the determination of deoxyribonu-cMc acid phosphorus. With very small amounts of tissue this requires considerable technical skill and the fractionation is tedious. For this reason the method has not been generally applied to the measurement of growth in tissue cultures although its theoretical desirability has been recognized. This communication describes a rapid and simple method for deoxyribonucleic acid determinations on amounts of tissue of the order of 1 mg. dry weight. The determination is essentially a direct application of the method of Ceriotti (5). The original method depended on the application of a modification of Dische's indole reaction (6, 7) for deoxyribonucleic acid to a tissue fraction obtained by the method of Ogur and Rosen (8). It was found that 797 the fractionation procedure could be replaced by simple extraction of the tissue with hot perchloric acid, deoxy-ribonucleic acid being estimated by applying Ceriotti's method to the extract. The final method adopted was as follows :1 Method The tissue is washed quickly with BSS to .remove excess medium. (This is usually done by mixing the cells with BSS and centrifuging to separate them from the washing, which is discarded.) Two ml. 1.0 N perchloric acid are added to the tissue in a centrifuge tube and the mixture incubated in a water bath at 70°C. for 20 minutes, stirring occa-1 Reagents: 1. Hanks's balanced salt solution (BSS). 2. 1.0 N perchloric acid. 3. 0.04 per cent indole solution (dissolved by warming). 4. Concentrated hydrochloric acid (36 per cent w/w). 5. Chloroform. 6. DNA standard solution. A few rag. of a pure specimen of DNA are dissolved in 1.0 N perchloric acid by heating at 70°C. Ultraviolet absorption and/or phosphorus content are determined on it. For use this stock standard solution is diluted with 1.0 N per-chloric acid to give a phosphorus content of about 1 #g. per ml. which corresponds to an optical density at 260 In# of about 0.275, a …