76 HEWSON SWIFT 



pared with other tissues in the same organism, the lowest class is also 

 present, and in some cases, such as pancreas and thymus, polyploid classes 

 are also found. The means of these classes, from different tissues, also 

 match each other closely even when the volumes of the different nuclei, 

 and thus the DNA concentrations, vary widely. Certainly the most plausi- 

 ble explanation of such data is that there is a constant relation between the 

 diploid chromosome set and the DNA content, so that each diploid nucleus 

 of a species contains a characteristic amount of DNA, and also that the 

 Feulgen intensity gives an accurate indication of the amount of DNA 

 present. This question is discussed further in Chapter 19. 



It is rather surprising that this relationship holds throughout the concen- 

 tration range encountered in mouse nuclei. In Fig. 6, a linear relationship 

 may be seen to hold even for concentrations of 0.3 g./ml. of DNA, about 

 300 times higher than the optimal concentration found by Lessler^" for 

 gelatin-DNA drops. The protein-DNA ratios must vary rather widely in 

 these nuclei. Evidence for protein interference as described from in vitro 

 studies,'"^ •'^^■"* however, is apparently absent, except possibly in the case 

 of the fixation and pycnosis effects discussed above. 



From these data, and also from data of several other laboratories (see 

 Swift*" for review) one may conclude that the Feulgen reaction can give 

 quantitative information of considerable accuracy. This does not mean it 

 should be accepted indiscriminately as a quantitative method. Where dis- 

 tributions of the type shown in Fig. 6 are obtained, one may conclude with 

 a high degree of probability that the Feulgen reaction is quantitative for 

 such material. If such a relation is not obtained, one will have to determine 

 whether the Feulgen reaction or the photometric technique is at fault, or a 

 real DNA vaiiation is involved. Many biochemical color reactions are 

 subject to variations when applied to different material, and the Feulgen 

 reaction is probably no exception. Anyone using the Feulgen reaction as a 

 quantitative method should obviously watch for variables in technique 

 that may affect some nuclei differently from others. 



In many of the quantitative studies that have been made with the Feul- 

 gen reaction, values have been expressed in arbitrary photometric units, 

 and not in absolute amounts of DNA. Where absolute amounts are desired, 

 in view of the many variables that cause slight alterations in intensity, the 

 simplest procedure is to mount the unknown beside a control tissue with 

 nuclei of known DNA content, such as rat or beef liver, and compare meas- 

 urements from the two sections. Fixation and handhng of the material 

 should be identical with the control. Even so, variation may occur with 

 fixative penetration unless freezing-drying or homogenates are used. Widely 

 different organisms may also show different hydrolysis optima, and so 



