yield dialdehydes (CHO-CHO). The resulting aldehydes are then local- 

 ized by their combination with leiicobasic juchsin (Schiff's reagent) to 

 give a red-colored dye complex. 



Lipids are most easily stained with jat-soluble dyes such as Sudan 

 black B (phospholipids), sudan red, and oil red (neutral fats). The 

 reduction of osmium tetroxide to black oxides by unsaturated fatty acids 

 is a standard method for the routine demonstration of lipids in the cell. 

 Neutral fats and fatty acids may also be demonstrated by the use of 

 Nile blue sulphate. Phospholipids are identified by the acid hematein 

 method which involves their staining with oxidized acid hematoxylin 

 solution following prolonged exposure to chromates. 



The methods used to demonstrate nucleic acids, particularly DNA, 

 are probably the most extensively investigated from the point of view of 

 their validity and mode of action. DNA is best demonstrated by means 

 of the Feulgen reaction. The basic dye, methyl green, is also specific for 

 DNA under specified conditions. The Feulgen reaction depends on the 

 mild acid hydrolysis of tissues to release aldehyde groups of the deoxy- 

 ribose sugar moiety of DNA which then react with Schifi's reagent. The 

 total reaction occurs in two steps: (1) removal of purines of DNA by 

 acid hydrolysis to permit the deoxyribose sugar rings to open and form 

 reactive aldehyde groups, and (2) reaction of the SchifTs reagent with 

 the aldehydes to give the colored complex. 



DNA can be measured quantitatively by photometric analysis of fixed 

 tissues stained by the Feulgen reaction. In this method, the intensity of 

 light transmitted by the Feulgen-stained specimen (/,) compared with 

 that transmitted by a blank part of the slide (/„) is measured by means 

 of a photoelectric cell (Figure 11-6). The ratio IJh X 100 represents 

 the per cent of light transmitted by the Fuelgen-stained specimen. From 

 this information the absorption or extinction coefficient (E) can be cal- 

 culated. The value of E can then be used to calculate the amount of 

 nuclear DNA in arbitrary units. 



In all photometric studies involving the quantitative measurement of 

 cell substances by virtue of their light absorption (Figure 11-7) it is 

 essential to show that the amount of light absorbed by the substance is 

 proportional to the number of absorbing molecules present per unit 

 thickness. This linear relationship between the amount of light absorbed 

 and the number of absorbing molecules is usually expressed as Beer- 

 Lambert's law. In the case of the Feulgen reaction, the Beer-Lambert 

 law appears to hold, indicating that the amount of light absorbed by 

 the stain is a reasonably accurate measurement of the amount of DNA. 



SURVEY OF CYTOLOGICAL TECHNIQUES / 221 



